Tumor-associated CD19^+ macrophages induce immunosuppressive microenvironment in hepatocellular carcinoma

Domain: chemistry
Published: 2026-02-26
Editorial outcome: minor_revision

Abstract
Tumor-associated macrophages are a key component that contributes to the immunosuppressive microenvironment in human cancers. However, therapeutic targeting of macrophages has been a challenge in clinic due to the limited understanding of their heterogeneous subpopulations and distinct functions. Here, we identify a clinically relevant CD19+ subpopulation of macrophages that is enriched in many types of cancer, particularly in hepatocellular carcinoma (HCC). The CD19+ macrophages exhibit increased levels of programmed cell death 1 ligand 1 (PD-L1) and CD73, enhanced mitochondrial oxidation, and compromised phagocytosis, indicating their immunosuppressive functions. Targeting CD19+ macrophages with anti-CD19 chimeric antigen receptor T (SCAR-T) cells inhibited HCC tumor growth. We identify Paired Box 5 (PAX5) as a primary driver of up-regulated mitochondrial biogenesis in CD19+ macrophages, which depletes cytoplasmic Ca2+, leading to lysosomal deficiency and consequent accumulation of CD73 and PD-L1. Inhibiting CD73 or mitochondrial oxidation enhanced the efficacy of immune checkpoint blockade therapy in treating HCC, suggesting great promise for CD19+ macrophage-targeting therapeutics.

Introduction
Liver cancer has emerged as the third leading cause of cancer-related death worldwide, with hepatocellular carcinoma (HCC) being the predominant pathological subtype1. Current therapies for treating HCC comprise surgical resection, liver transplantation, locoregional therapies, and systemic treatments including targeted therapies and immunotherapy2. Surgical resection and liver transplantation offer the best chance for a cure, but are only options for a small subset of patients diagnosed at an early stage. Locoregional therapies, including radiofrequency ablation and transarterial chemoembolization, are used for intermediate stages of HCC or when surgery is not feasible. Systemic treatments, like tyrosine kinase inhibitors (e.g., sorafenib and lenvatinib) and immune checkpoint inhibitors (e.g., atezolizumab, nivolumab and pembrolizumab), have expanded treatment options for the majority of patients with advanced HCC that misses the window for surgical intervention3. Despite these advancements, the 5-year overall survival rates for HCC remain below 20%4. These therapies face significant challenges in the clinic, including tumor heterogeneity, which leads to variable treatment responses, and the immunosuppressive tumor microenvironment (TME), which hampers the efficacy of immunotherapies5,6.

The immunosuppressive microenvironment of HCC is characterized by a complex interplay of various cell types that collectively inhibit effective anti-tumor immune responses. Key players include myeloid-derived suppressor cells (MDSCs), which accumulate and inhibit T cell activation7. Regulatory T cells (Tregs) suppress cytotoxic T cell function, while cancer-associated fibroblasts (CAFs) produce extracellular matrix components and cytokines that further support an immunosuppressive milieu8. Particularly, tumor-associated macrophages (TAMs) are the most abundant in HCC that promotes tumor growth and suppresses inflammation9. These various types of stroma cells interact through direct contact and the release of soluble factors, creating a TME that hinders immune-mediated tumor eradication10.

Macrophages exhibit different features in the presence of different inducers, such as lipopolysaccharide and interleukin-4, which lead to the two classic polarizations of macrophages, defining as M1 and M2 macrophages11. TAMs are believed to promote tumor progression by reprogramming local immunosuppression, inducing angiogenesis and drug resistance, and promoting tumor cell invasion. Phenotypically, despite being considered to have an M2-like polarization12, TAMs are noncanonical and dynamically altered in response to stimuli within the TME. Consequently, the binary classification of macrophages is inadequate for describing them within the highly heterogeneous TME, both spatially and temporally13. Studies have identified specific TAM subgroups, such as PD-1+ TAMs in colon cancer, TREM1+ TAMs in hepatocellular carcinoma, and CD169+ TAMs in glioblastoma14-16. These subgroups have diverse or even contradictory effects on tumor progression, suggesting that precise interventions targeting certain TAM subgroups, rather than the wholesale removal of M1, M2 or all TAMs in the TME, are likely to achieve better anti-tumor efficacy. Therefore, understanding the full spectrum of TAMs is crucial for developing therapeutic approaches that target macrophages in HCC.

In the present study, we identified a subgroup of CD19+ TAMs that are enriched in a number of solid tumor types, particularly in HCC. As a transmembrane glycoprotein, CD19 is considered as a biomarker and functionally crucial for the regulation of B cell receptor signaling in both the early and late stages of B cell development. Interestingly, a recent analysis of single-cell RNA sequencing (scRNA-seq) data defined a transdifferentiated cell types in the ischemic brain that was characterized by co-expression of B-cell markers and several macrophage markers17, suggesting a potential role of CD19 in macrophages. Here, we elucidated the clinical relevance, cellular functions, and molecular features of CD19+ TAMs and determined the underlying regulatory mechanisms of their immunosuppressive characteristics. Based on these findings, we propose that targeting CD19+ TAMs or mitigating their pro-tumoral effects could be a promising strategy for treating HCC.

Results
CD19+ macrophages are enriched in HCC
A group of tumor tissue samples (n = 30) from patients with HCC were analyzed with mass cytometry (CyTOF) for profiling their tumor immune microenvironment 6. Unexpectedly, we identified a subgroup of TAMs expressing CD19 at a level comparable to that in B cells in the tumors (Fig. 1A). To validate the presence of CD19+ TAMs, we further conducted immune profiling analysis of HCC samples in a 41-patient cohort. Approximately a half (29 out of 41) of the HCC samples had a considerable proportion (>20% of total TAMs) of CD19+ TAMs. The existence of a CD19+ subpopulation in the TAMs was further confirmed using immunofluorescence (Fig. 1B), ImageStream (Fig. 1C and Supplementary Fig.1A) and flow cytometry (Fig. 1D and Supplementary Fig.1B). Additionally, we applied multiplex immunohistochemical (mIHC) staining on tumors and their adjacent normal tissues using CD14, CD68, CD19, and CD20 markers to demonstrate CD19+ TAMs at the single-cell level (Fig. 1E). Conventional macrophage markers, CD11b and CD68/CD14, were used to confirm their macrophage identity. Notably, the proportion of CD19+ TAMs was much higher in HCC than in adjacent normal tissue or peripheral blood (Fig. 1B and 1D). In terms of cell size, the CD19+ TAMs had greater FSC/SSC (forward scatter/side scatter) values in flow cytometry than those of B cells, suggesting their macrophage cell lineage rather than a B cell lineage (Supplementary Fig.1C).

Next, we wanted to determine whether CD19+ TAMs exist in other types of solid tumors. In a total of six types of human solid tumors, including HCC, CD19+ TAMs are enriched (Fig. 1F) compared with those in peripheral blood and normal tissues. In particular, HCC, renal carcinoma, colorectal cancer, pancreatic cancer, and gastric cancer had a median proportion of CD19+ TAMs over 20% of total TAMs. Similarly, we detected CD19+ macrophages in various normal tissues and peripheral blood in mice (Supplementary Fig.1D). Moreover, the proportion of CD19+ macrophages did not correlate with that of B cells in these tissues (Supplementary Fig. 1E and 1F), further suggesting that they are not derived from B cells. The results collectively suggest that CD19+ macrophages naturally exist in the body but are enriched in solid tumors.

CD19+ TAMs are associated with poor clinical outcomes in HCC
As no previous study reported this subgroup of TAMs, we wanted to examine the clinical relevance of CD19+ macrophages in HCC. A human HCC tissue microarray was co-immunostained with anti-CD19 and anti-CD68 antibodies (Supplementary Fig. 2A). In a total of 191 tissue samples with valid co-immunostaining, we found that the abundance (shown as density) of CD19+ TAMs was positively correlated with tumor size (Supplementary Fig. 2B) and tumor cell differentiation (Supplementary Fig. 2C), but not with disease stages (Supplementary Fig. 2D), suggesting CD19+ TAMs may be recruited at early stage of tumor initiation for creating immunosuppressive TME. Importantly, HCC patients with greater density of CD19+ TAMs had worse overall survival (hazard ratio 3.12, 95% confidential interval 1.23–7.89) and disease-free survival (Fig. 2A and Supplementary Fig. 2E). By contrast, the density of total TAMs had no correlation with overall survival of these patients (Fig. 2B). Consistent with its clinical relevance, the density of CD19+ TAMs was positively correlated with tumor cell proliferation, as indicated by Ki-67 staining in human HCC samples (Fig. 2C). TME analysis of the HCC tissue microarray indicated that a high density of CD19+ TAMs was associated with increased infiltration of Tregs but reduced infiltration of CD8+ T cells (Supplementary Fig. 2F). Collectively, these results demonstrated that CD19+ TAMs correlate with poor clinical prognosis in patients with HCC, underlying a potential modification of TME.

CD19+ TAMs possess immunosuppressive abilities and promote HCC growth
To study the role of CD19+ TAMs in shaping the TME, we further analyzed the CyTOF data from the 41 patients with HCC (the same cohort analyzed in Fig. 1A). CD19+ TAMs represent a major subgroup of TAMs, accounting for approximately 30% of TAMs and 5% of CD45+ cells in the tumors (Supplementary Fig. 2G). The CD19+ and CD19- subsets of TAMs showed a sharp difference at the level of the M2 macrophage marker CD163 (Supplementary Fig. 2H). The proportion of CD19+ TAMs was also negatively correlated with the abundance of total T, CD4+ and CD8+ T cells (Supplementary Fig. 2I). In-depth analysis showed that the proportion of CD19+ TAMs was positively correlated with that of PD-1+ effector memory T cells (TEMs), but negatively correlated with that of CD127+ TEMs (Supplementary Fig. 2I). These data strongly suggest that CD19+ TAMs possess immunosuppressive abilities.

To determine the role of CD19+ macrophages in tumor growth in vivo, we isolated CD19+ and CD19- macrophages from mouse spleen and mixed them with mouse HCC cells (Hepa 1-6) to generate orthotopic syngeneic mouse models (Supplementary Fig. 3A). The presence of CD19+ macrophages significantly promoted tumor growth in comparison to CD19- macrophages (Fig. 2D). Meanwhile, the number of infiltrating CD8+ T cells was much lower, but Gr-1+ cells were more abundant in tumors with CD19+ macrophages (Supplementary Fig. 3B and 3C). To test the clinical potential of targeting CD19+ macrophages in HCC, we developed mouse chimeric antigen receptor T (CAR-T) cells specifically targeting mouse CD19 (see details in Supplementary Methods) for adoptive T cell therapy (Supplementary Fig. 3D). The growth of the tumors with CD19+ macrophages, but not with CD19- macrophages, was significantly inhibited when the tumor-bearing mice received anti-CD19 CAR-T cells. This inhibition was not observed in the mice treated with control CAR-T cells for tumors containing either CD19+ or CD19- macrophages (Fig. 2E and Supplementary Fig. 3E). To exclude the possibility that the inhibition is associated with B cells that are also positive for CD19, we tested the anti-tumor effect of the anti-CD19 CAR-T in muMT mice that lack mature B cells18. Low levels of B cells in muMT mice were verified by flow cytometry (Supplementary Fig. 3F). GFP+ anti-CD19 CAR-T cells were infiltrated into tumors in muMT mice (Supplementary Fig. 3G) and CD19+ macrophages were effectively depleted by the anti-CD19 CAR-T treatment (Supplementary Fig. 3H) The similar anti-tumor effect of anti-CD19 CAR-T cells was observed in the tumors with CD19+ macrophages (Fig. 2F), independent of the B cell levels in the mice. These results highlight the role of CD19+ macrophages in HCC progression and suggested CD19+ macrophages are a potential clinical target for HCC treatment.

CD19+ TAMs exhibit a distinct gene expression profile
To understand the molecular features of CD19+ macrophages, we isolated CD19+ TAMs, CD19- TAMs and B cells from three HCC patients, and conducted scRNA-seq analysis for a total of 52,749 cells (Fig. 3AC and Supplementary Fig. 4A). TAMs were annotated by a high expression level of CD11b, CD14 and CD68, while B cells were identified by CD45, CD19 and CD79A. CD19+ TAMs, CD19- TAMs and B cells were clustered (Fig. 3B). We next characterized the transcriptional profiles of the three types of cells (Fig. 3C). CD19+ TAMs exhibited a distinctive gene signature, among which ribonuclease 1 (RNASE1), selenoprotein P (SELENOP) and apolipoprotein E (APOE) are closely associated with cell metabolism. CD19- TAMs expressed high levels of genes encoding small calcium-binding proteins in the S100 family including S100A9, S100A8 and S100A4. Interestingly, CD19+ TAMs and B cells both express high levels of genes (IGHG1, IGLC1 and IGKC) encoding immunoglobin proteins. Furthermore, CD19+ TAMs appeared to be in proximity to M2-like macrophages, while CD19- TAMs were close to M1 macrophages to some extent, although no definite associations were found (Fig. 3D and Supplementary Fig. 4B). Genes in metabolic pathways and lysosome were significantly enriched in CD19+ TAMs (Fig. 3E). Specifically, CD19+ TAMs had higher expression levels of genes associated with mitochondrial functions and higher mitochondria scores (Fig. 3F and 3G). The gene expression profiling analysis demonstrated that CD19+ macrophages are a subgroup of cells distinct from CD19- macrophages and B cells.

Based on the scRNA-seq data analysis, we identified a 10-gene signature capable of distinguishing between CD19+ and CD19- macrophages. This signature includes five genes specific to CD19+ TAMs (RNASE1, APOE, APOA2, APOC1, SELENOP) and five genes specific to CD19- TAMs (S100A9, S100A8, S100A4, FCN1, VCAN) (Supplementary Fig. 4C). The mRNA expression profiles of these 10 genes effectively differentiate CD19+ TAMs from CD19- TAMs, aligning well with their protein levels determined by flow cytometry (Supplementary Fig. 4D and 4E). This 10-gene signature was also applied to analyze two public scRNA-seq datasets (Bioproject: PRJCA010606 and PRJCA007744) (Supplementary Fig. 5A-5C and 6A-6C). The defined CD19+ TAMs are highly enriched in HCC compared to normal adjacent tissues and show a higher level of mitochondrial activity than CD19- TAMs (Supplementary Fig. 5D, 5E and 6D, 6E).

CD19+ TAMs are highly proliferative and immunosuppressive
Given the high mitochondria score of CD19+ TAMs in HCC, we next analyzed the metabolic activity of mitochondria based on the expression levels of oxidative phosphorylation (OXPHOS)-related genes, including NADH dehydrogenase members, cytochrome c oxidase subunit members, and ATP synthase membrane subunit members in CD19+ TAMs and CD19- TAMs. Overall, a majority of these genes was significantly up-regulated in CD19+ TAMs (Fig. 4A). To further characterize the metabolic phenotype of CD19⁺ TAMs, we sorted CD19⁺ and CD19⁻ TAMs from human HCC tissues and subjected to mitochondrial mass staining and reactive oxygen species (ROS) staining. Results revealed that CD19+ TAMs exhibit enhanced mitochondrial mass, accompanied by increased ROS production relative to CD19⁻ TAMs (Supplementary Fig. 7A and 7B). Moreover, we found that CD19+ TAMs exhibit increased co-expression of COX-IV, a key subunit of mitochondrial complex IV, supporting the maintenance of robust OXPHOS activity in CD19+ TAMs (Supplementary Fig. 7C). Seahorse analysis of cell metabolism showed remarkably enhanced mitochondrial respiration of CD19+ macrophages, including both basal and maximal respiratory capacities (Fig. 4B). The energetic feature of CD19+ macrophages may lead to the alterations of their functions and cell activities. First, CD19+ TAMs showed much compromised phagocytotic activity (Fig.S 4C). Second, we found that the proportion of CD19+ TAMs was significantly higher in large tumors than in small ones in an HCC syngeneic mouse model (Supplementary Fig. 7D), consistent with their clinical relevance in human HCC. CD19+ TAMs showed higher levels of Ki-67 expression than CD19- TAMs from the same tumor, both in mice and patients (Supplementary Fig. 7E and 7F). The enhanced proliferation of CD19+ TAMs was confirmed using an orthotopic mouse model and 5-ethynyl-2′-deoxyuridine (EdU) assays (Supplementary Fig. 7G). Therefore, we reasoned that abundance of CD19+ TAMs in tumors, especially large ones, is likely due to their high proliferation rate.

As CD19+ TAMs are a biomarker for poor clinical outcomes of HCC, we found increased PD-L1 levels in CD19+ TAMs compared to CD19- TAMs (Fig. 4D). During the HCC metabolite analysis6, we were particularly interested in adenosine, a primary immunosuppressive metabolite presents in high levels in the TME of most solid tumors19. In the metabolite data from the analysis using liquid chromatography with tandem mass spectrometry (LC-MS/MS), we observed significant associations between the concentrations of adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine and the abundance of CD19+ TAMs in human HCC tumors (Supplementary Fig. 7H). Factors that contribute to adenosine production include hypoxia, high cell turnover, and particularly, the expression of CD73, a 5′-nucleotidase (NT5E) that converts autocrine and paracrine danger signals of ATP to anti-inflammatory adenosine20. Given its pivotal role in regulating the ADP/AMP/adenosine conversion21, CD73 has been an emerging therapeutic target for immune checkpoint blockade22, 23, and numerous CD73-targeted antibodies and small-molecule inhibitors are currently undergoing clinical testing for cancer therapy24. As expected, we observed significantly elevated levels of CD73 in CD19+ TAMs (Fig. 4D). Biochemical assay verified a high 5′-nucleotidase activity of CD73 in the CD19+ TAMs (Fig. 4E). As an indicator of their immunosuppressive functions, CD19+ macrophages significantly inhibited the proliferation of total T cells, CD4+ T cells, and CD8+ T cells when these cells were isolated from the clinical tumor samples and co-cultured in vitro in mixed lymphocyte reaction (MLR) assays (Fig. 4F).

PAX5 is a master regulator for CD19+ macrophages
CD19 is important for B cell development and differentiation25. Therefore, it is intriguing to determine whether CD19 expression is essential for the immunosuppressive activity of CD19+ TAMs. To this end, CD19△Mφ (CD19flox/flox;Lyz-Cre) mice were generated to establish orthotopic mouse HCC tumor models with macrophage-specific knockout of CD19. Unexpectedly, no differences in orthotopic tumor growth were observed between CD19△Mφ mice and their littermate controls (Fig. 5A), although CD19 expression was depleted in literally all the macrophages in the CD19△Mφ mice (Fig. 5B). These results suggest that CD19 can only be used as a biomarker for the CD19+ macrophages, but functionally dispensable for this subgroup of macrophages in the tumor. Among upstream regulators of CD19, PAX5 is the key transcription factor that governs the expression of CD1926, 27. We found that CD19+ TAMs indeed had up-regulated expression of PAX5 at both mRNA and protein levels (Fig. 5C-5E). Thus, we reasoned that PAX5 may play a role in the CD19+ macrophages. Exogenous overexpression of PAX5 in human THP-1 cells (a model for macrophage-related cell activities) induced the expression of CD19, CD73 and PD-L1 (Fig. 5F and 5G). Similar results were observed in mouse immortalized bone marrow-derived macrophages (iBMDMs) (Fig. 5H). Functionally, overexpression of PAX5 impaired phagocytosis in the mouse iBMDMs (Supplementary Fig. 8A). Collectively, PAX5 appears to be a master regulator for the essential features of CD19+ macrophages in the tumor.

In the human THP-1 cells, overexpression of PAX5 also profoundly enhanced mitochondrial respiration in terms of basal respiration, maximal respiration and ATP production (Fig. 5I). Electron microscopy analysis demonstrated that the number of mitochondria was much higher in the THP-1 cells with PAX5 overexpression (Fig. 5J). The increased number of mitochondria and decreased mitochondrial superoxide levels were detected by flow cytometry in these cells ( Fig. 5K). Importantly, PAX5 significantly enhanced the expression of PGC-1α, a potent inducer of mitochondrial biogenesis (Supplementary Fig. 8B)28. Additionally, PAX5-overexpressing THP-1 cells presented a higher level of adenosine in the culture medium than control cells, consistent with increased CD73 activity (Supplementary Fig. 8C and 8D). Since cellular metabolism is tightly related to macrophage functions15, we asked whether regulation of mitochondrial metabolism affected CD73 activity in CD19+ macrophages. Antimycin, an OXPHOS inhibitor, dramatically impaired CD73 activity (Supplementary Fig. 8D). Induction of reactive oxide species by diamide, rather than their removal by N-acetyl-cysteine (NAC), also inhibited CD73 activity. By contrast, blocking CD19 with neutralizing antibodies or inhibiting glycolysis with 2-deoxy-d-glucose (2-DG) had no effect on the CD73 activity (Supplementary Fig. 8D). Therefore, mitochondrial metabolism, but not CD19-associated cell signaling regulate the functions of CD19+ macrophages in the tumor. Consistent with the cell studies, conditional knocked out of Pax5 in mouse macrophages (Pax5flox/flox;Lyz-Cre, named as Pax5△Mφ) drastically inhibited the growth of orthotopically injected Hepa1-6-derived tumors (Fig.5L and Supplementary Fig. 8E-8G). As expected, depletion of PAX5 efficiently decreased the protein levels of CD19, CD73, and PD-L1 in the macrophages of these mice (Supplementary Fig. 8H). Collectively, these results suggested that PAX5, instead of CD19, plays a central role in the regulation of CD19+ macrophages in the tumor.

PAX5 induces PD-L1 and CD73 in a post-transcriptional manner
Interestingly, PAX5 overexpression in human THP1 cells did not significantly induce the mRNA levels of PD-L1 and CD73 (Fig. 6A), indicating post-transcriptional regulation of PAX5 in CD19+macrophages. We performed CUT&Tag (Cleavage Under Targets and Tagmentation) and RNA-seq assays on human THP-1 cells with or without PAX5 overexpression and identified global expression changes of genes that are transcriptionally regulated by PAX5 (Fig. 6B). Among these changes, genes associated with lysosome pathways were the second most significantly enriched, behind the genes involved in phagocytosis (Fig. 6C), suggesting that PAX5 may control target protein degradation via lysosome. Heat shock cognate 71-kDa protein (HSC70) is a chaperon protein that brings target proteins to lysosomes for degradation in cells29. We first performed mIHC on human HCC samples, specifically quantifying the protein co-expression patterns of PAX5, HSC70, CD19, PD-L1, and CD73 within CD19⁺TAMs. Results revealed that in CD19⁺TAMs, PAX5 expression exhibited significant positive correlations with the expression of CD19, PD-L1, and CD73. However, no correlation was observed between PAX5 and HSC70 protein levels in these cells (Supplementary Fig. 9A). These findings suggested that PAX5 is proposed to influence HSC70-mediated protein degradation by altering these interaction dynamics rather than changes in the total abundance of HSC70. We then identified three KFERQ-like motifs (CMA), which are potential binding sites to HSC70, in both PD-L1 and CD73 (Fig. 6D). Coimmunoprecipitation assays validated protein interactions between HSC70 and PD-L1 or CD73 (Fig. 6E), While KFERQ-like motif mutations in PD-L1 and CD73 disrupted their interactions with HSC70 compared to the wild type (WT) proteins (Supplementary Fig. 9B and 9C). In lysosome function assays, we observed that lysosomes were significantly inhibited upon PAX5 overexpression (Fig. 6F and Supplementary 6G). Meanwhile, both CD73 and PD-L1 were induced when PAX5-overexpresing THP-1 cells were treated with inhibitors of lysosomal proteolysis (NH4Cl and leupeptin) (Fig. 6H).

Because decreased number of lysosomes were seen in the PAX5-overexpressing THP-1 cells, we speculated that PAX5 may also regulate the activity of TFEB (Transcription Factor EB), a master regulator of lysosomal biogenesis (Supplementary Fig. 10A)30, in the nucleus. We found that nuclear TFEB levels were dramatically diminished in PAX5-overexpressing cells (Fig. 6I and Supplementary Fig. 10B). Knockdown of TFEB inhibited lysosomal functions and increased PD-L1 and CD73 protein levels, similar to the effects of PAX5 overexpression (Supplementary Fig.10C-10E). Overexpression of TFEB offset the effects of PAX5 overexpression, leading to reduced levels of PD-L1 and CD73 in the PAX5-overexpressing cells (Supplementary Fig. 10F and 10G). We sought to delineate the mechanism by which PAX5 regulates the nuclear translocation of TFEB. TFEB subcellular translocation is regulated primarily by Ca2+ signaling or mTOR-mediated phosphorylation31,32. While no change in mTOR expression was observed in the PAX5-overexpressing cells (data not shown), we reasoned that increasing calcium uptake by mitochondria, due to their up-regulated biogenesis in CD19+ TAMs, may impact Ca2+ signaling in the cytosol32. Using a fluorescent probe, we found an increased level of mitochondrial Ca2+ and, accordingly, a decreased level of cytosolic Ca2+ in PAX5-overexpressing THP-1 cells (Fig. 6J). Treatment with IACS-010759 (an OXPHOS inhibitor) or ionomycin (a Ca2+ ionophore) significantly increased the cytosolic level of Ca2+ and thus promoted the nuclear level of TFEB (Supplementary Fig. 10H and 10I). These results suggested that PAX5 modulates the Ca2+ shuttling between mitochondria and the cytosol, consequently inhibiting the nuclear translocation of TFEB and lysosomal activity, which eventually increases the levels of CD73 and PD-L1 by inhibiting their lysosomal degradation.

Blocking CD19+ TAMs enhances the efficacy of immune checkpoint blockade therapy
Given the role of CD19+ TAMs in HCC progression, we wanted to determine whether they could be a therapeutic target in treating HCC. In a syngeneic HCC tumor model, we first depleted macrophages in mice using clodronate liposomes every other day for four doses (Supplementary Fig. 11A-11C) prior to mouse Hepa1-6-derived tumor models establishment (Supplementary Fig. 11D)33,34. Control or Pax5-overexpressing iBMDMs were then infused into these mice to test their effects on tumor growth (Supplementary Fig. 11D). As expected, Pax5-overexpressing iBMDMs promoted greater tumor growth than wild-type iBMDMs (Supplementary Fig. 11E). In another orthotopic mouse model without systemic depletion of macrophages, Pax5-overexpressing iBMDMs also imposed a profound pro-tumor effect (Fig. 7A). Immunohistochemical analysis revealed that Pax5-overexpressing iBMDMs limited the tumor infiltration of CD8+ T cells (Fig. 7B), indicating an immunosuppressive TME.

CD19+ TAMs create an immunosuppressive tumor microenvironment with increased levels of PD-L1 and CD73. We next tested whether the combination of PD-L1 and CD73 blockade had a synergistic effect on tumor control (Fig. 7C). Anti-CD73 neutralizing antibodies or a small-molecule inhibitor of CD73 significantly enhanced the anti-tumor effect of anti-PD-L1 antibodies in an orthotopic HCC mouse model (Fig. 7D). These effects are solely dependent on PAX5 in the CD19+ macrophages, as PD-L1 and CD73 blockade did not affect tumor progression in the Pax5△Mφ mice (Supplementary Fig. 11F). In the presence of anti-PD-L1 antibodies, CD73 blockade significantly increased the tumor infiltration of CD8+ T cells, as well as CD4+ T cells and natural killer cells. Tumor cell proliferation was greatly suppressed, as indicated by reduced Ki-67 staining (Supplementary Fig. 12).

Because induced mitochondrial respiration is critical for the functions of CD19+ TAMs, we tested whether inhibiting mitochondrial respiration of CD19+ TAMs had a similar anti-tumor effect. The OXPHOS inhibitor IACS-010759 exhibited an improved anti-tumor effect when combined with anti-PD-L1 antibodies in the orthotopic mouse HCC tumor model (Fig. 7E). The combination of PD-L1 blockade and OXPHOS inhibition also significantly promoted tumor infiltration of immune cells, including CD4+ T cells and CD8+ T cells (Supplementary Fig. 13). Collectively, therapeutic approaches targeting CD73 or OXPHOS -when optimized to leverage the heightened dependency of CD19+ TAMs on these pathways show great promise for improving the anti-tumor efficacy of immunotherapy for HCC.

Methods
Ethics Statement
All animal experiments were approved by the Ethics Committee of the First Affiliated Hospital, School of Medicine, Zhejiang University (approval number:No. 2020-483). The maximal permitted tumor size/burden as stipulated by this ethics committee is a single tumor diameter of no more than 20 mm, or a total tumor volume per animal of no more than 2000 mm3; or tumor weight not exceeding 10% of the animal's body weight. All animals included in the study maintained tumor sizes/burden within the permitted limits, and no instances of exceeding the maximal tumor size/burden were recorded.

The collection and application of clinical samples were approved by the ethics committee of the First Affiliated Hospital, Zhejiang University School of Medicine (approval number:No. 2020-681). All patients’ informed consent was obtained.

A detailed description of the methods used in the study can be found in the Supplementary information.

Cell culture
THP-1 cells, Hepa1-6, Plat-E and HEK-293T cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA), immortalized murine bone marrow-derived macrophages (iBMDMs) were provided by Jingying Zhang at Zhejiang University. THP-1 cells were cultured in Roswell Park Memorial Institute medium (RPMI 1640, Gibco, Carlsbad, CA). Hepa1-6, Plat-E and HEK-293T cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco). Media were supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Sigma-Aldrich, Saint Louis, MO), and maintained at 37.0 ± 0.2 °C in a humidified incubator with 5.0% CO2. BMDMs were isolated from femurs and tibia of adult C57BL/6 mice (The Model Animal Research Center of Nanjing University, Nanjing, China) and cultured in RPMI 1640 medium containing 50 ng/ml recombinant murine macrophage colony-stimulating factor (M-CSF, Peprotech, Rocky Hill, NJ) for 5-day differentiation.

Mouse models
All mice were bred under pathogen-free conditions. Animal experiments were approved by the ethics committee of The First Affiliated Hospital, Zhejiang University School of Medicine. The maximal permitted tumour size/burden for the study was defined as a longest diameter of 20 mm or a tumour burden equivalent to 10% of the animal’s body weight, in accordance with the ethical approval. Throughout the experimental period, none of the animals exceeded these predefined maximal tumour size or burden thresholds. Lyz-Cre mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Homozygous muMT mice were provided by Prof. Chao Wang at Zhejiang University. Pax5flox/flox and CD19flox/flox mice were purchased from Cyagen Biosciences (Suzhou, China). C57BL/6 (CD45.2) mice were purchased from The Model Animal Research Center of Nanjing University (Nanjing, China). Mice at 6-12 weeks of age were used for animal experiments.

For in situ mice model, a 20 μl suspension consisting of 50% Matrigel Basement Membrane Matrix (Corning, Teterboro, NJ) and 50% phosphate buffered saline (PBS; Cienry, Hangzhou, China) with Hepa1-6 cells in combination with macrophages were injected into the liver of mice. For syngeneic model, Hepa1-6 cells with 100 μl PBS were inoculated subcutaneously into the left or right flanks of mice. Sacrificed tumors were processed for further experiments.

Transfection of lentiviral vectors, siRNA and plasmid
THP-1 cells and iBMDMs were seeded in 6-well plates at a density of 2 × 105 cells/ml and infected with lenti-PAX5, lenti-TFEB or lenti-NC (Jikai, Shanghai, China). Stably transduced cells were purified using fluorescence activated cell sorting for further experiments. For siRNA transfection, 1.5 × 105 per well THP-1 cells were seeded in 24-well plates and transfected with human TFEB siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) using lipofectamine RNAimax (ThermoFisher Scientific, Waltham, MA) for 48 hours according to manufacturer’s protocols. For plasmids transfection, HEK-293T cells were seeded in suitable density, the plasmids were transfected with Lipo8000TM Transfection Reagent (Beyotime, Shanghai, China) according to the manufacturer’s instructions. After incubation for 24 h, the transfected cells were subjected to indicated analysis.

Targeted anti-CD19 CAR-T cell therapy
CD19+/CD19- macrophages were isolated from the spleens of mice (CD45+CD11b+F4/80+CD19+), and were mixed with Hepa1-6 cells to establish subcutaneous xenograft model at the left (CD19+ macrophages) or right (CD19- macrophages) flank of C57BL/6 or muMT mice. Anti-CD19 CAR-T cells were prepared and expanded. Mice were randomly assigned into groups for T cell infusion (1 × 107 cells in 100 µL PBS), and were sacrificed after 14 days. Harvested tumors were processed for subsequent experiments.

ImageStream analysis
Single-cell suspensions were stained and observed using an ImageStream II system (Amnis Corp, Seattle, WA) to identify CD19+ macrophages, CD19- macrophages, and B cells. Totally 1 × 105 single and focus cells were collected and analyzed by IDEASTM 6.2 software (Amnis Corp).

Multiplex immunohistochemistry (mIHC)
HCC and para-tumor were prepared into slides with 4-μm thickness. mIHC was performed using Opal™ 6-Plex Detection Kits (Akoya Biosciences, Marlborough, MA) according to the manufacturer’s instructions.

Single-cell RNA sequencing (scRNA-seq) and RNA-seq
ScRNA-seq libraries were constructed using a Chromium Single Cell 3’ Library and Gel Bead Kit v3.1 (10 x Genomics) according to the manufacturer’s protocol. RNA-seq was performed using the Illumina Hiseq XTEN platform (Illumina, San Diego, CA, USA) at Novogene Co. Ltd (Beijing, China).

Statistical analysis
Statistical analysis was conducted using GraphPad Prism 8 (GraphPad Inc., La Jolla, CA).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In their study paper Junli Wang and colleagues identified a unique subgroup of TAMs, characterized by the positivity to CD19+, in several human tumors, including HCC. They found that in HCC the density of CD19+ TAM correlated with worse OS and DFS and with favourable response upon treatment with anti PDL1. They proved that CD19+ TAMs are similar to M2-like macrophages and exhibited immunosuppressive abilities. They concluded that the elimination or functional inhibition of CD19+ TAM can represent a novel therapeutic strategy for HCC. This is a very interesting paper, with a valid rationale, an accurate and well-argued scientific design, and significant conclusions strongly supported by data.\nTwo major aspects should be addressed by the authors.\n1. The administration of a treatment requires the preliminary evaluation of a biomarker feasible in routine diagnostic practice. The authors should clarify if this biomarker is CD19 or Pax5. They should show, using bright field immunohistochemistry, that CD19 or PAX5 is expressed and evaluable in human HCC. They should explain how, in the routine diagnostic workflow, these TAM can be differentiated from B cells. Finally they need to provide data demonstrating the cut-off to use for this biomarker.\n2. HCC can be classified into some molecular categories, somehow mirrored at phenotypical level by the expression of few antibodies, the authors should show if CD19+ TAMs are enriched in any specific category or in all HCC.", "reviewer_2": "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_3": "This manuscript investigates the unique CD19+ tumor-associated macrophages (TAMs) in hepatocellular carcinoma (HCC). The authors found that the CD19+ TAMs had higher levels of PD-L1 and CD73, and higher mitochondrial oxidation with weakened phagocytosis, resulting in immunosuppression for the tumor cell survival. The authors further used CAR-T cells specifically targeting the CD19+ macrophages to show that they could suppress HCC tumor growth. PAX5 was identified to induce PD-L1 and CD73 expression post-transcriptionally. Using overexpression of PAX5 in THP-1 cells (as model for TAM), the authors found that PAX5 overexpression promoted mitochondrial biogenesis, reduced cytoplasmic Ca2+ and suppressed lysosome. The PAX5-induced lysosome deficiency blocks the lysosomal degradation of PD-L1 and CD73 as they were brought to lysosome by heat shock cognate 71-kDa protein (HSC70). Via this post-transcriptional regulation, PAX5 was found to promote PD-L1 and CD73 expression and subsequent immunosuppression to drive tumor growth. Hence, the authors proposed the targeting of CD19+ TAM as a potential therapeutic of HCC.\nThe molecular mechanism that PAX5 as the upstream post-transcriptional regulator of CD19 and other immune checkpoint inhibitors like PD-L1 and CD73 protein expression is a novelty of this study. Overall, the work in this study was detailed, with a large amount of data supporting their claims. The manuscript is well written and clear.\nI have comments below, hoping to improve this proposed mechanistic story.\nComments:\n1. To support the mechanistic story that PAX5 in CD19+ TAM promotes PD-L1 and CD73 protein expression by blocking their lysosomal degradation after the docking and carriage by HSC70 to the lysosome, the authors should strengthen this point. They could provide data on the correlation of PAX5, HSC70, lysosome staining, CD19, PD-L1 and CD73 protein expression in clinical samples.\n2. The authors mentioned that PD-L1 and CD73 can be carried by HSC70 to the lysosome and their binding to HSC70 depends on the presence of the KFERQ motifs on them. The authors can perform some experiments upon by mutating the motif, to see if this will reverse all the phenomenon observed, e.g. will the mutation of such motif block the binding of them to HSC70 and reduce the accumulation of them in lysosomes?\n3. As this study is investigating CD19+ TAM, the use of THP-1 alone may not exactly recapitulate the tumor association of the macrophages. The THP-1 should at least be co-cultured with tumor cells or treated with conditioned media from cancer cell lines. What is the percentage of CD19+ cells in the THP-1 cell line and can the authors sort out CD19+ and CD19- cells from THP-1 cell line? Will CD19+ THP-1 cells have higher PAX5, PD-L1 and CD73 expression and mitochondrial biogenesis or activity than the CD19- counterpart?\n4. Regarding CD19-targeting CAR-T cells, since CD19 is a B cell marker, how to ensure that it is the CD19+ TAM but not the general B cells that got specifically inhibited by the anti-CD19 CAR-T cells?\n5. The authors pointed out that CD19+ TAMs had higher mitochondria-related gene expression as compared to CD19- counterparts. In Fig 3G, they showed the former had higher mitochondria score than the latter. The authors should clarify how the score was calculated and based on expression levels of what genes. Besides OCR, were there differences in the mitochondria mass or biogenesis between CD19+ TAM and CD19- TAM (evidence was only shown in THP-1 in Fig. 5J)? Do they have different ROS levels? Also, can the mitochondria-related gene-encoding proteins or mitochondria activity be quantified in clinical HCC samples to show any correlation with CD19+ TAM in HCC?\n6. PAX5 is a pro-B-cell factor and the above-mentioned previous study (DOI: 10.1038/s41467-022-33117-y) has reported that the deficiency of PAX5 could drive the macrophage differentiation. The authors should at least discuss how the current study can be related to that finding. Because here the current study found a positive role of PAX5 on the PD-L1 expression and tumor immunosuppression in the TAM. It looks like the findings from these two studies are counteracting.", "reviewer_4": "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_5": "In this study, the authors compellingly identified CD19⁺ TAMs as a novel, immunosuppressive subpopulation enriched in HCC and linked to poor prognosis. The findings are quite innovative and informative, and the paper is well written. There were several concerns that need to be solved.\nMajor：\n1. The authors should explicitly compare CD19⁺ TAMs with traditional M1/M2-like features on their metabolic, transcriptional, and functional identities. Is there a M2 subpopulation in the single cell mass cytometry (CyTOF) dataset? Are CD19+ TAMs a subpopulation of M2 macrophages, or phenotypically altered M2 macrophages? Please clarify the relationship between this novel cell population and traditional macrophage subtypes more directly.\n2. Could CD19+ macrophages be fused cells between macrophage and B cells, or macrophage with B cell endocytosed?\n3. Although the OXPHOS inhibitor IACS-010759 exhibited an improved anti-tumor effect, the authors could not attribute the function to CD19 macrophages, since tumor cells or other cells can also be targeted by this inhibitor.\n4. The last sentence “Collectively, therapeutic approaches targeting CD73 or OXPHOS in CD19 + TAMs show great promise for improving the anti-tumor efficacy of immunotherapy for HCC.” is not acceptable. The authors need to discuss more about how their findings could translate to therapeutical approaches. OXPHOS and CD73 are all non-specific to CD19 macrophages and might not be good targets for developing new therapies.\nMinor:\n1. In figure 4A, p-value needs to be provided.\n2. In figure 7A, the authors should clarify the differences between the upper and bottom panel.\n3. Gr1+ cells need to be introduced and the biological significance should be explained."}, "author_response": "Detailed Poin-by-Point Response to Reviewer Comments\nThank you for the editors and all reviewers’ valuable comments and suggestions on our\nmanuscript. Detailed point-by-point response to the reviewers are listed below and\nhighlighted in red. For all revisions, we provide specific locators (page, line, figure, or SI\nsection) to the revised manuscript.\n1. Regarding data access: We were not able to access to the data access you\ndeposited in ScienceDB. Could you please guide me on how to access this data? It\nis important that the data is accessible to the reviewers, in case we share your\nrevised version with them.\nResponse：We apologize for the data access inconvenience. Please use the direct link\nbelow or search the dataset title to access our data in ScienceDB:\nDirect Link: https://www.scidb.cn/s/b63uAr\nCSTR: https://cstr.cn/31253.11.sciencedb.11557\nDOI: https://doi.org/10.57760/sciencedb.11557\nSearch Method: On ScienceDB’s homepage, navigate to Advanced Search and enter the\ndataset title: \"Data for CD19 positive macrophages\".\nThank you for your patience. We hope these can help you access the data successfully.\nWe have provided detailed descriptions of the relevant CSTR and direct link in the\nData Availability section, ensuring that all supporting data can be accessed and\nreferenced appropriately. If you still have questions, please feel free to contact us.\n2. Regarding human data and clinical trial: We noticed that some of the human data\nin your study may be associated with a clinical trial (NCT03732547). Could you\nplease clarify the source of the human data used in your study? If any of the data\nis from a clinical trial, please confirm whether it has already been published.\nResponse：We are sorry for the confusion in our previous description and appreciate the\nopportunity to clarify the clinical trial-derived human data in our study. The clinical trial\nNCT03732547 was registered as titled Study of “PolyIC and PD-1 mAb in Subjects with\nUnresectable Hepatocellular Carcinoma”. This study aimed to evaluate the safety and\nefficacy of the combination of PolyIC and PD-1 mAb in unresectable late-stage HCC\npatients. Currently, the recruitment for this clinical study has been completed, and the\nrelated article is still in an unpublished state.\nWhile NCT03732547 primarily investigated the combination of PolyIC and PD-1\nmAb in HCC, our study focused exclusively on the monotherapy subgroup to\nisolate the role of CD19+TAMs in anti-PD-1 therapy response. Tumor paraffin sections\nwere collected from patients who received only anti-PD-1 antibody monotherapy and\nstained with CD68 and CD19 (performed to quantify CD19+TAMs). Corresponding\ncomputed Tomography (CT) imaging and clinical data were systematically collected\nand analyzed to evaluate tumor response to anti-PD-1 antibody monotherapy. Our\nanalysis revealed that a high density of CD19+TAMs (but not total TAMs) was significantly\nassociated with favorable tumor responses (defined as partial response or stable disease)\nto anti-PD-1 therapy, as shown in Figure 2D and 2E. This suggests that CD19+TAMs may\nmediate an immunosuppressive tumor microenvironment that can be reversed by\nimmunotherapy in HCC and supported our hypothesis that CD19⁺ TAMs contribute to an\nimmunosuppressive microenvironment in HCC. Notably, both the immunofluorescence\nand CT datasets used in this study are unpublished and original to the current work.\nWe hope this information addresses your concerns. Please do not hesitate to contact\nus if you require any further details.\nPoint -by- point response to reviewer comments\nReviewer #1：\nIn their study paper Junli Wang and colleagues identified a unique subgroup of TAMs,\ncharacterized by the positivity to CD19+, in several human tumors, including HCC. They\nfound that in HCC the density of CD19+ TAM correlated with worse OS and DFS and with\nfavourable response upon treatment with anti PD-L1. They proved that CD19+ TAMs are\nsimilar to M2-like macrophages and exhibited immunosuppressive abilities. They\nconcluded that the elimination or functional inhibition of CD19+ TAM can represent a novel\ntherapeutic strategy for HCC. This is a very interesting paper, with a valid rationale, an\naccurate and well-argued scientific design, and significant conclusions strongly supported\nby data.\nResponse: We would like to take this opportunity to express our sincere gratitude for your\nreview of our manuscript and your valuable feedback. The reviewer has provided an\naccurate summary of our work. We understand the significance of each comment and\nquestion raised, and we will spare no effort to address them in a thorough and systematic\nmanner. In the following responses, we have carefully considered reviewer’s suggestions,\nconducted additional analyses where necessary, and made substantial revisions to\nimprove the quality and rigor of our manuscript. Once again, thank you for your\nconstructive guidance, which has been invaluable in enhancing the scientific integrity and\nclarity of our research.\nTwo major aspects should be addressed by the authors.\nComment 1. The administration of a treatment requires the preliminary evaluation\nof a biomarker feasible in routine diagnostic practice. The authors should clarify if\nthis biomarker is CD19 or Pax5. They should show, using bright field\nimmunohistochemistry, that CD19 or PAX5 is expressed and evaluable in human\nHCC. They should explain how, in the routine diagnostic workflow, these TAM can\nbe differentiated from B cells. Finally, they need to provide data demonstrating the\ncut-off to use for this biomarker.\nResponse: We appreciate the reviewer’s critical comments regarding the biomarker\nevaluation and feasibility for routine diagnostic practice for CD19+TAMs. Below is our\ndetailed clarification:\n1) Identification of the Biomarker: CD19 vs. Pax5\nThe primary biomarker founded in our study is the expression CD19 on macrophages,\nwhich enabled visualization of CD19+TAMs via imaging flow cytometry and subsequent\nisolation by cell sorting for single-cell RNA sequencing (scRNA-seq). Further mechanistic\nanalysis revealed that CD19 mRNA expression was not high in CD19+ TAMs and CD19\nexpression in macrophages does not drive functional effects; instead, PAX5 acts as the\ncritical regulatory protein governing the functional phenotype of CD19+ TAMs. Despite this,\nCD19 remains the definitive biomarker for this population due to its cell-surface\nlocalization, which is more amenable to real-time detection and sorting in experimental\nand clinical settings. This distinction between surface marker utility and intracellular\nfunctional regulation underscores the translational value of CD19 for diagnostic evaluation,\nwhile PAX5 represents a potential therapeutic target for modulating TAM activity.\n2) Bright-Field IHC Validation of CD19 in Human HCC\nWe appreciate the opportunity to supplement our findings with representative images. The\nbright-field immunohistochemistry (IHC) staining for CD19 in human HCC tissues\n(Corresponding to Figure 1E, right) confirms detectable CD19 expression, with positive\nstaining localized to the cell membrane (brown color, nuclear counterstaining in blue) (For\nreviewer-Figure 1). This visual evidence aligns with our prior results, demonstrating that\nCD19 is expressed on the cell surface of macrophages in HCC microenvironments, which\nvalidates its utility as a surface biomarker for cell visualization and sorting.\nFor reviewer-Figure 1. Representative immunofluorescence images and corresponding\nbright-field immunohistochemistry (IHC) staining of CD19+ TAMs in HCC tissue. Red\narrows indicate CD19+ TAMs in IHC.\n3) Differentiation of CD19⁺ TAMs from B Cells in Routine Diagnostics\nTo distinguish CD19⁺ TAMs from B cells, we propose a multi-marker approach feasible in\nclinical labs:\nFor reviewer-Table 1. Differentiation of CD19⁺ TAMs from B Cells in Routine\nDiagnostics\nFeature CD19+ TAMs B cells Cut-off value of CD19 expression\nFlow CD45+CD11b+ CD45+CD11b- Macrophages were classified as\nCytometry CD68+CD19+ CD68-CD19+ CD19+ TAMs when their CD19\nfluorescence intensity matched\nthat of B cells.\nImmunofluore CD45+CD11b+ CD45+CD11b- CD19 expression was detectable\nscence CD14+CD19+ CD14-CD19+ with positive staining localized to\ncell surface.\nImaging Flow CD68+CD19+ CD68-CD19- Macrophages were classified as\nCytometry CD19+ TAMs when their CD19\nfluorescence intensity matched\nthat of B cells.\nmIHC CD11b+CD14+ CD11b-CD14- CD19 expression was detectable\nCD19+CD20- CD19+CD20+ with positive staining localized to\ncell surface.\nCyToF CD45+CD11b+ CD45+CD11b- Macrophages were classified as\nCD68+CD19+ CD68-CD19+ CD19+ TAMs when their\nnormalized CD19 signal intensity\nmatched that of B cells.\nWe believe these clarifications demonstrate the clinical feasibility of CD19 as a biomarker\nand its utility in routine diagnostic settings. Please let us know if further details or\nexperimental data are needed.\nComment 2. HCC can be classified into some molecular categories, somehow\nmirrored at phenotypical level by the expression of few antibodies, the authors\nshould show if CD19+ TAMs are enriched in any specific category or in all HCC.\nResponse: We sincerely appreciate the reviewer’s insightful suggestion regarding the\npotential association between CD19⁺ TAMs abundance and specific molecular subtypes\nof HCC, which is critical for clarifying the clinical relevance of our findings. Through a\ncomprehensive review of the relevant literature, we noted that the currently recognized\nmolecular classifications of HCC are diverse, primarily rely on RNA-seq, proteomics, or\nmulti-omics integration (PMID: 19723656, PMID: 31585088, PMID: 33290597).\nTo clarify the relevance of CD19⁺ TAMs across HCC molecular subtypes, we\nperformed molecular subtyping of 40 HCC samples from our cohort using transcriptional\nprofiles, following the established classification framework (PMID: 26054909) that\ndistinguishes proliferative and non-proliferative subtypes. Concurrently, we quantified\nCD19⁺ TAMs abundance via immunofluorescence staining, co-labeling with CD19 and\nCD68 to specifically identify and count CD19⁺ TAMs. Statistical analysis revealed no\nsignificant differences in CD19⁺ TAMs abundance across distinct molecular subtypes (For\nreviewer-Figure 2, p > 0.05). However, we acknowledge that this result may be influenced\nby the limited sample size in our cohort—specifically, one of the subtypes was represented\nby only 7 samples, which could reduce the statistical power to detect potential differences.\nFor reviewer-Figure 2. RNA-seq was performed on a cohort of 40 HCC specimens to\nclassify tumors into proliferative and non-proliferative molecular subtypes based on\nestablished transcriptional signatures. Consecutive tumor sections from the same\nspecimens were subjected to multiplex immunofluorescence staining for CD19 and CD68\nto identify CD19⁺ TAMs. Quantification of CD19⁺ TAMs abundance in progressive (n =7)\nand non-progressive (n =33) types. Statistical analysis was performed using (Mann-\nWhitney U test), with no significant difference observed between groups (p > 0.05).\nIn addition, we also attempted to validate this observation using public datasets.\nUnfortunately, due to the scarcity of HCC cohorts that simultaneously include high-\ndimensional immune phenotyping data (e.g., CyTOF for precise definition of CD19⁺ TAMs)\nand RNA-seq data (for molecular subtyping), we were unable to obtain sufficient external\nvalidation results.\nIn summary, we recognize that the sample size in our current analysis is modest,\nparticularly for proliferative subtypes, and we plan to expand our cohort with additional\nHCC samples in future studies to further verify this relationship. For transparency, the raw\ndata related to these analyses are available upon request from the corresponding author.\nThank you again for this valuable comment, which underscores the need for larger-scale\nstudies to clarify the subtype-specific relevance of CD19⁺ TAMs. We will prioritize this\nvalidation in our subsequent research to strengthen the clinical implications of our findings.\nReviewer #2:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review and\nto provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: Thank you for the thoughtful insights and constructive feedback contributed\nby both reviewers, which have been invaluable in helping us strengthen the rigor and\nclarity of our manuscript. We recognize the importance of such initiatives in fostering the\nnext generation of scientific reviewers, and we are grateful for the careful attention given\nto our work through this collaborative review process. We have responded all points raised\nin the reviews comprehensively, and we hope the revised manuscript meets the journal's\nstandards. Please let us know if any further clarifications are needed.\nReviewer #3:\nThis manuscript investigates the unique CD19+ tumor-associated macrophages (TAMs)\nin hepatocellular carcinoma (HCC). The authors found that the CD19+ TAMs had higher\nlevels of PD-L1 and CD73, and higher mitochondrial oxidation with weakened\nphagocytosis, resulting in immunosuppression for the tumor cell survival. The authors\nfurther used CAR-T cells specifically targeting the CD19+ macrophages to show that they\ncould suppress HCC tumor growth. PAX5 was identified to induce PD-L1 and CD73\nexpression post-transcriptionally. Using overexpression of PAX5 in THP-1 cells (as model\nfor TAM), the authors found that PAX5 overexpression promoted mitochondrial biogenesis,\nreduced cytoplasmic Ca2+ and suppressed lysosome. The PAX5-induced lysosome\ndeficiency blocks the lysosomal degradation of PD-L1 and CD73 as they were brought to\nlysosome by heat shock cognate 71-kDa protein (HSC70). Via this post-transcriptional\nregulation, PAX5 was found to promote PD-L1 and CD73 expression and subsequent\nimmunosuppression to drive tumor growth. Hence, the authors proposed the targeting of\nCD19+ TAM as a potential therapeutic of HCC.\nThe molecular mechanism that PAX5 as the upstream post-transcriptional regulator of\nCD19 and other immune checkpoint inhibitors like PD-L1 and CD73 protein expression is\na novelty of this study. Overall, the work in this study was detailed, with a large amount of\ndata supporting their claims. The manuscript is well written and clear.\nResponse: We sincerely appreciate your positive evaluation of our work. Your recognition\nof our study is deeply encouraging and reinforces our confidence in the significance of\nthese findings. We have carefully considered all suggestions to further refine our\nmanuscript. We acknowledge that the section addressing the mechanisms underlying\nCD73 and PD-L1 protein degradation is currently less robust. Following your valuable\nsuggestions, we have conducted additional experiments to provide more direct evidence\nfor the proposed regulatory mechanisms. Thank you again for your time and thoughtful\nfeedback, which have been invaluable in strengthening this study. We hope the revised\nmanuscript will meet the standards of the journal and contribute meaningfully to the field.\nComment 1. To support the mechanistic story that PAX5 in CD19+ TAM promotes\nPD-L1 and CD73 protein expression by blocking their lysosomal degradation after\nthe docking and carriage by HSC70 to the lysosome, the authors should strengthen\nthis point. They could provide data on the correlation of PAX5, HSC70, lysosome\nstaining, CD19, PD-L1 and CD73 protein expression in clinical samples.\nResponse: We greatly appreciate the reviewer’s critical input, which has been\ninstrumental in deepening the mechanistic understanding of how PAX5 in CD19⁺ TAMs\nregulated PD-L1 and CD73 via lysosomal pathways. To address this key point, we have\nperformed multiplex immunohistochemistry (mIHC) on human HCC samples, specifically\nquantifying the protein co-expression patterns of PAX5, CD19, HSC70, PD-L1, and CD73\nwithin CD19⁺ TAM (defined by the co-staining of CD19 and CD68).\nOur results revealed that in CD19⁺ TAMs, PAX5 expression exhibited significant\npositive correlations with CD19, PD-L1, and CD73. Notably, however, no significant\ncorrelation was observed between PAX5 and HSC70 protein levels in these cells (For\nreviewer-Figure 3, We have also added these findings as Supplementary Figure S9A,\n(Result Section, Page 14, Line 15)). As HSC70 typically functions as a housekeeping\nchaperone with stable basal expression across cell types, its role in cargo trafficking (e.g.,\ntargeting proteins to lysosomes) is primarily governed by dynamic interactions with\nsubstrates rather than total protein abundance (PMID: 31253954). These findings\nreinforce our proposed mechanism that PAX5 promotes the accumulation of PD-L1 and\nCD73 by inhibiting lysosomal degradative function, rather than by disrupting HSC70-\nmediated trafficking in CD19⁺ TAMs.\nFor reviewer-Figure 3. Representative images showing CD19⁺ TAMs with low and high\nPAX5 expression (cyan) in HCC tissues; corresponding expression levels of CD73\n(orange), PD-L1 (white), and HSC70 (yellow) are shown in the same microenvironment\n(upper). CD19⁺ TAMs are defined as CD19⁺CD68⁺ double-positive cells (White arrows).\nScale bar = 50 μm. Correlation analysis of PAX5 expression with CD73, PD-L1, and\nHSC70 expression specifically within CD19⁺ TAMs. Each dot represents an individual cell,\nwith correlation coefficients (r) and p-values calculated using Pearson’s correlation\n(bottom).\nWe sincerely apologize for the challenge in directly visualizing PAX5-lysosome\ninteractions within primary CD19⁺ TAMs of HCC tissues. This technical hurdle arises\nbecause lysosomal staining with LysoTracker typically requires live-cell imaging, whereas\nPAX5 detection necessitates permeabilization to allow antibody access, creating an\ninherent conflict in sample preparation. Despite extensive efforts to optimize protocols, we\nfound that permeabilization disrupts lysosomal integrity, precluding reliable co-localization\nanalysis. We are actively exploring alternative approaches to identify an optimized method\nfor dissecting the relationship between PAX5 and lysosomal dynamics, and we remain\ncommitted to addressing this aspect.\nOnce again, we thank you profoundly for highlighting this critical point, as it has\nsignificantly enhanced the rigor of our study. Please do not hesitate to inform us if further\nvalidation, such as additional experiments or alternative methodologies, would help\nstrengthen this mechanistic link; we are fully prepared to undertake such efforts to ensure\nthe robustness of our conclusions.\nComment 2. The authors mentioned that PD-L1 and CD73 can be carried by HSC70\nto the lysosome and their binding to HSC70 depends on the presence of the KFERQ\nmotifs on them. The authors can perform some experiments upon by mutating the\nmotif, to see if this will reverse all the phenomenon observed, e.g. will the mutation\nof such motif block the binding of them to HSC70 and reduce the accumulation of\nthem in lysosomes?\nResponse: We respectfully agree with your suggestion to validate the role of KFERQ\nmotifs in mediating HSC70 binding and lysosomal trafficking of PD-L1 and CD73. To\naddress this, we performed experiments using site-directed mutagenesis of the KFERQ-\nlike motifs in PD-L1 and CD73, followed by functional assays to assess their impact on\nHSC70 binding.\nWe generated mutant constructs of CD73 (VQ68-69AA/VD204-205AA/DR467-468AA)\nand PD-L1 (VQ76-77AA/DQ90-91AA/LQ106-107AA) to disrupt the KFERQ-like motifs,\nwhile preserving overall protein structure. Mutant or wild-type (WT) PD-L1 and CD73\nplasmids were transfected into HEK-293T cells. Co-immunoprecipitation (Co-IP) showed\nthat motif mutations reduced the interaction between HSC70 and PD-L1/CD73 compared\nto WT proteins (For reviewer-Figure 4). These data confirm that the KFERQ-like motifs\nin\nin PD-L1 and CD73 are critical for binding to HSC70, which mediates their trafficking to\nlysosomes. Disrupting the motifs reduces lysosomal targeting, making PD-L1 and CD73\nless sensitive to HSC70-mediated inhibition of lysosomal degradation.\nWe have added these findings as Supplementary Figure S9B and S9C (Result\nSection, Page 15, Line 1), with detailed methods in the Supplementary Methods\n（section in Transfection of lentiviral vectors, siRNA, and plasmid）. These\nexperiments strengthen the mechanistic link between HSC70-mediated trafficking,\nlysosomal degradation, and PAX5 regulation of PD-L1 and CD73.\nFor reviewer-Figure 4. (A) HEK-293T cells were co-transfected with empty vector or\nvector expressing Flag-CD73 or mutants and His-HSC70 for 24 h. The cell lysates were\nimmunoprecipitated with anti-Flag IgG beads and subjected to immunoblotting using the\nindicated antibodies. The KFERQ-like motif was marked in red. The mutant regions were\nmarked in yellow. EV: Empty vector. (B) HEK-293T cells were co-transfected with empty\nvector or vector expressing Myc-PD-L1 or mutants and His-HSC70 for 24 h. The cell\nlysates were immunoprecipitated with HSC-70 primary antibody and subjected to\nimmunoblotting using the indicated antibodies. The KFERQ-like motif was marked in red.\nThe mutant regions were marked in yellow. EV: Empty vector.\nPlease let us know if further validation would enhance the robustness of these\nconclusions. Thank you for this critical suggestion, which has significantly strengthened\nthe mechanistic rigor of our study.\nComment 3. As this study is investigating CD19+ TAM, the use of THP-1 alone may\nnot exactly recapitulate the tumor association of the macrophages. The THP-1\nshould at least be co-cultured with tumor cells or treated with conditioned media\nfrom cancer cell lines. What is the percentage of CD19+ cells in the THP-1 cell line\nand can the authors sort out CD19+ and CD19- cells from THP-1 cell line? Will\nCD19+ THP-1 cells have higher PAX5, PD-L1 and CD73 expression and\nmitochondrial biogenesis or activity than the CD19- counterpart?\nResponse：We deeply appreciate the reviewers’ critical insight regarding the modeling of\ntumor-macrophage interactions. Initially, we prioritized mimicking in vivo conditions to\ninduce CD19 expression in THP-1 cells, testing three biologically relevant strategies: co-\nculture with HepG2 cells, exposure to tumor-conditioned media (TCM), and treatment with\ntumor tissue fluid. However, none of these approaches significantly increased the\nfrequency of CD19⁺ THP-1 cells above baseline levels (For reviewer-Figure 5). This\nobservation suggested that CD19 induction in macrophages may depend on more\ncomplex, cell-intrinsic regulatory mechanisms rather than solely on soluble or contact-\ndependent signals from the tumor microenvironment (TME).\nFor reviewer-Figure 5. Representative flow cytometry histograms showing CD19\nexpression in THP-1 cells under different experimental conditions: isotype control\n(negative staining control), unstimulated control, co-culture with HepG2 cells, exposure to\nHepG2-derived tumor-conditioned media (TCM), and treatment with tumor tissue fluid\n(left). Quantitative analysis of CD19 expression levels in THP-1 cells across the five\ngroups. Data are presented as mean ± SEM from three independent experiments.\nStatistical analysis was performed using one-way ANOVA, NS indicating no significant\ndifferences.\nThis finding prompted us to shift to a mechanistic model centered on PAX5, a\ntranscription factor previously implicated in CD19 transcriptional regulation. Our results\nconfirmed that lentiviral overexpression of PAX5 in THP-1 cells not only drove CD19\nupregulation but also recapitulated key phenotypic features of CD19⁺ TAMs, including\nincreased expression of CD73 and PD-L1 and enhanced mitochondrial biogenesis.\nConsistent with the metabolic and functional properties of these cells in human HCC\ntissues. Notably, while PAX5 overexpression effectively induces CD19 in THP-1 cells, we\nacknowledge that no in vitro model fully replicates the in vivo tumor microenvironment.\nPAX5-induced system offers a controlled framework to test hypotheses about CD19⁺ TAM\nbiology, with findings that can then be validated in primary cells or preclinical models. This\napproach, we believe, presents a balance between physiological relevance and\nexperimental tractability, making it a valuable tool for identifying the role of CD19⁺ TAMs\nin tumor progression. To address this, we are currently performing experiments to clarify\nthe cellular origin of CD19+ TAMs with the aim of further elucidating the mechanisms\nunderlying CD19 induction in macrophages in our ongoing studies.\nThank you again for highlighting this important consideration, which has helped us\nframe a more rigorous and contextually grounded experimental strategy.\nComment 4. Regarding CD19-targeting CAR-T cells, since CD19 is a B cell marker,\nhow to ensure that it is the CD19+ TAM but not the general B cells that got\nspecifically inhibited by the anti-CD19 CAR-T cells?\nResponse: We deeply appreciate the reviewer’s critical concern regarding the specificity\nof anti-CD19 CAR-T cells for CD19+ TAMs versus normal B cells. This is a pivotal\nconsideration, as CD19 serves as a canonical B cell marker, and potential off-target\neffects on B cells could lead to immunosuppression or other adverse events.\nOur preclinical investigations addressed this by using genetically modified μMT mice,\nwhich lack mature B cells due to a mutation in the immunoglobulin μ heavy chain gene. In\nthese models, administration of anti-CD19 CAR-T cells significantly inhibited\nhepatocellular carcinoma (HCC) progression. Flow cytometric analysis confirmed that this\nanti-tumor effect was attributed to the selective depletion of CD19⁺ TAMs within the tumor\nmicroenvironment (TME), with no confounding contributions from B cells. (Figure S3F).\nThese findings provide direct evidence that CD19-targeted CAR-T cells can exert anti-\ntumor effects via TAM depletion independent of B cell presence, supporting the feasibility\nof TAM-specific targeting.\nWhile μMT mice provide genetic validation of TAM-specific targeting, we acknowledge\nthe translational challenge of B cell toxicity in clinical settings. To address this, we are\nimplementing the following complementary strategies:\nA. Dual-signal CAR-T design: Engineering CAR-T cells to require a second signal (e.g.,\ntumor-specific cytokines or checkpoint molecules) that is only present in the TME.\nB. AND-gate CARs: Developing dual-targeting CARs (e.g., CD19 + TAM-specific markers\nsuch as CD163 or MARCO) to reduce off-target B cell depletion.\nC. Hypoxia-activated CARs: Exploiting the low-oxygen tumor microenvironment to trigger\nCAR-T activity selectively.\nD. Intratumoral injection: Directly administering CAR-T cells into tumors (e.g., via\nultrasound or CT-guided intratumoral injection) to enhance local targeting and minimize\nsystemic exposure.\nWe are committed to translating these approaches to the clinic and providing a\nprecision treatment regimen for HCC patients. Thank you again for highlighting this critical\nconsideration, which has guided our focus on refining CAR-T specificity for clinical\napplicability. Please let us know if further details on any of these strategies would\nstrengthen this aspect of our work.\nComment 5. The authors pointed out that CD19+ TAMs had higher mitochondria-\nrelated gene expression as compared to CD19- counterparts. In Fig 3G, they\nshowed the former had higher mitochondria score than the latter. The authors\nshould clarify how the score was calculated and based on expression levels of what\ngenes. Besides OCR, were there differences in the mitochondria mass or\nbiogenesis between CD19+ TAM and CD19- TAM (evidence was only shown in THP-\n1 in Fig. 5J)? Do they have different ROS levels? Also, can the mitochondria-related\ngene-encoding proteins or mitochondria activity be quantified in clinical HCC\nsamples to show any correlation with CD19+ TAM in HCC?\nResponse: We sincerely appreciate the reviewer’s insightful questions regarding\nmitochondrial characterization in CD19+ TAMs. We address each concern systematically:\n1) Clarification of Mitochondrial Score Calculation (Fig. 3G)\nThe mitochondrial score was derived from a curated set of 13 identified mitochondrial-\nencoded genes identified in single-cell RNA-seq data, include MT-ND1, MT-ND2, MT-\nCO1, MT-CO2, MT-ATP8, MT-ATP6, MT-CO3, MT-ND3, MT-ND4L, MT-ND4, MT-ND5,\nMT-ND6, MT-CYB.\nThis metric was calculated using the PercentageFeatureSet function in Seurat v4.3.0,\nwhich quantifies the proportion of transcripts derived from these 13 mitochondrial-encoded\ngenes relative to the total number of expressed genes in each cell. We will include the\nabsolute expression values and the full gene list with official symbols of these\nmitochondrial genes in the revised manuscript’s Supplementary Methods (see also\nSupplementary Table 9 for gene annotations).\n2) Mitochondrial Mass, Biogenesis, and ROS Levels\nBeyond oxygen consumption rate (OCR), we provide additional evidence:\nA. MitoTracker: CD19+ TAMs and CD19- TAMs were sorted from human HCC specimens,\nCD19+ TAMs showed higher MitoTracker Deep Red fluorescence intensity than CD19-\nTAMs (For reviewer-Figure 6).\nFor reviewer-Figure 6. Representative confocal microscopy images of CD19⁺ and CD19⁻\nTAMs sorted from HCC tissues, stained with MitoTracker (red) to label mitochondria.\nNuclei are counterstained with DAPI (blue). Scale bar = 5 μm. Fluorescence intensity\nprofile analysis of MitoTracker staining along the white lines for CD19⁺ and CD19⁻ TAMs.\nThe graph shows overlapping peaks of MitoTracker fluorescence intensity across the\nlinear profiles, reflecting mitochondrial distribution and density within the cells.\nB. qPCR: in Fig 4A, CD19+ TAMs exhibited elevated mRNA expression of mitochondrial\nbiogenesis markers.\nC. Reactive Oxygen Species (ROS)\nDCFH-DA Assay: CD19+ TAMs isolated from primary human HCC displayed lower ROS\nlevels than CD19- TAMs (For reviewer-Figure 7). This correlates with scRNA-seq data\nshowing 5-fold higher expression of antioxidant genes (SOD3, GPX3) in CD19+ TAMs\n(Supplementary Table 8).\nFor reviewer-Figure 7. Representative confocal microscopy images of CD19⁺ and CD19⁻\nTAMs sorted from HCC tissues, stained with DCFH-DA (Green) to label ROS. Nuclei are\ncounterstained with DAPI (blue). Scale bar = 5 μm. Fluorescence intensity profile analysis\nof DCFH-DA staining along the white lines for CD19⁺ and CD19⁻ TAMs. The graph shows\noverlapping peaks of DCFH-DA fluorescence intensity across the linear profiles, reflecting\nROS distribution and density within the cells.\n3) Clinical Validation in HCC Samples\nWe performed multiplex immunofluorescence staining on HCC specimens to assess the\nexpression of Complex IV, a key component of the mitochondrial respiratory chain and a\nmarker of OXPHOS activity. Quantitative analysis revealed that CD19⁺ TAMs exhibited\napproximately 3-fold higher COX IV expression compared to other CD19⁻ TAMs within\nthe same microenvironment (For reviewer-Figure 8).\nFor reviewer-Figure 8. Representative images showing CD19⁺ TAMs and CD19⁺ TAMs\nwith COX IV expression (orange) in HCC tissue sections. CD19⁺ TAMs are defined as\nCD19⁺CD68⁺ double-positive cells (White arrows). Scale bar = 50 μm. Quantitative\nanalysis of COX IV expression levels in CD19⁺ TAMs and CD19⁺ TAMs. Data are\npresented as mean ± SEM. Statistical analysis was performed using unpaired two-tailed\nt-test, ****P < 0.0001.\nCollectively, these data demonstrate that CD19+ TAMs exhibit enhanced mitochondrial\nbiogenesis, mass, and metabolic activity compared to CD19− TAMs in both in vitro models\nand clinical HCC samples. We had integrated these additional analyses and statistical\ncontrols into the revised manuscript (Result Section, Page 10, Line 23 and.\nSupplementary Figure 7A-C) to strengthen the mechanistic and translational\nsignificance of our findings.\nComment 6. PAX5 is a pro-B-cell factor and the above-mentioned previous study\n(DOI: 10.1038/s41467-022-33117-y) has reported that the deficiency of PAX5 could\ndrive the macrophage differentiation. The authors should at least discuss how the\ncurrent study can be related to that finding. Because here the current study found\na positive role of PAX5 on the PD-L1 expression and tumor immunosuppression in\nthe TAM. It looks like the findings from these two studies are counteracting.\nResponse: We appreciate the reviewer’s insightful perspective regarding the relationship\nbetween our identified CD19+ TAMs and previously reported pro-B-cell-derived\nmacrophages (B-MFs). This comparison provides a valuable framework to contextualize\nour findings, and we thank the opportunity to clarify key distinctions and connections:\nWhile prior work has elegantly demonstrated that cancers can induce trans-differentiation\nof bone marrow-derived B-cell precursors—specifically Csf1R⁺ Pax5Low pre-B cells and\nimmature IgM⁺ B cells—into TAMs (termed B-MFs) via M-CSF signaling. A defining\nfeature of B-MFs is the downregulation of PAX5 alongside upregulation of macrophage-\nassociated genes (CD68, Csf1r, Cebpb, Cebpg, Ccl2, Csf1), coupled with enhanced\nphagocytic and immunosuppressive functions. Notably, these B-MFs are nearly absent\nin μMT mice (which lack mature B cells), consistent with their origin from B-cell\nlineage precursors.\nIn our study, single-cell RNA sequencing (scRNA-seq) analysis revealed that CD19+\nTAMs exhibit co-expression of B-cell (IGHM, IGHG) and macrophage (CD68, CSF1R)\ngene modules (Fig 3C). Furthermore, in vivo studies demonstrate that CD19⁺ TAMs exist\nat low frequencies under physiological conditions (see Figure S1D), including in μMT mice\n(Figure S3H), and are enriched during tumor progression, where they promote\nimmunosuppression.\nA key mechanistic distinction lies in the role of PAX5. In B-MFs, PAX5 is downregulated\nas part of the trans-differentiation process from B-cell precursors to macrophages. In our\nstudy, however, PAX5 functions not as a lineage repressor but as a transcriptional\nactivator within CD19⁺ TAMs: PAX5 overexpression in macrophages (THP-1 cells)\ninduces CD19 expression without suppressing macrophage markers (e.g., CD68, CSF1R).\nFar from being contradictory, our findings and the prior report on B-MFs collectively\nhighlight the context-dependent functionality of PAX5 in regulating hematopoietic cell fate\nand plasticity. While PAX5 loss drives myeloid commitment in progenitors, its\noverexpression in mature macrophages promotes an immunosuppressive, mitochondrial-\nrich TAM phenotype. We had expanded the Discussion section (Page 18, Line 11) to\nexplicitly address this context-dependent functionality and its implications for TAM biology.\nFurthermore, our preliminary tracing studies suggest that CD19⁺ macrophages in\ntumors originate from two sources: peripheral blood monocytes and tissue-resident\nmacrophages (For reviewer-Figure 9). However, the relative contribution of each\npopulation remains to be quantified. We will perform more in vivo and in vitro models to\nclarify the origination and cell differentiation of CD19+ macrophages in the future.\nFor reviewer-Figure 9. (A) Representative flow cytometry gating strategy to identify\nmonocyte-derived CD19⁺ TAMs (Mo-CD19⁺ TAMs) and tissue-resident CD19⁺ TAMs (TR-\nCD19⁺ TAMs) in tumor tissues of CD45.1 and CD45.2 bone marrow chimeric mice. Cells\nwere first gated on live, single cells (excluding doublets/debris) and CD45⁺ leukocytes.\nWithin CD45⁺ cells, Mo-Macs were further defined as CD11bhighF4/80⁺, while TRMs were\ndefined asCD11blowF4/80⁺ (gating based on PMID: 26287683). CD19⁺ TAMs were\nidentified by co-expression of CD19 within the Mo-Mac and TRM gates, yielding Mo-\nCD19⁺ TAMs and TR-CD19⁺ TAMs, respectively. (Right) Quantification of CD45.1⁺ and\nCD45.2⁺ cellular origins in Mo-CD19⁺ TAMs and TR-CD19⁺ TAMs. The graph shows the\npercentage of CD45.1⁺ (red) and CD45.2⁺ (green) cells within CD19⁺ TAM subset. Data\nare derived from n = 5 chimeric mice. (B) Schematic overview of the experimental strategy:\nKitMerCreMer;LSL-YFP mice were administered tamoxifen at embryonic day 7.5 (E7.5) to\ninduce Cre-mediated recombination, permanently labeling tissue-resident macrophage\nprecursors with YFP. Upon adulthood, orthotopic liver tumors were established, and\ntumor-infiltrating CD19⁺ TAMs were analyzed by flow cytometry to determine their YFP\nexpression status. Representative flow cytometry plots showing gating for CD19⁺ TAMs\nin tumor tissues, followed by assessment of YFP expression. YFP⁺ cells represent tissue-\nresident origin. (Right) Quantification of YFP⁺ and YFP⁻ subsets within CD19⁺ TAMs. The\ngraph shows the percentage of YFP⁺ (green) and YFP⁺ (gray) cells within CD19⁺ TAM\nsubset. Data are derived from n = 5 mice.\nThank you again for this critical comparison, which enriches our understanding of\nCD19⁺ TAM biology. Please let us know if further functional assays would help strengthen\nthis contextualization.\nReviewer #4：\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review and\nto provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: We appreciate your thoughtful and constructive feedback, helping us identify\nkey areas for improvement and enhancing the quality and robustness of our manuscript.\nSuch initiatives are crucial for fostering the growth of emerging scientists in the peer review\nlandscape, and we are pleased that our work could be part of this developmental process.\nWe have strived to addressed all the points raised in the reviews, and we hope the revised\nmanuscript will meet the journal's expectations. Please do not hesitate to contact us if any\nadditional clarifications are required.\nReviewer #5：\nIn this study, the authors compellingly identified CD19⁺ TAMs as a novel,\nimmunosuppressive subpopulation enriched in HCC and linked to poor prognosis. The\nfindings are quite innovative and informative, and the paper is well written. There were\nseveral concerns that need to be solved.\nResponse: We sincerely value your positive assessment of our study. We take your\nconcerns seriously and are committed to addressing them thoroughly to strengthen the\nrigor and impact of our work. Please find below our detailed responses to each concern,\nalong with the specific revisions we have implemented (or plan to implement) in the revised\nmanuscript. We hope the revised manuscript will meet your expectations.\nMajor：\nComment 1. The authors should explicitly compare CD19⁺ TAMs with traditional\nM1/M2-like features on their metabolic, transcriptional, and functional identities. Is\nthere a M2 subpopulation in the single cell mass cytometry (CyTOF) dataset? Are\nCD19+ TAMs a subpopulation of M2 macrophages, or phenotypically altered M2\nmacrophages? Please clarify the relationship between this novel cell population\nand traditional macrophage subtypes more directly.\nResponse: We appreciate the reviewer’s interest in clarifying the relationship between\nCD19⁺/CD19⁻ TAMs and classical M1/M2 polarization states. To address this, we\nsystematically compared these subsets as detailed.\nTo compare the classification of CD19⁺/CD19⁻ macrophages with M1/M2 polarization\nstates, we re-analyzed scRNA-seq data to profile their M1/M2 subtype markers,\ninflammatory, metabolic, proliferative, and immune functional signatures. While CD19⁺\nmacrophages exhibited transcriptional proximity to M2-like macrophages, no strong\ncorrelative associations were observed (Supplementary Table 8; see also Figure 3D).\nCyTOF analysis further confirmed the presence of a classical M2-like subpopulation\n(CD163⁺) within HCC tumor-associated macrophages (TAMs), which is not entirely\nidentical to CD19⁺ TAMs (For reviewer-Figure 10).\nFor reviewer-Figure 10. Visualized tSNE representation of the immune cell subtypes in\nhuman HCC tissues, including CD19+ TAMs, CD19- TAMs, and B cells (left) and CD19\nprotein expression levels in these three clusters. Clusters are differently color-coded and\nannotated in the figure. Visualized tSNE representation M2-like subpopulation (CD163+,\nright). Data were generated from CyTOF data.\nIn addition, flow cytometry validation of canonical M1/M2 markers revealed that\nCD19⁺ TAMs expressed higher levels of M2-related markers than CD19⁻ TAMs (For\nreviewer-Figure 11). However, no definite associations were found between the CD19+/\nCD19- TAMs and M1/M2 classifications.\nFor reviewer-Figure 11. The expression of classical markers of M1 and M2-polarized\nmacrophages were analyzed for CD19+ TAMs and CD19- TAMs from human HCC\nsamples. Representative flow cytometry histograms are shown.\nThese data indicated that the CD19⁺/CD19⁻ axis represents a distinct dimension of\nmacrophage heterogeneity orthogonal to traditional M1/M2 polarization. Their unique co-\nexpression of B cell lineage markers, PAX5 dependency, and mitochondrial hyperactivity\nfurther distinguish them as a functionally discrete TAM subset. We are conducting\nadditional in vivo and in vitro investigations to systematically characterize their\ndevelopmental provenance and lineage commitment processes, and clarify the positioning\nof CD19⁺ macrophages within the established M1/M2 polarization spectrum.\nComment 2. Could CD19+ macrophages be fused cells between macrophage and B\ncells, or macrophage with B cell endocytosed?\nResponse: We fully acknowledge the critical nature of your concerns and have addressed\nthe potential fusion or endocytosis hypotheses through a combination of single-cell\nimaging, flow cytometry, and genetic modeling. These experiments now provide\nconclusive evidence that CD19+ TAMs arise from transcriptional reprogramming rather\nthan cell fusion.\n1) Fluorescence Distribution and Marker Analysis\nSingle-cell imaging flow cytometry staining and mIHC: High-resolution imaging revealed\nnon-uniform co-distribution of CD14 (macrophage marker) and CD19 within CD19⁺ TAMs.\nNotably, CD19 fluorescence was not encapsulated by CD14 structures, ruling out\nendocytosis of intact B cells, which would likely show membrane-bound CD19 signals (Fig\n1C). Furthermore, CD19⁺ macrophages were uniformly negative for CD20, another B cell-\nspecific markers (Fig 1E).\n2) Cell Size and Phenotypic Validation\nFlow Cytometry Gating: Back-gating analysis of CD19⁺ macrophages exhibit a distinct\nFSC/SSC values distribution from B cells (Fig S1C).\n3) Genetic Model Validation\nIn B cell-deficient uMT mouse models, CD19⁺ TAMs were still detected, demonstrating\ntheir development is independent of B cell presence (Fig S3H). This eliminates the\npossibility that CD19⁺ expression arises from B cell fusion or phagocytosis, as both\nprocesses would require intact B cells in the microenvironment. In WT mice, CD19⁺ TAMs\nfrequencies showed no correlation with B cell counts across tumor models (Fig. S1F),\nfurther excluding B cell dependence.\nTogether, these results indicated that CD19⁺ macrophages were not derived from\nmacrophage-B cell fusion or B cell endocytosis. Their CD19 expression likely reflects a\nunique transcriptional program, as supported by their distinct metabolic, transcriptional,\nand functional profiles. Instead, CD19 expression arises from PAX5-driven transcriptional\nreprogramming in myeloid cells.\nComment 3. Although the OXPHOS inhibitor IACS-010759 exhibited an improved\nanti-tumor effect, the authors could not attribute the function to CD19 macrophages,\nsince tumor cells or other cells can also be targeted by this inhibitor.\nResponse： We sincerely appreciate the reviewer's insightful comment concerning the\npotential outside-target effects of IACS-010759. As a pharmacological OXPHOS inhibitor,\nIACS-010759 may indeed exert effects not only on CD19+ TAMs but also on other cell\ntypes within the tumor microenvironment (TME), including tumor cells and stromal\npopulations.\nHowever, previous studies have indicated that M2-TAMs exhibit high OXPHOS activity\nand are characterized by an enhanced spare respiratory capacity, which supports their\nimmunosuppressive function (PMID: 33407885, 26649578). In contrast, tumor cells in\nhypoxic regions, particularly in hepatocellular carcinoma (HCC), primarily rely on\nglycolysis for energy metabolism (PMID: 29171040, 33398194). More importantly, our\ndata demonstrate that OXPHOS inhibition combined with anti-PD-L1 treatment\nsignificantly remodels the immune microenvironment (Supplementary figure S12),\nspecifically by reducing CD19+ TAMs (For reviewer-Figure 12) and enhancing antitumor\nimmunity. These findings suggest that targeting CD19+ TAMs is a major contributor to the\nobserved antitumor response.\nFor reviewer-Figure 12. Representative images showing CD19⁺ TAMs abundance in\nHCC tissue sections from control mice (n=5) and mice treated with OXPHOS inhibition\ncombined with anti-PD-L1 (n=5). CD19⁺ TAMs are identified as CD19⁺CD68⁺ double-\npositive cells (white arrows). Scale bar = 50 μm. (Right) Quantitative analysis of CD19⁺\nTAMs abundance in the two groups. Data are presented as mean ± SEM. Statistical\nanalysis was performed using unpaired two-tailed t-test, **P < 0.01.\nWhile we cannot definitively ascribe the entire therapeutic effect exclusively to CD19+\nTAMs, we revised the wording as “Collectively, therapeutic approaches targeting\nCD73 or OXPHOS -when optimized to leverage the heightened dependency of\nCD19+ TAMs on these pathways show great promise for improving the anti-tumor\nefficacy of immunotherapy for HCC.” in the manuscript to ensure more precise\ndescriptions of this relationship. Additionally, we plan to conduct further experiments to\ndissect the cell type-specific effects of IACS-010759 in future studies, which will help\nclarify the contributions of different cell populations to the therapeutic outcome.\nComment 4. The last sentence “Collectively, therapeutic approaches targeting\nCD73 or OXPHOS in CD19 + TAMs show great promise for improving the anti-tumor\nefficacy of immunotherapy for HCC.” is not acceptable. The authors need to discuss\nmore about how their findings could translate to therapeutical approaches.\nOXPHOS and CD73 are all non-specific to CD19 macrophages and might not be\ngood targets for developing new therapies.\nResponse: We sincerely appreciate the reviewer’s critical and insightful comments, which\nhighlight a key consideration for translating our findings into therapeutic strategies. We\nfully agree that OXPHOS and CD73 are not exclusive to CD19⁺ TAMs, and their non-\nspecificity poses challenges for the development of targeted therapies.\nWhile OXPHOS and CD73 are not unique to CD19⁺ TAMs, our data reveal that CD19⁺\nTAMs in HCC exhibit a selectively heightened dependency on these pathways to exert\ntheir immunosuppressive functions. Specifically, CD19⁺ TAMs display significantly higher\nOXPHOS activity and CD73 expression compared to CD19⁻ TAMs, whereas HCC tumor\ncells primarily rely on glycolysis—consistent with their hypoxic microenvironment. Notably,\nCD19⁺ TAMs and HCC cells show comparable levels of CD73 and PD-L1 expression (For\nreviewer-Figure 13), our functional data highlight a distinct role of CD19⁺ TAMs in\nmediating the effects of these pathways: in vivo studies using Pax5△MΦ mice (which\nspecifically deplete CD19⁺ TAMs) demonstrated that blockade of PD-L1 and CD73 no\nlonger impacted tumor progression (Figure S10F). This suggests that CD19⁺ TAMs are\ncritical mediators of the immunosuppressive effects exerted by these molecules.\nFor-reviewer-Figure 13. The expression of CD73 and PD-L1 on CD19+ TAMs and HCC\ncells.\nMoreover, reports indicate that mitochondrial drugs containing triphenylphosphine\n(TPP⁺) exert effects not only on tumor cells but also on other immunosuppressive cells\nwithin the tumor microenvironment (TME), such as myeloid-derived suppressor cells\n(MDSCs) and regulatory T cells (Tregs) (PMID: 34189437, 37172686). Therefore, we\npropose that small-molecule inhibitors typically act on multiple cell types in vivo; however,\nthe intensity of their biological effects varies significantly depending on the expression\nabundance of the target in different cells and the degree of cellular dependence on the\ntarget’s function.\nWe acknowledge that our original conclusion overstated the direct therapeutic potential\nof these targets without sufficiently addressing their broad expression profiles. We will\nrevise it to: “Collectively, therapeutic approaches targeting CD73 or OXPHOS -when\noptimized to leverage the heightened dependency of CD19⁺ TAMs on these\npathways show great promise for improving the anti-tumor efficacy of\nimmunotherapy for HCC.” In addition, our results from the preclinical HCC model\nindicate significant translational potential for CD19-targeted CAR-T therapy in treating\nsolid tumors with high levels of CD19+ TAMs independent of B cell presence. We are\ncurrently launching an investigator-initiated trial (IIT) to test the efficacy of anti-CD19 CAR-\nT cells for HCC treatment.\nThank you again for highlighting this critical point, which will significantly strengthen\nthe translational rigor of our manuscript.\nMinor:\n1. In figure 4A, p-value needs to be provided.\nResponse: We appreciate the reviewer’s attention to statistical clarity. For this figure\n(CD19⁺ Mφ/CD19⁻ Mφ mitochondrial gene expression ratios), the y - axis represents the\nfold-change in expression of CD19⁺ macrophages relative to CD19⁻ macrophages for\neach mitochondrial gene. As observed, most mitochondrial genes display a trend of\nrelatively higher expression in CD19⁺ TAMs, consistent with our initial observation.\nWe acknowledge that only a limited number of these genes reach statistical\nsignificance. However, the consistent trend of upregulated mitochondrial gene expression\nin CD19⁺ TAMs still served as a critical preliminary observation. This collective pattern\nprovided a strong rationale to pursue subsequent functional studies exploring whether\nCD19⁺ TAMs possess distinct mitochondrial properties, which we further investigated and\nvalidated in downstream experiments.\nThank you for highlighting this important consideration, which has helped us present\nthe data more transparently.\nComment 2. In figure 7A, the authors should clarify the differences between the\nupper and bottom panel.\nResponse: We appreciate the reviewer’s suggestion to clarify the figure. In Figure 7A:\nThe upper panel shows in situ images of tumors within the liver (i.e., tumors remain\nattached to the liver parenchyma without dissection). The lower panel displays images of\nthe same tumors after complete dissection and isolation from the liver tissue. This\ndistinction helps visualize both the natural tumor location within the organ and the intact\ntumor morphology post-dissection. We have revised the figure legend to explicitly state\nthis difference:\nRevised Figure 7A Legend:\nHepa1-6 cells (5× 105) were mixed with iBMDMs (with or without Pax5 overexpression) at\na ratio of 1:1, and orthotopically injected into the livers of C57BL/6 mice. Tumor images\nand sizes were showed at 15 days post-inoculation. Tumor sites were indicated by red\ndotted line in liver. Upper panel: Tumors in situ (attached to liver tissue). Lower panel: The\nsame tumors after complete dissection from the liver. n = 5 per group.\nThank you for prompting this clarification to improve figure interpretability.\n3. Gr1+ cells need to be introduced and the biological significance should be\nexplained.\nResponse： We appreciate the reviewer’s suggestion to clarify the role of Gr1⁺ cells in\nour model. Gr1⁺ cells are a heterogeneous population of immunosuppressive myeloid\ncells that typically inhibit T cell activation and promote tumor immune evasion. Clinically,\nhigh infiltration of Gr1⁺ cells correlated with poor responses to immunotherapies such as\nanti-PD-1 antibodies, making them critical regulators of the tumor microenvironment.\nIn orthotopic syngeneic mouse models, Gr-1+ cells were more abundant in tumors with the\npresence of CD19+ TAMs (Figures S3C). In our comparative analysis of Pax5flox/flox-Lyz-\ncre mice and wild-type (WT) controls mice, we also observed that a higher abundance of\nCD19⁺ TAMs was concomitant increased infiltration of Gr1⁺ cells in TME (For reviewer-\nFigure 14). These findings suggests that CD19⁺ TAMs may recruit Gr1⁺\nimmunosuppressive cells to foster a pro-tumorigenic TME.\nFor reviewer-Figure 14. Correlation analysis between CD19+ TAMs and Gr-1+ cells in\nmice HCC tissues. n = 14. Pearson correlation test was used.\nWe acknowledge that our initial focus on CD19⁺ TAMs and T cell interactions led us\nto underemphasize Gr1⁺ cells. and this will be a focus of our Discussion section (Page\n19, line 17) and follow-up studies. Please do not hesitate to contact us if you have any\nfurther questions or require additional clarification. We are happy to provide more details\nto address any concerns.\nThank you again for all reviewers’ guidance and support for our manuscript. We believe\nthese revisions have substantially improved the manuscript’s scientific validity, clarity, and\nsuitability for publication in Nature Communications. We look forward to your favorable\nevaluation of the revised manuscript and happy to provide further clarification,\nadditional\ndata, or minor adjustments promptly as needed.\nSincerely,\nQi Zhang, M.D.\nAssociated Professor of Hepatobiliary and Pancreatic Surgery,\nThe First Affiliated Hospital of Zhejiang University School of Medicine\nHangzhou, China\nEmail: qi.zhang@zju.edu.cn\nTingbo Liang, M.D., Ph.D.\nProfessor of Hepatobiliary and Pancreatic Surgery,\nThe First Affiliated Hospital of Zhejiang University School of Medicine\nHangzhou, China\nEmail: liangtingbo@zju.edu.cn\nDear Editor and Reviewers,\nWe sincerely appreciate the editorial board for providing us with the valuable\nopportunity to revise our manuscript. We also wish to express our heartfelt\ngratitude to all reviewers for their careful and rigorous evaluation of our work\namid their busy schedules, as well as for their constructive comments and\nsuggestions. We have carefully studied and fully addressed each comment.\nDetailed point-by-point responses to the reviewers’ comments are provided\nbelow.\nReviewer #1 - Remarks to Author\nThe authors, supporting the clinical applicability of the biomarker, present a\nlow-quality image that makes it difficult to determine which cells are truly\nimmunoreactive, and even more so to distinguish between macrophages and other\nstromal cells. Furthermore, despite being requested, they do not provide a cutoff point\nfor considering the marker positive in clinical practice. The suggested diagnostic\napproach seems more suitable for analysis with flow cytometry rather than on human\ntissues.\nResponse: Thank you again for your critical and constructive comments, which have\nguided us to clarify key details of our research. We have carefully addressed your\nconcerns regarding image quality and the cutoff value for CD19⁺ macrophage\nidentification, incorporating specific experimental contexts and supplementary\nmaterials as follows:\n1) We fully acknowledge your observation that the image quality in the submission\nhindered the identification of immunoreactive cells and cell type distinction. To meet\nthe file size limit for manuscript attachments, we had to compress the original images,\nwhich inadvertently compromised the clarity of cellular morphological details.We have\nsupplied original high-resolution Figures as attachments (in PDF format). These\nuncompressed images and annotated figures ensure that the morphology of CD19⁺\nmacrophages and their distinction from other cells are readily observable, especially\nin Figure 1.\n2) Regarding your request for a cutoff value for CD19 positivity in clinical practice, we\nwould like to clarify that a universal fixed cutoff is not feasible due to inherent\nvariability in experimental systems, e.g., batch differences in antibody affinity,\nvariations in tissue fixation time across clinical samples, or subtle adjustments to\nstaining protocols. We have established a contextual, clinically validated identification\nstandard based on relative expression and cell type co-localization of CD68 and\nCD19. The specific criteria are as follows: CD19⁺macrophage or CD19 expression\nin macrophages (pre-identified by CD68 positivity, large irregular cytoplasm,\nand eccentric nuclei) is defined by comparing it to the \"gold standard\" of CD19\nexpression in B cells. This standard is directly applied in our ongoing phase I/II\nCD19 CAR-T trial for advanced liver cancer (NCT03732547). Prior to CD19 CAR-T\ninfusion, we perform multi-color IHC (CD68/CD19/DAPI) on patients’ tumor tissue\nsamples to quantify CD19⁺ macrophages. Tissues are sectioned and stained,\nCD19⁺ macrophages are strictly counted as CD68⁺ cells meeting the above CD19\nexpression (relative to B cells).\nAligns with routine clinical pathology workflows: FFPE sections are standard for\ntumor diagnosis, and our multi-color IHC protocol can be implemented in most clinical\nlaboratories with basic immunostaining equipment.\nWe deeply appreciate your patience and guidance in helping us refine these\ncritical details. The supplementary original images and clarified identification criteria\nwill significantly enhance the reproducibility and clinical relevance of our work. We\nhope they fully addresses your concerns and look forward to your further feedback.\nReviewer #2 - Remarks to Author\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training in\npeer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nResponse: We appreciate the thorough and constructive feedback provided by both\nreviewers, which has helped strengthen our manuscript. We have carefully addressed\nall comments and revised the manuscript accordingly, with detailed responses\nincluded in the rebuttal letter. Please let us know if any further information or\nclarification is required.\nReviewer #3 - Remarks to the Author\nThe authors have sufficiently addressed all my comments except one minor point. In\ntheir point-by-point response pdf (p.13) addressing our comment 2, for their \"For\nreviewer Figure 4\" panel B's WB for PDL1, it looks like that upon the pulldown of\nHSC70, it failed to show altered binding for the PD-L1 mutants. I am not sure if they\nhave pasted a wrong WB by mistake, as the leftmost lane for \"IP\" also has band for\nPD-L1 but the \"IB\" one does not.\nResponse: Thank you for your careful observation. You are correct in noting the band\npattern in the WB for PD-L1 in \"For reviewer Figure 4\" panel B. During Co-IP\nexperiments, we initially attempted to pulldown HSC70 using PD-L1 as the bait but\nwere unable to obtain interpretable WB results, likely due to the presence of multiple\nmodified forms of PD-L1 leading to complex co-precipitated protein mixtures. We\ntherefore switched to pulldown of PD-L1 using HSC70 as the bait (as presented in the\nfigure). As shown, mutation of the PD-L1 CMA-motif significantly reduces its binding\nto HSC70, which aligns with our conclusion. We apologize for any confusion caused\nby the initial experimental design. Please let us know if further clarification is needed.\nReviewer #5 - Remarks to Author\n1. According to the scRNA-seq data presented in Reviewer Figure 10, CD19⁺\nmacrophages exhibited substantial overlap with traditional M2 macrophages,\nalthough they were not entirely identical. This observation may diminish the novelty of\nthe study, given that these CD19⁺ macrophages were also found to be functionally\nsimilar to M2 macrophages in mediating immunosuppression. Consequently, the key\nfindings of this study—the upregulation of Pax5 and CD19 in this macrophage\nsubpopulation—may represent merely new markers for an M2-like subtype. However,\nthese markers lack sufficient specificity, as they demonstrate poor selectivity in\ndistinguishing these cells from B cells.\nResponse: We greatly appreciate your insightful comment on the scRNA-seq data\nand the novelty of our study. Your observation regarding the overlap between CD19⁺\nmacrophages and traditional M2 macrophages, as well as the specificity of\nPax5/CD19 markers, has prompted us to conduct a more in-depth analysis and\nclarification, which we present in detail below:\n1) We fully agree with your finding that CD19⁺macrophages show substantial overlap\nwith traditional M2 macrophages. However, we emphasize that CD19⁺\nmacrophages are not merely a \"subset of M2 macrophages\" but a distinct\nsubpopulation with unique molecular and functional features that distinguish\nthem from canonical M2 macrophages, which is a key basis for the novelty of our\nstudy.\nNumerous single-cell analyses of human and mouse samples have revealed that\nthe status of macrophages is not a simple M1/M2 dichotomy as in vitro, but a\ncontinuous spectrum. In some cases, M1 and M2 phenotypes of macrophages may\nexpress “opposing” markers at the same time in vivo. CD19+ macrophages were\nobserved to co-express established M1 and M2 markers and the scRNA-seq data\nhighlighted the involvement of both CD19+ macrophage-specific and B cell-related\ntranscription factors in their differentiation. We belive that CD19⁺macrophages are\na functionally distinct subpopulation, not just a \"M2-like subtype\", their unique\ntranscriptional and functional traits provide new insights into the heterogeneity\nof immunosuppressive macrophages in the tumor microenvironment.\n2) We acknowledge your concern that Pax5 and CD19 alone lack sufficient\nspecificity to distinguish CD19⁺macrophages from B cells (given that both markers\nare canonical B cell lineage markers). However, in our study, when PAX5 was\noverexpressed in B cells, we observed that it failed to upregulate the expression\nlevels of CD73 and PD-L1. While the function of CD19 expression in\nmacrophages remains unclear to us at present, the upregulation of Pax5 and\nCD19 in macrophages is not merely a \"new marker for M2-like cells\", it\nrepresents a novel lineage reprogramming event in macrophages that has not\nbeen reported previously in HCC. We are currently engaged in active exploration of\nthe associated differentiation between PAX5/CD19 expression and macrophages.\nFor reviewer Figure #2-1. B cells were overexpressed with Pax5, the relative protein\nexpression of PAX5, CD19, CD73 and PD-L1 were showed.\nIn summary, while CD19⁺macrophages share some immunosuppressive\nfeatures with M2 macrophages, their unique transcriptional program, functional\nmechanisms, and Pax5/CD19-driven lineage reprogramming distinguish them as a\nnovel subpopulation. The multi-marker identification panel we established fully\naddresses the specificity concern for Pax5/CD19 markers. We believe these\nclarifications further highlight the novelty and clinical relevance of our study.Thank you\nagain for your valuable feedback, which has helped us strengthen the rigor and clarity\nof our work. We look forward to your further comments.\n2. Furthermore, the translational significance of this study remains unclear. The\nauthors propose targeting OXPHOS and CD73; however, these targets are not\nexclusive to CD19⁺ TAMs. Although the authors attempted to eliminate CD19⁺\nmacrophages using CD19 CAR-T cells in mature B cell-depleted mice, this approach\nitself is not ideal for translation, as CD19 CAR-T therapy carries significant risks and is\nnot targeted.\nResponse: Thank you for your valuable comments on the translational significance of\nour study. We fully understand your concerns about the specificity of OXPHOS/CD73\ntargets and the translational feasibility of CD19 CAR-T therapy, and we would like to\nclarify and supplement the relevant details based on our research data and ongoing\nclinical practice:\n1) We acknowledge that OXPHOS and CD73 are not exclusive to CD19⁺\nmacrophages, but our study confirms that macrophages (especially CD19⁺\nmacrophages) are the main contributors to OXPHOS activity and CD73 expression in\nthe tumor microenvironment (TME) of liver cancer, which provides a practical basis for\nthe translational potential of targeting these molecules.\nAs shown in For-reviewer-Figure 12 and 13, although OXPHOS and CD73 are\nnot exclusive to CD19⁺macrophages, targeting them will primarily affect CD19⁺\nmacrophages (the main functional contributors in TME) . This \"functional specificity\"\nis sufficient to support their translational value, especially in combination with\nimmunotherapy to further enhance targeting accuracy.\n2) We acknowledge that traditional CD19 CAR-T cell therapy carries risks, such as\noff-target effects on B cells. However, our research and ongoing clinical trials focus on\noptimizing the application of CD19 CAR-T cell therapy in solid tumors (particularly\nHCC) by regulating infusion doses; in the future, dual-CAR strategies may also be\nexplored. This approach aims to minimize B cell-associated toxicity while ensuring\ntherapeutic efficacy against CD19⁺macrophages, proposing a key practical\nexploration to advance its clinical translation.\nIn conclusion, based on the dominant role of CD19⁺macrophages in TME's\nOXPHOS activity and CD73 function, targeting these molecules can primarily\naffect CD19⁺macrophages, providing a feasible strategy for reversing TME\nimmunosuppression. Through preclinical dose optimization and ongoing\nclinical trials, CD19 CAR-T can safely and effectively target CD19⁺\nmacrophages in HCC, providing a new personalized treatment option for\npatients with high CD19⁺ macrophages levels.\nWe believe that these explorations address the key issues of translational\nfeasibility and provide a solid foundation for the clinical application of our findings.\nThank you again for your valuable comments, which have helped us further improve\nthe rigor and clinical relevance of our research. We look forward to your further\nfeedback."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors, supporting the clinical applicability of the biomarker, present a low-quality image that makes it difficult to determine which cells are truly immunoreactive, and even more so to distinguish between macrophages and other stromal cells. Furthermore, despite being requested, they do not provide a cutoff point for considering the marker positive in clinical practice.\nThe suggested diagnostic approach seems more suitable for analysis with flow cytometry rather than on human tissues.", "reviewer_2": "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_3": "the authors have sufficiently addressed all my comments except one minor point. In their point-by-point response pdf (p.13) addressing our comment 2, for their \"For reviewer Figure 4\" panel B's WB for PDL1, it looks like that upon the pulldown of HSC70, it failed to show altered binding for the PD-L1 mutants. I am not sure if they have pasted a wrong WB by mistake, as the leftmost lane for \"IP\" also has band for PD-L1 but the \"IB\" one does not.", "reviewer_4": "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_5": "In the revised manuscript, the authors solved most of my questions. However, some major concerns still remain.\n1.According to the scRNA-seq data presented in Reviewer Figure 10, CD19⁺ macrophages exhibited substantial overlap with traditional M2 macrophages, although they were not entirely identical. This observation may diminish the novelty of the study, given that these CD19⁺ macrophages were also found to be functionally similar to M2 macrophages in mediating immunosuppression. Consequently, the key findings of this study—the upregulation of Pax5 and CD19 in this macrophage subpopulation—may represent merely new markers for an M2-like subtype. However, these markers lack sufficient specificity, as they demonstrate poor selectivity in distinguishing these cells from B cells.\n2.Furthermore, the translational significance of this study remains unclear. The authors propose targeting OXPHOS and CD73; however, these targets are not exclusive to CD19⁺ TAMs. Although the authors attempted to eliminate CD19⁺ macrophages using CD19 CAR-T cells in mature B cell-depleted mice, this approach itself is not ideal for translation, as CD19 CAR-T therapy carries significant risks and is not targeted.\nGiven these concerns, I strongly recommend that the authors reconsider the claimed novelty of their study and more clearly highlight the translational potential of their findings."}, "author_response": ""}]

Decision letter