Palmitic acid-triggered B7H3 palmitoylation promotes immune escape

Domain: chemistry
Published: 2026-01-19
Editorial outcome: major_revision

Abstract
Colorectal cancer responds poorly to immune checkpoint blockade in most patients with microsatellite-stable (MSS) tumors, highlighting the need for alternative targets. B7H3 (CD276) is an immune checkpoint protein that is frequently overexpressed in tumors, but how it is maintained at high protein levels is unclear. Here we show that palmitic acid (PA) promotes B7H3 palmitoylation by the palmitoyltransferase ZDHHC24 at cysteine 496. This modification prevents B7H3 from binding to sequestosome 1 (SQSTM1, also called p62), limiting autophagic degradation and stabilizing B7H3, which suppresses CD8+ T cell antitumor activity. Disrupting this pathway by mutating cysteine 496, or by deleting Zdhhc24 in a colitis-associated colorectal cancer (CAC) mouse model, enhances CD8+ T cell responses. We also develop a cell-penetrant peptide that blocks the ZDHHC24–B7H3 interaction, boosts antitumor immunity, and synergizes with blockade of programmed cell death protein 1 (PD-1). These findings identify B7H3 palmitoylation as a targetable metabolic–immune node for colorectal cancer immunotherapy.

Introduction
Colorectal cancer is the third most common cancer worldwide1. Traditionally, surgery has been the primary treatment, but recent advances suggest that immunotherapy may offer durable and potentially curative outcomes2. Immune checkpoints, such as programmed death-1 (PD-1), programmed death-ligand 1 (PD-L1), and cytotoxic T lymphocyte–associated antigen 4 (CTLA-4), play crucial roles in maintaining self-tolerance and regulating antitumor immunity3–5. These checkpoint pathways involve receptor–ligand interactions that deliver co-inhibitory signals to T cells, thereby suppressing their activation and effector function. Immune check point inhibitors (ICIs) targeting PD-1/PD-L1 are effective primarily in microsatellite instability-high (MSI-H) colorectal cancers, which constitute only about 15% of cases6–9. In contrast, these treatments show limited success in the majority of colorectal cancers that are microsatellite-stable (MSS)10,11. Therefore, it is imperative to conduct an intensive search for alternative immune checkpoints to apply in the treatment of MSS tumors.

The B7 family, comprising a group of structurally related transmembrane proteins, including B7-1, B7-2, B7-H1 (also known as PD-L1), B7-H3, and others, interacts with CD28 family receptors to either stimulate or suppress immune response12–14. B7H3 (CD276) has recently emerged as a compelling immunotherapy target due to its role in suppressing cytotoxic T cell and natural killer cell activity15–18. B7H3 is highly expressed in various malignancies, and its elevated expression is associated with poor prognosis and adverse clinical outcomes19–22. Although B7H3 mRNA is widely detectable across various tissues and cell types, protein expression is comparatively limited, suggesting that posttranscriptional mechanisms tightly regulate its abundance23. Posttranslational modifications (PTMs) of B7H3 remain underexplored, yet they are likely to play important roles in modulating its function in cancer. While phosphorylation and ubiquitination sites have been predicted, N-glycosylation plays a pivotal role in stabilizing B7H3 and ensuring its stable expression on the plasma membrane, thereby facilitating its immunosuppressive function24–26. However, it remains mostly unknown whether other post-translational modifications apart from glycosylation contribute to regulating the expression or function of B7H3. Elucidating these unexplored modifications and their regulatory mechanisms will be critical for developing more effective B7H3-targeted therapies.

S-acylation (often referred to as S-palmitoylation or simply palmitoylation) is an important post-translational modification in which a thioester bond is formed between the sulfhydryl group of a cysteine residue and, most commonly, a 16-carbon palmitate27–30. This PTM leads to an increase in the lipophilicity of the modified target protein, which can affect its stability, function, or subcellular trafficking31–35. Palmitoylation is mainly catalyzed by a relatively conserved family of zinc-finger DHHC (Asp-His-His-Cys) type-containing (ZDHHC) protein acyl-transferases (PATs)36,37. Accumulating evidence suggests that immune-related molecules such as PD-L138 and NOD1/239 can be palmitoylated, indicating that palmitoylation plays an important role in the immune response.

Protein palmitoylation is regulated not only by ZDHHC family enzymes but also by the availability of its active substrate, palmitoyl-coenzyme A. Palmitoyl-coenzyme A, the active metabolite of palmitic acid (PA), reportedly initiates palmitoylation modifications in a variety of proteins40–42. PA is a naturally occurring saturated fatty acid that has been reported to play important roles in immune evasion, metabolism, and inflammation43–45. However, whether PA contributes to the establishment of an immunosuppressive microenvironment in MSS colorectal cancer remains unclear.

In this work, we identify that the palmitoylation of B7H3 triggered by PA is a key driving factor in the formation of the immune-suppressive microenvironment in colorectal cancer. Mechanistically, we show that ZDHHC24 catalyzes B7H3 palmitoylation at cysteine 496 (C496), preventing its interaction with the autophagy receptor SQSTM1/p62 and blocking its autophagic degradation. Biologically, we demonstrate that palmitoylation of B7H3 C496 remodels the immune microenvironment and specifically promotes tumor immune escape by inhibiting the antitumor effects of CD8+ T cells. Based on these findings, we design a peptide (E-peptide) that binds competitively to ZDHHC24. Both in vivo and in vitro experiments demonstrate that E-peptide reduces the palmitoylation of B7H3, promotes its autophagic degradation, and ultimately enhances the anti-tumor activity of CD8+ T cells. Notably, combining E-peptide with anti-PD-1 therapy produces a synergistic effect, significantly improving therapeutic outcomes. These findings provide important insights into immunotherapy for colorectal cancer and identify B7H3 palmitoylation as a promising therapeutic target.

Results
PA-triggered B7H3 palmitoylation enhances B7H3 protein stability
To identify the key immune checkpoints regulating the tumor microenvironment in MSS colorectal cancer, we performed an immunohistochemical analysis of B7 family members using formalin-fixed paraffin-embedded (FFPE) tumor samples from both MSS and MSI-H colorectal cancer patients (cohort 1, Table S1). The results indicated that the B7H3 protein was markedly overexpressed in MSS tumors (Figs. 1A, B and S1A). As an immune checkpoint molecule, B7H3 is expressed not only on tumor cells but also on various immune cell populations. Immunofluorescence stainings showed strong co-localization of B7H3 with the tumor marker epithelial cell adhesion molecule (EPCAM) in tumor tissues (Fig. S1B), indicating prominent expression in tumor cells. Flow cytometry further confirmed that more than 95% of tumor cells are B7H3–positive, compared to about 30% of immune cells (Fig. S1C, D) (cohort 2, Table S2). When normalized to 100,000 total cells, B7H3⁺ tumor cells accounted for approximately 90,000 (Fig. S1E), highlighting tumor cells as the predominant source of B7H3 in MSS tumors. Interestingly, subsequent qPCR analysis revealed no significant alterations in B7H3 mRNA levels in MSS samples compared to MSI-H samples (Fig. S1F) (cohort 3, Table S3), suggesting that PTMs likely play a critical role in regulating B7H3 expression specifically in MSS tumors. Since it has been reported that the FUT8-catalyzed glycosylation of B7H3 can stabilize B7H3 and ensure its stable expression on the plasma membrane25, we next assessed FUT8 expression in MSS and MSI-H colorectal tumor samples via immunohistochemistry. However, no significant difference in FUT8 expression was observed between these groups (Fig. S1G, H). To better understand the underlying mechanisms, we performed RNA sequencing using fresh colon tumor samples (cohort 4, Table S4). Gene Ontology (GO) enrichment analysis revealed that several genes regulating metabolites, such as carboxylic acids, fatty acids, and vitamins, were aberrantly expressed in MSS tumors (Fig. 1C and Supplementary Data 1). Therefore, we speculated that there were underlying metabolic abnormalities in these two types of tumors. We then collected fresh CRC samples for nontargeted metabolite analysis (Figs. 1D and S1I) (cohort 5, Table S5 and Supplementary Data 2) and conducted Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the metabolomics data (Fig. 1E). For the metabolites increased in MSS CRC, we observed the enrichment of pathways related to fatty acid degradation (Fig. 1F). Notably, fatty acid metabolism-associated pathways were also enriched in the GSEA results of the RNA-seq data (Fig. S1J–L). The findings suggest that abnormalities in fatty acid metabolism may be characteristic of MSS tumors. On the basis of those findings, we treated RKO cells with five metabolites associated with fatty acid degradation—glutaric acid, palmitoyl carnitine, hexadecanal, Palmitic acid (PA), and coenzyme A—identified in our metabolomic analysis. Among these, only PA significantly increased B7H3 expression (Fig. 1G, H). To further confirm this observation, we performed dose-dependent experiments in RKO cells and found that increasing PA concentrations led to a concentration-dependent increase in B7H3 protein levels (Fig. S1M). Since PA has been widely reported to trigger palmitoylation, we employed an acyl-biotin exchange (ABE) assay46 to determine whether B7H3 undergoes palmitoylation (Fig. S1N). Our results demonstrated that B7H3 palmitoylation levels increased in response to higher concentrations of PA (Fig. 1I). Moreover, treatment with 2-bromopalmitate (2-BP), a pan-palmitoylation inhibitor, significantly reduced B7H3 palmitoylation (Figs. 1J and S1O). Like that of another member of the B7 family, B7H1 (PD-L1)38, the protein level of B7H3 was closely related to its palmitoylation status (Fig. S1P). Notably, the qPCR results indicated that neither 2-BP nor palmostatin B (an inhibitor of depalmitoylation) affected B7H3 transcript levels (Fig. S1Q). When 2-BP was applied to inhibit palmitoylation, we observed a dose- and time-dependent decrease in B7H3 expression, whereas palmostatin B treatment led to the opposite effect (Figs. 1K and S1R). Given that palmitoylation can also affect protein membrane association, we conducted cytoplasmic and membrane fractionation assays, which revealed a reduction in B7H3 expression within both the cytoplasmic and membrane fractions following 2-BP treatment (Fig. S1S). Immunofluorescence analysis confirmed this decrease in B7H3 expression (Fig. S1T). Additionally, using a cycloheximide (CHX) chase assay, we assessed the impact of B7H3 palmitoylation on protein stability. Treatment with 2-BP markedly accelerated B7H3 degradation (Fig. 1L, M), whereas inhibition of depalmitoylation with palmostatin B slowed down B7H3 degradation (Fig. S1U, V). Taken together, these findings suggest that the PA-triggered palmitoylation contributes to B7H3 stability.

B7H3 C496 palmitoylation inhibits SQSTM1/p62-mediated autophagic degradation
B7H3, as a membrane protein, can in principle be degraded via the ubiquitin–proteasome system (UPS), autophagy–lysosome pathway, or endocytosis to lysosomes47,48. We hypothesized that palmitoylation primarily modulates one of these routes to stabilize B7H3. Therefore, we applied pathway-specific modulators together with 2-BP. Pharmacological agents that disrupt distinct stages of the autophagy–lysosome pathway-3-MA (which inhibits class III PI3K and blocks autophagosome initiation), bafilomycin A1 (BafA1) and chloroquine (both block lysosomal degradation)—effectively blocked the 2-BP-induced degradation of B7H3, whereas the proteasome inhibitor MG132 had no such effect (Fig. 2A, B). Moreover, autophagy induction via EBSS significantly accelerated B7H3 degradation, which was further enhanced by 2-BP (Fig. S2A, B). Together, these observations indicate that B7H3 degradation is predominantly mediated by the autophagy–lysosome pathway. The essential autophagy proteins ATG5 and BECN1 are required for autophagosome formation, and B7H3 degradation was markedly reduced in ATG5- or BECN1-knockout (KO) cells (Figs. 2C, D and S2C, D). Impaired autophagy also counteracted the 2-BP-induced reduction in B7H3 expression (Fig. S2E, F). Selective autophagy relies on cargo receptors that transport specific substrates to autophagosomes for degradation49,50. Among the several cargo receptors tested (SQSTM1, OPTN, NDP52, NBR1, TOLLIP, and NIX), B7H3 exhibited a strong affinity for SQSTM1, as shown by immunoprecipitation assays (Fig. 2E). Additionally, cycloheximide (CHX) chase assays revealed that B7H3 degradation was nearly completely halted in RKO cells lacking SQSTM1 (Fig. 2F, G) and that SQSTM1 knockout prevented the 2-BP-induced degradation of B7H3 (Fig. 2H). These findings collectively demonstrate that B7H3 degradation occurs primarily through SQSTM1-mediated selective autophagy in the cell models examined. To identify the specific palmitoylation site on the B7H3 protein, we examined all 13 of its cysteine residues, five of which were predicted to be palmitoylated using GPS-palm51 and GPS-lipid52 (Fig. 2I). Analysis with the Swiss-Palm database, based on known palmitoyl-proteomes, also revealed a palmitoylation site (Fig. 2I). To validate these predictions experimentally, we generated site-specific cysteine-to-alanine mutants and assessed their palmitoylation levels using ABE assays. Among the mutants, the palmitoylation of C496A was almost undetectable compared with that of wild-type B7H3 (Fig. 2J), highlighting the essential role of C496. C496 is located in the intracellular domain of B7H3 (Fig. S2G) and is highly conserved across species based on multiple sequence alignment analysis performed in Jalview53 (Fig. 2K). Consistently, C496A mutation at the B7H3 palmitoylation site also showed greater affinity for the SQSTM1/p62 cargo receptor than WT B7H3 (Fig. 2L). To explore the functional role of C496 palmitoylation, we utilized CRISPR/Cas9 to generate B7H3-knockout RKO cells (RKOKO) (Fig. S2H) and subsequently established stable cell lines expressing the C496A mutant (RKOC496A) (Fig. S2I). ABE assays confirmed that B7H3 palmitoylation was scarcely detectable in RKOC496A cells compared with that in wild-type (RKOWT) cells (Fig. 2M). A CHX assay revealed that B7H3 protein degradation was accelerated in RKOC496A cells (Fig. 2N, O), similar to the effects of palmitoylation inhibition by 2-BP. Immunofluorescence staining further demonstrated a marked decrease in fluorescence intensity in the RKOC496A cells (Fig. S2J). In addition, we found that treatment with rapamycin, which inhibits mTORC1 activity and thereby induces autophagy, led to accelerated degradation of B7H3 protein in RKOWT cells (Fig. S2K). In contrast, treatment with 3-MA, bafilomycin A1, or chloroquine effectively prevented B7H3 degradation in RKOC496A cells (Fig. S2L). Moreover, confocal microscopy revealed that RKOC496A cells displayed greater colocalization of B7H3 with LC3 and formed more distinct B7H3–SQSTM1/p62 aggregates compared with RKOWT cells (Figs. 2P and S2M), indicating that C496 palmitoylation restrains the autophagic degradation of B7H3. In conclusion, these findings demonstrate that the palmitoylation of B7H3 at cysteine 496 reduces its interaction with the cargo receptor SQSTM1/p62, thereby hindering its selective autophagic degradation.

B7H3 palmitoylation inhibits the antitumor activity of CD8+ T cells
To investigate the function of B7H3 palmitoylation in vivo, we generated B7h3-knockout CT26 (CT26KO) and MC38 (MC38KO) cell lines of mouse colon cancer (Fig. S3A). Since murine B7H3 comprises only a single pair of IgC-like and IgV-like domains24, we engineered C278A mutants (CT26C278A and MC38C278A) that stably express palmitoylation-deficient B7H3, corresponding to the human modification site (Fig. S3B). Palmitoylation in these mutants was barely detectable, as confirmed by ABE assays (Fig. S3C). We then transplanted these modified mouse colon cancer cells subcutaneously into mice. In immunocompetent BALB/c mice, tumor progression was significantly inhibited in the palmitoylation-deficient B7H3 (CT26C278A) group compared with the CT26 WT group (Figs. 3A, B and S3D). However, in highly immunodeficient NCG (NOD ShiLtJ Gpt Prkdcem26Cd52 Il2rgem26Cd22/Gpt) mice, no significant differences in tumor growth were observed between CT26 WT and CT26C278A, as evidenced by comparable tumor weights and volumes (Figs. 3C, D and S3E). Similar results were obtained with the MC38 model, where there was no significant difference between MC38 WT and MC38C278A in NCG mice (Fig. S3F–H). These findings suggest that B7H3 palmitoylation does not affect the proliferation of tumor cells directly; rather, the antitumor effects of B7H3 palmitoylation deficiency depend on the host immune system.
To investigate the impact of B7H3 palmitoylation on the tumor microenvironment (TME), we conducted a comprehensive analysis of CD45+ immune cells infiltrating two types of CT26 tumors in BALB/c mice using spectral flow cytometry. In summary, we identified 14 unique cell subpopulations (Fig. 3E, F). The results revealed that some immunosuppressive cells, including monocytic myeloid-derived suppressor cells (M-MDSCs) and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), were reduced in palmitoylation-deficient (C278A) tumors (Figs. 3G and S3I). In contrast, some cell populations with antitumor effects, including CD8+ T, CD4+ T, and natural killer (NK) cells, were increased in C278A tumors (Figs. 3G and S3I). These results indicate that the mutation of B7H3 palmitoylation substantially alters the immune landscape within the TME, particularly by promoting CD8+ T cell infiltration (Figs. 3G, H and S3I). To test whether CD8⁺ T cells are essential mediators of antitumor effects caused by B7H3 palmitoylation deficiency, we depleted CD8⁺ T cells in vivo using an anti-CD8α antibody (Fig. 3I). Strikingly, depletion of CD8⁺ T cells completely abrogated the tumor growth difference between wild-type B7H3 and the palmitoylation-deficient C278A mutant (Figs. 3J, K and S3J), indicating that the antitumor effect associated with loss of B7H3 palmitoylation is primarily dependent on CD8⁺ T cells. To validate these findings, we subcutaneously transplanted the MC38 B7h3 knockout cell line (MC38KO) and the stable MC38 WT and MC38C278A cell lines into C57BL/6J mice. The results revealed that the mutation of the palmitoylation site significantly suppressed tumor growth, an effect that was even more pronounced in the B7h3 knockout (MC38KO) group (Figs. 3L, M and S3K). An analysis of CD8+ T cell infiltration revealed that the MC38C278A group presented a significantly greater proportion of CD8+ T cells than the MC38 WT group did, a trend that was further amplified in MC38KO tumors (Figs. 3N and S3L). Immunofluorescence staining of tumor tissues corroborated those findings (Fig. 3O, P). Furthermore, the expression of effector molecules of CD8+ T cells, such as granzyme B (GZMB) and interferon-gamma (IFN-γ), was higher in MC38C278A tumors compared with MC38 WT tumors (Figs. 3Q, R and S3M, N). To confirm these observations, we performed a T cell-mediated tumor-killing assay by coculturing activated human primary T cells with the RKOKO, RKOWT, and RKOC496A cell lines. The RKOC496A mutation markedly enhanced the cytotoxicity of T cells to tumor cells (Figs. 3S and S3O). Consistent with the flow cytometry findings, the lactatedehydrogenase (LDH) cytotoxicity assay yielded similar results (Fig. 3T). Finally, using tissue microarrays of colorectal cancer samples, we examined the correlation between B7H3 expression and CD8+ T cell infiltration. Patients with high B7H3 expression had shorter overall survival and significantly fewer CD8+ T cells than did those with low B7H3 expression (Fig. S3P, Q). Consistently, Kaplan–Meier Plotter analysis further confirmed that high B7H3 expression is associated with poorer overall survival (Fig. S3R), and TIMER database analysis showed that high B7H3 (CD276) expression correlated with lower CD8⁺ T cell infiltration (Fig. S3S). Furthermore, gene set enrichment analysis (GSEA) based on RNA-seq data from colorectal tumor samples (cohort 4, Fig. 1C) revealed that the gene set negative regulation of T cell activation was significantly enriched in MSS tumors (Fig. S3T). In contrast, pathways related to T cell function, including CD8-positive, alpha-beta T cell differentiation and the T cell receptor signaling pathway, were negatively enriched in MSS tumors (Fig. S3U, V), suggesting impaired cytotoxic T cell functionality. Collectively, these findings suggest that B7H3 palmitoylation enhances its immunosuppressive function by limiting CD8+ T cell infiltration and activity, thereby contributing to tumor immune evasion.

ZDHHC24 mediates the palmitoylation of B7H3 at C496 and is correlated with poor survival in CRC patients
To identify which ZDHHC(s) catalyze B7H3 palmitoylation, we co-expressed each of the 23 FLAG-tagged ZDHHC enzymes with HA-B7H3. ZDHHC24 significantly increased the palmitoylation level of B7H3, whereas ZDHHC3, ZDHHC8, and ZDHHC13 only slightly increased the B7H3 palmitoylation level (Fig. S4A). Subsequent transfection of RKO cells with ZDHHC3, ZDHHC8, ZDHHC13, or ZDHHC24 indicated that only ZDHHC24 substantially increased B7H3 expression (Fig. S4B–E). Those findings led us to hypothesize that ZDHHC24 is the primary enzyme responsible for B7H3 palmitoylation. To confirm the interaction between B7H3 and ZDHHC24, we conducted coimmunoprecipitation (co-IP) assays, which revealed a physical interaction at the endogenous level in RKO and SW480 cells (Fig. 4A). Consistent with these findings, we observed the colocalization of B7H3 and ZDHHC24 through immunofluorescence (Fig. 4B). To assess the functional role of ZDHHC24, we used short hairpin RNA (shRNA) to stably knockdown ZDHHC24 in human RKO cells and Zdhhc24 in murine MC38 cells (Fig. S4F, G). ABE assays subsequently revealed a significant decrease in B7H3 palmitoylation levels following ZDHHC24 or Zdhhc24 knockdown (Figs. 4C and S4H). In line with the above results, ZDHHC24 or Zdhhc24 knockdown also led to a reduction in endogenous B7H3 protein expression (Fig. 4D, E), an effect further validated by confocal microscopy (Fig. 4F). Notably, the transcript levels of B7H3 showed minimal changes following ZDHHC24 knockdown (Fig. S4I). To confirm this, we performed a CHX assay and found that ZDHHC24 knockdown accelerated the degradation of B7H3 protein (Fig. 4G, H). Additionally, when Flag-ZDHHC24 was transfected into RKOWT and RKOC496A cells, an increase in endogenous B7H3 expression was observed exclusively in RKOWT cells but not in palmitoylation-deficient RKOC496A cells (Fig. 4I). Moreover, we assessed the palmitoylation levels of RKOWT and RKOC496A following ZDHHC24 transfection. The results indicated that ZDHHC24 increased palmitoylation of RKOWT, but not that of the palmitoylation site-deficient RKOC496A variant (Fig. 4J). Interestingly, co-IP experiments also revealed that in the palmitoylation-deficient mutant RKOC496A, B7H3 exhibited a stronger interaction with SQSTM1/p62 proteins compared to RKOWT, and that ZDHHC24 reduced this interaction in RKOWT but not in RKOC496A cells (Fig. 4K). In addition, ZDHHC24 knockdown promoted punctate accumulation and colocalization of B7H3 and p62, as demonstrated by immunofluorescence (Fig. 4L). This enhanced interaction was further confirmed by proximity ligation assay (PLA) (Fig. 4M). An analysis of the Gene Expression Profiling Interactive Analysis (GEPIA) database revealed substantial ZDHHC24 overexpression in multiple tumor types, particularly in colorectal cancer (Fig. S4J). Tissue microarray analysis of colorectal cancer specimens revealed that patients with high ZDHHC24 expression had shorter overall survival (Fig. 4N), a finding further supported by Kaplan–Meier Plotter database analysis, which linked high ZDHHC24 expression to worse relapse-free survival in colon cancer (Fig. S4K). Moreover, ZDHHC24 expression was markedly higher in tumor tissues than in adjacent normal tissues and was positively correlated with clinical tumor stage (Fig. 4O, P). It is worth noting that no significant differences in B7H3 or ZDHHC24 protein levels were detected between MSS and MSI-H subtypes in colorectal cancer cell lines (Fig. S4L). However, patient-derived tumor specimens showed that ZDHHC24 expression was significantly higher in MSS tumors than in MSI-H tumors (Fig. S4M). This suggests that the in vivo tumor microenvironment, which is absent in standard cell culture systems, may modulate ZDHHC24 expression. Importantly, ZDHHC24 expression was positively correlated with B7H3 levels in tissue microarray samples (Fig. 4Q). Together, these findings highlight the critical role of ZDHHC24-mediated B7H3 C496 palmitoylation in maintaining B7H3 protein stability and underscore its association with poor survival outcomes in patients with colorectal cancer.

ZDHHC24 promotes immunosuppression in colorectal cancer
To examine the biological function of ZDHHC24 in vivo, we generated Zdhhc24 floxed (Zdhhc24fl/fl) mice (Fig. S5A) and crossed them with Villin-Cre transgenic animals (Fig. S5B) to generate intestinal epithelial cell (IEC) specific Zdhhc24-deficient (Zdhhc24ΔIEC) models (Fig. S5C). The efficacy of Cre recombinase was confirmed by crossing the Villin-Cre line with Rosa26-CAG-LSL-tdTomato reporter (Fig. S5D) mice. As expected, intestinal epithelial cells presented specific red fluorescence under the microscope (Fig. S5E). We further isolated IECs to verify the specific deletion of Zdhhc24 in Zdhhc24ΔIEC mice (Fig. S5F). Subsequently, we investigated the role of ZDHHC24 in CRC malignancy by establishing a colitis-associated colorectal (CAC) carcinogenesis mouse model (Fig. 5A). During CAC induction, Zdhhc24ΔIEC mice presented less weight loss than Zdhhc24fl/fl mice did (Fig. 5B). At the end of the observation period, the Zdhhc24fl/fl mice had slightly shorter colons than their Zdhhc24ΔIEC littermates did (Figs. 5C and S5G). Additionally, Zdhhc24ΔIEC mice developed fewer tumors (Fig. 5D, E) and presented significantly fewer large tumors (>~1 mm) (Fig. 5F, G). We collected tumor tissues from Zdhhc24ΔIEC and Zdhhc24fl/fl and assessed B7H3 protein expression and palmitoylation levels. B7H3 protein expression and palmitoylation were both reduced in Zdhhc24ΔIEC mice (Fig. S5H, I). Histological examination revealed that Zdhhc24ΔIEC mice presented reduced colon thickening, inflammation, hyperplasia, and injury in the affected regions (Fig. 5H). Immunofluorescence analysis also revealed increased CD8+ T cell infiltration in tumors from Zdhhc24ΔIEC mice (Fig. 5I). These findings suggest that ZDHHC24 modulates antitumor immunity during colon tumor development. To validate these results, we transplanted Zdhhc24-knockdown mouse colon cancer cells (MC38) subcutaneously into C57BL/6J mice. Tumor growth was markedly reduced in the Zdhhc24-knockdown group compared with the control group, as shown by tumor growth curves and final tumor weights (Figs. 5J, K and S5J). Flow cytometry of single-cell suspensions from these tumors revealed a substantial increase in CD8+ T cell infiltration in the Zdhhc24-knockdown group (Figs. 5L and S5K), an effect that was corroborated by immunofluorescence analysis (Fig. S5L, M). Furthermore, the levels of functional markers, including GZMB and IFN-γ, were significantly greater in tumor-infiltrating CD8+ T cells in the Zdhhc24-knockdown group than in those in the control group (Figs. 5M, N and S5N, O). We subsequently established an in vitro cytotoxic T-cell killing assay for validation. Compared with control cells, cells with shRNA-mediated ZDHHC24 knockdown displayed increased susceptibility to apoptosis when activated T cells derived from human peripheral blood mononuclear cells were exposed (Fig. S5P, Q). This finding was supported by increased LDH levels in the culture supernatants of the respective cell populations (Fig. S5R). Additionally, IHC analysis of CRC tissue microarrays revealed that patients with high ZDHHC24 expression had lower CD8+ T cell counts in tumor tissues (Fig. 5O). A three-dimensional scatter plot further illustrated the correlation among ZDHHC24, B7H3, and CD8+ T cells (Fig. 5P). Collectively, these results suggest that ZDHHC24 promotes immunosuppression in colorectal cancer by inhibiting CD8+ T cell-mediated antitumor responses.

B7H3 palmitoylation is necessary for the ZDHHC24-mediated suppression of tumor immunity
To further investigate the relationship between B7H3 palmitoylation and ZDHHC24 in modulating the tumor immune microenvironment, we stably overexpressed Zdhhc24 in MC38WT and MC38C278A cells (Fig. S6A) and subcutaneously implanted these cells into C57BL/6J mice. ZDHHC24 significantly promoted tumor growth in the MC38WT group; however, this effect was nearly absent in the MC38C278A group (Figs. 6A, B and S6B). An analysis of immune cell infiltration revealed a greater proportion of CD8+ T cells in MC38C278A tumors, whereas ZDHHC24 overexpression reduced CD8+ T cell levels in MC38WT tumors but had no effect on MC38C278A tumors (Figs. 6C and S6C). These results were consistent with the immunofluorescence findings (Fig. 6D, E). Furthermore, CD8+ T cells expressing GZMB and IFN-γ were more common in MC38C278A tumors than in MC38WT tumors (Fig. 6F, H). While ZDHHC24 decreased the levels of GZMB- and IFN-γ-expressing CD8+ T cells in MC38WT tumors, it had no effect on MC38C278A tumors (Fig. 6F–I). In vitro, cytotoxic T cell assays with RKOWT and RKOC496A cells overexpressing ZDHHC24 (Fig. S6D) demonstrated that ZDHHC24 did not affect apoptosis in the absence of T cells (Figs. 6J and S6E). However, in the presence of T cells, ZDHHC24 significantly reduced apoptosis in RKOWT cells but not in RKOC496A cells (Figs. 6J, K and S6E). Collectively, these findings confirm that ZDHHC24-mediated tumor immunosuppression depends on B7H3 palmitoylation.

Inhibition of B7H3 palmitoylation with synthetic peptides
Given the critical role of ZDHHC24-mediated palmitoylation of B7H3 at C496 in promoting an immunosuppressive microenvironment in colorectal cancer, we further investigated the therapeutic potential of targeting B7H3 palmitoylation. To better understand the binding interaction between ZDHHC24 and B7H3, we analyzed the structural domains of B7H3. As an immune checkpoint protein, the extracellular Ig-like domain of B7H3 likely contributes to antigen recognition and immunomodulation. Thus, we hypothesized that the primary binding sites for ZDHHC24 are located within the transmembrane and intracellular regions. To test this hypothesis, we generated B7H3 constructs with targeted deletions in specific structural domains and conducted immunoprecipitation with ZDHHC24 (Fig. 7A). Our results revealed that amino acids 488–497 in the B7H3 sequence are essential for its interaction with ZDHHC24 (Fig. 7B). Notably, this region is highly conserved in human and mouse and encompasses the palmitoylation site in B7H3, underscoring its functional relevance. We subsequently designed a peptide (E-peptide) corresponding to the B7H3 488–497 amino acid sequence, and a control peptide (C-peptide) in which the same amino acids were randomly scrambled to disrupt the sequence-specific structure and abolish potential binding, following a previously described strategy54. Both peptides were fused with an N-terminal transcriptional activator of transcription (TAT) tag to facilitate cellular uptake (Fig. 7C). We assessed the cytotoxicity of the peptides in vitro and determined that cytotoxicity was observed only at concentrations exceeding 50 μM (Fig. S7A). We then attached a fluorescent motif to the first lysine of both the E-peptide and C-peptide (Fig. S7B) and confirmed its cellular entry via fluorescence microscopy (Fig. S7C). To evaluate the functionality of the synthesized peptides, we treated cells with E-peptide and C-peptide. In contrast to C-peptide, E-peptide significantly inhibited the binding of ZDHHC24 to B7H3, suggesting that E-peptide effectively competes with B7H3 for binding to ZDHHC24 (Fig. 7D). In parallel, we employed surface plasmon resonance (SPR) analysis to confirm the direct interaction between the E-peptide and ZDHHC24 (Fig. S7D, E). We further assessed B7H3 palmitoylation levels and found a significant reduction in palmitoylation with E-peptide treatment but not with C-peptide treatment (Fig. 7E). Notably, the decrease in B7H3 protein levels was dose dependent in RKOWT cells treated with E-peptide, whereas neither peptide affected B7H3 expression in RKOC496A cells (Fig. 7F). Additionally, E-peptide, but not C-peptide, markedly reduced B7H3 palmitoylation in RKO control cells, with limited effects in ZDHHC24 knockdown cells (Figs. 7G and S7F). Similarly, E-peptide treatment also reduced B7H3 protein levels in RKO control cells, but the decrease was not significant in ZDHHC24 knockdown cells (Fig. 7H, I). Moreover, co-immunoprecipitation analysis showed that E-peptide treatment enhanced the interaction between B7H3 and p62 (Fig. 7J). To determine the impact of synthesized peptides on immune function in vitro, RKO cells were treated with either E-peptide or C-peptide and subsequently coincubated with activated human CD8+ T cells. Both flow cytometry and lactate dehydrogenase toxicity assays demonstrated that E-peptide effectively enhanced CD8+ T cell killing (Figs. 7K, L and S7G). In conclusion, these data suggest that E-peptide inhibits B7H3 palmitoylation and promotes its degradation by disrupting the interaction between B7H3 and ZDHHC24, thereby effectively enhancing CD8+ T cell-mediated cytotoxicity in vitro.

E-peptide enhances antitumor immunity in vivo
To further evaluate the in vivo efficacy of the synthesized small-molecule peptides, we conducted a toxicity assessment. Mice received five different concentrations of peptides via intraperitoneal injection (Fig. S8A). Only one mouse died at each of the 25 and 50 mg/kg doses, while all others survived throughout the observation period (Fig. S8B). At the end of the experiment, the surviving mice were sacrificed, and peripheral blood and organ tissues were collected for analysis. Hematoxylin–eosin (HE) staining revealed mild hepatic steatosis at the 50 mg/kg dose, but no significant histological abnormalities were observed in other organs (Fig. S8C). Additionally, no notable changes were observed in the levels of creatinine (CRE), alanine aminotransferase (ALT) or aspartate aminotransferase (AST) in the peripheral blood (Fig. S8D–F), indicating minimal systemic toxicity at lower concentrations. To evaluate hemocompatibility, we performed in vitro hemolysis assays and measured prothrombin time (PT) and activated partial thromboplastin time (APTT). The E-peptide did not induce hemolysis (Fig. S8G, H) or prolong PT or APTT (Fig. S8I, J), demonstrating good blood compatibility under the tested conditions. Next, we subcutaneously transplanted MC38 cells into C57BL/6J mice and administered the peptides intraperitoneally (10 mg/kg) after transplantation, with normal saline serving as a control (Fig. 8A). To verify intratumoral peptide infiltration, a separate cohort received 5′-FITC-conjugated peptides via intraperitoneal injection. Immunofluorescence of tumor sections confirmed effective infiltration of the peptides into the tumor, establishing a spatial basis for their biological activity (Fig. S8K). We then assessed the effect of E-peptide treatment on B7H3 expression and its palmitoylation in tumor tissues. E-peptide administration significantly reduced both total B7H3 protein and its palmitoylation levels (Fig. S8L, M). Importantly, compared with NS and C-peptide, E-peptide significantly slowed tumor growth (Fig. 8B–D), and similar results were observed in BALB/c mice (Fig. 8E–G). Flow cytometry analysis of CD8+ T cells in BALB/c mice revealed a significant increase in CD8+ T cell infiltration within the tumor microenvironment following E-peptide treatment, whereas no such increase was observed with C-peptide (Figs. 8H and S8N). Moreover, the proportion of IFN-γ+ CD8+ T cells and GZMB+ CD8+ T cells—key indicators of tumor-killing activity—was significantly elevated after E-peptide treatment (Fig. 8I–L). To further evaluate the therapeutic efficacy of the E-peptide in a human immune context, we employed immune-reconstituted humanized mice bearing subcutaneous SW480 human colorectal tumors. Mice were treated with either the control C-peptide or E-peptide (Fig. 8M). E-peptide treatment significantly suppressed tumor growth (Fig. 8N–P), enhanced CD8⁺ T cell infiltration (Fig. 8Q, R), and increased the expression of cytotoxic effector molecules GZMB and IFN-γ in the tumor microenvironment (Figs. 8S–V). Together, these results demonstrate that the E-peptide effectively enhances CD8⁺ T cell-mediated antitumor immunity in vivo.

The combination of E-peptide and anti-PD-1 therapy exerts a significant antitumor effect
A single immune checkpoint inhibitor may be insufficient to address the complexities of the tumor immune microenvironment. Increasing evidence suggested that combination therapies involving immune checkpoint inhibitors yield better clinical outcomes than monotherapies do55,56. Therefore, we hypothesized that E-peptide synergizes with PD-1 blockade, leading to more effective tumor suppression. We initially subcutaneously injected BALB/c mice with luciferase-expressing CT26 cells, followed by the intraperitoneal administration of anti-PD-1, E-peptide, or a combination of anti-PD-1 and E-peptide, with the IgG group serving as the control (Fig. S9A). The results demonstrated that monotherapies with either E-peptide or anti-PD-1 antibodies had moderate antitumor activity. However, the combination treatment significantly enhanced tumor inhibition, reducing the tumor size to a minimal level (Fig. 9A, B). To validate these findings, we expanded the sample size for each group and conducted a 60-day observation. The results confirmed that, compared to E-peptide or anti-PD-1 monotherapy, the combination therapy significantly inhibited tumor growth and even led to complete tumor regression in one mouse (Fig. 9C–G). Survival data (Fig. 9G) show a mean survival of 18.1 days in the IgG control, versus 24.4 days with anti-PD-1 (+34.8%) and 25.2 days with E-peptide (+39.2%). The combination of E-peptide and anti-PD-1 extended mean survival to 40.5 days (~124% over IgG). To assess interaction under the independent-action assumption, we estimated the Bliss-expected combination effect for E-peptide plus anti-PD-1 from their single-agent effects (defined as the normalized improvement in mean survival over IgG). This yielded an expected additive improvement of 60.4%, corresponding to a predicted mean survival of 29.0 days. The observed mean survival (40.5 days) exceeds the Bliss expectation, consistent with a synergistic interaction. Additionally, we validated the findings in C57BL/6J mice, which yielded results consistent with those observed in the BALB/c model (Figs. 9H, I and S9B). Moreover, flow cytometry analysis was performed to assess the impact of different treatments on tumor-infiltrating CD8+ T cells. The results demonstrated that the combination of E-peptide and anti-PD-1 antibodies significantly increased the number of CD8+ T cells and enhanced their functional activity (Figs. 9J–L and S9C–E). In summary, our findings emphasize the pivotal role of ZDHHC24-mediated B7H3 palmitoylation in promoting immune escape in colorectal cancer and demonstrate that targeting this modification could enhance antitumor immune responses, suggesting a promising strategy for improving the efficacy of immunotherapy in patients with colorectal cancer.

Methods
Cell lines and cell culture
The human colon cancer cell lines RKO and SW480, the human embryonic kidney cell line HEK293T, and the murine colon cancer lines MC38 and CT26 were purchased from the American Type Culture Collection (ATCC). RKO and HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA), whereas SW480 cells were cultured in Leibovitz’s L-15 medium (Gibco, USA). MC38 and CT26 cells were cultured in RPMI-1640 medium (Gibco, USA). All culture media were supplemented with 10% fetal bovine serum (Thermo Fisher, USA). RKO, HEK293T, MC38, and CT26 cells were maintained at 37°C in a humidified incubator with 5% CO2, whereas SW480 cells were maintained at 37°C in a CO2-free atmosphere. All cell lines were routinely tested for mycoplasma contamination using a mycoplasma PCR detection kit (BMC1040, Abbkine).

Antibodies and chemicals
The following commercially available primary antibodies were used: anti-B7H3 (ab134161, abcam, [WB: 1:200]; ab227679, abcam [IF: 1:100]; 66481-1-Ig, proteintech, [WB: 1:1000; IHC: 1:2500]); anti-ZDHHC24 (ARP55989_P050, aviva systems biology, [WB: 1:1000]; HPA016619, Atlas antibodies, [IHC: 1:50]; and ABIN6743942, antibodies online, [WB: 1:1000]); anti-Flag tag (14793S, cell signaling technology, [WB: 1:1000]); anti-HA tag (3724T, cell signaling technology, [WB: 1:1000]); anti-GAPDH (sc-47724, santa cruz biotechnology, [WB: 1:200] and A19056, ABclonal, [WB: 1:5000]); caveolin-1 (A19006; ABclonal, [WB: 1:5000]); anti-PD-L1 (29122, cell signaling technology, [WB: 1:1000]); horseradish peroxidase (HRP)-streptavidin (A21000, abbkine, [WB: 1:10000]); anti-SQSTM1/p62 (HA721171, HUABIO, [WB: 1:5000; IHC: 1:1000]); anti-CD326/EpCAM (A25344, ABclonal, [IF: 1:200]); anti-ATG5 (ET1611-38, HUABIO, [WB: 1:5000]); anti-Beclin 1 (R381896, zenbioscience, [WB: 1:1000]). The following secondary anti-bodies were used in Western blot assays: goat anti-rabbit IgG secondary antibody (HRP) (SSA004; Sino Biological Inc), goat anti-mouse IgG secondary antibody (HRP) (SSA007; Sino Biological Inc), HRP-conjugated goat anti-mouse IgG light chain (AS0-62; ABclonal), and HRP-conjugated mouse anti-rabbit IgG light chain (AS061; ABclonal). All Western blot secondary antibodies were used at a dilution of 1:5000. The secondary antibodies used for immunofluorescence (IF) assays were as follows: Cy3, goat anti-rabbit IgG (A22220, Abbkine); Cy3, goat anti-mouse IgG (A22210, Abbkine); D-light 488, goat anti-rabbit IgG (A23220, Abbkine) and DyLight 488, goat anti-mouse IgG (A23210, Abbkine); goat anti-human IgG H&L (FITC) (HY-P83649, MCE); goat anti-human IgG H&L (AF594) (HY-P83648, MCE). All IF secondary antibodies were used at a dilution of 1:200. Antibodies for flow cytometry were listed: Zombie NIR Fixable Viability Kit (423106, BioLegend), BD Pharmingen™ FITC mouse anti-human CD45 (560976, BD), BD OptiBuild™ BV421 mouse anti-human NCAM-1 (CD56) (744217, BD), BD OptiBuild™ BV510 mouse anti-human CD11b (742638, BD), BD OptiBuild™ BV650 mouse anti-human CD14 (740633, BD), PE anti-human CD68 antibody (333808, BioLegend), BD pharmingen™ PerCP-Cy™ 5.5 mouse anti-human CD3 (552852, BD), BD OptiBuild™ RB780 mouse anti-human CD276 (B7-H3) (755486, BD), BD OptiBuild™ R718 mouse anti-human HLA-DR (752501, BD), BD horizon™ BV605 mouse anti-human CD19 (569363, BD), BD OptiBuild™ BV786 mouse anti-human CD16 (741042, BD), BD pharmingen™ APC mouse anti-human CD326 (EpCAM) (566842, BD), BD OptiBuild™ BV650 Mouse Anti-Human CD8 (743067, BD), APC anti-human/mouse Granzyme B (372204, BioLegend), BD pharmingen™ PE mouse anti-human IFN-γ (554552, BD), brilliant violet (BV) 785-conjugated anti-mouse CD45 antibody (103149, BioLegend), BV510-conjugated anti-mouse CD3 antibody (100234, BioLegend), BV785-conjugated anti-mouse CD3 antibody (100232, BioLegend), BV650-conjugated anti-mouse CD8 antibody (100742, BioLegend), PE/cyanine7-conjugated anti-human/mouse Granzyme B recombinant antibody (372214, BioLegend), PerCP/Cyanine5.5-conjugated anti-mouse IFN-γ antibody (505822, BioLegend), FITC-conjugated anti-active Caspase-3 (559341, BD), PE hamster anti-mouse γδ T-cell receptor (553178, BD), Alexa Fluor™ 647 rat anti-mouse CD206 (568808, BD), PE-CF594 rat anti-mouse CD49b (562453, BD), BUV661 rat anti-mouse CD93 (741574, BD), BUV737 rat anti-CD11b (612800, BD), BV510 rat anti-mouse F4/80 (743280, BD), PE-Cy7 rat anti-mouse Ly-6G (560601, BD), R718 rat anti-mouse Ly-6C (566987, BD), BUV496 rat anti-mouse CD4 (741050, BD), BUV563 hamster anti-mouse CD3e (741235, BD), BUV805 rat anti-mouse CD45 (748370, BD), BV421 rat anti-mouse Foxp3 (562996, BD), V450 rat anti-mouse CD45R (560472, BD), BV711 rat anti-mouse CD86 (740688, BD), BV650 hamster anti-mouse CD11c (564079, BD), Zombie Yellow™ fixable viability kit (423104, BioLegend), FITC anti-mouse Ly-6G/Ly-6C (Gr-1) antibody (108406, BioLegend), PE/Fire™ 640 anti-mouse I-A/I-E antibody (107664, BioLegend), PE/cyanine7 anti-mouse CD8a antibody (100722, BioLegend). For flow cytometry staining, viability dyes were used at 1:500 (per 2×106 cells in 100 μL), and all other fluorochrome-conjugated antibodies were used at 2.5 μL per 1×106 cells in 100 μL. The reagents used as follows: phenylmethylsulfonyl fluoride (PMSF) (HY-B0496; MCE), phosphatase inhibitor cocktail I (HY-K0021; MCE), phosphatase inhibitor cocktail II (HY-K0022; MCE), chloroquine (HY-17589A; MCE) 2-bromohexadecanoic acid (HY-111770; MCE), palmostatin B (HY-120911; MCE), N-ethylmaleimide (04260; Millipore Sigma), hydroxylamine solution (467804; Millipore Sigma), 3-MA (HY-19312; MCE), MG-132 (HY-13259; MCE), cycloheximide (HY-12320; MCE), EZ-LinkTM BMCC-biotin (21900; Thermo Fisher Scientific), NH4Cl (HY-Y1269; MCE), anti-fade reagent (with DAPI) (HY-K1048; MCE), PMA (HY-18739; MCE), ionomycin (HY-13434A; MCE), monensin (HY-N0150; MCE), brefeldin A (HY-16592; MCE).

Plasmids
23 Flag-tagged ZDHHC family protein-encoding plasmids (n=23) were obtained from Miao Ling Bio, China (catalog numbers: P44768, P44768, P44770, P41465, P43454, P44771, P43506, P43467, P44775, P44767, P44779, P44777, P44769, P44783, P44773, P44780, P44390, P41826, P43407, P44772, P41821, P41823, P44-776). Expression vectors encoding pcDNA3.1-HA-B7H3, pcDNA3.1-Flag-SQSTM1/p62, pcDNA3.1-Flag-OPTN, pcDNA3.1-Flag-NDP52, pcDNA3.1-Flag-TOLLIP, pcDNA3.1-Flag-NIX and pcDNA3.1-Flag-NBR1 plasmids were constructed by insertion of the indicated DNA into the indicated vector. The pcDNA3.1-HA-B7H3-C496A was performed by using MutExpress II Fast Mutagenesis Kit V2 (C21401 Vazyme) according to the manufacturer’s instructions. The sequences are listed in Table S6 (Supplementary Information). All plasmids were confirmed by sequencing. Plasmids were first transformed into competent DH5α Escherichia coli (TSC-C14, Beijing Tsingke Biotech Co., Ltd.), and plasmid DNA for transfection was prepared using a plasmid miniprep kit (D6904, OMEGA) according to the manufacturer’s instructions. Cells cultured in dishes were starved with serum-free medium for 8 h. The indicated plasmids and transfection reagents (HighGene plus, RM09014P, abclonal) were then mixed well and waited for 20 min before being uniformly transferred to the culture dish. After 8 h, the complete medium containing serum was replaced and waited for 48 h.

RNA sequencing
Total RNA was isolated using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol. The RNA’s purity and concentration were assessed via a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), while RNA integrity was confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). For transcriptome sequencing, libraries were prepared using the VAHTS Universal V6 RNA-seq Library Prep Kit as per the manufacturer’s guidelines. Sequencing was conducted on an Illumina Novaseq 6000 platform, generating 150 bp paired-end reads. Initial raw reads in FASTQ format underwent quality filtering with fast to yield clean reads. Clean reads were aligned to the reference genome using HISAT2, with gene expression levels quantified in fragments per kilobase million (FPKM) and gene read counts obtained through HTSeq-count. Differential expression analysis was performed using DESeq2, identifying differentially expressed genes (DEGs) with a threshold of p<0.05 and fold change ≥2; genes with base man <10 were excluded. Functional enrichment analysis of DEGs was performed using a hypergeometric test to identify significantly enriched Gene Ontology (GO) terms.

Non-targeted metabolomics analysis
Non-targeted LC–MS-based metabolomics was performed on 18 colorectal tumor samples (MSS, n=8; MSI-H, n=10), each representing an independent biological replicate. In addition, pooled quality-control (QC) samples were prepared by combining equal aliquots of all extracts and were injected at regular intervals to monitor instrument stability by principal component analysis (PCA). All tumor samples used for non-targeted metabolomic analysis were collected under strictly standardized conditions to ensure experimental consistency. Tissues were rapidly excised, snap-frozen in liquid nitrogen, and stored at –80°C until analysis. To minimize batch-related variability, all samples were processed simultaneously and analyzed within a single LC–MS/MS run. This unified workflow effectively eliminated the need for additional batch effect correction in downstream data analysis. Weigh 30 mg of sample into a 1.5 mL EP tube, followed by the addition of two small steel beads and 400 μL of methanol-water (4:1, v/v) containing an internal standard (4 μg/mL). The sample was pre-cooled at −40°C for 2 min, then ground at 60 Hz for 2 min. After grinding, the sample underwent ultrasonic extraction in an ice-water bath for 10 min and was then left to stand at −40°C for 2 h. The mixture was centrifuged for 20 min at 15,000 × g and 4°C. Subsequently, 150 μL of the supernatant was drawn with a syringe and transferred to an LC injection vial. Liquid chromatography–mass spectrometry (LC–MS) analysis was conducted using a Waters ACQUITY UPLC I-Class plus/Thermo QE HF ultra-high-performance liquid chromatography (UHPLC) tandem high-resolution mass spectrometer (HRMS) at OE Biotech, Inc., Shanghai, China. The system was equipped with a heated electrospray ionization (ESI) source (Thermo Fisher Scientific, Waltham, MA, USA) to analyze metabolic profiles in both positive and negative ESI modes. An ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 × 100 mm) was used for both ionization modes; the column temperature was maintained at 45°C, and the flow rate was 0.35 mL/min. Gradient elution was performed with solvent A (water with 0.1% formic acid, v/v) and solvent B (acetonitrile with 0.1% formic acid, v/v) using the following program: 0–2 min, 95% A/5% B; 2–4 min, linear to 70% A/30% B; 4–8 min, linear to 50% A/50% B; 8–10 min, linear to 20% A/80% B; 10–14 min, 0% A/100% B; 14–15 min, 0% A/100% B; 15.1–16 min, re-equilibration at 95% A/5% B. Samples were kept at 10°C during analysis, with an injection volume of 4 μL. Detection was performed over a mass range of 70–1050 m/z, using a primary scan resolution of 60,000 and a secondary scan resolution of 15,000. Collision energies of 10, 20, and 40 eV were applied. The mass spectrometer’s operating conditions were as follows: spray voltage of 3800 V (positive mode) and 3200 V (negative mode); sheath gas flow rate of 35 units; auxiliary gas flow of 8 units; capillary temperature of 320°C; auxiliary gas heater temperature of 350°C; and S-lens RF level of 50. Data preprocessing was performed using Progenesis QI v3.0 (Nonlinear Dynamics, Newcastle, UK) for baseline correction, peak identification, integration, retention time alignment, and intensity normalization across samples. Following missing value imputation and filtering, the data were further normalized via log-transformation to approximate a Gaussian distribution and reduce heteroscedasticity, ensuring suitability for downstream statistical analysis. Compound identification was based on accurate mass-to-charge ratios (m/z), secondary fragments, and isotopic distributions, utilizing databases such as the Human Metabolome Database (HMDB), Lipidmaps (V2.3), Metlin, and self-built databases. Peaks with over 50% missing values (ionic intensity = 0) were excluded, and zero values were replaced by half the minimum value. Data from positive and negative ion modes were merged into a single data matrix for further analysis. Differential metabolites between MSS and MSI-H groups were selected using Student’s t test with P<0.05 and fold change (FC) ≥1.2 or ≤1/1.2.

Construction of stable cell lines
The lentiviral plasmids pLVX-ZDHHC24 or pLVX-Zdhhc24 were constructed by cloning ZDHHC24 or Zdhhc24 genes into the pLVX vector. We used the pLVX, pMD2.G, and psPAX2 three-plasmid packaging systems to generate the highly expressed viruses. Short hairpin RNA (shRNA) targeting the human ZDHHC24 or murine Zdhhc24 genes was expressed in the pLKO.1 vector to obtain knockdown (KD) clones. The pLKO.1-shRNAs, pMD2.G, and psPAX2 triple packaging systems were then used to construct silencing expression lentiviruses. The sequences are listed in Table S7 (Supplementary Information). Similarly, the three of them were mixed in 500 μL of serum-free medium and 40 μL of Lipofectamine 2000 in a mass ratio of 2:1:2. The plasmid mixture was then transfected into HEK293T cells. Harvested viral supernatant was filtered through a sterilized polyethersulfone PES syringe filter (SFPES025022SI, membrane solutions LLC) and used to infect the indicated cells. Stabilize cell lines by screening with 1 μg/ml puromycin for 48 h or 0.8 mg/mL G418 for 20 days. The CRISPR/Cas9 system was used to generate B7H3, SQSTM1, ATG5, and BECN1 KO cells. The single guide RNAs were designed from a public website, CHOPCHOP, and then cloned into the PX459 plasmid (62988, Addgene) or the PX462 plasmid (62987, Addgene). The sgRNA sequences are listed in Table S7 (Supplementary Information).

Cellular membrane and cytoplasmic protein isolation
Separation of cytosolic and cytoplasmic proteins of the indicated cells was performed using the ExKine™ membrane and cytoplasmic protein extraction kit (KTP3005; Abbkine).

Cell counting kit-8 assay
To assess peptide cytotoxicity in vitro, cells were seeded into 96-well plates (100 μL complete medium) and allowed to adhere overnight. Cells were then treated with E-peptide or C-peptide at the indicated concentrations (2.5, 5, 10, 25, 50, 100, and 200 μM) for 24 h. Subsequently, 10 μL of cell counting kit-8 (C6005, new cell and molecular biotech) reagent was added to each well and incubated at 37°C for 1–4 h protected from light. Absorbance was measured at 450 nm using a microplate reader. Wells containing medium plus reagent without cells served as blanks, and cell viability was normalized to the vehicle-treated control (set to 100%).

Mouse serum biochemistry
Peripheral blood was collected from mice and allowed to clot at room temperature for 30 min. Serum was separated by centrifugation at 4°C at 3000 × g for 10 min, and analyzed immediately or stored at −80°C until use. Serum creatinine (CRE) was measured using the creatinine (CRE) content assay kit (AKNM008M, Beijing Boxbio Science and Technology Co., Ltd). The microplate reader was pre-warmed for ≥30 min and set to 546 nm. Reactions were carried out at 37°C according to the manufacturer’s protocol, absorbance was recorded, and CRE concentration was calculated against the kit standard/blank using the provided formula. Serum alanine aminotransferase (ALT/GPT) and aspartate aminotransferase (AST/GOT) activities were determined using the GPT activity assay kit (AKAM006M, Beijing Boxbio Science and Technology Co., Ltd) and the GOT activity assay kit (AKAM019M, Beijing Boxbio Science and Technology Co., Ltd), respectively. The instrument was pre-warmed for ≥30 min and set to 505 nm. A matched control and a standard curve were included as instructed. After incubation and color development at 37°C, absorbance was measured, and enzyme activities were calculated and reported as U/L according to the kit-provided formula.

Immunoprecipitation
Different magnetic beads (HAK21042, HAK21011, HUABIO) were selected according to the experimental needs. If the magnetic beads (HY-K0202, MCE) did not have a coupled antibody, it was necessary to mix the beads with the corresponding primary antibody for half an hour at room temperature or 2 h at 4°C before harvesting the cells. Subsequently, start lysing the cells to harvest the proteins. Proteins are extracted in NP40 cell lysis buffer containing the protease cocktail inhibitor for 30 min. Next, the magnetic beads that have been coupled to the antibody are transferred to the supernatant into the cell lysate and rotated at 4°C overnight. The following day, the supernatant was discarded, and the magnetic beads were washed five times with 500 μL of NP40 cell lysis buffer. Between each wash, magnetic separation gently. Finally, the supernatant was discarded, and 40 μl SDS loaded buffer was mixed with magnetic beads and boiled in a water bath for 10 min before being used for Western blotting.

Protein extraction and Western blotting
For cultured cells, discard the medium and wash the cells three times with cold PBS. Cells were lysed for 30 min using NP-40 buffer containing phenylmethylsulfonyl fluoride (HY-B0496, MCE) and cocktail protease inhibitor (HY-K0010, MCE). For tissue samples, total protein was extracted using the Total Protein Extraction Kit (EX1100, Solarbio) according to the manufacturer’s instructions. Lysates were centrifuged at 15,000 × g for 15 min at 4°C. Supernatants were collected, and protein concentrations were quantified by BCA kit (23227; Thermo Fisher). Subsequently, the protein supernatants acquired were mixed with 5× concentrated sample buffer containing 250 mM Tris-HCl (pH 6.8), 5% β-mercaptoethanol, 10% sodium dodecyl sulfate (SDS), 30% glycerol, and bromophenol blue, followed by heating at 95–100°C for 10 min. Protein samples were separated by electrophoresis in 8–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the separated protein bands were transferred to a polyvinylidene fluoride (PVDF) membrane. Subsequently, the membrane was then blocked by immersing in 5% skimmed milk in TBST or rapid blocking buffer (SW162-01, Seven, Beijing, China). Next, the membranes were incubated overnight at 4°C with primary antibodies and then exposed to HRP-linked secondary antibodies for 2 h at ambient temperature. Finally, the membrane bands were washed three times with TBST and then visualized using an efficient chemiluminescence kit (34096; Thermo Fisher).

Acyl-biotin exchange (ABE) assay
The ABE assay was performed based on established protocols33,39,46, and the key experimental steps are detailed below. 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 1% NP-40, 10% glycerol, protease inhibitor, and phosphatase inhibitor were added to NP-40 to configure the lysis buffer. Fresh N-ethylmaleimide (NEM) solution was prepared and added to the lysis buffer to a final concentration of 50 mM. The indicated cells were lysed in a buffer containing 50 mM NEM for 1 h on ice. Collect the cell suspension and mix it with magnetic beads pre-coated with the appropriate antibody for 3 h at 4°C. After 5 washes with lysis buffer (pH 7.5), the beads were washed again 3 times with lysis buffer (pH 7.2). Fresh hydroxylamine (HAM) solution was prepared in lysis buffer at pH 7.2 to a final concentration of 1 M. The beads were then incubated with lysis buffer containing 1 M HAM for 1 h at room temperature. Wash 3 times with Lysis Buffer (pH 6.2) to remove residual HAM. Subsequently, the beads were incubated with lysis buffer (pH=6.2) containing Biotin-BMCC (5 μM) at 4°C for 1 h. The beads were washed twice with buffer (pH=6.2) and then three times with lysis buffer (pH 7.5). Excess buffer was removed and added to 50 μL sample buffer (5% SDS, 5% glycerol, 125 mM Tris-HCL (pH 6.8), 0.01% bromophenol blue) containing 5 mM DL-Dithiothreitol (DTT). Samples were boiled at 80°C for 10 min and then cooled to room temperature for 15 min.

Immunofluorescence assays
Cells were inoculated onto slides of 24-well culture plates, allowed to adhere to the wall, and then subjected to various treatments. The slides were rinsed three times with precooled PBS, followed by fixation in 4% paraformaldehyde for 20 min at room temperature. After removing the paraformaldehyde, the slides were washed three times with PBS. Added 0.3% Triton-X-100 to the wells and permeabilized for 15 min at room temperature. The slides were blocked with 3% BSA for 1 h at room temperature after washing three times with PBS. Subsequently, they were incubated with primary antibodies at 4°C overnight. The following day, fluorochrome-labeled secondary antibodies were applied in the dark for 2 h at room temperature, and the samples were then stained with DAPI for 10 min. Tissue samples were treated similarly as described above. Finally, we obtained representative images using a Japanese Olympus FLUOVIEW FV1000 confocal scanning microscope or a Japanese Nikon ECLIPSE Ti2 fluorescence microscope.

Tumor-infiltrating lymphocyte isolation and flow cytometry analysis
For tumor-infiltrating immune cells, all mice were isolated immediately after sacrifice at the indicated times, and tumors were placed on ice and carefully cut into small pieces of approximately 1 mm. These fragmented tumor tissues were then digested in serum-free 1640 medium containing collagenase IV (1 mg/ml, 17104019; Invitrogen), deoxyribonuclease I (100 μg/ml, HY-108882; MCE), and hyaluronidase (250 μg/ml, BS171; Biosharp) for 90 min at 37°C. The cells suspension was then filtered using a cell strainer. A Percoll gradient cell separation solution (17089101, Cytiva) was used to isolate tumor cells and immune cells infiltrating the tumor. For cytokine analysis, the obtained immune cells were incubated with PMA, Brefeldin A, monensin, and ionomycin for 6 h and then stained with the corresponding antibodies. Cytofix/Cytoperm™ fixation and permeabilization solution (554722, BD) was used for intracellular staining. Transcription factor buffer set (562574, BD) used for nuclear staining. Raw FCS files were analyzed using FlowJo software (v10.8.1).

T-cell killing assay
The experimental methodology was adapted from published protocols75,76, and the main procedures are described below. Peripheral blood mononuclear cells (PBMCs) were isolated from anticoagulated whole blood using human lymphocyte separation medium (7111011, Dakewe). Briefly, whole blood was diluted 1:1 with PBS and carefully layered onto the separation medium. Samples were centrifuged at room temperature at 800 × g for 30 min using a horizontal rotor with low acceleration and deceleration settings. After centrifugation, the opaque mononuclear cell layer at the plasma-medium interface was carefully collected into a new tube. Cells were washed 2 times with RPMI-1640 by centrifugation at 20°C and 250 × g for 10 min, and the cell pellet was resuspended in normal saline or appropriate culture medium for downstream isolation. Human CD8+ T cells and human DCs were isolated from PBMCs using the CD8+ T cell isolation kit, human (130-096-495, Miltenyi Biotec), and the Dynabeads® Human DC Enrichment Kit (11308D, Thermo Fisher Scientific), respectively. The isolated DCs were first co-incubated with the indicated tumor cells, and then the pretreated DCs were then used to activate CD8+ T cells. To evaluate the tumor-killing effect mediated by T cells, the preconditioned CD8+ T cells were reactivated with anti-CD3 antibody (2 μg/ml), anti-CD28 antibody (2 μg/ml), and IL-2 (100 IU/ml). The activated CD8+ T cells were then co-incubated with the indicated tumor cells at a ratio of 10:1 for 24–48 h. Detection of T-cell mediated cytotoxicity using caspase-3 cleavage assay and lactatedehydrogenase (LDH) release assay. After centrifugation, the supernatant of each well was collected, and the amount of LDH released in the culture medium was determined using the LDH Cytotoxicity Assay Kit (HY-K1090, MCE). Cell clusters obtained after centrifugation were washed three times with PBS and then stained with FITC-coupled activated caspase-3 antibody, followed by flow cytometric analysis.

Immunohistochemistry (IHC)
The embedded tissue specimens were cut into sections at a thickness of 4 μm. Following sequential processes of dewaxing, rehydration, antigen retrieval, and blocking, the sections were treated with the specified primary antibody overnight and then incubated with the secondary antibody for 1 h at room temperature. Immunohistochemical results were quantified by the density and intensity of positive cells. The density of positive staining was categorized into 4 grades based on the intensity of positive staining as follows: 1 (<5%), 2 (5–30%), 3 (30–70%), and 4 (>70%). The intensity of positive staining was categorized into 4 grades, 0 (unstained), 1 (weak), 2 (moderate), and 3 (strong). The density and intensity scores were then multiplied to obtain the IHC score, which ranged from 0 to 12. To evaluate the CD8 staining score, three random fields of view were chosen from each tissue section and analyzed at a magnification of ×400. The mean number of positively stained cells observed in these fields was calculated to represent each case. IHC staining was independently scored by two experienced pathologists, and the final IHC score for each case was defined as the average of the two scores.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
RNA was initially extracted from cells using AG RNAex Pro RNA extraction reagent (AG21101, Accurate Biotechnology (Hunan) Co., Ltd) following the manufacturer’s protocol. The extracted RNA was then reverse-transcribed into cDNA using the Reverse Transcription Kit (HiScript III RT SuperMix for qPCR, Vazyme). Finally, qPCR was performed using the SupRealQ Ultra Hunter SYBR qPCR Master Mix (U+) (Q713-02, Vazyme Biotech Co., Ltd) reagent on QuantStudio 3 instrument (Thermo Fisher). All primer sequences are provided in Table S8.

Animal study
The mice used in this study (Mus musculus) included C57BL/6J, BALB/c, NCG, Zdhhc24fl/fl, Villin-cre, and Rosa26-CAG-LSL-tdTomato, which were purchased from GemPharmatech (Nanjing, China). The Zdhhc24fl/fl mouse line was generated using CRISPR/Cas9 on a C57BL/6J genetic background (gRNA sequences are provided in Table S9) and maintained in our facility. The huHSC-C-NKG-ProF mice were generated by Cyagen Biosciences (Suzhou, China). All animals used in this study were female. Unless otherwise indicated in the figure legends, mice were 6 weeks old at the start of each experiment, and the number of animals used for each experiment (n) and the corresponding strain/substrain are reported in the figure legends. Sex was not included as a biological variable because all experiments were performed using female mice to reduce variability and maintain consistency across cohorts; therefore, no sex-based comparisons were performed, and this choice was made solely for experimental design and reproducibility considerations and does not reflect any sex bias or discrimination. All animal experiments were conducted with approval from the Animal Care and Use Committee of Tongji Hospital (TJH-202303006). Mice were housed under specific pathogen-free conditions with a 12 h light/12 h dark cycle, an ambient temperature of 22–24°C, and relative humidity of 40–60%, with food and water available ad libitum. For xenograft tumor assays, cells were digested, washed three times with PBS, and mixed with Matrigel (abs9493, Absin) at a 1:1 ratio to a final density of 2×106 cells/mL. A total of 100 μL of cells suspension was injected subcutaneously into 6-week-old female mice. Tumor volume was measured at the indicated time points and calculated as V=L×D2/2 (L: long axis, D: short axis). In the approved protocol, the maximal tumor burden was a tumor volume of 2000 mm3, and mice were euthanized when tumors reached this limit or when other humane endpoints were met. In addition to the predefined maximal tumor burden (tumor volume of 2000 mm3), mice were euthanized if any of the following humane endpoint criteria were met: (i) ulceration, necrosis, or bleeding at the tumor site; (ii) impaired mobility, inability to access food or water, or persistent abnormal posture/behavior; (iii) rapid weight loss (≥20% of baseline) or severe dehydration; (iv) signs of systemic distress such as labored breathing, hypothermia, or marked lethargy; or (v) any other condition judged by the animal care staff/veterinarian to require euthanasia to prevent undue suffering. Mice were monitored at least every 1–2 days, and more frequently when tumors approached the endpoint. Overall, the study did not substantially exceed the permitted limit. In the CT26 survival experiment (Fig. 9C–G), a small number of mice showed calculated tumor volumes slightly above 2000 mm3 at the scheduled measurement time points, reflecting caliper measurement variability and the estimated nature of the volume formula, together with rapid tumor growth between two consecutive monitoring time points; mice were euthanized immediately upon detection in accordance with the approved protocol. For therapeutic agents, anti-PD-1 antibody (10 mg/kg), E-peptide, and C-peptide (10 mg/kg) were injected into mice by intraperitoneal injection at the indicated time intervals. For Zdhhc24fl/fl mice, CRISPR/Cas9 technology was used to modify the Zdhhc24 gene (gRNA sequences are provided in Table S9). Briefly, the CRISPR/Cas9 system and donor were microinjected into fertilized eggs of C57BL/6J mice. Transplantation of fertilized eggs yielded positive F0 mice, which were confirmed by PCR and sequencing. Positive F0 generation mice were backcrossed with C57BL/6J for at least two generations to obtain a stable Zdhhc24fl/fl mouse model. Villin-cre and Rosa26-CAG-LSL-tdTomato are well-established commercial mice. Validation of PCR primers for Zdhhc24fl/fl and Villin-cre mice is provided in Table S10.

Hemolysis assay
Approximately 0.6 mL of blood was collected from anesthetized mice via cardiac puncture into a centrifuge tube pre-coated with sodium citrate as an anticoagulant. The sample was centrifuged at 2500 × g for 10 min to remove the plasma. The remaining red blood cells (RBCs) were washed repeatedly with normal saline until the supernatant became colorless. After discarding the supernatant, the RBCs were resuspended in 1 mL of normal saline to obtain an RBC suspension. Then, 100 μL of RBC suspension was added to 1 mL of E-peptide solution (dissolved in saline) at various concentrations. Saline alone was used as a negative control, and distilled water was used as a positive control. The mixtures were incubated at 37°C for 2 h, followed by centrifugation at 2500 × g for 10 min. The optical density (OD) of the supernatant was measured at 541 nm. The hemolysis rate was calculated using the following formula:

Hemolysis (%) = (ODsample − ODnegative) / (ODpositive − ODnegative) × 100%

Coagulation assays: prothrombin time (PT) and activated partial thromboplastin time (APTT)
PT and activated partial thromboplastin time APTT were measured using commercial assay kits (Bioswamp Life Science Lab, Wuhan, China) according to the manufacturer’s instructions. Briefly, whole blood was centrifuged at 2500 × g for 10 min to obtain platelet-poor plasma. Two hundred microliters of plasma was mixed with 10 μL of E-peptide solution (dissolved in normal saline) to achieve final peptide concentrations of 5, 10, 25, and 50 μM. An equal volume of normal saline was added to the plasma as the control. For PT testing, 70 μL of peptide-treated or control plasma and 140 μL of PT reagent were pre-incubated separately in a 37°C water bath for 3 min. The two components were then mixed rapidly, and the clotting time was recorded upon cessation of flow during gentle tilting of the reaction tube. For APTT testing, 100 μL of peptide-treated or control plasma was mixed with 100 μL of APTT reagent (containing ellagic acid) and incubated at 37°C for 5 min. Subsequently, 100 μL of 25 mM CaCl2 solution (pre-warmed to 37°C) was added to initiate coagulation. The tube was gently agitated in a 37°C water bath, and the time to visible fibrin formation was recorded as the APTT value.

Peptide synthesis
The C-peptide (YGRKKRRQRRRRKWEISQCKC) and E-peptide (YGRKKRRQ-RRRCWRKIKQSCE) were synthesized by QYAOBlO (China Peptides Co., Ltd.) using standard Fmoc-based solid-phase peptide synthesis (SPPS) protocols on 2-chlorotrityl chloride resin. The synthesis followed a stepwise procedure involving resin swelling in DMF, sequential coupling of Fmoc-protected amino acids using HBTU and DIEA, and Fmoc deprotection with 20% piperidine in DMF. Coupling efficiency was monitored by Kaiser tests. Upon completion of chain assembly, the peptide was cleaved from the resin using a TFA-based cleavage cocktail (TFA/H2O/EDT/TIS, 95:2:2:1, v/v) and precipitated in cold ether. The crude product was purified by preparative reversed-phase high-performance liquid chromatography (RP-HPLC) on a C18 column. Final peptide identity and purity were confirmed by mass spectrometry (MS) and analytical HPLC. The purified peptide was lyophilized and stored at −20°C until use.

Proximity ligation assay (PLA)
Proximity ligation assay was detected using the Duolink® in situ red starter kit mouse/rabbit (Sigma-Aldrich) following the manufacturer’s protocol. Briefly, fixed and permeabilized samples on glass slides were blocked with Duolink® Blocking Solution at 37°C for 60 min, then incubated overnight at 4°C with primary antibodies: anti-CD276 (Abcam, ab134161) and anti-SQSTM1/p62 (CST, 88588). After washing in 1× Wash Buffer A, PLUS and MINUS PLA probes (1:5 dilution) were applied for 1 h at 37°C, followed by ligation (1:40 ligase, 30 min at 37°C) and amplification (1:80 polymerase, 100 min at 37°C, in the dark). Slides were washed, mounted with Duolink® in situ mounting medium containing DAPI, and imaged using a confocal microscope (LSM900 Airyscan).

Surface plasmon resonance (SPR)
Binding measurements between ZDHHC24 and E-peptide were performed by surface plasmon resonance (SPR) on a Biacore 8K instrument (GE Healthcare/Cytiva) using CM5 sensor chips, based on established protocols77,78 with all key experimental parameters provided below. In brief, experiments were conducted at 25°C with a blank activated/deactivated reference flow cell. ZDHHC24 was immobilized on CM5 via amine coupling using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS), and residual esters were blocked with ethanolamine. The running/immobilization buffer was 1× phosphate-buffered saline with surfactant P20 (PBS-P+, pH 7.4). Interaction analyses were carried out in PBS-P+ (pH 7.4) supplemented with 5% (v/v) dimethyl sulfoxide (DMSO); an eight-point solvent-correction curve bracketing 5.0% DMSO (prepared by mixing 4.5% and 5.8% DMSO stocks) was used for bulk-refractive index correction. E-peptides were injected as analytes from low to high concentration (0.3125–10 μM) at a flow rate of 30 μL min−1 with a 150 s association phase, followed by surface regeneration with 10 mM glycine–HCl (pH 2.0) for 5 min between injections. Binding parameters were obtained by global fitting of double-referenced, solvent-corrected sensorgrams to a 1:1 Langmuir model in Biacore Insight (Cytiva), yielding the equilibrium dissociation constant (Kd).

Statistics and reproducibility
All assays were performed in at least three independent experiments. Sample sizes were determined based on prior experience or published literature. No statistical method was used to predetermine sample size. No data were excluded from the analyses. Human specimens diagnosed with colorectal cancer were randomly selected from eligible hospital patients. Animals were randomly assigned to control and experimental groups using a simple randomization method. Investigators were blinded to group allocation during data collection and analysis, including outcome assessment. Data are presented as mean ± SD. Statistical analyses were performed using GraphPad Prism (version 9.5) and Origin (version 2021). Before statistical analysis, tests for normality and homogeneity of variance were performed. Two-tailed unpaired or paired Student’s t tests were used for comparisons between two groups. For multiple-group comparisons, one-way ANOVA with Tukey’s or Dunnett’s multiple-comparisons tests, or two-way ANOVA with Tukey’s multiple-comparisons test were used. Categorical variables were analyzed using chi-square tests. Correlations were assessed using Pearson’s correlation (two-tailed). Survival analyses were performed using the Kaplan–Meier method and compared by the log-rank test. Statistical significance was set at p<0.05.

Ethics statement
The clinical specimens were obtained from the Department of Gastrointestinal Surgery, Tongji Hospital. All patients were informed about the study and provided written informed consent prior to surgery. Consent was obtained for the acquisition, reporting, and sharing of patient clinical information at the individual level. No participant compensation was provided. Patients’ microsatellite status (MSS or MSI-H) and clinical characteristics were obtained from their medical records. Patient information, including age and sex, is shown in Tables S1–S5 (Supplementary Information). Tissue microarrays of colorectal tumors and paracancers were purchased from the Shanghai Outdo Biotech Co. Ltd (HColA180Su21). For these commercial tissue microarrays, demographic information was provided by the vendor and is reported in the Source Data. Sex- and/or gender-stratified analyses were not performed because the study was not designed to detect sex/gender differences, and the human analyses primarily focused on tumor molecular subtype (MSS vs MSI-H). The study was approved by the Ethics Committee of Tongji Hospital (TJ-IRB20220723). All animal experiments were conducted with approval from the Animal Care and Use Committee of Tongji Hospital (TJH-202303006).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors of this study investigate how the palmitoyltransferase ZDHHC24 participates in regulating immunosuppression by influencing the palmitoylation of B7H3. They demonstrate that the palmitoylation of B7H3 at cysteine 496 (C496) catalyzed by ZDHHC24, which inhibits its binding to the autophagy receptor SQSTM1/p62 and prevents its degradation, could impair the anti-tumor activity of CD8+T cells. Besides, they also develop a peptide to block the binding of ZDHHC24 and B7H3, whose combination with anti-PD-1 antibody significantly inhibits tumor growth in vivo. Although the palmitoylation of PD-L1 has been reported, no one has found the palmitoylation of B7H3 and its biological function yet, and this study has a certain innovation. However, some of the conclusions are not well supported by the data and there are multiple issues.\nOverall Comments:\n1. If the authors wish to show that T cells mediate the anti-tumor effect after inhibition of B7H3 palmitoylation, then at the very least the T cells should have been deleted.\n2. The result of Fig3G showed many immune cell populations (such as CD8+T, CD4+T, NKT, NK, MDSC and so on) were significantly changed by the inhibition of B7H3 palmitoylation, but only the CD8+T was discussed in the following research. The other populations should also be concerned.\n3. Although cleaved caspase 3 looks good in both human and mouse in vitro co-culture systems, the overall survival data of tumor cells should be shown, for the tumor cells may not only undergo apoptosis.\n4. All the immunoblotting results in this study lack labels of protein molecular weight.\n5. In the Fig4B, the background of IgG group is particularly clean compared with both Input and IP groups, and it seems like lack of protein sample. Please repeated this experiment and provide corresponding internal control protein.\n6. In the FigS4a, Flag-ZDHHC9/ZDHHC16/ZDHHC22 did not successfully express after transfection, and whether they participate in the palmitoylation of B7H3 is unknown. Additional experiments are needed to rule out their effects.\n7. Some of the flow cytometry analyses only provide statistical charts, and the mode charts should also be shown.", "reviewer_2": "This study reveals a novel mechanism underlying the lower response rate to immune checkpoint blockade (ICB) therapy in microsatellite stable (MSS) colorectal cancer (CRC) patients compared to microsatellite instability-high (MSI-H) patients, highlighting the role of B7-H3 overexpression in tumor cells. Using patient tumor tissue samples and multi-omics technologies such as transcriptomics and metabolomics, they discovered that B7-H3 expression is significantly higher in MSS patient tissues compared to MSI patients. Furthermore, they elucidated that the key molecular mechanism involves palmitic acid-mediated palmitoylation modification of B7-H3. In detail, they discovered that ZDHHC24 mediates the palmitoylation of B7H3 at Cys496, which hinders its interaction with autophagy cargo receptor-p62 to inhibit its protein degradation. Based on their molecular mechanism, they designed a small mimic peptide of B7H3, which can enhance CD8+ T cell function by blocking the interaction between ZDHHC24 and B7H3. This strategy also elevated the sensibility and responsibility of CRC cells to the ICB therapy. Overall, this study is highly interesting and innovative, with rigorous experimental design and reliable results. It offers new perspectives and a material foundation for tackling the unresponsiveness of most CRC patients to ICB therapy in clinical settings. The major limitations of the study, however, are as follows:\nMajor comments:\n1. As an immune checkpoint, B7-H3 not only expresses in tumor cells, but also in immune cells, including monocytes, dendritic cells, myeloid-derived suppressor cells (MDSCs), neutrophils, macrophages, B cells, and activated T cells. So, is B7H3 only highly expressed in tumor cells of MSS type tumor tissues? The author didn’t make any exclusion experiments. If not, does the molecular mechanism also suit in other immune cells?\n2. The authors explored their molecular mechanism mainly in RKO cells (MSI), SW480 cells (MSS), CT26 cells (MSS) and MC38 cells (MSI). I wonder whether B7H3 is also highly expressed in MSS CRC cell lines compared to the MSI ones. If this post-translational modification of B7-H3 exists in both MSS and MSI-type tumor cells, then is the high expression of B7-H3 in MSS tissues primarily due to differences in ZDHHC24 expression or the distribution of palmitic acid, or both? If it is due to ZDHHC24, they should provide more details of its protein expression in MSS and MSI tumor tissues and cells. If it is due to palmitic acid, it would be interesting to know why there is a higher abundance of palmitic acid in MSS tumors. At least, it could be a good content for the discussion part.\n3. Whether ZDHHC24 enhances the protein stability of B7H3 should be detected.\n4. As they mentioned, E-peptide works through interrupting the binding of B7H3 and ZDHHC24. They should give more details about the working mechanism. For example, whether E-peptide binds with ZDHHC24? If so, surface plasmon resonance (SPR) assay should be utilized to obtain kinetic parameters. Besides, whether E-peptide enhances the binding of B7H3 with p62 should be detected.\n5. The authors have tested the anti-tumor effect of E-peptide synthesized according to the human B7H3 amino acids sequence in the mouse tumor models. Is there significant sequence conservation of this peptide segment between humans and mice? It is necessary to verify whether the E-peptide reduces the protein expression of B7H3 and impacts its palmitoylation in mice tumors. To enhance the clinical relevance of the results, it is recommended to evaluate the anti-tumor effects of the peptide using immunologically reconstituted humanized mice.\nMinor comments:\n1. In Figure 5, the authors investigated the tumor-promoting role of ZDHHC24 in the AOM/DSS-induced CRC mouse model using ZDHHC transgenic mice. However, since they have not confirmed that ZDHHC24 functions through palmitoylating B7H3 in this model, this section appears somewhat disconnected from the central topic. It is recommended to either relocate this part to the supplementary material or provide additional evidence linking ZDHHC24's activity to B7H3 palmitoylation to maintain coherence with the main narrative.\n2. To make sure whether ZDHHC24 interrupt the interaction of B7H3 with p62, proximity ligation assay should also be used besides Co-IP assay.", "reviewer_3": "B7H3\nIt this manuscript Rao et al. have analysed a member of the B7 family and found that B7H3 shows increased expression in MSS tumours. They go on showing that B7H3 undergoes S-acylation on Cys-496, and that this slows down the turnover rate of B7H3. They propose that degradation of the non-acylated form occurs via autophagy. They show that ZDHHC24 is the major acyltransferase involved in modifying B7H3. They subsequently address the role of B7H3 acylation in tumour progression and finally design a peptide that inhibits B7H3 acylation, showing that it inhibits tumour growth.\nThe finding that preventing B7H3 acylation can have therapeutic benefit is clearly of interest. However the manuscript lack a rigour of thinking/writing that affects its quality.\nI have focused my review on S-acylation, since the cancer immunology aspects are outside of my expertise. I do wish to mention that the effects of the e-peptide and anti-PD-1 antibodies by no means seem synergistic. The data indicates that they are somewhat additive, although not fully.\nFrom my perspective, the paper is written in a manner that is superficial and lacks the necessary rigour to make the reader confident of the quality of the work. Reference to the literature and what is really demonstrated in the literature is insufficient and again superficial. While clearly the interest of the authors lies in the therapeutic application, I regret that there is no questioning regarding the mechanistic aspect. This gives the impression of a pseudo-mechanistic insight.\nHere are a series of more specific comments, this is non-exhaustive:\n- Line 38: p62 is not a cargo protein\n- Line 51: for non-experts, having three acronyms in the first sentence is difficult.\n- Define B7 family, provide some background information\n- Line 70: I think that what the author call “membrane localization” is plasma membrane localization”. Indeed, B7H3 is a transmembrane protein, that’s what determines its membrane localization.\n- Line 78: the correct term is S-acylation, often referred to as S-palmitoylation , or just Palmitoylation.\n- Line 79: there is very little evidence so far that S-acylation affects protein structure. This is thus incorrect.\n- Line 87: reword “irreplaceable”, this is likely true for any PTM.\n- Abstract and lines 95, 163 and others places in the manuscript: Palmitic acid does not “mediate” palmitoylation in the context of this work, but triggers it. All palmitoylation is by definition palmitate-mediated.\n- Line 155: separating cytosolic from membrane by fractionnation when looking at a membrane protein seems rather meaning less. These type of experiments seed doubts in the reader’s mind as to the mechanistic understanding of the authors regarding the protein under study.\n- Line 160: the authors are monitoring total B7H3 levels, not palmitoylated B7H3. This is an interpretation of the data, not a description.\n- Line 166: the two protein degradation “sites” in the cell are lysosomes and the proteasome. Autophagy is a pathway to lysosomal degradation. However, the default routes for a transmembrane protein like B7H3 are 1) the proteasome if it fails to fold properly during its biogenesis in the ER (this requires retro translocation as part of the ERAD pathway) and 2) trafficking to lysosomes. Given its role in the interaction with T-cells, B7H3 acts at the plasma membrane and thus its route to degradation would be endocytosis and trafficking to lysosomes via the classical route. Autophagy is not the most evident pathways as opposed to what the authors suggest, and thus the approach appears to have a significant bias.\nThat acylation affects the endocytosis of plasma membrane transmembrane proteins has been shown for a variety of proteins. B7H3 is likely to be as others surface proteins, its endocytosis is negatively regulated by S-acylation, thus acylation deficient mutants will undergo accelerated endocytosis and lysosomal degradation. There is a priori no need to bring up autophagy.\n- line 170: Baf A1 and chloroquine are not autophagy inhibitors, they affect the pH of lysosomes, thus inhibit lysosomal enzymes, thus affect the end point of autophagy.\n- Line 193: C496A has a mutation of te acylation site, not a deletion\n- Line 204: microscopy can never inform about the ability to bind. This is an interpretation.\n- Line 272: ZDHHC24 has been reported to be an ER localized acyltransferase. The authors should comment on the fact that their localization at the plasma membrane is in disagreement with the literature. Also a quantification of the localization would be required.\n- Molecular weight markers are missing on all blots\n- Quantifications of Fig. 1G and many other blots are missing.", "reviewer_4": "In this research article, Rao et al. study the mediation of the palmitoylation of the immune checkpoint protein B7H3, demonstrating that deletion of palmitoylation site restores CD8+ T cell antitumor activity. The authors further developed a peptide that enhances CD8+ T cell function by blocking the interaction between ZDHHC24 and B7H3, which works synergistically with anti-PD-1 therapy. The work presents a relevant contribution for cancer therapy, but some concerns need to be addressed:\n1) The authors discussed the rationale of the design of the “E-peptide”, in which the amino acids 488-497 in the B7H3 sequence were found to be essential for the interaction with ZDHHHC24. However, the rationale of the “C-peptide” is not explained. Why did the authors use this sequence? The authors compare the E-peptide to C-peptide, but the reason for C-peptide being a control is unclear.\n2) Did the authors check if the C-peptide is also internalized by confocal microscopy?\n3) The authors should provide the synthesis protocol and characterization for all the compounds, including the data that supports the assignment of structure (NMR and mass spectrometry) and purity, such as HPLC.\n4) What was the solvent used to prepare the solutions of E- and C- peptide?\n5) The authors tested the peptides in vivo. Was the hemocompatibility and proteolytic stability of the peptide assessed?\nAlso, does the peptides form aggregates? The aggregation could limit the application of larger concentrations.", "reviewer_5": "The manuscript titled \"Palmitic Acid Mediated Palmitoylation of B7H3 Promotes Immune Escape\" presents an important exploration into immune evasion mechanisms in MSS colorectal cancer, highlighting the palmitoylation of B7-H3 by ZDHHC24 as a critical modulator of tumor immune suppression. While the study is extensive and covers multiple experimental systems, several conceptual, methodological, and analytical aspects limit the current impact and clarity of the findings.\nAlthough the authors attribute the observed immune escape mechanism primarily to B7-H3 palmitoylation, the broader biological effects of palmitic acid are not fully addressed. Palmitic acid is a well-known inducer of ER stress, oxidative stress, activation of pro-inflammatory pathways (e.g., NF-κB, JNK), and lipotoxicity. Given that the concentration used for cell treatments (100 μM) is relatively high, there remains a concern that some of the observed effects could result from these pleiotropic actions rather than palmitoylation alone\n. While the manuscript includes experiments with 2-bromopalmitate (2-BP) to demonstrate palmitoylation dependence, additional controls such as genetic knockdown of ZDHHC24 or CRISPR-mediated editing of B7-H3 palmitoylation sites could more conclusively isolate the specific role of this modification.\nThe metabolomics analysis is presented without sufficient methodological clarity regarding normalization procedures or batch effect correction. Visualizations such as MDS plots, PLS-DA models with VIP scores, or clear volcano plots would help to more effectively communicate metabolic shifts between groups\n. Moreover, the connection between the identified metabolic changes and the functional outcomes of B7-H3 palmitoylation could be explored in greater detail to strengthen the mechanistic narrative.\nSimilarly, the GO term enrichment results are overly broad and lack clear prioritization of critical pathways. Application of GSVA or GSEA could help focus the analysis on pathways most relevant to tumor immune evasion and metabolic reprogramming. Integrating these pathway analyses with experimental data on immune cell infiltration and function would provide a more cohesive story.\nThe single-cell analysis described in Figure 3H lacks objective statistical comparisons. While clustering results are visually presented, there is an absence of quantitative analyses comparing immune cell subtypes and functional states across treatment groups. Including violin plots, annotated UMAP/t-SNE plots, and appropriate statistical tests (e.g., Wilcoxon rank-sum test) would enhance the rigor and clarity of these analyses.\nIn the clinical relevance section, the manuscript touches briefly on correlations between ZDHHC24 expression and patient outcomes but does not deeply explore publicly available datasets such as TCGA or CPTAC to support these findings. A more thorough analysis correlating ZDHHC24 and B7-H3 expression with clinical parameters such as CD8+ T cell infiltration and overall survival would significantly strengthen the translational impact of the study.\nAlthough the therapeutic E-peptide is a novel approach, the current data regarding its pharmacokinetics, biodistribution, and toxicity remain limited. The authors mention minimal toxicity at doses up to 50 mg/kg, but detailed pharmacodynamic data and long-term safety evaluations are lacking\n. Without this information, it is difficult to assess the true translational potential of this therapeutic strategy. Additionally, exploring combination therapies with standard-of-care immune checkpoint inhibitors in more clinically relevant models would help to further demonstrate the value of this approach.\nThe manuscript would also benefit from a more detailed discussion on the potential redundancy or compensatory mechanisms involving other immune checkpoint molecules, such as PD-L1 and B7-H4. Exploring whether inhibition of B7-H3 palmitoylation has additive or synergistic effects when combined with inhibitors targeting these molecules would provide valuable insights into the broader therapeutic landscape.\nFinally, several figures require improvements in resolution, annotation of statistical significance, and clearer figure legends to enhance interpretability. Language polishing throughout the manuscript would also improve readability and professionalism.\nIn conclusion, while this study presents a compelling case for targeting B7-H3 palmitoylation as a novel therapeutic strategy in MSS colorectal cancer, substantial clarifications, additional experimental controls, and deeper clinical data analyses are needed to fully support the authors’ conclusions.", "reviewer_6": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "RESPONSE TO REVIEWERS' COMMENTS (NCOMMS-25-17666-T)\nNote to Reviewers: We sincerely appreciate the reviewers’ time and thoughtful evaluation of our manuscript. We are grateful for the constructive comments and suggestions, which have substantially improved the quality of our work. A point-by-point response to each comment is provided below. For the convenience of review, key data are presented as response figures in this letter, and we indicate here the corresponding figure/panel and text locations in the revised manuscript. We have submitted two versions of the revised manuscript: (i) a clean version and (ii) a marked-up version. In the marked-up version, added text is highlighted in red with underlining, and deleted text is shown in dark blue with strikethrough.\nREVIEWER COMMENTS\nReviewer #1:\nThe authors of this study investigate how the palmitoyltransferase ZDHHC24 participates in regulating immunosuppression by influencing the palmitoylation of B7H3. They demonstrate that the palmitoylation of B7H3 at cysteine 496 (C496) catalyzed by ZDHHC24, which inhibits its binding to the autophagy receptor SQSTM1/p62 and prevents its degradation, could impair the anti-tumor activity of CD8+T cells. Besides, they also develop a peptide to block the binding of ZDHHC24 and B7H3, whose combination with anti-PD-1 antibody significantly inhibits tumor growth in vivo. Although the palmitoylation of PD-L1 has been reported, no one has found the palmitoylation of B7H3 and its biological function yet, and this study has a certain innovation. However, some of the conclusions are not well supported by the data and there are multiple issues.\nResponse：We sincerely thank you for the thoughtful summary of our study and for commending the novelty of our findings, particularly the identification of B7H3 palmitoylation and its functional relevance in tumor immune evasion. We acknowledge that certain conclusions require further clarification and experimental support. In response to your comments, we have carefully revised the manuscript, conducted additional experiments, and refined our interpretations to strengthen the overall rigor and clarity of the study. Detailed point-by-point responses are provided below. Once again, we sincerely thank you for your valuable time, insightful comments, and efforts in helping us improve the quality of our work.\nMajor comments:\nQ1: If the authors wish to show that T cells mediate the anti-tumor effect after inhibition of B7H3 palmitoylation, then at the very least the T cells should have been deleted.\nResponse: We sincerely thank you for this insightful and important suggestion. As recommended, we performed CD8⁺ T cell depletion experiments to determine whether the anti-tumor effect observed upon inhibition of B7H3 palmitoylation is mediated by CD8+ T cells. Specifically, anti-CD8α monoclonal antibody (mAb) was administered intraperitoneally one day prior to subcutaneous implantation of MC38 tumor cells, followed by two additional doses at weekly intervals (three injections in total) (Response Figure.1a; Fig. 3I in the manuscript). Strikingly, depletion of CD8⁺ T cells completely abolished the difference in tumor growth between wild-type B7H3 and the palmitoylation-deficient C278A mutant, indicating that the anti-tumor effect conferred by B7H3 palmitoylation deficiency is indeed CD8⁺ T cell-dependent (Response Figure.1b-d; Fig. 3J, 3K, and Fig. S3J in the manuscript). We truly appreciate your suggestion, which led us to perform this critical experiment and significantly enhanced the rigor and completeness of our study.\nResponse Figure.1 CD8⁺ T cell depletion abolishes the anti-tumor effect driven by B7H3 palmitoylation deficiency.\nQ2: The result of Fig3G showed many immune cell populations (such as CD8+T, CD4+T, NKT, NK, MDSC and so on) were significantly changed by the inhibition of B7H3 palmitoylation, but only the CD8+T was discussed in the following research. The other populations should also be concerned.\nResponse: We sincerely thank you for the thoughtful and professional comment regarding the broader impact of B7H3 palmitoylation on various immune cell populations, as shown in Figure 3G. Firstly, among the immune subsets analyzed—including CD8⁺ T cells, CD4⁺ T cells, NKT cells, NK cells, and MDSCs—the change in CD8⁺ T cells was the most pronounced following the loss of B7H3 palmitoylation, as shown in Figure 3G and 3H. Secondly, CD8⁺ T cells are widely recognized as the principal effectors that directly mediate tumor cell killing and play a pivotal role in shaping the overall effectiveness of antitumor immune responses[1, 2]. In line with this, numerous studies have demonstrated that both the abundance and functional state of CD8⁺ T cells serve as reliable indicators of effective antitumor immunity[3, 4]. Furthermore, B7H3 is a member of the B7 family, sharing structural and functional similarities with immune checkpoint molecules such as PD-L1[5]. Based on this analogy, we hypothesize that B7H3 palmitoylation, similar to PD-L1, regulates antitumor immunity primarily through its impact on CD8⁺ T cell function. To functionally validate the importance of CD8⁺ T cells in this context, we performed depletion experiments using CD8⁺ T-depleting antibodies, which revealed that the enhanced antitumor effects associated with B7H3 palmitoylation deficiency were abolished following CD8⁺ T cell removal, as shown in Response Figure 1. These results further underscore the indispensable role of CD8⁺ T cells in mediating the observed immune response. Taken together, these findings provide a rationale for our decision to center the mechanistic investigation on CD8⁺ T cells. While we fully acknowledge that other immune cell types, including CD4⁺ T cells, NKT cells, NK cells, and MDSCs, may also contribute meaningfully to the tumor immune microenvironment, we believe that prioritizing CD8⁺ T cells in this study is both scientifically sound and supported by our data. Once again, we are grateful to you for this valuable suggestion, which has helped us strengthen the depth and clarity of our work.\nReference:\n[1] RASKOV H, ORHAN A, CHRISTENSEN J P, et al. Cytotoxic CD8+ T Cells in Cancer and Cancer Immunotherapy[J]. British Journal of Cancer, 2021, 124(2): 359–367.\n[2] GILES J R, GLOBIG A M, KAECH S M, et al. CD8+ T Cells in the Cancer-Immunity Cycle[J]. Immunity, 2023, 56(10): 2231–2253.\n[3] EJ W. T Cell Exhaustion[J]. Nature immunology, 2011, 12(6).\n[4] HEGDE P S, CHEN D S. Top 10 Challenges in Cancer Immunotherapy[J]. Immunity, 2020, 52(1): 17–35.\n[5] B Z, H L, Y X, et al. Immune Checkpoint of B7-H3 in Cancer: From Immunology to Clinical Immunotherapy[J]. Journal of hematology & oncology, 2022, 15(1).\nQ3: Although cleaved caspase 3 looks good in both human and mouse in vitro co-culture systems, the overall survival data of tumor cells should be shown, for the tumor cells may not only undergo apoptosis.\nResponse: We sincerely appreciate your insightful comment. While cleaved caspase-3 staining provides clear evidence of apoptosis in both human and mouse in vitro co-culture systems, we fully agree that additional data assessing overall tumor cell survival are necessary. In fact, we had already performed LDH release assays in parallel with cleaved caspase-3 flow cytometry. LDH, as a stable cytosolic enzyme released upon loss of membrane integrity, reflects both apoptotic and necrotic cell death. These results have been presented in Figure 3T, Figure S5R, and Figure 6K. In addition, in response to your suggestion, we have now performed crystal violet staining to further assess the overall survival of tumor cells after T cell–mediated killing. Quantification of the remaining adherent tumor cells by absorbance at 570 nm revealed that both ZDHHC24 knockdown and the C496A mutation of B7H3 significantly enhanced CD8⁺ T cell–mediated tumor cell killing) (Response Figure.2). These findings provide further support for the functional relevance of B7H3 palmitoylation in immune evasion. We are grateful for your constructive feedback, which has helped us enhance the completeness and robustness of our study.\nResponse Figure.2 B7H3 C496A mutation and ZDHHC24 knockdown enhance CD8⁺ T cell–mediated tumor cell killing.\nQ4: All the immunoblotting results in this study lack labels of protein molecular weight.\nResponse: We sincerely thank you for pointing this out. In response to your suggestion, we have carefully reviewed all the immunoblotting figures and have now added the appropriate protein molecular weight markers to each blot.\nQ5: In the Fig4B, the background of IgG group is particularly clean compared with both Input and IP groups, and it seems like lack of protein sample. Please repeated this experiment and provide corresponding internal control protein.\nResponse: We sincerely thank the reviewer for the careful observation and valuable suggestion. We agree that the IgG group in the original Figure 4B appeared to have a particularly clean background compared to the Input and IP groups. We believe this was likely due to the relatively short incubation time (0.5h at room temperature) between the protein lysate and antibody-conjugated magnetic beads, which may have reduced nonspecific binding. Additionally, a shorter exposure time was used for this blot, which may have further contributed to the clean background. In response to your suggestion, we have repeated the Co-IP experiment with improved conditions. Specifically, we extended the incubation time between the antibody-conjugated beads and the lysates to 8 hours at 4°C and increased the exposure time during imaging. The updated results are now shown in the revised manuscript (Response Figure.3; Fig. 4A in the manuscript). Regarding the internal control protein, we would like to clarify that conventional housekeeping proteins such as GAPDH or β-actin are not typically suitable for Co-IP experiments. This is because Co-IP aims to detect specific protein–protein interactions from a limited immunoprecipitated fraction rather than from total lysates, and the sample input is not standardized in the same way as conventional Western blots. Therefore, internal loading controls like β-actin are generally not informative in this context. Instead, we included the IgG heavy chain as an internal reference to indicate successful immunoprecipitation. However, in specific experiments such as the pull-down of B7H3 using a rabbit anti-ZDHHC24 antibody, the corresponding IgG was also rabbit-derived. Since B7H3 was detected using a mouse primary antibody and an anti-mouse secondary antibody, the rabbit IgG heavy chain was not visualized on the blot. This is due to the species-specific detection in Western blot, where the anti-mouse secondary antibody does not recognize rabbit IgG. We greatly appreciate the reviewer’s attention to this detail, which prompted us to improve both the experimental design and the clarity of data presentation.\nResponse Figure 3. Co-immunoprecipitation analysis of the interaction between ZDHHC24 and B7H3.\nQ6: In the FigS4a, Flag-ZDHHC9/ZDHHC16/ZDHHC22 did not successfully express after transfection, and whether they participate in the palmitoylation of B7H3 is unknown. Additional experiments are needed to rule out their effects.\nResponse: We sincerely thank you for this insightful comment. As suggested, we performed the transfection experiments for Flag-ZDHHC9, ZDHHC16, and ZDHHC22 to further assess their potential involvement in B7H3 palmitoylation. Although the expression of these constructs appeared weak in Figure S4a, we believe this is attributable to their inherently low expression levels and distinct subcellular localizations—ZDHHC9 localizes to the ER and Golgi, ZDHHC16 to the ER, and ZDHHC22 to both the ER and Golgi—which may reduce their detectability under our initial screening conditions. To clarify this, we individually transfected each of these ZDHHCs and examined their effects on B7H3 protein levels. Consistently, we did not observe any appreciable change in B7H3 expression following overexpression of ZDHHC9, ZDHHC16, or ZDHHC22 (Response Figure.4). Although ABE assays were not conducted in this specific follow-up experiment, our previous data have established a strong correlation between B7H3 palmitoylation and its protein stability. Therefore, the lack of effect on B7H3 levels suggests that these ZDHHC enzymes are unlikely to catalyze its palmitoylation. We appreciate your suggestion, which prompted us to further validate the specificity of ZDHHC24 in regulating B7H3 palmitoylation.\nResponse Figure 4. Evaluation of ZDHHC9, ZDHHC16, and ZDHHC22 in regulating B7H3 expression.\nQ7: Some of the flow cytometry analyses only provide statistical charts, and the mode charts should also be shown.\nResponse: We sincerely thank you for the thoughtful comment. We quiet understand the importance of including representative flow cytometry plots to support quantitative analyses. For all flow cytometry-based experiments presented in the manuscript, representative plots (mode charts) have already been included alongside the corresponding statistical summaries. Specifically, these representative plots can be found in the following figures: Main Figures – Fig. 6G, Fig. 6I, Fig. 8J, Fig. 8L; Supplementary Figures – Fig. S3L–O, Fig. S5K, S5N–P, Fig. S6C, S6E, Fig. S7F, Fig. S8N, Fig. S9C–E. In addition, for the newly added figures in this version of the manuscript, we have also included the corresponding representative flow cytometry plots where appropriate (Fig. S1C, Fig. 8R, Fig. 8T, and Fig. 8V). Furthermore, we have provided gating strategy plots in the supplementary Figures S10 and S11.We have carefully reviewed all relevant figures again to ensure that the flow cytometry plots are clearly presented and appropriately labeled. We would be pleased to provide any further plots or clarifications that could assist in the review. Thank you again for pointing this out.\nReviewer #2:\nThis study reveals a novel mechanism underlying the lower response rate to immune checkpoint blockade (ICB) therapy in microsatellite stable (MSS) colorectal cancer (CRC) patients compared to microsatellite instability-high (MSI-H) patients, highlighting the role of B7-H3 overexpression in tumor cells. Using patient tumor tissue samples and multi-omics technologies such as transcriptomics and metabolomics, they discovered that B7-H3 expression is significantly higher in MSS patient tissues compared to MSI patients. Furthermore, they elucidated that the key molecular mechanism involves palmitic acid-mediated palmitoylation modification of B7-H3. In detail, they discovered that ZDHHC24 mediates the palmitoylation of B7H3 at Cys496, which hinders its interaction with autophagy cargo receptor-p62 to inhibit its protein degradation. Based on their molecular mechanism, they designed a small mimic peptide of B7H3, which can enhance CD8+ T cell function by blocking the interaction between ZDHHC24 and B7H3. This strategy also elevated the sensibility and responsibility of CRC cells to the ICB therapy. Overall, this study is highly interesting and innovative, with rigorous experimental design and reliable results. It offers new perspectives and a material foundation for tackling the unresponsiveness of most CRC patients to ICB therapy in clinical settings.\nResponse: We sincerely thank you for the thoughtful and comprehensive evaluation of our manuscript. We are especially grateful for your appreciation of the novelty and scientific rigor of our study, as well as the translational relevance of our findings. Your encouraging comments on our experimental design, multi-omics analyses, and therapeutic strategy targeting B7H3 palmitoylation are deeply appreciated and highly encouraging. At the same time, we highly value the constructive suggestions regarding the limitations of our work. Your insights have guided several important improvements that have further strengthened the clarity and impact of the manuscript. Thank you again for your time, support, and helpful feedback.\nMajor comments:\nQ1: As an immune checkpoint, B7-H3 not only expresses in tumor cells, but also in immune cells, including monocytes, dendritic cells, myeloid-derived suppressor cells (MDSCs), neutrophils, macrophages, B cells, and activated T cells. So, is B7H3 only highly expressed in tumor cells of MSS type tumor tissues? The author didn’t make any exclusion experiments. If not, does the molecular mechanism also suit in other immune cells?\nResponse: We sincerely thank you for your important and insightful comment. We fully agree that B7H3 can be expressed not only in tumor cells but also in various immune cell populations. In response to this important point, we conducted additional experiments using freshly collected clinical tumor samples. We performed immunofluorescence staining with EPCAM (a tumor cell marker, in green) and B7H3 (in red, Cy5), and observed extensive co-localization of EPCAM and B7H3 in tumor tissues, indicating that B7H3 is predominantly expressed in tumor cells (Response Figure.5a; Fig. S1B in the manuscript). Furthermore, flow cytometry analysis revealed that while approximately 30% of immune cells in the tumor microenvironment are B7H3-positive, the B7H3 positivity rate in tumor cells is more than 95% (Response Figure.5b-c; Fig. S1C-S1D in the manuscript). When normalized to 100,000 total cells, the number of B7H3⁺ tumor cells were estimated to be around 90,000 (Response Figure.5d; Fig. S1E in the manuscript), further supporting the notion that tumor cells are the dominant source of B7H3 expression in the MSS tumor context. While we do not exclude the possibility that the molecular mechanism we proposed could also be relevant in immune cells, we believe that, within the tumor microenvironment, the overwhelming expression of B7H3 in tumor cells suggests that the described palmitoylation-dependent regulatory mechanism primarily functions in tumor cells. We appreciate the reviewer’s thoughtful suggestion, which prompted us to perform these additional validations and helped further strengthen the conclusions of our study.\nResponse Figure 5. B7H3 expression is predominantly localized to tumor cells in MSS colorectal cancer tissues.\nQ2: The authors explored their molecular mechanism mainly in RKO cells (MSI), SW480 cells (MSS), CT26 cells (MSS) and MC38 cells (MSI). I wonder whether B7H3 is also highly expressed in MSS CRC cell lines compared to the MSI ones. If this post-translational modification of B7-H3 exists in both MSS and MSI-type tumor cells, then is the high expression of B7-H3 in MSS tissues primarily due to differences in ZDHHC24 expression or the distribution of palmitic acid, or both? If it is due to ZDHHC24, they should provide more details of its protein expression in MSS and MSI tumor tissues and cells. If it is due to palmitic acid, it would be interesting to know why there is a higher abundance of palmitic acid in MSS tumors. At least, it could be a good content for the discussion part.\nResponse: We sincerely thank you for your thoughtful and insightful comment. In response to your suggestion and to further improve the completeness of our study, we examined the expression of B7H3 and ZDHHC24 in a panel of human colorectal cancer cell lines representing both MSS and MSI-H subtypes. Notably, we did not observe consistent differences in the protein levels of B7H3 or ZDHHC24 between MSS and MSI-H cell lines (Response Figure.6a; Fig. S4L in the manuscript). However, analysis of patient-derived tumor tissues revealed that ZDHHC24 expression is significantly elevated in MSS tumors compared to MSI-H tumors (Response Figure.6b; Fig. S4M in the manuscript). This discrepancy between cell lines and clinical samples may reflect the influence of the in vivo tumor microenvironment, which is absent in standard in vitro models. In addition to the elevated expression of ZDHHC24, our metabolomic profiling showed that palmitic acid abundance is also higher in MSS tumors than in MSI-H tumors (Fig. 1D in the manuscript). These findings suggest that both increased ZDHHC24 expression and enhanced palmitic acid availability may cooperatively promote B7H3 palmitoylation and stabilization in MSS tumors. We have expanded the Discussion section to explore potential mechanisms underlying the elevated palmitic acid levels observed in MSS tumors (Page 19-20, line 565-576). We are grateful for this valuable suggestion, which helped us refine our interpretation and explore a broader biological context for our findings.\nResponse Figure 6. Expression of B7H3 and ZDHHC24 in colorectal cancer cell lines and ZDHHC24 expression in patient tumor tissues.\nQ3: Whether ZDHHC24 enhances the protein stability of B7H3 should be detected.\nResponse: We sincerely thank you for this insightful comment, which helped us further improve the completeness of our study. Following your suggestion, we performed additional experiments to investigate whether ZDHHC24 affects the protein stability of B7H3. We found that knockdown of ZDHHC24 accelerated the degradation of B7H3 protein, indicating that ZDHHC24 enhances the stability of B7H3 (Response Figure.7a-b; Fig. 4G-4H in the manuscript). We appreciate your valuable suggestion, which enabled us to further elucidate the regulatory role of ZDHHC24 in our study.\nResponse Figure 7. Knockdown of ZDHHC24 reduces the stability of B7H3\nQ4: As they mentioned, E-peptide works through interrupting the binding of B7H3 and ZDHHC24. They should give more details about the working mechanism. For example, whether E-peptide binds with ZDHHC24? If so, surface plasmon resonance (SPR) assay should be utilized to obtain kinetic parameters. Besides, whether E-peptide enhances the binding of B7H3 with p62 should be detected.\nResponse: We sincerely thank you for this thoughtful and constructive comment. In line with your suggestion, we conducted a surface plasmon resonance (SPR) assay to assess the direct interaction between the E-peptide and ZDHHC24. The results confirmed that the E-peptide binds directly to ZDHHC24 with measurable kinetic parameters, supporting our proposed model (Response Figure.8a; Fig. S7D in the manuscript). Furthermore, we examined whether E-peptide treatment affects the interaction between B7H3 and p62. Co-immunoprecipitation analysis revealed that E-peptide treatment enhanced the association between B7H3 and p62 (Response Figure.8b; Fig. 7J in the manuscript). These findings are consistent with our hypothesis that the E-peptide disrupts the interaction between B7H3 and ZDHHC24, thereby reducing B7H3 palmitoylation and promoting its binding to p62. We sincerely appreciate your insightful suggestions, which greatly enhanced the mechanistic depth and experimental rigor of our work.\nResponse Figure 8. E-peptide binds to ZDHHC24 and enhances the interaction between B7H3 and p62.\nQ5: The authors have tested the anti-tumor effect of E-peptide synthesized according to the human B7H3 amino acids sequence in the mouse tumor models. Is there significant sequence conservation of this peptide segment between humans and mice? It is necessary to verify whether the E-peptide reduces the protein expression of B7H3 and impacts its palmitoylation in mice tumors. To enhance the clinical relevance of the results, it is recommended to evaluate the anti-tumor effects of the peptide using immunologically reconstituted humanized mice.\nResponse: We sincerely thank you for your insightful and constructive comment. As you correctly pointed out, it is important to evaluate the sequence conservation of the E-peptide between humans and mice. We compared this region using the UniProt database and found that it exhibits a high degree of sequence conservation between the two species (Response Figure 9a). We acknowledge that this point was not clearly highlighted in the original version of the manuscript. To avoid potential misunderstanding, we have now added explicit annotations and explanatory text (Page 15, line 441-443) in the revised manuscript to clarify this rationale. Following your recommendation, we conducted additional experiments to assess the effect of E-peptide on B7H3 expression and palmitoylation in mouse tumors. The results showed that treatment with the E-peptide led to a marked reduction in both B7H3 protein levels and its palmitoylation status in mouse tumor tissues (Response Figure 9b–9c; Fig. S8L-S8M in the manuscript). Furthermore, to enhance the clinical relevance of our findings, we evaluated the anti-tumor efficacy of the E-peptide in immunologically reconstituted humanized mice. Specifically, human hematopoietic stem cells derived from umbilical cord blood were transplanted into C-NKG mice via the superficial temporal vein. After 12 weeks, successful engraftment was confirmed by flow cytometric analysis of human immune cell markers in retro-orbital blood samples (Response Figure 9d; Fig. 8M in the manuscript). Humanized mice were then subcutaneously inoculated with human colorectal cancer cells (SW480), and treated with either C-peptide or E-peptide (administered four times). The E-peptide significantly inhibited tumor growth (Response Figure 9e–9g; Fig. 8N-8P in the manuscript), increased the infiltration of CD8+ T cells (Response Figure 9h–9i; Fig. 8Q-8R in the manuscript), and markedly enhanced the expression of cytotoxic effector molecules GZMB and IFN-γ within the tumor microenvironment (Response Figure 9j–9m; Fig. 8S-8V in the manuscript). Together with our previous results in immunocompetent mice, these findings confirm that the E-peptide exhibits anti-tumor activity in both murine and humanized immune environments. We sincerely appreciate your thoughtful suggestions, which have helped us to strengthen the translational value and mechanistic depth of our study.\nResponse Figure 9. E-peptide reduces B7H3 expression and palmitoylation in mouse tumors and exhibits anti-tumor activity in humanized mice.\nMinor comments:\nQ1: In Figure 5, the authors investigated the tumor-promoting role of ZDHHC24 in the AOM/DSS-induced CRC mouse model using ZDHHC transgenic mice. However, since they have not confirmed that ZDHHC24 functions through palmitoylating B7H3 in this model, this section appears somewhat disconnected from the central topic. It is recommended to either relocate this part to the supplementary material or provide additional evidence linking ZDHHC24's activity to B7H3 palmitoylation to maintain coherence with the main narrative.\nResponse: We sincerely thank you for your thoughtful and constructive comment. In our study, ZDHHC24 serves as the palmitoyltransferase that catalyzes the palmitoylation of B7H3, a modification we found to be crucial for maintaining B7H3 protein stability. To our knowledge, the role of ZDHHC24 in colorectal cancer has not been previously reported, making it a novel and mechanistically important focus of our work. Based on this, we believe that investigating its function in an in vivo CRC model is necessary and relevant to the main theme of the manuscript. As you rightly pointed out, our original manuscript did not provide direct evidence linking ZDHHC24 activity to B7H3 palmitoylation in the AOM/DSS-induced CRC model, which could weaken the connection between this part and the central narrative. In response to your suggestion, we conducted additional experiments using this model. After successful induction of CRC, we collected tumor tissues from Zdhhc24△IEC and control mice, and assessed B7H3 protein expression and palmitoylation levels. The results showed that both B7H3 expression and its palmitoylation were reduced in Zdhhc24△IEC mice (Response Figure 10a–b; Fig. S5H-S5I in the manuscript), highlighting the in vivo relevance of ZDHHC24 in modulating B7H3 function, thereby reinforcing the study’s core mechanistic framework. We truly appreciate your valuable feedback, which helped us clarify and strengthen this section.\nResponse Figure 10. Loss of Zdhhc24 reduces B7H3 expression and palmitoylation in the AOM/DSS-induced colorectal cancer model.\nQ2: To make sure whether ZDHHC24 interrupt the interaction of B7H3 with p62, proximity ligation assay should also be used besides Co-IP assay.\nResponse: We sincerely thank you for your insightful suggestion. As you recommended, we performed additional experiments to validate whether ZDHHC24 interferes with the interaction between B7H3 and p62. First, immunofluorescence staining and confocal microscopy showed that knockdown of ZDHHC24 led to increased co-localization and puncta formation of B7H3 and p62 (SQSTM1) (Response Figure 11a; Fig. 4L in the manuscript). To further validate this observation, we performed a proximity ligation assay (PLA), which provides direct evidence of protein–protein interactions. The PLA results confirmed that the interaction between B7H3 and p62 was significantly enhanced following ZDHHC24 knockdown (Response Figure 11b; Fig. 4M in the manuscript). We sincerely appreciate your valuable recommendation, which helped us reinforce the mechanistic insights of our study.\nResponse Figure 11. ZDHHC24 knockdown enhances the interaction between B7H3 and p62.\nReviewer #3:\nIt this manuscript Rao et al. have analysed a member of the B7 family and found that B7H3 shows increased expression in MSS tumours. They go on showing that B7H3 undergoes S-acylation on Cys-496, and that this slows down the turnover rate of B7H3. They propose that degradation of the non-acylated form occurs via autophagy. They show that ZDHHC24 is the major acyltransferase involved in modifying B7H3. They subsequently address the role of B7H3 acylation in tumour progression and finally design a peptide that inhibits B7H3 acylation, showing that it inhibits tumour growth.\nThe finding that preventing B7H3 acylation can have therapeutic benefit is clearly of interest. However the manuscript lack a rigour of thinking/writing that affects its quality.\nI have focused my review on S-acylation, since the cancer immunology aspects are outside of my expertise. I do wish to mention that the effects of the e-peptide and anti-PD-1 antibodies by no means seem synergistic. The data indicates that they are somewhat additive, although not fully. From my perspective, the paper is written in a manner that is superficial and lacks the necessary rigour to make the reader confident of the quality of the work. Reference to the literature and what is really demonstrated in the literature is insufficient and again superficial. While clearly the interest of the authors lies in the therapeutic application, I regret that there is no questioning regarding the mechanistic aspect. This gives the impression of a pseudo-mechanistic insight.\nResponse: We sincerely thank you for your careful and detailed evaluation of our manuscript, and for noting the potential therapeutic interest of targeting B7H3 palmitoylation. We fully acknowledge your concerns regarding the need for greater scientific rigour, particularly in mechanistic depth and clarity of presentation. Following your suggestions, we have substantially revised the Introduction and Discussion sections to improve the manuscript’s structure, clarity, and logical flow. We have also strengthened the mechanistic narrative by incorporating additional experimental data, and expanded the discussion of B7H3 palmitoylation within the context of relevant literature to better frame our findings. Moreover, we have explicitly acknowledged the limitations of our study and proposed directions for future investigation. We sincerely hope that these revisions and the efforts we have made based on your suggestions will meet with your approval. We also greatly appreciate your insightful observation regarding the combined effect of the E-peptide and anti-PD-1 therapy. To further clarify this point, we have provided a brief mechanistic rationale and quantitative analysis of their interaction. Mechanistically, the E-peptide disrupts the interaction between B7H3 and ZDHHC24, leading to reduced palmitoylation and surface expression of B7H3, thereby relieving the immunosuppressive signal delivered by B7H3 to CD8⁺ T cells. In contrast, anti-PD-1 therapy directly reinvigorates CD8⁺ T cells by blocking the inhibitory PD-1/PD-L1 pathway. Although both interventions enhance CD8⁺ T cell function via distinct upstream mechanisms converging on the same effector population, our quantitative analyses indicate that their combined effects exceed simple additivity. Specifically, survival data (Fig.9G) show that the mean survival in the IgG group was 18.1 days, compared to 24.4 days for the anti-PD-1 group (a 34.8% increase) and 25.2 days for the E-peptide group (a 39.2% increase). The combination therapy extended mean survival to 40.5 days (~124% improvement over IgG). We applied the Bliss independence model—a standard approach for quantifying interaction effects under the assumption of independent action—to estimate the expected additive outcome based on the effects of each intervention alone. This analysis indicated that the expected additive improvement would be approximately 60.4%, corresponding to a predicted mean survival of 29.0 days. The observed survival benefit of the combination therapy therefore substantially exceeds the predicted additive effect, consistent with a synergistic interaction. Similarly, Bliss independence analysis shows that reductions in tumor volume (Fig.9H) in the combination group exceed predicted additive effects, supporting a synergistic enhancement of anti-tumor immunity. Taken together, these findings are consistent with a synergistic interaction between E-peptide and anti-PD-1 therapy in improving therapeutic outcomes. We hope the additional analyses and explanations help to clarify the potential mechanistic and functional interplay between these two interventions. While our initial focus was primarily on the therapeutic potential of targeting B7H3, we fully agree that mechanistic rigor is essential for a convincing scientific argument. Your comments have been invaluable in guiding these improvements, and we are truly grateful for your thoughtful feedback.\nQ1: Line 38: p62 is not a cargo protein.\nResponse: Thank you very much for pointing this out. We agree that the term \"cargo protein\" was not used accurately in this context. P62 (also known as SQSTM1) is more appropriately described as a selective autophagy receptor, which binds to ubiquitinated proteins and facilitates their delivery to the autophagosome for degradation. We have revised the wording in the manuscript to reflect this distinction more precisely and now refer to p62 as an “autophagy adaptor protein” instead of a cargo protein (Page 2, line 38). We appreciate your correction, which helped us improve the accuracy of our terminology.\nQ2: Line 51: for non-experts, having three acronyms in the first sentence is difficult.\nResponse: We sincerely thank you for the constructive suggestion. We fully agree that using multiple acronyms in the opening sentence may hinder readability for non-expert readers. To address this, we have revised the sentence in the revised manuscript to spell out the full names upon first mention. The revised sentence now reads: “Immune checkpoints, such as programmed death-1 (PD-1), programmed death-ligand 1 (PD-L1), and cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4), play a crucial role in maintaining self-tolerance and regulating anti-cancer immunity.” (Page 2, line 52-55). We hope this change improves clarity and accessibility. Thank you again for pointing this out.\nQ3: Define B7 family, provide some background information.\nResponse: Thank you for your suggestion. In the revised manuscript, we have added a brief definition and background of the B7 family in the Introduction (Page 3, line 64-66) to address this comment. Specifically, we included the following sentence: “The B7 family, comprising a group of structurally related transmembrane proteins, including B7-1, B7-2, B7-H1 (PD-L1), B7-H3, and others, interact with CD28 family receptors to either stimulate or suppress immune activity”. We sincerely appreciate your helpful suggestion, which allowed us to improve the clarity and completeness of the background information in our manuscript.\nQ4: - Line 70: I think that what the author call “membrane localization” is plasma membrane localization”. Indeed, B7H3 is a transmembrane protein, that’s what determines its membrane localization.\nResponse: We sincerely thank you for this helpful suggestion. We agree that the term “membrane localization” could be misleading or too broad in this context, as B7-H3 is a type I transmembrane protein whose immunosuppressive function depends on its stable expression on the plasma membrane, rather than on intracellular membranes. In line with the your comment and the original reference (PMID: 33976130), we have revised the sentence to more accurately reflect this mechanism (Page 3, line 74-77). The revised sentence now reads: “While phosphorylation and ubiquitination sites have been predicted, N-glycosylation plays a pivotal role in stabilizing B7H3 and ensuring its stable expression on the plasma membrane, thereby facilitating its immunosuppressive function.” We appreciate the reviewer’s careful reading and constructive feedback, which has helped us improve the clarity and precision of our manuscript.\nQ5: Line 78: the correct term is S-acylation, often referred to as S-palmitoylation , or just Palmitoylation.\nResponse: Thank you for your correction. We acknowledge that “S-acylation” is the more accurate and comprehensive term, with “S-palmitoylation” or “palmitoylation” being commonly used to describe the most prevalent form of this modification. In response to your suggestion, we have revised the sentence to improve clarity and terminology accuracy. Specifically, we have replaced “Palmitoylation (usually referred to as S-palmitoylation)” with “S-acylation (often referred to as S-palmitoylation or simply palmitoylation)” in the manuscript (Page 3, line 82). We appreciate your helpful feedback, which has helped us to refine the scientific language of the manuscript.\nQ6: Line 79: there is very little evidence so far that S-acylation affects protein structure. This is thus incorrect.\nResponse: We thank the reviewer for this important and constructive comment. Our original intention was to describe the biochemical nature of palmitoylation, specifically the formation of a thioester bond between the sulfhydryl group of a cysteine residue and a 16-carbon palmitate molecule. While some studies report that S-acylation can directly or indirectly induce conformational changes. For example, S-acylation has been shown to regulate the opening and closing of membrane channels[1–3], or modulate the exit of LRP6 from the endoplasmic reticulum by tilting transmembrane domains[4]. However, we acknowledge that the phrase “is commonly used to modify protein structure” may be misleading. To avoid this ambiguity and improve scientific accuracy, we have removed the phrase from the sentence. The revised version (Page 3, line 82-85) now reads: “S-acylation (often referred to as S-palmitoylation or simply palmitoylation) is an important post-translational modification in which a thioester bond is formed between the sulfhydryl group of a cysteine residue and, most commonly, a 16-carbon palmitate.” We appreciate your careful reading and helpful feedback, which have helped us enhance the clarity and precision of our manuscript.\nReference:\n[1] F B, M M, KJ S. Palmitoylation Influences the Function and Pharmacology of Sodium Channels[J]. Proc Natl Acad Sci U S A, 2011, 108(50).\n[2] Z P, Y X, J M, et al. Cardiac Sodium Channel Palmitoylation Regulates Channel Availability and Myocyte Excitability with Implications for Arrhythmia Generation[J]. Nat Commun, 2016, 7.\n[3] S C, C V R, A B G, et al. Palmitoylation of Voltage-Gated Ion Channels[J]. Int J Mol Sci, 2022, 23(16).\n[4] L A, B K, I I, et al. Palmitoylation and Ubiquitination Regulate Exit of the Wnt Signaling Protein LRP6 from the Endoplasmic Reticulum[J]. Proc Natl Acad Sci U S A, 2008, 105(14).\nQ7: Line 87: reword “irreplaceable”, this is likely true for any PTM.\nResponse: We thank the reviewer for this thoughtful comment. We agree that the term “irreplaceable” may overstate the specificity of palmitoylation, as many post-translational modifications (PTMs) contribute uniquely to immune regulation. To improve precision and avoid overgeneralization, we have replaced the word “irreplaceable” with “important” (Page 3, line 90) to avoid overstatement and improve accuracy. We appreciate the reviewer’s careful reading and helpful suggestion, which has helped us refine the clarity and balance.\nQ8: Abstract and lines 95, 163 and others places in the manuscript: Palmitic acid does not “mediate” palmitoylation in the context of this work, but triggers it. All palmitoylation is by definition palmitate-mediated.\nResponse: We sincerely thank the reviewer for this insightful comment. We understand that the term “palmitic acid-mediated palmitoylation” could be confusing, as palmitoylation is enzymatically catalyzed and palmitic acid serves as the lipid donor rather than the direct mediator. Our original intention was to convey that palmitic acid functions as an upstream stimulus that increases B7H3 palmitoylation, rather than being the direct enzymatic mediator of the modification. In accordance with your suggestion, we have carefully revised the text in the Abstract and throughout the manuscript (Title, Abstract, Lines 99, 122, 161, 181, 558, 561, and 635), replacing “mediates” with the more appropriate term such as “triggers,” “promotes,” or “facilitates”, depending on the context, in order to ensure scientific clarity and precision. We appreciate the reviewer’s thoughtful feedback, which has helped improve the accuracy of our terminology.\nQ9: Line 155: separating cytosolic from membrane by fractionnation when looking at a membrane protein seems rather meaning less. These type of experiments seed doubts in the reader’s mind as to the mechanistic understanding of the authors regarding the protein under study.\nResponse: Thank you for your valuable and critical comment. We fully recognize that B7H3 is a type I transmembrane protein. However, our rationale for performing cytoplasmic and membrane fractionation assays was not to confirm its localization, but rather to understand the mechanism behind the observed reduction in B7H3 protein levels following palmitoylation inhibition. By altering the hydrophobicity of proteins, palmitoylation can influence their stability, intracellular trafficking, functional activity, and plasma membrane localization[1–3]. We hypothesized that the reduced total B7H3 levels after 2-BP treatment could result either from mislocalization—such as impaired membrane targeting and cytosolic accumulation—or from a global degradation of B7H3 across both compartments. To distinguish between these possibilities, we conducted cytoplasmic and membrane fractionation assays. Our results showed that 2-BP treatment led to a consistent decrease in B7H3 levels in both membrane and cytosolic fractions (Fig. S1S), indicating that the loss is not due to redistribution but reflects an overall downregulation of the protein. We sincerely appreciate your thoughtful comment, which allowed us to clarify the experimental rationale and strengthen the mechanistic interpretation of our findings.\nReference:\n[1] LU Y, ZHENG Y, COYAUD É, et al. Palmitoylation of NOD1 and NOD2 Is Required for Bacterial Sensing[J]. Science (New York, N.Y.), 2019, 366(6464): 460–467.\n[2] LINDER M E, DESCHENES R J. Palmitoylation: Policing Protein Stability and Traffic[J]. Nature Reviews Molecular Cell Biology, 2007, 8(1): 74–84.\n[3] S MESQUITA F, ABRAMI L, LINDER M E, et al. Mechanisms and Functions of Protein S-Acylation[J]. Nature Reviews Molecular Cell Biology, 2024, 25(6): 488–509.\nQ10: Line 160: the authors are monitoring total B7H3 levels, not palmitoylated B7H3. This is an interpretation of the data, not a description.\nResponse: We sincerely thank the reviewer for this insightful comment. We agree that it is necessary to distinguish between total B7H3 levels and palmitoylated B7H3. You are absolutely right that the experiments shown in Figures 1K and 1L assessed total B7H3 protein levels under DMSO and 2-BP treatment conditions, rather than directly quantifying the palmitoylated form of B7H3. Our original intent was to evaluate how inhibition of palmitoylation affects B7H3 protein stability over time. Given that 2-BP is a well-established inhibitor of palmitoylation, and B7H3 is expected to be palmitoylated under DMSO treatment, we initially described the observation as a comparison of degradation rates between palmitoylated and palmitoylation-impaired (2-BP-treated) B7H3. However, we agree with you that this wording could be misleading, as it implies that the assay directly measured palmitoylated B7H3. To address this concern and more objectively describe the experimental result, we have revised the wording (Page 6, line 179-180) to read: “Treatment with 2-BP markedly accelerated B7H3 degradation (Fig. 1L and 1M)” We sincerely appreciate your careful reading and constructive feedback, which has helped us improve the accuracy and clarity of our manuscript.\nQ11: Line 166: the two protein degradation “sites” in the cell are lysosomes and the proteasome. Autophagy is a pathway to lysosomal degradation. However, the default routes for a transmembrane protein like B7H3 are 1) the proteasome if it fails to fold properly during its biogenesis in the ER (this requires retro translocation as part of the ERAD pathway) and 2) trafficking to lysosomes. Given its role in the interaction with T-cells, B7H3 acts at the plasma membrane and thus its route to degradation would be endocytosis and trafficking to lysosomes via the classical route. Autophagy is not the most evident pathways as opposed to what the authors suggest, and thus the approach appears to have a significant bias. That acylation affects the endocytosis of plasma membrane transmembrane proteins has been shown for a variety of proteins. B7H3 is likely to be as others surface proteins, its endocytosis is negatively regulated by S-acylation, thus acylation deficient mutants will undergo accelerated endocytosis and lysosomal degradation. There is a priori no need to bring up autophagy.\nResponse: Thank you for this insightful comment. We agree that most transmembrane proteins are normally cleared via endocytosis and lysosomal trafficking or by ERAD/proteasome when misfolded. However, emerging evidence suggests that certain plasma membrane–localized transmembrane proteins can also undergo degradation via selective autophagy under specific conditions. For example, Yamamoto et al. demonstrated that the major histocompatibility complex class I (MHC-I) (Single-pass type I membrane protein) is degraded via a selective autophagy pathway in pancreatic ductal adenocarcinoma[1]. Similarly, Liu et al. reported that PD-L1 (a type-I transmembrane glycoprotein) can undergo selective autophagic degradation mediated by the cargo receptor p62/SQSTM1[2]. Notably, Zhang et al. showed that demonstrated that B7H3 is degraded via selective autophagy in glioma cells, as its degradation was blocked by autophagy inhibitors and involved interaction with p62 through LC3-interacting region motifs[3]. These studies indicate that autophagy can target plasma membrane proteins in certain contexts. Our findings are consistent with these observations. In the original manuscript, we showed that treatment with 3-MA (an autophagy inhibitor that blocks the formation of the phagophore by inhibiting class III PI3K) rescued the reduction of B7H3 levels induced by 2-BP (Fig. 2A), suggesting the involvement of autophagy in B7H3 degradation. To further confirm this, we conducted further experiments to validate that B7H3 degradation occurs via the autophagy pathway. Specifically, we found that treatment with rapamycin—which inhibits mTORC1 activity and thereby induces autophagy—led to a marked reduction of B7H3 protein levels in RKOWT cells (Response Figure 12a; Fig. S2K in the manuscript). Conversely, inhibition of autophagy using 3-MA (a PI3K inhibitor that blocks autophagosome formation), bafilomycin A1, or chloroquine (both of which disrupt lysosomal acidification and block autophagic flux) effectively prevented the degradation of B7H3 in the palmitoylation-deficient mutant RKOC496A cells (Response Figure 12b; Fig. S2L in the manuscript). These results strongly support our conclusion that B7H3 is subject to degradation via the autophagy–lysosome pathway in our system. While we fully acknowledge that S-acylation can influence endocytosis, such as PD-L1[4], we believe that in our context, autophagy plays a significant and experimentally validated role. We appreciate your thoughtful comment, which prompted us to provide a more thorough explanation.\nResponse Figure 12. B7H3 undergoes autophagy-mediated degradation in RKO cells.\nReference:\n[1] YAMAMOTO K, VENIDA A, YANO J, et al. Autophagy Promotes Immune Evasion of Pancreatic Cancer by Degrading MHC-I[J]. Nature, 2020, 581(7806): 100–105.\n[2] LIU D, LI M, ZHAO Z, et al. Targeting the TRIM14/USP14 Axis Enhances Immunotherapy Efficacy by Inducing Autophagic Degradation of PD-L1[J]. Cancer Research, 2024, 84(17): 2806–2819.\n[3] ZHANG M, ZHANG H, FU M, et al. The Inhibition of B7H3 by 2-HG Accumulation Is Associated With Downregulation of VEGFA in IDH Mutated Gliomas[J]. Frontiers in Cell and Developmental Biology, 2021, 9.\n[4] H Y, J L, C L, et al. Inhibiting PD-L1 Palmitoylation Enhances T-Cell Immune Responses against Tumours[J]. Nature biomedical engineering, 2019, 3(4).\nQ12: - line 170: Baf A1 and chloroquine are not autophagy inhibitors, they affect the pH of lysosomes, thus inhibit lysosomal enzymes, thus affect the end point of autophagy.\nResponse: We sincerely thank you for this insightful comment. You are absolutely right that Bafilomycin A1 and chloroquine do not inhibit autophagy initiation directly. Instead, they interfere with lysosomal acidification, thereby impairing the activity of lysosomal enzymes and blocking the terminal stage of autophagy. In our original text, we used the term “autophagy inhibitors” in a broad sense, which may have unintentionally caused confusion about their precise mode of action. In response to your suggestion, we have revised the sentence to more accurately reflect the mechanisms of these compounds. The updated sentence now reads: “Interestingly, pharmacological agents that disrupt different stages of the autophagy pathway—3-MA (an early-stage autophagy inhibitor), and BafA1 and chloroquine (which impair lysosomal degradation)—effectively blocked the 2-BP-induced degradation of B7H3, whereas the proteasome inhibitor MG132 had no such effect.” (Page 7, line 191-195). Thank you again for helping us improve the accuracy and clarity of our manuscript.\nQ13: - Line 193: C496A has a mutation of te acylation site, not a deletion.\nResponse: Thank you very much for your careful reading and valuable comment. You are absolutely right—referring to the C496A mutant as having a “deletion” of the palmitoylation site was inaccurate, as the site was mutated rather than deleted. In response to your suggestion, we have corrected the description by replacing “deletion” with “mutation” to accurately reflect that C496A is a point mutant of the acylation site (Page 8, line 219). We truly appreciate your attention to detail and thank you again for helping us improve the precision of our manuscript.\nQ14: - Line 204: microscopy can never inform about the ability to bind. This is an interpretation.\nResponse: Thank you very much for this insightful comment. You are absolutely right that confocal microscopy alone does not directly measure binding affinity, and our original wording may have overstated the interpretation of the imaging data. In response to your suggestion, we have revised the sentence to describe the microscopy results more accurately, focusing on the observed colocalization patterns rather than implying a direct binding interaction. The updated sentence now reads: “Moreover, confocal microscopy revealed that RKOC496A cells displayed greater colocalization of B7H3 with LC3 and formed more distinct B7H3–SQSTM1/p62 aggregates compared with RKOWT cells.” (Page 8, line 233-235). We sincerely appreciate your thoughtful feedback, which helped us improve the accuracy and clarity of our description.\nQ15: - Line 272: ZDHHC24 has been reported to be an ER localized acyltransferase. The authors should comment on the fact that their localization at the plasma membrane is in disagreement with the literature. Also a quantification of the localization would be required.\nResponse: Thank you very much for your valuable comment. As you rightly pointed out, ZDHHC24 has been previously reported by Bu et al. to localize predominantly to the endoplasmic reticulum[1]. However, their study also detected partial localization at the plasma membrane, which is consistent with our findings in Figure 4B. In our study, we observed endogenous ZDHHC24 localization at both the plasma membrane and in the cytoplasm of RKO tumor cells. This distribution aligns with Bu et al.’s results, although they described prominent cytoplasmic aggregates that may result from ZDHHC24 overexpression in 293T cells. In contrast, our analysis was conducted under endogenous expression conditions, which may better reflect its physiological localization in cancer cells. Furthermore, subcellular annotations from UniProt (Q6UX98) support the possibility of multi-compartment localization: the UniProt annotation indicates localization to the plasma membrane (Response Figure 13a), while the Gene Ontology (GO) annotation suggests localization to the ER and Golgi apparatus (Response Figure 13b). This dual annotation supports our observation of ZDHHC24 presence in both the cytoplasm and plasma membrane, and suggests that its localization may vary depending on cellular context. In response to your suggestion, we repeated the confocal microscopy experiments and confirmed the co-localization of ZDHHC24 and B7H3 in both the cytoplasm and plasma membrane (Response Figure 13c). To quantitatively support these findings, we performed fluorescence intensity profile analysis of the merged signals (Response Figure 13d). These results provide further evidence that ZDHHC24 is not restricted to the endoplasmic reticulum and may function at multiple subcellular compartments, including the plasma membrane. We appreciate your thoughtful feedback, which helped us strengthen our manuscript.\nResponse Figure 13. ZDHHC24 subcellular localization and quantitative analysis.\nReference:\n[1] L B, H W, S Z, et al. Targeting APT2 Improves MAVS Palmitoylation and Antiviral Innate Immunity[J]. Molecular cell, 2024, 84(18).\nQ16: - Molecular weight markers are missing on all blots\nResponse: We sincerely apologize for the oversight and thank you for pointing this out. In response to your comment, we have now included molecular weight markers on all immunoblot images in the revised figures. We appreciate your attention to this important detail, which has helped us improve the clarity and completeness of our data presentation.\nQ17: - Quantifications of Fig. 1G and many other blots are missing.\nResponse: Thank you very much for your helpful comment. We sincerely appreciate your suggestion regarding the quantification of immunoblot data. In the revised manuscript, we have now added the quantification analysis for Figure 1G as you recommended (Fig. 1H in the manuscript). As part of our submission, we had already included quantifications for several key immunoblots, including Figure 1M, Figure S1V, Figure 2B, Figure 2D, Figure 2G, Figure 2O, Figure S2B, Figure S2D, Figure 4H, and Figure 7I. For certain other blots, where the differences in band intensity were visually prominent and unambiguous, we did not perform additional quantification, as the visual evidence was considered sufficient for supporting the conclusion. We appreciate your thoughtful feedback, which has helped us improve the completeness and clarity of our data presentation.\nReviewer #4:\nIn this research article, Rao et al. study the mediation of the palmitoylation of the immune checkpoint protein B7H3, demonstrating that deletion of palmitoylation site restores CD8+ T cell antitumor activity. The authors further developed a peptide that enhances CD8+ T cell function by blocking the interaction between ZDHHC24 and B7H3, which works synergistically with anti-PD-1 therapy. The work presents a relevant contribution for cancer therapy, but some concerns need to be addressed:\nResponse: Thank you very much for your thoughtful and encouraging overall assessment of our work. We are grateful that you noted the relevance of our study and its potential contribution to cancer therapy, particularly in relation to palmitoylation-mediated regulation of B7H3 and its impact on CD8⁺ T cell function. We also sincerely appreciate your constructive comments and suggestions. We have carefully addressed each of your concerns in the revised manuscript, as detailed in our point-by-point responses. Your feedback has been invaluable in helping us improve the clarity, rigor, and impact of our study.\nQ1: - The authors discussed the rationale of the design of the “E-peptide”, in which the amino acids 488-497 in the B7H3 sequence were found to be essential for the interaction with ZDHHHC24. However, the rationale of the “C-peptide” is not explained. Why did the authors use this sequence? The authors compare the E-peptide to C-peptide, but the reason for C-peptide being a control is unclear.\nResponse: Thank you very much for your insightful comment. We apologize for not clearly explaining the rationale behind the design of the C-peptide in the original manuscript. As you correctly noted, the E-peptide corresponds to amino acids 488–497 of B7H3, which we identified as essential for binding to ZDHHC24. To generate a proper control, we designed the C-peptide by randomly scrambling the amino acid sequence of the 488–497 region. This approach is intended to abolish specific binding to ZDHHC24 by disrupting the sequence-specific structure and function, while maintaining peptide length and composition, as has been demonstrated in previous studies[1]. Both peptides contain an N-terminal TAT tag to ensure cellular uptake. We have now revised the main text to clarify this rationale (Page 15, line 440-443). Thank you again for your valuable suggestion, which helped us improve the clarity and completeness of our experimental design.\nReference:\n[1] ZHANG W, WANG G, XU Z G, et al. Lactate Is a Natural Suppressor of RLR Signaling by Targeting MAVS[J]. Cell, 2019, 178(1): 176-189.e15.\nQ2: - Did the authors check if the C-peptide is also internalized by confocal microscopy?\nResponse: Thank you very much for your insightful comment. Initially, we assumed that the cellular uptake of both peptides would primarily rely on the TAT transduction domain, and therefore only assessed the internalization of the E-peptide. We acknowledge that this assumption was not sufficiently rigorous. In response to your suggestion, we have now performed confocal microscopy to examine the internalization of the C-peptide as well. The results confirmed that the C-peptide is also effectively taken up by the cells (Response Figure 14a; Fig. S7C in the manuscript). We sincerely thank you for pointing this out, which has helped us improve the scientific rigor and completeness of our study.\nResponse Figure 14. Confocal microscopy analysis showing intracellular localization of the C-peptide in RKO cells.\nQ3: -The authors should provide the synthesis protocol and characterization for all the compounds, including the data that supports the assignment of structure (NMR and mass spectrometry) and purity, such as HPLC.\nResponse: We sincerely thank you for your insightful suggestion. We have now included the synthesis protocol for the peptide in the revised Methods section of the supplement information (Page 13, Line 369-381). The synthesis steps for the peptides are as follows: The C-peptide (YGRKKRRQRRRRKWEISQCKC) and E-peptide (YGRKKRRQRRRCWRKIKQSCE) were synthesized by QYAOBlO (ChinaPeptides Co., Ltd.) using standard Fmoc-based solid-phase peptide synthesis (SPPS) protocols on 2-chlorotrityl chloride resin. Briefly, after resin swelling in DMF (15 mL/g, 1 h), the first Fmoc-protected amino acid (C-terminal residue) was coupled using 3 equivalents of amino acid and 10 equivalents of DIEA, followed by methanol capping. Fmoc deprotection was carried out with 20% piperidine in DMF in two steps (5 min + 15 min). After each coupling and deprotection step, a Kaiser test was performed to monitor the reaction progress. Each subsequent amino acid was coupled using 3 equivalents of Fmoc-amino acid, 3 equivalents of HBTU, and 10 equivalents of DIEA in DMF for 45 minutes. The synthesis proceeded from the C- to N-terminus via iterative cycles of coupling, deprotection, and washing, with reaction progress monitored by the Kaiser test. Following chain assembly, the resin was washed (DMF, DCM, methanol) and dried under vacuum. Peptides were cleaved from the resin using a cleavage cocktail (TFA:H₂O:EDT:TIS = 95:2:2:1) for 3 hours, precipitated in cold ether, washed, and dried. Crude peptides were purified by preparative RP-HPLC on a C18 column, using 0.1% TFA in water and acetonitrile as mobile phases. Final products were confirmed by mass spectrometry and analyzed for purity by analytical HPLC. Peptides with >98% purity were lyophilized and stored at –20°C. In addition, we have provided the relevant MS and HPLC data supporting the structural assignment and purity assessment of the E-peptide (Response Figure 15) and C-peptide (Response Figure 16). We appreciate your helpful comments, which have improved the clarity and completeness of our manuscript.\nResponse Figure 15. High-performance liquid chromatography (HPLC) and Mass spectrometry (MS) analysis of the E-peptide.\nResponse Figure 16. High-performance liquid chromatography (HPLC) and Mass spectrometry (MS) analysis of the C-peptide.\nQ4: - What was the solvent used to prepare the solutions of E- and C- peptide?\nResponse: We sincerely thank you for this important question. To prepare the peptide solutions, both E- and C-peptides were first aliquoted into individual sterile EP tubes at 2 mg per tube and briefly centrifuged. We initially added 20 μL of 0.1% formic acid in water to pre-dissolve the peptide powder, followed by the addition of 1980 μL of sterile normal saline (0.9% NaCl). The mixture was then sonicated and vortexed repeatedly until the peptide was fully dissolved. The resulting solution had a final concentration of 1 mg/mL (approximately 354 μM, based on the peptide molecular weight of 2822.38 Da). If other concentrations are required, the volume of normal saline can be adjusted according to the molecular weight. All solutions were prepared fresh before use and filtered through a 0.22 μm sterile filter. We sincerely appreciate your careful and rigorous review, and thank you for raising this important point, which helped us clarify and improve the description of our experimental procedures.\nQ5: - The authors tested the peptides in vivo. Was the hemocompatibility and proteolytic stability of the peptide assessed?\nAlso, does the peptides form aggregates? The aggregation could limit the application of larger concentrations.\nResponse: Thank you very much for your thoughtful and important comments. As you suggested, we assessed the hemocompatibility of the peptide using in vitro hemolysis assays as well as PT and APTT coagulation tests. The results showed that E-peptide did not cause hemolysis (Response Figure 17a-b; Fig. S8G-S8H in the manuscript) or prolong PT/APTT times (Response Figure 17c-d; Fig. S8I-S8J in the manuscript), indicating that the peptide possesses good hemocompatibility under the tested conditions. Regarding the proteolytic stability, we performed a preliminary pharmacokinetic (PK) analysis following intraperitoneal administration. At multiple time points, serum samples were collected and quantified by LC-MS. These results are summarized in the updated PK curve (Response Figure 17e), which shows that E-peptide reaches its peak plasma concentration at around 30 minutes and remains detectable for at least 6 hours. This pharmacokinetic profile suggests that E-peptide exhibits moderate in vivo stability. We acknowledge that our current knowledge in pharmacology is relatively limited, and the experiments we conducted represent only an initial assessment of the pharmacological behavior of the E-peptide. In future studies, we plan to collaborate with pharmacology experts to further optimize and comprehensively evaluate the pharmacokinetics, tissue distribution, and metabolic stability of E-peptide. Moreover, we will explore potential strategies—such as peptide modification or formulation engineering—to enhance its half-life and bioavailability. As for peptide aggregation, we fully agree that aggregation, especially at higher concentrations, could significantly impact biological activity and practical application. Due to time and experimental constraints, we were not able to perform physical characterization techniques such as dynamic light scattering (DLS), size-exclusion chromatography (SEC), or electron microscopy (TEM/AFM). Instead, we conducted full-scan LC-MS analysis at different concentrations to indirectly evaluate the aggregation behavior. However, as you may be aware, LC-MS is not ideally suited for aggregation detection since aggregates often dissociate during ionization or are poorly ionized, which can lead to underestimation. In our measurements, the dominant species was observed at 2820 Da with a high deconvolution score (DScore = 94.94), and no significant signal loss or abnormal high–molecular–weight species were detected in the mass (Da) spectrum (Response Figure 17f-i). These results suggest that the peptide exists predominantly in its monomeric form under our test conditions. However, we acknowledge that this approach cannot fully exclude the presence of weakly ionizing or transient aggregates. We truly appreciate your insightful suggestion and will consider employing orthogonal techniques such as DLS or SEC-HPLC in future studies to more thoroughly evaluate peptide aggregation behavior. Thank you again for your valuable input and support in improving our manuscript.\nResponse Figure 17. Evaluation of the hemocompatibility and pharmacokinetic profile of E-peptide.\nReviewer #5:\nThe manuscript titled \"Palmitic Acid Mediated Palmitoylation of B7H3 Promotes Immune Escape\" presents an important exploration into immune evasion mechanisms in MSS colorectal cancer, highlighting the palmitoylation of B7-H3 by ZDHHC24 as a critical modulator of tumor immune suppression. While the study is extensive and covers multiple experimental systems, several conceptual, methodological, and analytical aspects limit the current impact and clarity of the findings.\nResponse:\nThank you very much for your thoughtful and constructive overall assessment of our manuscript. We are grateful for your positive view on the significance of our study in uncovering a novel mechanism of immune evasion in MSS colorectal cancer. We also fully acknowledge the conceptual, methodological, and analytical concerns you raised. Your comments were insightful and highly valuable in helping us improve the clarity and impact of our work. In response, we have carefully revised the manuscript to address each of your concerns in detail, and we sincerely thank you for your efforts in guiding us toward a more rigorous and comprehensive presentation of our findings.\nQ1: - Although the authors attribute the observed immune escape mechanism primarily to B7-H3 palmitoylation, the broader biological effects of palmitic acid are not fully addressed. Palmitic acid is a well-known inducer of ER stress, oxidative stress, activation of pro-inflammatory pathways (e.g., NF-κB, JNK), and lipotoxicity. Given that the concentration used for cell treatments (100 μM) is relatively high, there remains a concern that some of the observed effects could result from these pleiotropic actions rather than palmitoylation alone. While the manuscript includes experiments with 2-bromopalmitate (2-BP) to demonstrate palmitoylation dependence, additional controls such as genetic knockdown of ZDHHC24 or CRISPR-mediated editing of B7-H3 palmitoylation sites could more conclusively isolate the specific role of this modification.\nResponse:\nThank you very much for your insightful and constructive comment. We fully agree that palmitic acid exerts a variety of pleiotropic effects—such as inducing ER stress, oxidative stress, inflammation, and lipotoxicity—and that these effects could potentially confound the interpretation of immune escape mechanisms. We appreciate your concern regarding the specificity of the observed phenotype. To specifically address this issue, we employed multiple strategies to isolate the effect of B7-H3 palmitoylation from other actions of palmitic acid, as you suggested. First, we identified Cys496 as the palmitoylation site on B7H3 (Fig. 2J) and generated a C496A mutant cell line using CRISPR-Cas9 (Fig. S2I), enabling us to directly assess the functional relevance of this modification (Fig. 3). To identify the palmitoyltransferase responsible for B7H3 palmitoylation, we performed a comprehensive screening approach, which revealed ZDHHC24 as the candidate enzyme (Fig. S4A). We subsequently knocked down ZDHHC24 using shRNA (Fig. S4F and S4G) and further validated its role in vivo using intestinal epithelium-specific Zdhhc24 knockout mice (Fig. 5), confirming that ZDHHC24 mediates B7-H3 palmitoylation in this context. Importantly, we further demonstrated in Fig. 6 that the immune escape phenotype induced by ZDHHC24 was abolished in the B7H3 palmitoylation mutant background, providing strong evidence that the observed effects are indeed dependent on B7H3 palmitoylation rather than non-specific consequences of palmitic acid treatment. We sincerely appreciate your thoughtful feedback, which has helped us strengthen the mechanistic clarity and overall rigor of our study.\nQ2: - The metabolomics analysis is presented without sufficient methodological clarity regarding normalization procedures or batch effect correction. Visualizations such as MDS plots, PLS-DA models with VIP scores, or clear volcano plots would help to more effectively communicate metabolic shifts between groups. Moreover, the connection between the identified metabolic changes and the functional outcomes of B7-H3 palmitoylation could be explored in greater detail to strengthen the mechanistic narrative.\nResponse: Thank you very much for your constructive suggestions regarding our metabolomics analysis. We appreciate your attention to the methodological clarity and the visualization of data interpretation. To minimize potential batch-related variability, all tumor samples used for metabolomic analysis were collected under identical conditions, rapidly snap-frozen in liquid nitrogen, and stored at –80 °C. Importantly, all samples were processed and analyzed within a single LC-MS/MS run. Therefore, batch effect correction was not required, as the experimental design itself effectively controlled for inter-batch variation. Regarding data normalization, we applied a two-step approach. Initially, during the qualitative processing stage, normalization was performed using Progenesis QI v3.0 software (Nonlinear Dynamics, Newcastle, UK), which standardizes raw intensity values across samples. Subsequently, after preprocessing steps such as missing value imputation and filtering, log-transformation was applied to the data to further normalize the distribution and reduce heteroscedasticity. These procedures have now been more clearly described in the revised Methods section of the supplement information (Page 5-6, Line 139-179). In response to your helpful suggestion, we have now included a volcano plot to better illustrate the differential metabolites between groups (Response Figure 18a; Fig. S1I in the manuscript). Moreover, we appreciate your suggestion to further explore the mechanistic link between the observed metabolic changes and the functional consequences of B7H3 palmitoylation. While this is an important direction, we would like to note that inferring immune-related functional outcomes directly from untargeted metabolomics data has inherent limitations. Metabolomics primarily captures global small-molecule profiles, which reflect a complex interplay of metabolic processes but do not directly indicate immune cell activation states, cytokine signaling, or receptor-ligand interactions. Furthermore, standard pathway enrichment tools for metabolomics (e.g., KEGG) are less well-curated for immune-specific pathways compared to transcriptomic databases. As a result, the resolution of immunological insights derived from metabolomics data remains relatively low. Thus, we have included GSEA results based on RNA-seq data from MSS and MSI-H tumors to help link the observed metabolic alterations to the functional consequences of B7H3 palmitoylation, thereby strengthening the mechanistic narrative (Response Figure 18b; Fig. S3T in the manuscript). Once again, thank you for your valuable feedback, which helped us improve the rigor and clarity of this part of the study.\nResponse Figure 18. Volcano plot of metabolomics and GSEA analysis of RNA-seq comparing MSS and MSI-H tumors.\nQ3: - Similarly, the GO term enrichment results are overly broad and lack clear prioritization of critical pathways. Application of GSVA or GSEA could help focus the analysis on pathways most relevant to tumor immune evasion and metabolic reprogramming. Integrating these pathway analyses with experimental data on immune cell infiltration and function would provide a more cohesive story.\nResponse: Thank you very much for your insightful comment. We agree that the initial GO enrichment analysis was too broad and lacked focus on key pathways. Following your suggestion, we applied targeted GSEA to highlight pathways specifically relevant to tumor immune evasion and metabolic reprogramming. Our results show that lipid metabolism pathways, including “Long-Chain Fatty Acid Transporter Activity,” “Long-Chain Fatty Acid Binding,” and “Fatty Acid Binding,” are positively enriched in MSS tumors, indicating increased lipid metabolic activity in this group (Response Figures 19a-c; Figs. S1J-S1L in the manuscript). Regarding immune evasion, the gene set “Negative Regulation of T Cell Activation” is enriched in MSS tumors (Response Figure 19d; Fig. S3T in the manuscript), suggesting a more immunosuppressive environment. Conversely, “CD8-Positive, Alpha-Beta T Cell Differentiation” and “T Cell Receptor Signaling Pathway” are negatively enriched in MSS tumors (Response Figures 19e-f; Figs. S3U-3V in the manuscript), implying impaired cytotoxic T cell function. We sincerely appreciate your suggestion, which has helped enhance the mechanistic clarity and relevance of our study.\nResponse Figure 19. GSEA analysis comparing MSS and MSI-H colorectal tumors\nQ4: - The single-cell analysis described in Figure 3H lacks objective statistical comparisons. While clustering results are visually presented, there is an absence of quantitative analyses comparing immune cell subtypes and functional states across treatment groups. Including violin plots, annotated UMAP/t-SNE plots, and appropriate statistical tests (e.g., Wilcoxon rank-sum test) would enhance the rigor and clarity of these analyses.\nResponse: Thank you very much for your constructive feedback. We fully agree that integrating more objective and quantitative comparisons would strengthen the interpretation of our single-cell analysis. In our original submission, we presented a UMAP visualization of the immune landscape (Figure 3E), a compositional analysis of immune cell types (Figure 3F), and the relative proportions of immune subpopulations across groups (Figure 3G). Following your valuable suggestion, we have now added violin plots to quantitatively illustrate the distribution of specific immune cell states between groups and applied the Wilcoxon rank-sum test (Mann–Whitney U test) for statistical analysis (Response Figures 20a; Figs. S3I in the manuscript). We sincerely appreciate your insightful comments, which have helped us improve the rigor and clarity of this part of our study.\nResponse Figure 20. Violin plots showing the comparison of different immune cell types between the WT and C278A groups.\nQ5: - In the clinical relevance section, the manuscript touches briefly on correlations between ZDHHC24 expression and patient outcomes but does not deeply explore publicly available datasets such as TCGA or CPTAC to support these findings. A more thorough analysis correlating ZDHHC24 and B7-H3 expression with clinical parameters such as CD8+ T cell infiltration and overall survival would significantly strengthen the translational impact of the study.\nResponse: Thank you very much for your thoughtful and important comment. We completely agree that a more in-depth exploration of publicly available datasets would strengthen the clinical relevance of our findings. In response, we performed additional analyses using the TIMER database (https://cistrome.shinyapps.io/timer/) to investigate the relationship between ZDHHC24 and B7H3 (CD276) expression and CD8⁺ T cell infiltration. We found that higher expression levels of both genes were significantly associated with reduced CD8⁺ T cell infiltration in colorectal cancer, supporting their role in immune evasion (Response Figure 21a; Fig. S3S in the manuscript). Moreover, we utilized the Kaplan–Meier Plotter database (https://kmplot.com/analysis/) to examine the correlation between gene expression and clinical outcomes. Specifically, we found that high CD276 expression was associated with poorer overall survival (Response Figure 21b; Fig. S3R in the manuscript), while high ZDHHC24 expression was linked to worse relapse-free survival in colon cancer patients (Response Figure 21c; Fig. S4K in the manuscript). We sincerely thank you for your insightful feedback, which greatly helped us strengthen the clinical relevance and overall impact of our study.\nResponse Figure 21. Public dataset analysis shows that ZDHHC24 and CD276 expression negatively correlates with CD8⁺ T cell infiltration and patient survival in colorectal cancer.\nQ6: - Although the therapeutic E-peptide is a novel approach, the current data regarding its pharmacokinetics, biodistribution, and toxicity remain limited. The authors mention minimal toxicity at doses up to 50 mg/kg, but detailed pharmacodynamic data and long-term safety evaluations are lacking. Without this information, it is difficult to assess the true translational potential of this therapeutic strategy. Additionally, exploring combination therapies with standard-of-care immune checkpoint inhibitors in more clinically relevant models would help to further demonstrate the value of this approach.\nResponse: Thank you very much for your insightful and forward-looking comments. We completely agree that a thorough understanding of the pharmacokinetics, biodistribution, and long-term safety of the E-peptide is essential for evaluating its translational potential. In response to your suggestion, we performed a preliminary pharmacokinetic (PK) study by collecting plasma samples at multiple time points following intraperitoneal injection of E-peptide (10 mg/kg) in mice. LC-MS analysis revealed that E-peptide reached peak plasma concentration at approximately 30 minutes and remained detectable for at least 6 hours (Response Figure 22a). Regarding long-term safety evaluation, we performed a one-month short-term safety evaluation in vivo, during which E-peptide was administered at doses up to 50 mg/kg. At the working dose of 10 mg/kg, we did not observe any apparent signs of toxicity, such as mouse mortality, liver or kidney dysfunction, or histopathological abnormalities in major organs (Figure S8B-S8F). These observations suggest that the peptide exhibits an acceptable preliminary safety profile under the tested conditions. In future studies, we plan to collaborate with pharmacology experts to further investigate its PK properties, tissue distribution, and chronic safety assessment. In our current work, we also explored the therapeutic benefit of combining the E-peptide with anti–PD-1 antibodies. This combination showed significantly enhanced anti-tumor efficacy compared to monotherapy (Figure 9), supporting its potential clinical utility. As you rightly pointed out, validation in more clinically relevant models is essential. Due to time and funding constraints, we further extended our analysis to a humanized mouse model treated with E-peptide monotherapy, in which we observed a marked reduction in tumor burden compared to C-peptide (Response Figure 22b-k; Fig. 8M-8V in the manuscript). We fully agree that more detailed PK, biodistribution, and long-term toxicity studies are essential, and we are actively planning follow-up work to support the further development of this peptide-based therapeutic strategy. We sincerely appreciate your valuable feedback, which has greatly helped us refine our study and plan more comprehensive future investigations.\nResponse Figure 22. Pharmacokinetic analysis of E-peptide following intraperitoneal injection and its therapeutic efficacy in a humanized mouse model.\nQ7: - The manuscript would also benefit from a more detailed discussion on the potential redundancy or compensatory mechanisms involving other immune checkpoint molecules, such as PD-L1 and B7-H4. Exploring whether inhibition of B7-H3 palmitoylation has additive or synergistic effects when combined with inhibitors targeting these molecules would provide valuable insights into the broader therapeutic landscape.\nResponse: Thank you very much for this valuable suggestion. We fully agree that a more comprehensive discussion of the potential redundancy or compensatory mechanisms among immune checkpoint molecules would enhance the depth of our manuscript. In the revised Discussion (Page 20-22, Line 591-634), we have now added a more in-depth exploration of these mechanisms. Specifically, we highlight that inhibition of B7H3 can lead to compensatory upregulation of PD-L1 on myeloid cells, as shown in previous studies[1, 2], and that PD-L1–negative tumors such as certain breast cancers may escape immune surveillance by upregulating B7-H4[3]. These findings underscore how alternative immunosuppressive pathways can undermine the effectiveness of monotherapies. We also elaborate on our rationale for combining the E-peptide with anti–PD-1 therapy, supported by our experimental findings showing enhanced antitumor efficacy with this combination. Furthermore, we now address the potential for synergy with CTLA-4 blockade, given its distinct role in targeting Treg-mediated suppression[1, 4], and we highlight other checkpoint targets such as LAG-3, TIM-3, and TIGIT as promising candidates for future combinatorial approaches. These additions aim to provide a broader therapeutic perspective and emphasize the translational potential of our findings. We truly appreciate your suggestion, which helped us improve the depth and clarity of this important discussion.\nReference:\n[1] SHI W, WANG Y, ZHAO Y, et al. Immune Checkpoint B7-H3 Is a Therapeutic Vulnerability in Prostate Cancer Harboring PTEN and TP53 Deficiencies[J]. Science Translational Medicine, 2023, 15(695): eadf6724.\n[2] GUO Y, WANG X, ZHANG C, et al. Tumor Immunotherapy Targeting B7-H3: From Mechanisms to Clinical Applications[J]. ImmunoTargets and Therapy, 2025, 14: 291–320.\n[3] ALTAN M, KIDWELL K M, PELEKANOU V, et al. Association of B7-H4, PD-L1, and Tumor Infiltrating Lymphocytes with Outcomes in Breast Cancer[J]. NPJ breast cancer, 2018, 4: 40.\n[4] WING K, ONISHI Y, PRIETO-MARTIN P, et al. CTLA-4 Control over Foxp3+ Regulatory T Cell Function[J]. Science, 2008, 322(5899): 271–275.\nQ8: - Finally, several figures require improvements in resolution, annotation of statistical significance, and clearer figure legends to enhance interpretability. Language polishing throughout the manuscript would also improve readability and professionalism.\nResponse: Thank you very much for your helpful suggestion. In response, we have carefully revised all relevant figures to improve resolution and added annotations to indicate statistical significance where applicable. We have also rewritten the corresponding figure legends to provide clearer and more informative descriptions. Additionally, to improve the overall readability and professionalism of the manuscript, we have utilized a professional English language editing service to polish the entire text. We sincerely appreciate your constructive suggestions, which have greatly helped us enhance the quality and clarity of our work.\nReviewer #6:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nResponse:\nWe sincerely appreciate the time and effort you invested in reviewing our manuscript. Your professional comments and feedback have been invaluable in helping us improve the quality of the manuscript. Thank you once again for your thoughtful and constructive suggestion.\nRESPONSE TO REVIEWERS' COMMENTS (NCOMMS-25-17666-A)\nNote to Reviewers: We sincerely thank the reviewers for the time and effort they devoted to reviewing our manuscript and for the insightful comments that helped us further improve it. A point-by-point response to each comment is provided below. For ease of review, we include key data as response figures in this letter and indicate the corresponding figure/panel and the locations of the relevant text in the revised manuscript. We have submitted two versions of the revision: (i) a clean version and (ii) a marked-up version. In the marked-up file, additions are shown as red underlined text, and deletions are shown in dark blue with strikethrough.\nREVIEWER COMMENTS\nReviewer #1:\nThe revised manuscript has undergone extensive modifications, thoroughly addressing my concerns.\nResponse：We sincerely thank you for the positive and encouraging feedback. We are pleased that the revisions have satisfactorily addressed your concerns. Your constructive suggestions helped us improve the quality and clarity of the manuscript, and we are very grateful for your time and thoughtful evaluation.\nReviewer #2:\nThanks for the author's response. They have canceled most of my concerns. However, there is one issue about the result of SPR as followed.\nThe surface plasmon resonance (SPR) experiments conducted by the authors require further refinement, particularly since the dissociation constant (Kd) was not determined. Besides, in SPR experiments, a typical and reliable binding curve should demonstrate a saturation plateau as the analyte concentration increases. This serves as one of the key criteria for determining whether the experiment is successful and the data are credible. However, this is not observed in the data the authors shown. As a result, the reliability of the data obtained under these conditions remains uncertain. It is advisable to consult with an SPR specialist to optimize the experimental setup and validate the results.\nResponse: Thank you for the careful review. We take the concern seriously regarding the lack of a reported 𝐾 and the absence of an obvious saturation plateau in our initial SPR data. In response, we repeated the SPR experiments with an expanded concentration range (0.3125–10 µM), longer association/equilibration times, and increased flow rates to minimize mass-transport effects. We also optimized the running buffer to reduce non-specific binding. The revised dataset (sensorgrams and steady-state binding curve) yields a 𝐾 = 2.37 𝜇M from a 1:1 Langmuir steady-state fit (Response Figure 1a-b; Fig. S7D-S7E in the manuscript). In addition, the steady-state binding curve exhibits a clear saturable trend and approaches a plateau within the tested concentration range. We sincerely appreciate your professional and constructive feedback. Your guidance has materially improved the rigor, clarity, and reliability of our SPR analysis and presentation.\nResponse Figure.1 SPR of E-peptide binding to ZDHHC24. a. Representative SPR sensorgrams. b. Steady-state binding curve.\nReviewer #4:\nThe authors addressed all the concerns.\nResponse: We sincerely thank you for the positive assessment. Your suggestions helped improve the clarity and rigor of the manuscript, and we are grateful for your time and thoughtful evaluation.\nReviewer #6:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nResponse: Thank you very much for co-reviewing our work. We are grateful for your time and guidance, which greatly helped us improve the manuscript.\nReviewer #7:\nI have no further comments.\nResponse:\nThank you very much for your time and evaluation, and we are grateful for the constructive and supportive comments you previously provided.\nReviewer #8:\nMy comments are focused on the response to Reviewer#3’s critique. The revision and the authors’ rebuttal satisfactorily address most of the points raised in the initial review. The revised manuscript had additional data, more clearly conveyed the results and interpretation of the data and improved the accessibility of the manuscript to non-specialists. There are, however, there are several issues that would benefit from additional attention.\nResponse: We sincerely appreciate the time and effort you devoted to this careful review. Your valuable comments have greatly helped us improve the manuscript. In response to your additional suggestions, we have carefully revised the manuscript, provided point-by-point responses, and made the corresponding changes in the text. Your guidance is very important to us, and we sincerely hope that the current revision meets your expectations. Thank you again for your time and constructive feedback.\nMajor comments:\nQ1: - A major point raised in the review was the issue of whether the peptide inhibitor and anti-PD-L1 therapy act synergistically. The abstract states definitively that their combined actions are synergistic. In the manuscript, the authors provide a plausible mechanism to account for synergism, but do not directly provide support. In the rebuttal, however, they report their analysis using the Bliss independence model to support synergism. Adding the analysis to the text would clearly strengthen the manuscript.\nResponse: We sincerely thank you for this insightful comment. We completely agree that directly including the synergistic analysis in the manuscript text would significantly strengthen our study. In response to this valuable suggestion, we have now integrated the description of the Bliss independence analysis and its results into the revised manuscript (Page 18-19, line 534-542). We hope that these additions, now incorporated into the main text, offer clearer and more direct support for the proposed synergism between the E-peptide and anti–PD-1 therapy, and help improve the manuscript. Once again, we are grateful for this constructive suggestion.\nQ2: - Q9 concerns the separation of the cytosolic and membrane fractions. In the response, the rationale for performing the fractionation was “not to confirm its localization”. But lines 173- 176 on p.6 state, “Given that palmitoylation can also affect protein localization, we conducted cytoplasmic and membrane fractionation assays,…” Subcellular fractionation does not address localization in different compartments, but rather the distribution of a protein in particulate (membrane) fractions vs. the soluble (cytoplasmic) fraction. I suggest either omitting the experiment or replacing “localization” with “membrane association” in the text.\nResponse: Thank you very much for your careful and constructive suggestion. We agree that “localization” is potentially misleading in this context, and—as recommended—we have replaced “localization” with “membrane association” in the text (Page 6, line 174). Thank you again for your helpful guidance.\nQ3: - Q11- the rebuttal makes a strong argument for focusing on autophagy vs. the endocytic pathway to the lysosome, but this information is not conveyed in the text. The first sentence (line 188) is factually correct by not excluding other mechanisms, but nonetheless is jarring to a cell biologist. Providing additional context (using the information from the rebuttal) would be helpful in this regard.\nResponse: Thank you for this thoughtful and constructive comment. We agree that our rationale for focusing on autophagy should be stated explicitly in the text. We acknowledge that our previous opening sentence (“Autophagy and the ubiquitin–proteasome system (UPS) are two primary protein degradation pathways in eukaryotic cells”) was abrupt because it was not sufficiently connected to the surrounding context. In line with your suggestion, we added a brief bridging explanation to improve logical continuity. Specifically, we revised the first sentence to: “B7H3, as a membrane protein, can in principle be degraded via the ubiquitin–proteasome system (UPS), autophagy–lysosome pathway, or endocytosis to lysosomes (Page 7, line 187-189).” We then briefly clarify why our data support the autophagy–lysosome pathway as the predominant route in our system: 3-MA (which inhibits class III PI3K and blocks autophagosome initiation), and bafilomycin A1/chloroquine (which block lysosomal degradation) blocked the 2-BP–induced degradation of B7H3 (Page 7, line 191-195). Moreover, EBSS-induced autophagy also accelerated its degradation (Page 7, line 195-197). Therefore, we conclude that in our system, B7H3 degradation is predominantly mediated by the autophagy–lysosome pathway (Page 7, line 197-199). We are sincerely grateful for your guidance, and it has substantially improved the clarity, rigor, and readability of this section.\nAdditional comments:\nQ4: - The statements below are repetitive with the former “suggest” being more appropriate than “demonstrate” at this point in the manuscript. p.7, lines 181-182 – “These results suggest that palmitoylation contributes to stability” p. 7, lines 182 -184 – \"These findings demonstrate that the PA-triggered palmitoylation enhances stability”.\nResponse: Thank you for pointing this out. We agree that the two statements were repetitive, and we have removed one of them. In line with your suggestion, we retained the more cautious wording “suggest.” The revised text now reads: “Taken together, these findings suggest that the PA-triggered palmitoylation contributes to B7H3 stability (Page 7, line 182–183).” Once again, thank you for your careful and constructive comments.\nQ5: - On p.22, line 639 – “a transmembrane peptide effectively enhances the antitumor effects….”What is meant by transmembrane? If referring to the tat-peptide fusion, then it is a membrane-permeating peptide. If it is referring to the e-peptide sequence itself as transmembrane, then this contradicts the diagram in Figure 7A. The authors might want to comment in the text on p.15 that the palmitoylation site is included in the peptide sequence.\nResponse: Thank you for your careful and professional comment. We agree that the term “transmembrane peptide” is potentially misleading in this context. To avoid confusion, the term “transmembrane peptide” on page 22, line 650 (originally p.22, line 639) has been revised to “membrane-permeating peptide.” In addition, following your suggestion, we have added a note clarifying that the E-peptide sequence includes the palmitoylation site (Page 15, line 440-441). Thank you again for your careful and constructive feedback.\nRESPONSE TO REVIEWERS' COMMENTS (NCOMMS-25-17666- B)\nNote to Reviewers: We sincerely thank the reviewers for their time and effort in assessing our revised manuscript and for their constructive comments throughout the review process. We greatly appreciate your support and consideration.\nREVIEWER COMMENTS\nReviewer #2:\nThe author's revisions regarding the SPR experiments have fully addressed my concerns. I have no further comments.\nResponse：We sincerely thank you for your positive assessment and for the time and effort you have devoted to reviewing our manuscript.\nReviewer #6:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We sincerely thank you for co-reviewing our manuscript. We truly appreciate the time and effort you have devoted to this process."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised manuscript has undergone extensive modifications, thoroughly addressing my concerns.", "reviewer_2": "Thanks for the author's response. They have canceled most of my concerns. However, there is one issue about the result of SPR as followed.\nThe surface plasmon resonance (SPR) experiments conducted by the authors require further refinement, particularly since the dissociation constant (Kd) was not determined. Besides, in SPR experiments, a typical and reliable binding curve should demonstrate a saturation plateau as the analyte concentration increases. This serves as one of the key criteria for determining whether the experiment is successful and the data are credible. However, this is not observed in the data the authors shown. As a result, the reliability of the data obtained under these conditions remains uncertain. It is advisable to consult with an SPR specialist to optimize the experimental setup and validate the results.", "reviewer_4": "The authors addressed all the concerns.", "reviewer_6": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_7": "I have no further comments.", "reviewer_8": "My comments are focused on the response to Reviewer#3’s critique. The revision and the authors’ rebuttal satisfactorily address most of the points raised in the initial review. The revised manuscript had additional data, more clearly conveyed the results and interpretation of the data and improved the accessibility of the manuscript to non-specialists. There are, however, there are several issues that would benefit from additional attention.\n1. A major point raised in the review was the issue of whether the peptide inhibitor and anti-PD-L1 therapy act synergistically. The abstract states definitively that their combined actions are synergistic. In the manuscript, the authors provide a plausible mechanism to account for synergism, but do not directly provide support. In the rebuttal, however, they report their analysis using the Bliss independence model to support synergism. Adding the analysis to the text would clearly strengthen the manuscript.\n2. Q9 concerns the separation of the cytosolic and membrane fractions. In the response, the rationale for performing the fractionation was “not to confirm its localization”. But lines 173- 176 on p.6 state, “Given that palmitoylation can also affect protein localization, we conducted cytoplasmic and membrane fractionation assays,…” Subcellular fractionation does not address localization in different compartments, but rather the distribution of a protein in particulate (membrane) fractions vs. the soluble (cytoplasmic) fraction. I suggest either omitting the experiment or replacing “localization” with “membrane association” in the text.\n3. Q11- the rebuttal makes a strong argument for focusing on autophagy vs. the endocytic pathway to the lysosome, but this information is not conveyed in the text. The first sentence (line 188) is factually correct by not excluding other mechanisms, but nonetheless is jarring to a cell biologist. Providing additional context (using the information from the rebuttal) would be helpful in this regard.\nAdditional comments -\n1. The statements below are repetitive with the former “suggest” being more appropriate than “demonstrate” at this point in the manuscript.\np.7, lines 181-182 – “These results suggest that palmitoylation contributes to stability”\np. 7, lines 182 -184 – \"These findings demonstrate that the PA-triggered palmitoylation enhances stability”.\n2. On p.22, line 639 – “a transmembrane peptide effectively enhances the antitumor effects….”\nWhat is meant by transmembrane? If referring to the tat-peptide fusion, then it is a membrane-permeating peptide. If it is referring to the e-peptide sequence itself as transmembrane, then this contradicts the diagram in Figure 7A. The authors might want to comment in the text on p.15 that the palmitoylation site is included in the peptide sequence."}, "author_response": "RESPONSE TO REVIEWERS' COMMENTS (NCOMMS-25-17666-A)\nNote to Reviewers: We sincerely thank the reviewers for the time and effort they devoted to reviewing our manuscript and for the insightful comments that helped us further improve it. A point-by-point response to each comment is provided below. For ease of review, we include key data as response figures in this letter and indicate the corresponding figure/panel and the locations of the relevant text in the revised manuscript. We have submitted two versions of the revision: (i) a clean version and (ii) a marked-up version. In the marked-up file, additions are shown as red underlined text, and deletions are shown in dark blue with strikethrough.\nREVIEWER COMMENTS\nReviewer #1:\nThe revised manuscript has undergone extensive modifications, thoroughly addressing my concerns.\nResponse：We sincerely thank you for the positive and encouraging feedback. We are pleased that the revisions have satisfactorily addressed your concerns. Your constructive suggestions helped us improve the quality and clarity of the manuscript, and we are very grateful for your time and thoughtful evaluation.\nReviewer #2:\nThanks for the author's response. They have canceled most of my concerns. However, there is one issue about the result of SPR as followed.\nThe surface plasmon resonance (SPR) experiments conducted by the authors require further refinement, particularly since the dissociation constant (Kd) was not determined. Besides, in SPR experiments, a typical and reliable binding curve should demonstrate a saturation plateau as the analyte concentration increases. This serves as one of the key criteria for determining whether the experiment is successful and the data are credible. However, this is not observed in the data the authors shown. As a result, the reliability of the data obtained under these conditions remains uncertain. It is advisable to consult with an SPR specialist to optimize the experimental setup and validate the results.\nResponse: Thank you for the careful review. We take the concern seriously regarding the lack of a reported 𝐾 and the absence of an obvious saturation plateau in our initial SPR data. In response, we repeated the SPR experiments with an expanded concentration range (0.3125–10 µM), longer association/equilibration times, and increased flow rates to minimize mass-transport effects. We also optimized the running buffer to reduce non-specific binding. The revised dataset (sensorgrams and steady-state binding curve) yields a 𝐾 = 2.37 𝜇M from a 1:1 Langmuir steady-state fit (Response Figure 1a-b; Fig. S7D-S7E in the manuscript). In addition, the steady-state binding curve exhibits a clear saturable trend and approaches a plateau within the tested concentration range. We sincerely appreciate your professional and constructive feedback. Your guidance has materially improved the rigor, clarity, and reliability of our SPR analysis and presentation.\nResponse Figure.1 SPR of E-peptide binding to ZDHHC24. a. Representative SPR sensorgrams. b. Steady-state binding curve.\nReviewer #4:\nThe authors addressed all the concerns.\nResponse: We sincerely thank you for the positive assessment. Your suggestions helped improve the clarity and rigor of the manuscript, and we are grateful for your time and thoughtful evaluation.\nReviewer #6:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nResponse: Thank you very much for co-reviewing our work. We are grateful for your time and guidance, which greatly helped us improve the manuscript.\nReviewer #7:\nI have no further comments.\nResponse:\nThank you very much for your time and evaluation, and we are grateful for the constructive and supportive comments you previously provided.\nReviewer #8:\nMy comments are focused on the response to Reviewer#3’s critique. The revision and the authors’ rebuttal satisfactorily address most of the points raised in the initial review. The revised manuscript had additional data, more clearly conveyed the results and interpretation of the data and improved the accessibility of the manuscript to non-specialists. There are, however, there are several issues that would benefit from additional attention.\nResponse: We sincerely appreciate the time and effort you devoted to this careful review. Your valuable comments have greatly helped us improve the manuscript. In response to your additional suggestions, we have carefully revised the manuscript, provided point-by-point responses, and made the corresponding changes in the text. Your guidance is very important to us, and we sincerely hope that the current revision meets your expectations. Thank you again for your time and constructive feedback.\nMajor comments:\nQ1: - A major point raised in the review was the issue of whether the peptide inhibitor and anti-PD-L1 therapy act synergistically. The abstract states definitively that their combined actions are synergistic. In the manuscript, the authors provide a plausible mechanism to account for synergism, but do not directly provide support. In the rebuttal, however, they report their analysis using the Bliss independence model to support synergism. Adding the analysis to the text would clearly strengthen the manuscript.\nResponse: We sincerely thank you for this insightful comment. We completely agree that directly including the synergistic analysis in the manuscript text would significantly strengthen our study. In response to this valuable suggestion, we have now integrated the description of the Bliss independence analysis and its results into the revised manuscript (Page 18-19, line 534-542). We hope that these additions, now incorporated into the main text, offer clearer and more direct support for the proposed synergism between the E-peptide and anti–PD-1 therapy, and help improve the manuscript. Once again, we are grateful for this constructive suggestion.\nQ2: - Q9 concerns the separation of the cytosolic and membrane fractions. In the response, the rationale for performing the fractionation was “not to confirm its localization”. But lines 173- 176 on p.6 state, “Given that palmitoylation can also affect protein localization, we conducted cytoplasmic and membrane fractionation assays,…” Subcellular fractionation does not address localization in different compartments, but rather the distribution of a protein in particulate (membrane) fractions vs. the soluble (cytoplasmic) fraction. I suggest either omitting the experiment or replacing “localization” with “membrane association” in the text.\nResponse: Thank you very much for your careful and constructive suggestion. We agree that “localization” is potentially misleading in this context, and—as recommended—we have replaced “localization” with “membrane association” in the text (Page 6, line 174). Thank you again for your helpful guidance.\nQ3: - Q11- the rebuttal makes a strong argument for focusing on autophagy vs. the endocytic pathway to the lysosome, but this information is not conveyed in the text. The first sentence (line 188) is factually correct by not excluding other mechanisms, but nonetheless is jarring to a cell biologist. Providing additional context (using the information from the rebuttal) would be helpful in this regard.\nResponse: Thank you for this thoughtful and constructive comment. We agree that our rationale for focusing on autophagy should be stated explicitly in the text. We acknowledge that our previous opening sentence (“Autophagy and the ubiquitin–proteasome system (UPS) are two primary protein degradation pathways in eukaryotic cells”) was abrupt because it was not sufficiently connected to the surrounding context. In line with your suggestion, we added a brief bridging explanation to improve logical continuity. Specifically, we revised the first sentence to: “B7H3, as a membrane protein, can in principle be degraded via the ubiquitin–proteasome system (UPS), autophagy–lysosome pathway, or endocytosis to lysosomes (Page 7, line 187-189).” We then briefly clarify why our data support the autophagy–lysosome pathway as the predominant route in our system: 3-MA (which inhibits class III PI3K and blocks autophagosome initiation), and bafilomycin A1/chloroquine (which block lysosomal degradation) blocked the 2-BP–induced degradation of B7H3 (Page 7, line 191-195). Moreover, EBSS-induced autophagy also accelerated its degradation (Page 7, line 195-197). Therefore, we conclude that in our system, B7H3 degradation is predominantly mediated by the autophagy–lysosome pathway (Page 7, line 197-199). We are sincerely grateful for your guidance, and it has substantially improved the clarity, rigor, and readability of this section.\nAdditional comments:\nQ4: - The statements below are repetitive with the former “suggest” being more appropriate than “demonstrate” at this point in the manuscript. p.7, lines 181-182 – “These results suggest that palmitoylation contributes to stability” p. 7, lines 182 -184 – \"These findings demonstrate that the PA-triggered palmitoylation enhances stability”.\nResponse: Thank you for pointing this out. We agree that the two statements were repetitive, and we have removed one of them. In line with your suggestion, we retained the more cautious wording “suggest.” The revised text now reads: “Taken together, these findings suggest that the PA-triggered palmitoylation contributes to B7H3 stability (Page 7, line 182–183).” Once again, thank you for your careful and constructive comments.\nQ5: - On p.22, line 639 – “a transmembrane peptide effectively enhances the antitumor effects….”What is meant by transmembrane? If referring to the tat-peptide fusion, then it is a membrane-permeating peptide. If it is referring to the e-peptide sequence itself as transmembrane, then this contradicts the diagram in Figure 7A. The authors might want to comment in the text on p.15 that the palmitoylation site is included in the peptide sequence.\nResponse: Thank you for your careful and professional comment. We agree that the term “transmembrane peptide” is potentially misleading in this context. To avoid confusion, the term “transmembrane peptide” on page 22, line 650 (originally p.22, line 639) has been revised to “membrane-permeating peptide.” In addition, following your suggestion, we have added a note clarifying that the E-peptide sequence includes the palmitoylation site (Page 15, line 440-441). Thank you again for your careful and constructive feedback."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The author's revisions regarding the SPR experiments have fully addressed my concerns. I have no further comments.", "reviewer_6": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "RESPONSE TO REVIEWERS' COMMENTS (NCOMMS-25-17666- B)\nNote to Reviewers: We sincerely thank the reviewers for their time and effort in assessing our revised manuscript and for their constructive comments throughout the review process. We greatly appreciate your support and consideration.\nREVIEWER COMMENTS\nReviewer #2:\nThe author's revisions regarding the SPR experiments have fully addressed my concerns. I have no further comments.\nResponse：We sincerely thank you for your positive assessment and for the time and effort you have devoted to reviewing our manuscript.\nReviewer #6:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We sincerely thank you for co-reviewing our manuscript. We truly appreciate the time and effort you have devoted to this process."}]

Decision letter