A CD8αβ co-receptor modified to contain an intracellular CD28 signaling tail enhances TCR-engineered T cell function independent of solid-tumor-associated co-stimulatory ligands

Domain: biology
Published: 2026-01-03
Editorial outcome: major_revision

Abstract
Adoptive transfer of T cells engineered with tumor-specific T cell receptors (TCRs) has shown limited efficacy in solid tumors, hindered by insufficient persistence, tumor trafficking, and dependence on tumor-associated co-stimulatory ligands. In a phase I trial (NCT04639245) for patients with metastatic MAGE-A1-expressing tumors and adequate organ function; one participant received treatment, which was well-tolerated. In this case and NSG murine models, infusion of CD4/CD8 T cells co-expressing a class-I MAGE-A1-specific TCR and CD8αβ, failed to control tumor progression. To enhance function downstream of TCR signaling, here we investigate the adaptability of TCR components to synthetic modification. Leveraging the obligate co-expression of CD8αβ required for class-I TCR function in CD4 T cells, we identify CD8β as a tractable site for engineering without loss of function. In vitro screening demonstrates incorporation of the CD28 intracellular tail, yielding a CD8/CD28 chimeric co-receptor, most effectively enhances cytokine production, T cell persistence, and tumor control in immunodeficient murine models while preserving stem-like transcriptional features compared to native CD8β. Further rational modification of the CD28 binding motifs improves tumor control in vivo with increased intratumoral accumulation and reduced exhaustion. This benefit also extends to PRAME and WT1-specific TCRs in vitro supporting generalizability.

Introduction
Utilizing T cell receptors (TCRs) transduced into patient T cells is an attractive therapeutic approach as it enables immunorecognition of tumors, with high sensitivity for low peptide/HLA densities of a diverse range of intracellularly expressed antigens1. However, TCR-transduced T cells have shown modest efficacy against non-hematologic malignancies with sporadic anti-tumor effects2,3. Larger clinical trials have yielded overall response rates of 44% in rare indications, such as synovial sarcoma, and as low as 9% in other solid tumors4. Multiple barriers have been shown to contribute to this lack of efficacy: in addition to tumor heterogeneity limiting antigenic encounter5 and the presence of immunosuppressive populations6 the solid tumor microenvironment (TME) is typically rich in inhibitory cytokines7, suppressive checkpoint ligands8 and the general lack of positive co-stimulatory signals required for robust T cell activation8. Together, these obstacles underscore the imminent need to improve T cell trafficking and function within the solid tumor TME.

TCR-based therapies have primarily exploited CD8 T cells with cytotoxic activity that recognize tumor antigens presented by HLA class I1. However, the enhanced antitumor activity provided by concurrent CD4 T cell responses is well documented in murine models9 and upon co-transfer of tumor-reactive CD4 T cells with CD19-targeting chimeric antigen receptor-engineered (CAR) T cells10. Although most class I-restricted TCRs require concurrent CD8 co-receptor binding for optimal target avidity and downstream signaling11, antigen-specific recognition can also be conveyed to CD4 T cells by co-expressing the class I-restricted TCRs with the CD8αβ co-receptor12. This strategy is being explored in clinical trials to promote increased cytotoxic function and ultimately anti-tumor activity13.

Beyond functionally engaging CD4 T cells, effective T cell activation and associated proliferation typically also requires positive co-stimulation8,14. As an immune evasion tactic, tumor cells commonly downregulate positive and upregulate inhibitory co-stimulatory ligands that compromise T cell activation15. As a result, antigen-specific T cells are deleted and/or become dysfunctional after encountering tumors, resulting in failure to sustain tumor control16. Unlike CAR constructs that integrate a co-stimulatory domain, TCRs require independent co-stimulatory binding for co-receptor triggering17. Strategies employing fusion proteins combining an inhibitory ectodomain with a costimulatory endodomain are being explored18,19, however, these strategies depend on sufficient ligand density within the TME20.

Here, we engineered a synthetic co-stimulatory signal directly into the class-I TCR complex by modifying the CD8β chain to include a CD28 intracellular domain, enabling ligand-independent co-stimulation with TCR engagement. This design enhances cytokine production, persistence, and intra-tumoral accumulation of both CD4 and CD8 T cells while preserving stem-like features and reducing exhaustion. Further modification of CD28 signaling motifs yields an optimized variant with increased in vivo anti-tumor activity. Together, these findings establish a generalizable framework to strengthen TCR-engineered T cell function, integrating synthetic co-stimulation into diverse TCR-based therapies.

Results
Five TCR clonotypes targeting the HLA-A2-restricted MAGE-A1 278-286 (KVLEYVIKV) epitope21, were identified (TCR MA1 1-5) following in vitro stimulation and isolation using RACE-PCR22, and synthesized as codon-optimized P2A-linked constructs in a lentiviral backbone previously used for clinical transduction23 (see Methods). CD8 T cells were transduced with each TCR and demonstrated peptide/HLA multimer (p/HLA) binding with similar mean fluorescence intensities (MFIs) (range 2672-3059) (Supplementary Fig. 1A) and produced TNFα and IFNγ when exposed to supraphysiologic levels of cognate peptide (Supplementary Fig. 1B). Concurrently transduced CD4 T cells had lower p/HLA binding (MFIs 960-2131) and limited cytokine production, suggesting reduced p/HLA binding without the CD8 co-receptor and functional TCR engagement24. TCR MA1 5 had a similar functional avidity towards the wild-type peptide compared to TCR MA1 2, 3 and 4 (range 48.1-85.8 nM) (Supplementary Fig. 1C), and also recognized the previously described K278T mutated MAGE-A1 278-286 peptide (Supplementary Fig. 1D)25. Based on these characteristics, TCR MA1 5 was selected for future studies.

To assess the impact of the CD8αβ interaction with HLA Class I in TCR-transduced T cells, transduction with TCR MA1 5 alone (T TCR-MA1) or co-transduction with CD8αβ (T TCR-MA1-CD8αβ) using a single multi-cistronic vector construct (Fig. 1A) produced CD4 T TCR-MA1, CD4 T TCR-MA1-CD8αβ and CD8 T TCR-MA1-CD8αβ (CD8 T TCR-MA1 were not generated as addition of CD8αβ to CD8 T cells does not impact function12). Compared with CD8 T cells, CD4 T TCR-MA1-CD8αβ in part rescued the ability of CD4 T to produce IFNγ, TNFα, and IL-2 upon cognate peptide exposure (mean EC50 of 1119.5, 807.25, and 1464.5, respectively, for CD4 T compared with 20.65, 9.91, and 43.52 for CD4 T TCR-MA1-CD8αβ) (Fig. 1B).

To determine the impact of CD4 and CD8 T TCR-MA1-CD8αβ both alone and combined, in vitro survival of the MAGE-A1 and HLA-A*0201-expressing ME275 melanoma cell line was assessed in a real-time killing assay where T cells were repeatedly challenged with tumor (Fig. 1C left panel, Supplementary Table 1 and Supplementary Fig. 2). Although CD8 T TCR-MA1-CD8αβ remained superior at repeated tumor killing compared to CD4+ T TCR-MA1-CD8αβ, the 1:1 combination of CD4 and CD8 T TCR-MA1-CD8αβ (with each subset contributing half of the total cell number) significantly outperformed an equal total number of CD8-only or CD4-only T TCR-MA1-CD8αβ at 144h and 192h (Fig. 1C, right panel). At 144h, CD4 and CD8 T TCR-MA1-CD8αβ remaining in wells within the combination group were ~30x more abundant whereas CD4 and CD8 T TCR-MA1-CD8αβ alone were ~3x more abundant (Fig. 1D). CD4 and CD8 T TCR-MA1-CD8αβ combination group continued to control tumor for 13 days (Supplementary Fig. 3A).

To assess the proliferative capacity, CD4 and CD8 T TCR-MA1-CD8αβ were exposed once to ME275 and their divisions assessed 7 days later (Supplementary Fig. 3B–G). CD8 T TCR-MA1-CD8αβ divided more in the presence of CD4 T TCR-MA1-CD8αβ (Supplementary Fig. 3B–D), while CD4 T TCR-MA1-CD8αβ divided less when combined with CD8 T TCR-MA1-CD8αβ (Supplementary Fig. 3E–G). Despite the increased ability of TCR-transgenic CD4 T cells co-transduced with CD8αβ to secrete helper cytokines when exposed to supraphysiological levels of peptide12 and proliferate after tumor exposure (Supplementary Fig. 3E), they showed reduced tumor killing upon repeated challenge (Fig. 1C). In contrast, CD8 T cells divided less after single tumor exposure but were more effective at repeated killing (Supplementary Fig. 3B, 3E, Fig. 1C). Together, CD4 and CD8 T cells sustained tumor control (Supplementary Fig. 3A), likely through CD4-derived cytokine support that enhances CD8 T cells proliferation and cytotoxicity. The reduced proliferation of CD4 T cells in co-culture (Supplementary Fig. 3F), may reflect rapid tumor clearance by CD8 T cells and/or sequestration of CD4 T cell-derived cytokines.

To examine this in vivo, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were engrafted with 1×106 ME275 cells (Fig. 1E). CD4, CD8, or combined CD4/CD8 T TCR-MA1-CD8αβ (1:1 ratio, 1×107 total) mediated tumor control, whereas an irrelevant TCR (T TCR-Irr-CD8αβ) did not, confirming specificity. We next engineered A375 melanoma cell lines to express both MAGE-A1 and HLA-A*0201 (A375F) (Supplementary Table 1 and Supplementary Fig. 2), which reached ~60 mm3 by 13 days post engraftment (Fig. 1F). In this model, CD4+ or CD8+ T TCR-MA1-CD8αβ alone slowed but did not prevent tumor growth while the CD4/CD8 combination (1:1 ratio, 1×107 total) had significantly reduced tumor volumes compared to controls (Fig. 1F and G), supporting advancement of the combined approach to clinical translation.

Thirty-two patients were screened (Supplementary Fig. 4 – consort diagram) and one adult with MAGEA1-expressing non-small cell lung cancer (NSCLC) (Supplementary Fig. 5A, Supplementary Note. 1) received two T TCR-MA1-CD8αβ infusions (109 cells, ratio CD4:CD8 1:1) 7 months apart. Infused T TCR-MA1-CD8αβ expressed the CD28, CD62L and CD127 memory-associated markers (Fig. 2A) and produced IFNγ and TNFα upon cognate peptide exposure (Fig. 2B). Only low-grade adverse events (i.e. CTCAE grade ≤2) were documented with no post-infusion evidence of cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity (ICANS) (Supplementary Table 2). The patient progressed 60 days after infusions #1 and #2 (RECIST 1.1 criteria26) and succumbed to his disease 4 months later. T TCR-MA1-CD8αβ reached 1.2 and 2% of total CD3+ cells 1 day after each infusion but were detected at minimal levels 28 days later (Fig. 2C). Given the limitation of a single patient, this result cannot be conclusive but suggests additional measures may be needed to improve efficacy. Indeed, mice infused with half doses of CD4 plus CD8 T TCR-MA1-CD8αβ (5×106 vs. 1×107 total cells) (Fig. 2D) failed to control tumor growth (Fig. 2E). In the combined infusion group, only intratumoral CD4+ exhibited reduced expression of markers exhaustion-associated markers (Tim3, PD-1, CD39, and 2B4)27–29, compared to either subset infused alone (Supplementary Fig. 5B, Fig. 2F), yet all T TCR-MA1-CD8αβ subsets-either alone or combined-produced <10% median IFNγ and TNFα upon cognate peptide exposure (Fig. 2G). There were no Treg populations (CD4+ FOXP3+) identified in either CD4-containing group (Supplementary Fig. 5C). These results show that in a more stringent murine model, the observed reduced expression of exhaustion-associated markers in CD4 occurred without gains in function or tumor control, highlighting the need for additional T cell optimization.

T cells can receive co-stimulation either naturally through ligands in the TME or synthetically via immunomodulatory fusion proteins (IFPs) that couple inhibitory receptor ectodomains to costimulatory endodomains20. The CD28 co-stimulatory molecule was naturally expressed on T TCR-MA1-CD8αβ infused in a patient (Fig. 2A) and in our in vivo murine models30 (Supplementary Fig. 6A), indicating their capacity to respond to positive co-stimulation. Unlike in hematologic malignancies, costimulatory ligands are less consistently present in solid tumors31. To examine the expression of both positive and negative co-stimulatory ligands across human TMEs, in silico analysis of previously published single-cell RNA sequencing (scRNA-seq) datasets from NSCLC (n=42 patients) and melanoma patients (n=8 patients)32,33 was performed. Cell subsets were grouped and annotated according to prior reports32,33 (Supplementary Fig. 6B and C). Differential gene expression of curated positive and inhibitory ligands (Supplementary Table 3) in the NSCLC and melanoma datasets (Fig. 2H and I, green and red boxes) revealed low/absent tumor-specific expression relative to myeloid and endothelial cells, with a positive co-stimulatory ligand gene score predominantly enriched in non-tumor cells (Fig. 2J and K). Surface expression of CD86, CD80 and 4-1BB-L was also low or absent in A375F and ME275 tumor lines (Supplementary Fig. 6D), confirming positive co-stimulatory ligands are rare in the TME and that a single IFP strategy is unlikely to be broadly applicable.

To achieve ligand-independent signaling, we tethered positive intracellular costimulatory domains to the requisite CD8αβ co-receptor chains, based on findings that TCR chain modifications impaired function (Supplementary Fig. 7A–C) and prior evidence that CD8α chain modifications can transmit activation signals34–36. CD8α provides an LCK-binding domain, whereas CD8β provides a proximal palmitoylation site promoting membrane-lipid rafts association and downstream signaling30,37. Although CD8α expression alone can drive signaling via dimerization38, the CD8αβ heterodimer has ≥100 fold greater LCK affinity, favoring effective TCR activation and downstream signaling39. First, we assessed CD8α alone and a chimeric CD8α-CD4 that retained the CD8α extracellular and transmembrane domains tethered to the intracellular CD4 tail, containing both LCK-binding and palmitoylation sites37 (Supplementary Fig. 8A). TCR CD4 T cells transduced with CD8α or CD8α-CD4 had similar surface expression as cells transduced with both CD8α and CD8β (Supplementary Fig. 8B). However, the functional avidity was lower for CD8α and CD8α-CD4 (EC50 169.9, 103.2, respectively, vs 7.58 for CD8αβ) (Supplementary Fig. 8C) and lytic capacity was reduced (Supplementary Fig. 8D), confirming that retaining the CD8β chain is necessary for T TCR-MA1 function.

Next, to assess the impact of intracellular domain (ICD) modifications, we designed chimeric CD8αβ co-receptors (CCR) retaining the CD8α/β extracellular and transmembrane domains but replacing the entire CD8α ICD or CD8β ICD downstream of the palmitoylation site (Supplementary Table 4)40. Substitution of the CD8α ICD reduced CD8 surface expression, whereas CD8β ICD modifications preserved it (Supplementary Fig. 8E). CCRs incorporating the ICDs of CD28, 4-1BB, ICOS, OX40 or GITR tethered to the CD8β chain downstream of the palmitoylation site were generated (Fig. 3A; Supplementary Tables 5 & 6). To standardize TCR expression, constructs were first titrated in CD4+ T cells, selected for their lack of endogenous CD8αβ expression, thus forcing the use of CCRs for class I-restricted responses, ensuring comparable transduction efficiency and transgene expression (Fig. 3B, first 3 panels to the left). Transduced T cells were then sorted on similar p/HLA+ binding and expanded (Fig. 3B, right panel).

CCRs incorporating CD28, 4-1BB, ICOS or OX40 showed functional avidities (EC50 = 93.2, 128.6, 145.1 and 181.2, respectively) comparable to CD8αβ (125.2), whereas GITR and CD8α exhibited lower avidities (488.95 and 724.45) (Fig. 3C). All constructs produced similar levels of IFNγ after peptide exposure (1.02-1.19 fold vs T TCR-MA1-CD8αβ) (Fig. 3D, left panel), but only CD28, 4-1BB, ICOS, and OX40 suggested sustained IFNγ secretion after sequential exposure to irradiated ME275 and peptide (1.53- 2.16 fold vs T TCR-MA1-CD8αβ) (Fig. 3D, right panel). Among these, the CD8/28 co-receptor (CD8/28) most effectively restrained ME275 tumor growth in repeated killing assays (Fig. 3E, left and middle) and yielded greater live total remaining T cells in wells at 384h (16 days) (Fig. 3E, right). No differences in tumor-killing were observed between T TCR-MA1-CD8/28 with or without CRISPR knockout of endogenous CD8β (Supplementary Fig. 8F) and the construct did not exhibit tonic signaling (Supplementary Fig. 9A). As such, subsequent adoptive transfer studies used CD4 and CD8 T cells transduced with TCR 5 and CD8/28 (T TCR-MA1-CD8/28).

To assess T cell-tumor interactions, we repeated the stringent xenograft model where 5×106 T TCR-MA1-CD8αβ (ratio CD4:CD8 1:1) failed to control tumor growth (Fig. 2D). Mice in the T TCR-MA1-CD8/28 group markedly reduced tumor size 9 days post-transfer (average tumor volume at euthanasia 37 mm3) (Fig. 4A). Tumors from T TCR-MA1-CD8/28 treated mice contained significantly more CD4 and CD8 T cells per mm2 (Fig. 4B, quantified in Supplementary Fig. 9B), with higher T cell-to-tumor ratios overall (Fig. 4C), and within CD4 (Fig. 4D) and CD8 (Fig. 4E) T cell subsets. The CD4:CD8 ratio in this group approached 1 (mean 0.872) versus 0.381 in the T TCR-MA1-CD8αβ group where CD8 T cells predominated (Fig. 4F). T TCR-MA1-CD8/28 cells showed reduced expression of surface markers associated with exhaustion (PD1+/Tim3+, CD39+/Tim3+, and PD1+/TIGIT+) (Fig. 4G, Supplementary Fig. 9C) and produced significantly more cytokines upon cognate peptide exposure ex vivo (Fig. 4H). Collectively, this data shows that T TCR-MA1-CD8/28 were detected at a higher frequency in tumors, potentially due to either preferential tumor accumulation or an earlier burst of intratumoral expansion. Notably, the remaining cells displayed fewer exhaustion-associated features, and exhibited enhanced function compared to T TCR-MA1-CD8αβ.

While the survival of all mice infused with T TCR-MA1-CD8/28 on day 13 after A375F engraftment was only modestly prolonged compared to controls (T TCR-Irr-CD8/28) (Supplementary Fig. 9D), when administered on day 6 into mice with measurable tumors (Supplementary Fig. 9E), T TCR-MA1-CD8/28 significantly extended survival compared with T TCR-MA1-CD8αβ (Fig. 4I).

To profile transcriptional programs, we performed scRNAseq on tumor-infiltrating T cells 9 days after infusion of CD4 or CD8 T TCR-MA1-CD8αβ alone or combined (combo) CD4 and CD8 T TCR-MA1-CD8αβ or T TCR-MA1-CD8/28 (Figs. 2D, 4A). A total of 3,266 T cells were identified (2,445 CD8 and 821 CD4). Because CD4 T cells were relatively scarce – particularly in the T TCR-MA1-CD8αβ group (Supplementary Fig. 9B and Fig. 4F), we aggregated them across groups for pseudo-bulk analysis. CD4 T cells from the CD8αβ-only and combo CD8αβ groups clustered together, marked by expression of activation (IRF7, ISG20, IFITM1, ISG15, CD38, IFI6, CCL3) and exhaustion (LAG3, TIGIT) gene signatures. By contrast, CD4 cells from the combo CD8/28 group formed a distinct cluster, enriched for replication-associated genes (VIM, TUBB, TUBB4B) and IL7R (self-renewal) without expression of activation- and exhaustion-associated signatures (Supplementary Fig. 10A).

Unsupervised clustering of CD8 T cells across groups revealed six main clusters (Fig. 5A): proliferative clusters (2, 5) defined by proliferation-related (MKI67, TOP2A), tubulin- and histone-related (TUBA1B, HIST1H2AC, HIST1H1B) genes; cytotoxic clusters (0, 1) defined by cytotoxicity-related (CTSA, CTSW, FCER1G, GNLY), interferon-associated (IFNG, ISG15) as well as exhaustion-associated (TIGIT, DUSP2)41,42 genes; early Effector-Memory/Early Tem clusters (3, 4) defined by central-memory/stem-like (LEF1, IL7R, SELL, NELL2) and effector (GZMK, CCL5) genes, indicating cells with self-renewing capacity transitioning to a more effector-like state (Fig. 5A–C, Supplementary Fig. 10B). A distinct “exhausted” cluster was not identified, consistent with prior studies showing that exhaustion markers can also be expressed in activated T cells, complicating their distinction in scRNA-seq data-sets43–45. Relativeto CD8-only cells, CD8 T cells in the combo CD8αβ group showed enriched cytotoxic and proliferative but reduced stem-like signatures (Fig. 5D), aligning with their failure to achieve tumor volume reduction (Fig. 5E). In contrast, CD8 T cells in the combo CD8/28 group showed enriched cytotoxic and stem-like but reduced proliferative signatures (Fig. 5F), correlating with tumor volume reduction (Fig. 5G). Although speculative, the lower proliferative signature may reflect diminished antigen availability after tumor shrinkage, favoring the acquisition of self-renewing programs. Comparisons across groups suggest that CD8-only and CD8 combo CD8/28 proliferate and differentiate less than CD8 combo CD8αβ cells, thereby maintaining stem-like features. For CD8-only, this may reflect absence of CD4+ help (less proliferative stimulation), whereas for CD8 combo CD8/28 this may be linked to tumor burden reduction (Supplementary Fig. 10C). Cell proportions (Supplementary Fig. 10D) and absolute numbers (Supplementary Fig. 10E) further support this model: CD8 in the combo CD8/28 group showed a predominance of cytotoxic and stem-like rather than proliferative signatures (tumor burden reduction), whereas the CD8 in the combo CD8αβ group displayed cytotoxicity and proliferative rather than stem-like signatures (no tumor burden reduction compared to control).

Takentogether, these observations suggest that combining CD8 and CD4 T cells enhances proliferation of both subsets, while CD28 co-stimulation promotes cytotoxic signatures, consistent with their increased ability to produce cytokines (Fig. 4H) and preserves early memory programs, consistent with their reduced expression of exhaustion-associated surface markers (Fig. 4G).

Based on studies demonstrating that modifications CARs co-stimulatory motif scan enhance function46,47, we generated T TCR-MA1-CD8/28 constructs with distinct CD28 intracellular domain (ICD) motif mutation combinations. These included LL>GG shown to increase CD28 CAR surface expression48, and YMNM>AMNM, PYAP>AYAA and PRRP>ARRA shown to abrogate PI3K30, LCK49 and ITK49 binding sites, respectively (Supplementary Fig. 11A). CD4 T cells were co-transduced with one of 16 constructs (Supplementary Table 7). TNFα production after cognate peptide exposure was similar across constructs (EC50 12.93–56.98 nM) (Supplementary Fig. 11B), suggesting preserved functional avidity. However, differences emerged after repeated A375F tumor challenge (Supplementary Fig. 11C). CD4 T with all four domains mutated (E7, hereafter T TCR-MA1-CD8/28-mut), demonstrated superior tumor control relative to other variants (Supplementary Fig. 11C). Grouped analysis showed LL>GG and PYAP>AYAA enhanced tumor control and T cell proliferation, YMNM>AMNM and PRRP>ARRA showed mixed effects (Supplementary Fig. 11D, E), and concurrent YMNM+PRRP mutations significantly improved tumor control compared to other combinations (Supplementary Fig. 11E). Overall, T TCR-MA1-CD8/28-mut showed the strongest activity and was selected for further study (Fig. 6A).

T TCR-MA1-CD8/28mut and T TCR-MA1-CD8/28 had similar functional avidities measured by production of TNFα and IFNγ after exposure to decreasing concentrations of cognate peptide (Fig. 6B, Supplementary Fig. 12A) and mediated similar A375F tumor control in the stringent in vivo model (Fig. 6C). However, when the total infused dose was halved (2.5 × 106 T cells), T TCR-MA1-CD8/28mut achieved more robust tumor burden reduction relative to controls (Fig. 6D, E). Cells preferentially localized to tumors while remaining detectable in concurrent spleens (Supplementary Fig. 12B). CD4 and CD8 T TCR-MA1-CD8/28mut had higher expression of the Ki-67 proliferation marker than T TCR-MA1-CD8/28 by IHC (Supplementary Fig. 12C). T TCR-MA1-CD8/28mut also suppressed growth of the cell death-resistant HLA-A2+ MAGE-A1+ SK-MEL-37 melanoma cell line in vitro (Supplementary Fig. 2, Fig. 6F)50, and mediated superior tumor reduction in vivo in a MISTRG mouse model (see Methods) at 83 days compared to T TCR-MA1-CD8/28 (Fig. 6G). Thus, T TCR-MA1-CD8/28mut conferred modest but consistent improvements in both short- (10 days) and long-term (83 days) tumor control, potentially due to an increased proliferative capacity.

To test generalizability, we applied the CD28-mut strategy to PRAME- and WT1-specific TCRs (Supplementary Fig. 13). T TCR-PRAME-CD8/28mut displayed slightly improved functional avidity (EC50 IFNγ 86.78 vs 91.79; TNFα 24.2 vs 38.37 nM) compared with T TCR-PRAME-CD8/28 (Supplementary Fig. 13A, B), while T TCR-WT1-CD8/28mut showed similar functional avidity to T TCR-WT1-CD8/28 (EC50 IFNγ 2.25 vs 0.57; TNFα 1.92 vs 0.26 nM) (Supplementary Fig. 13C, D). However, both T TCR-PRAME-CD8/28mut and T TCR-WT1-CD8/28mut achieved superior tumor control after repeated tumor challenge in vitro against HLA-A2+ PRAME+ PANC1 (pancreatic ductal adenocarcinoma), HLA-A2+ PRAME+ A375F, and HLA-A2+ WT1+ SW480 (colon adenocarcinoma) lines compared with their non-mutated counterparts (Supplementary Fig. 13E–G). These findings indicate that CD28 tail mutations may enhance efficacy beyond TCR MA1, across to other TCR therapies.

To assess short-term effects of the mutant CD28 tail, we exposed CD4+ T cells to the cognate peptide and performed bulk RNA sequencing (RNAseq) with Gene Set Enrichment Analysis (GSEA). Compared to CD8αβ, both CD28 and CD28mut conditions showed similar enrichment in cytokine-related pathways including Th1/Th2 signaling (Fig. 7A), indicating comparable rapid activation of cytokine-driven programs. Exposure to cognate peptide-loaded TAP-deficient T2 B lymphoblastoid cell lines (T2-BLCL) revealed higher phosphorylated downstream TCR and CD28 signaling molecules (p)-ERK and (p)-AKT in T TCR-MA1-CD8/28mut than T TCR-MA1-CD8/28 at 5 minutes, with both exceeding T TCR-MA1-CD8αβ (Supplementary Fig. 14A), consistent with more pronounced early CD28-mediated costimulatory signaling and TCR-driven signaling51.

To evaluate the long-term effect(s), CCR-expressing cells were challenged with irradiated tumor cells every 7 days for 21 days and analyzed by bulk RNA seq with or without peptide stimulation (Fig. 7B). Principal component analysis (PCA) showed transcriptional separation along PC1, distinguishing T TCR-MA1-CD8αβ (Fig. 7C, brown ellipse) from the CD28 and CD28mut groups (Fig. 7C, green and purple ellipses) irrespective of peptide stimulation (Fig. 7C, Supplementary Fig. 14B). Differential gene expression and unsupervised clustering separated CD28mut (Fig. 7D, left side of the heatmap) from CD28 (right side of the heatmap), with each grouping together independent of stimulation, suggesting greater similarities within each construct, regardless of stimulation. Unstimulated T TCR-MA1-CD8/28mut showed no/low activation/proliferation gene expression, which increased after peptide stimulation, while T TCR-MA1-CD8/28 displayed higher basal activation further enhanced by peptide stimulation (Fig. 7D, insert). Z-score analysis of selected genes (Supplementary Table 8) revealed upregulation of proliferation and exhaustion programs in both CD28 and CD28mut, but at comparatively lower levels in CD28mut pre- and post-stimulation (Fig. 7E, F), along with reduced apoptosis gene expression (Fig. 7G). T TCR-MA1-CD8/CD28mut also showed lower PD1/Tim3 co-expression (Fig. 7H), indicating reduced cell exhaustion, while IFNγ and TNFα production remained comparable to CD28 (Fig. 7I). Together these results suggest that T TCR-MA1-CD8/CD28mut retains responsiveness to stimulation while limiting basal activation, reduces exhaustion under repeated stimulation, and maintains self-renewal features, potentially supporting more durable anti-tumor activity than T TCR-MA1-CD8/CD28wt.

Methods
Isolation of TCR MA1 and lentiviral vector construction
T cell lines recognizing MAGE-A1 peptide (KVLEYVIKV) 278-86 were generated from 12 donors, cloned by limiting dilution, and screened for high functional avidity for an HLA-A*0201+ TAP-deficient B-lymphoblastoid cell line [T2 B-LCL] pulsed with the MAGE-A1 278-86 class-I-HLA peptide65,66. The 5 clones selected as TCR donors, TCR 1-TCR 5, demonstrated the lower K values for tetramer binding. TCR 1-TCR 5 Vα1.3 and Vβ17 TCR chains were isolated using Rapid Amplification of cDNA Ends-Polymerase Chain Reaction (RACE-PCR)22 and expanded and cloned by limiting dilution. Based on the wild-type sequences, the final construct was synthesized by GeneArt (Regensburg, Germany) in a codon-optimized format to promote high-level protein expression in human cells as previously described23.

Functional avidity screening of 5 different clones of TCR MA1
Donor PBMC were thawed and rested in RPMI-1640 supplemented with 10% immune cell serum (A2596101, Gibco) overnight before transducing with the five clones. T cells were identified by flow cytometry using HLA-A2:MAGEA1 peptide/HLA-multimer-APC (p/HLA-multimer)(1:200, KVLEYVIKV, FHCRC in-house production), anti-CD8-FITC (1:200, RPA-T8, BioLegend, 301050), and anti-CD4-BV421 (1:200, RPA-T4, BioLegend, 300532).

Intracellular cytokine staining (ICS) was performed by surface staining with anti-CD4-APC-Cy7 (1:200, OKT4, BioLegend, 300518), anti-CD8-FITC (1:200 RPA-T8, BioLegend, 301050), and Live/Dead-BV510 (1:1000, Invitrogen, L34957). Cells were fixed and permeabilized with Cytofix buffer (BD Biosciences, 554722) and Perm/Wash (BD Biosciences, 554723) per manufacturer's protocol and stained with anti-TNFα-PE-Cy7 (1:200, MAb11, BioLegend, 502930) and anti-IFNγ-APC (1:200, B27, BioLegend, 506510), then analyzed by flow cytometry.

Production of TCR-deficient Jurkat cells and Nur77-mNeonGreen reporter cells
0.625 μL of 160 μM CRISPR RNAs targeting TRAC (sequence: AGAGTCTCTCAGCTGGTACA, IDT) and TRBC1/2 (sequence: GGA GAATGACGAGTGGACCC, IDT) were each duplexed with 1.25 µL of tracrRNAs (IDT, 1072533) in duplex buffer (IDT, 11-01-03-01) for 30 minutes at 37°C. Resulting duplexes were then incubated with Cas9 protein (61 μM, 1 μL, IDT) and polyglutamic acid (100 mg/mL, 1 μL) for 15 minutes at 37°C. Following this, 4 μL of the mixture was added to 17 μL of P3 buffer (Lonza) and used to resuspend 1×106 Jurkat cells (ATCC, TIB-152). The 20 μL cell suspension was then electroporated using the 4D-Nucleofector X Unit (Lonza, V4SP-3096) with the EH-115 program. Subsequent CD3-negative cells were sorted by FACS. To generate Nur77 reporter cells, the CD3-KO Jurkat cells were further modified by knocking out class-I HLA genes using CRISPR with a pan-gRNA (sequence: AAAAGGAGGGAGCTACTCTC), overexpressing codon-optimized CD8β-P2A-CD8α (pRRLSIN backbone) via lentiviral transduction, and knocking in mNeonGreen into the NR4A1 (Nur77) locus using CRISPR/homology-directed repair (guide: ATGAAGATCTTGTCAATGAT).

Recognition of MAGE-A1-K278T variant
Nur77 reporter Jurkat cells were transduced with the 5 TCR clones (TCR 1-5) using protamine sulfate (1:1000, 10 μg/mL stock, MP Biomedicals, S4480). TCR transduced Jurkats were cocultured with peptide-loaded T2 B-LCLs (1 µM MAGE-A1 or MAGE-A1 K278T) overnight and analyzed by flow cytometry for Nur77-mNeonGreen reporter activation.

Selection of the MA1-specific TCR for clinical translation
While other somatic mutations exist within the MAGE-A1 gene, only the K278T missense mutation falls within the targeted epitope (MAGE-A1₍₂₇₈₋₂₈₆₎). The K278T mutation has been in publicly available datasets and is included in the breast cancer cell line HCC100825. Of the 5 isolated TCRs and to choose one for clinical translation, we proceeded by first excluding the least avid TCR 1 (mean EC50 of 134 nM). The remaining 4 TCRs had very similar functional avidity (ranging from 48.1-85.8 nM). TCR 5 was chosen based on its ability to recognize the wild-type and mutated peptide, thus broadening the targeted epitope to include the K278T mutation. All 5 TCRs (TCR 1-5) were isolated from healthy HLA-A2+ donors. To avoid safety concerns, none were modified or affinity enhanced.

Generation of TCR MA1 lentivirus used in pre-clinical studies
Sequenced TCR 5 was synthesized (Genscript) and cloned into 3rd generation lentivirus vector pRRLSIN.cPPT.MSCV/TCRMA1β-P2A-CD8α-P2A-CD8β-P2A-TCRMA1α/wPRRE (in-house generation, FHCRC). Lentivirus was generated by transfecting HEK293T cells (ATCC, CRL-3216) with the TCR 5 lentiviral construct using Effectene Transfection Reagent (Qiagen, 301425). The virus was concentrated using the Lenti-X Concentrator (Takara Bio, 631231).

Generation of T TCR-MA1: T cell isolation, activation and lentiviral transduction
PBMCs were thawed and rested for 1 h in X-VIVO 15 media (Lonza, 04-418Q) supplemented with 5% fetal bovine serum (FBS)(Cytiva, SH30071.02), 5% CTS (Gibco, A2596101), and 1% antibiotic-antimycotic (Gibco, 15240062). CD4 or CD8 T cells were isolated using the CD4+ (Miltenyi, 130045101) or CD8+ T cell (Miltenyi, 130045201) positive selection kit per the manufacturer's protocol. The cells were activated for 48 h using TransAct (1:50, Miltenyi, 130111160) at 2×106 cells/mL with 50 IU/mL recombinant interleukin-2 (IL-2) (Peprotech, 200-02) in X-VIVO 15 media supplemented with 10% FBS and 1% antibiotic-antimycotic solution. Activated CD4 and CD8 T cells were lentivirally transduced using protamine sulfate (1:1000, 10 μg/mL stock) and supplemented with IL-2 (50 IU/mL). CD4 and CD8 T cells were expanded for one week, then stained with p/HLA-multimer-APC, anti-CD8-FITC and anti-CD4-PE (1:200, SK3, BioLegend, 980804), then FACS sorted by BD Symphony S6 sorter and cryopreserved with Cryostor CS10 (Stemcell, 07930). Chimeric Co-receptor (CCR) construct generation: chimeric co-receptor sequences replaced the CD8β chain but retained the first 6 amino acids (Supplementary Table 6) and were transduced into CD4 or CD8 T cells. CCR T cells were validated with anti-CD8α (1:200, BV421, RPA-T8, BioLegend, 301035), CD8β (1:200, PE, H35-17.2, Invitrogen, 12-0083-82) and p/HLA-multimer-APC. T cell identification with surface antibodies follows the surface gating strategy in the supplementary information (Supplementary Fig. 15A).

T cell rapid expansion protocol (REP)
CD4 and CD8 T cells were FACS sorted using p/HLA-multimer, anti-CD8-FITC, and anti-CD4-PE65 (Supplementary Fig. 15C). For each condition to expand, 5×104 cells were combined with 10×106 mixed PBMCs (3 healthy donors, irradiated with 40 Gy) and 2×106 TM-LCL cells65 (Irradiated with 80 Gy) into a 6-well GREX plate (Wilson Wolf). Each well was supplemented with anti-OKT3 (30 ng/ml, Miltenyi, 130-093-387), IL-2 (50 IU/ml, PeproTech, 200-02), IL-7 (10 ng/ml, PeproTech, 200-07), and IL-15 (10 ng/ml, PeproTech, 200-15). Cells were cultured for 10–12 days and then checked for purity with the p/HLA-multimer-APC, anti-CD4-PE, and anti-CD8-FITC as described above. T CD28 expression assessment: REP T cells were stained with anti-CD28 (1:200, PE-Cy7, 28.2, Invitrogen, 25-0289-42), anti-CD4-APC-Cy7, and anti-CD8α-BV421.

Generation of research tumor cell lines
A375 (ATCC, CRL-1619), ME275 (Research Resource Identifier [RRID]: CVCL_S597), and SK-MEL-37 (RRID: CVCL_3878) melanoma cell lines were obtained from ATCC and Ludwig Institute for Cancer Research and used as target cell lines for T cells. The A375 cell line was modified to increase expression of HLA-A2 and MAGE-A1 (Supplementary Fig. 2A). Briefly, A375 cells were transduced with the pRRLSIN.cPPT.MSCV/HLAA2-MAGEA1/wPRE (in-house generation, FHCRC) lentiviral vector and later stained with anti-HLA-A2-FITC (1:200, BB7.2, BioLegend, 343303) and FACS sorted on HLA-A2hi and subsequently referred to as A375F. A375F and ME275 were stained with anti-41BBL-PE (1:200, 5F4, BioLegend, 311503), anti-CD80-BV650 (1:200, 2D10, BioLegend, 305227), and anti-CD86-BV421 (1:200, GL-1, BioLegend, 105031) to confirm costimulatory ligand expression. PANC-1 and SW480 were obtained from ATCC (CRL-1469 and CCL-228) and used as a target of T TCR-PRAME or T TCR-WT1, respectively, in Incucyte killing assays. Tumor cell lines were transduced with luciferase-GFP and mCherry in the 3rd generation lentivirus vector (see above) for Incucyte killing assays and in vivo experiments.

T cell cytotoxicity assays: Intracellular Cytokine Staining
T cells were seeded at 1×105 cells/well in 100 µl X-VIVO-15 medium supplemented with 5% CTS and 5% FBS with 6 different peptide concentrations for 18 h (10 µM, 1 µM, 100 µM, 10 µM, 1 µM, 0 µM). Golgistop (BD Biosciences, 554724) and Golgi plug (BD Biosciences, 555092) were added 18 h before surface staining with anti-CD4 (1:200, PE, SK3, BioLegend, 980804), anti-CD8 (1:200, FITC, RPA-T8, BioLegend, 301050), and Live/Dead-BV510 (1:1000, Invitrogen, L34957) (Supplementary Fig. 15B). Cells were analyzed with BD Fortessa-X50.

Real-Time Killing Assay
CD4 and/or CD8 T cells purity was confirmed following the REP protocol using anti-CD4-PE, anti-CD8-FITC, and p/HLA-multimer-APC, and were seeded at 1×104 T cells/well in a clear, 96-well tissue-treated flat-bottom plate with 5 × 103 ME275 cells expressing mCherry. Kinetic image analysis was performed using the Incucyte S3 (Sartorious, 51540). Images were recorded every 2 h, and 5 × 103 ME275 cells were added every 48 h, depending on tumor killing efficiency. Serial rounds of tumor challenges were repeated until loss of tumor control was identified. Other tumor cell lines used in this study, A375F, SK-MEL-37, PANC-1, and SW480, were seeded as before with respective CCR TCR-T cells (TCR MA1, TCR PRAME, TCR WT1). E:T ratio varied from 2:1 to 10:1 depending on the effector and target cell types. 5 × 103 tumor cells were added between 48 and 72 h, depending on the lytic efficiency of the T cells. Analysis of T cell counts in Real-time killing assays: At indicated time points remaining T cells in the wells were counted via flow cytometry with count bright counting beads (Invitrogen, C36950).

CFSE proliferation assay
CD4⁺ or CD8⁺ T cells were labeled with 5 µM CFSE (carboxyfluorescein diacetate succinimidyl ester; Invitrogen, C34554) in PBS for 20 min at 37°C, followed by quenching with complete culture medium and washing twice. Labeled cells were then stimulated with ME275 cells at an effector-to-target ratio of 5:1 in complete X-VIVO 15 media. Half of the media was changed 3 days after, and cells were analyzed by flow cytometry 8 days after. Proliferation was assessed by sequential dilution of CFSE intensity in viable CD4⁺ or CD8⁺ T cells.

In vivo murine models
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice (NSG) were bred in-house at the Fred Hutchinson Cancer Center from mice obtained from The Jackson Laboratory (Strain#: 00555, RRID: IMSR_JAX:005557) and M-CSFh/h IL-3/GM-CSFh/h hSIRPAtg TPOh/h Rag2-/- Il2rg-/- (MISTRG) (VG#5097/5090/5155/ 5089 / 5079 / 5078, Regeneron)64 were bred in Rongvaux lab (Fred Hutch Cancer Center) and provided by Rongvaux lab. All mice were maintained in pathogen-free conditions at the Fred Hutchinson Cancer Center (Seattle, WA), and acclimatized for at least 2 days before experimentation, which was performed per Institutional Animal Care and Use Committee guidelines. All experimental mice were female and ranged in age from 8 to 12 weeks. All mouse experiments were approved by the Institut for Animal Care and Use Committee (IACUC Protocol #50898 and #2400037) at the Fred Hutchinson Cancer Center. All mice were euthanized using CO2 according to American Veterinary Medical Association (AVMA) guidelines.

1×106 ME275 or 1×105 A375F modified tumor cells were injected subcutaneously (SC) in 100 µl of solution containing 75% PBS, and 25% Matrigel (Corning, 356237). Tumors were monitored for 13 days or 22 days for A375F or ME275 respectively, at which point tumor volumes were recorded and mice were randomized accordingly. 2.5×106, 5×106, and 1×107 T cells with different TCR MA1 constructs were intravenously injected into mice between days 9–12. Groups included an irrelevant TCR (TCR Irr.-CD8αβ, TCR Irr.-CD8/28) as negative controls. After infusion, tumors were measured 3 times a week and mice were euthanized 10–14 days later. Tumors, spleens, and blood were harvested at approximately day 21 for A375F and day 33 for ME275. Infiltrated T Cells were isolated from tissues by the Human Tumor Dissociation Kit (Miltenyi, 130095929). ACK Lysis Buffer (Gibco, A1049201) was used to remove erythrocytes. TILs were counted with CountBright absolute counting beads (Invitrogen) from the tumor single cell suspension. The single cell suspensions were analyzed with flow cytometry (see In vivo experiment flow cytometry panel) or cryopreserved for 10X sc-RNA seq preparation. Undigested tumors were fixed in 10% neutral buffered formalin (Azer Scientific, PFNBF-20) and paraffin-embedded for immune-histochemistry staining and analysis. Survival experiments: tumors were monitored for 6 days until all palpable. A total of 5×106 T cells were injected on day 6, and tumor growth rates were recorded until reaching 500 mm3 in volume. Mouse tumor measurement guidelines: All mice engrafted with tumors were monitored 3 times per week and the every day after tumors became palpable (5-7 mm in diameter). In compliance with institutional (Fred Hutchinson Ethics Committee) regulations, tumors were measured using digital calipers and not allowed to exceed the maximum size of 20 mm in largest diameter (2000 mm3) for 1 implanted tumor, or 15 mm in diameter for 2 implanted tumors (1500 mm3). Tumor volume was determined by digital caliper measurement with tumor length x width x depth (ME275 and A375F models) and tumor length x width x ½ width (SKMEL-37 model).

Clinical protocol
The clinical trial (NCT04639245) was approved by the Fred Hutchinson Cancer Center (FHCC) Institutional Review Board (FWA00001920 / IORG0000057) under protocol FHCC #10420/RG1007463 (Supplementary Note 1), the U.S. Food and Drug Administration, and the Recombinant DNA Advisory Committee. The study design and conduct complied with all relevant regulations regarding the use of human study participants and was conducted in accordance with the criteria set by the Declaration of Helsinki. HLA A*0201 (HLA-A2)-expressing patients with triple negative breast cancer (TNBC), non-small cell lung cancer (NSCLC) or urothelial carcinoma were eligible for treatment. All participants provided informed consent.

Patient selection
HLA-A2 genotype was confirmed by high-resolution typing and MAGE-A1 expression was confirmed by immunohistochemistry defined as any tumor with >10%, 1+ or higher staining intensity, membranous, nuclear or cytoplasmic expression of MAGE-A1, prior to enrollment. Inclusion criteria further included measurable disease by RECISIT 1.1 criteria, ≥18 years old, ECOG performance status ≤2. Exclusion criteria included autoimmune diseases requiring immunosuppressive therapy and other tumor-specific concurrent treatments.

Treatment plan
The study was originally designed first as a Phase I 3+3 dose escalation (Supplementary Note 1). The first participant enrolled in each cohort was planned to receive 1 × 109 T TCR-MA1 alone, standard lymphodepleting chemotherapy of cyclophosphamide and fludarabine could be administered to subsequent patient within the cohort. Participants were eligible to receive a first infusion of 1 × 109 T TCR-MA1 with acceptable organ function (eGFR >30 mL/min, tBili <3.0 mg/dL, AST, and ALT <5x upper limit of normal, SaO2 ≥92% on ambient air, left ventricular ejection fraction (LVEF) ≥35%, ANC >500 cells/mm3). Second infusions at the same dose level could be administered as soon as 12 weeks after the first and if no dose limiting toxicities had occurred. If indicated, the protocol allowed for interval bridging treatments

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, the authors present a technically elegant and conceptually innovative approach to enhancing the efficacy of TCR-engineered T cells by integrating co-stimulatory signals through a chimeric CD8/CD28 receptor. They provide several in vitro, in vivo, and transcriptional analyses showing evidence for improved function and persistence of modified T cells. However, several important limitations should be addressed to strengthen the translational value of this work:\n1. The Introduction section lacks sufficient clarity regarding the well-established limitations of T cells genetically modified with T cell receptors in the treatment of solid tumors. The authors should briefly outline the key mechanisms underlying this limited efficacy, such as tumor antigen heterogeneity, immune suppressive microenvironments, and poor T cell infiltration, and then use this background to clearly articulate their central hypothesis and the rationale for their proposed strategy to overcome these barriers. Providing this context would greatly enhance the coherence and scientific relevance of the study.\n2. The presentation of the experimental findings in the Results section lacks coherence and narrative flow. In some places, the transitions between paragraphs are abrupt, and the rationale linking each experimental step is insufficiently developed. As a result, it is difficult for the reader to follow the logical progression of the study or to understand why specific experiments were performed at each stage.\n3. The Discussion section largely reiterates the results without offering a focused or integrative analysis of their broader implications. It would benefit from a more critical interpretation of the findings in the context of existing literature, highlighting how this work advances the field. Moreover, key limitations, such as the reliance on a single clinical case, limited T cell persistence, and the use of immunodeficient mouse models, are not adequately addressed. A more balanced discussion acknowledging these weaknesses, along with suggestions for future directions, would strengthen the manuscript considerably.\n4. The authors engineered TCR-transduced CD4+ and CD8+ T cells targeting MAGE-A1/HLA-A2 and showed enhanced cytokine production and tumor cell killing in vitro, as well as antitumor activity in vivo. Based on these findings, they treated a patient with MAGE-A1＋ NSCLC, demonstrating safety but no clinical efficacy. Although the authors explain the reason for the evaluation of only one case, a single-patient case limits interpretability, and additional cases are needed to draw meaningful conclusions regarding therapeutic potential.\n5. Fig. 1D: What is the explanation for the higher proliferative responses observed in the CD4+/CD8+ TTCR-MAI-CD8αβ group? Does the \"coordinated response\" contribute to enhanced cytokine expression (what cytokines?), thereby promoting proliferation?\n6. Page 7, line 120: Consider replacing “lacks tumor control” with “fails to control tumor growth” for improved clarity and precision.\n7. Fig. 2H–J: It is unclear whether the reported scRNA-seq data are representative of samples from multiple patients and whether the findings are reproducible. Have the authors conducted similar analyses using samples from their own patient cohort to validate the findings?\n8. Page 7, lines 128–129: The statement “Patient tumors have limited and inconsistent expression of both positive and negative co-stimulatory binders” refers to a well-recognized limitation of CAR T cell therapy in solid tumors, unlike in hematologic malignancies, where co-stimulatory ligands are more consistently present (PMID: 24667962). For clarity, the authors should first provide a brief background on this issue to orient readers before presenting the scRNAseq findings.\n9. Importantly, the study lacks translational evidence demonstrating the effectiveness of TCR-engineered T cells incorporating co-stimulatory signaling via a chimeric CD8/CD28 receptor in human patients with solid tumors. Including such data would greatly enhance the clinical relevance of the findings.", "reviewer_2": "The study investigates strategies to improve HLA-A2-restricted, MAGE-A1-specific TCR-engineered T cell therapy for solid tumors by enhancing CD4+ T cell function through TCR modification and co-stimulatory signaling. The authors found that redirecting CD4+ T cells—engineered to express a tumor-specific TCR and co-expressing the CD8αβ co-receptor—is not sufficient to achieve robust anti-tumor responses, likely due to limited co-stimulatory signals within the tumor microenvironment (TME).\nTo address this, they designed a synthetic construct by tethering the intracellular signaling domain of CD28 to CD8β. Modifying key signaling motifs within CD28 (LL, YMNM, PRRP, and PYAP) significantly enhanced T cell functionality and in vitro tumor cell killing. These modifications also rendered the engineered T cells more resistant to exhaustion. Furthermore, Zhang et al. demonstrated that the CD8β/CD28Mut construct enhances the cytotoxic activity of T cells engineered with other tumor-specific TCRs, such as those targeting PRAME and WT1, broadening the potential application of this strategy.\nThe data support the use of engineered CD8β/CD28 fusion proteins to improve both the durability and efficacy of T cell therapies in the solid tumor setting.\nOverall, the study is well-conducted and the conclusions are supported by the data. However, the absence of in vivo evidence showing that T cells expressing the CD8β/CD28Mut construct provide superior tumor control compared to CD8β/CD28 wild-type (WT) limits the impact of the findings.\nMajor Points:\n1. The authors should clarify the proportion of engineered CD4+ and CD8+ T cells used in the in vivo experiments.\n2. In Figure 4B, the tumor growth curves for mice treated with irrelevant TCR or MAGE-A1 TCR plus CD8αβ appear identical to those shown in Figure 2. The authors should either consolidate this data into a single figure or present an independent experiment with a new control group.\n3. An in vivo comparison of tumor growth in mice treated with MAGE-A1 TCR-engineered T cells co-expressing CD8β/CD28WT versus CD8β/CD28Mut is essential to validate the enhanced efficacy observed in vitro.\n4. It would also be important to assess in vivo persistence of the transferred T cells and the overall survival of treated mice.", "reviewer_3": "• The authors present an interesting manuscript whereby CD8β is tethered to the costimulatory domain of CD28 and thereby proposed to enhance the activity of TCR-engineered T cells. They claim this results in reduced exhaustion, enhanced tumor infiltration, and improved murine tumor control. Further modifications of a WT or mutated CD28 intracellular domain produced a mutant CD8β-CD28 construct that conferred superior therapeutic control across tumor models.\nCritiques below.\n• Abstract\no The abstract was not clearly written and led to some confusion especially as it relates to describing CD4 T cells. A clearer and more concise description of describing their strategy to engineer CD8αβ into CD4 T cells to improve TCR-T cell would be helpful.\n• Introduction\no A point for clarity. The authors state that “modifying the intracellular TCR chain domains decreased TCR function, likely by blocking the initiation of TCR signaling”. However, the publication they cited used a single-chain TCR that wouldn’t engage TCR signaling in the first place as such their rationale seems unfounded.\n• Results\no Figure 1\n  What is the significance of the K278T mutation in MAGE-A1 peptide for selecting TCRMA15 as TCR of interest? How does this relate to the modifications made to the T cell. Is this a higher affinity version? If so, would one want to use a lower affinity version to demonstrate the ability to alter the TCR activation threshold with said modifications?\n  Figure 1D:\n• It is not entirely clear whether the proliferation was determined via incucyte or a separate stimulation. It will be helpful offer a clear description of how they enumerated the CD4 vs CD8 T cells.\n• Were the number of CD4 only and CD8 only T cells seeded the same on their own as combined or reduced to half for their 1:1 (or are they doubling the total number of T cells in the combination group)? Could this effect fold-change numbers when determining expansion? One could assume reduced numbers but as written this is point is ambiguous.\n• Measuring T cell numbers over 6 days seems too brief. I suggest extending this time to a couple of weeks (at least). How was survival impacted? Please consider using a proliferation dye to visualize the extent of cell division (vs increased numbers due to cell survival).\n  Figure 1E\n• The numbers for the CD4 only and CD8 only treatments vs combined is ambiguous here too.\no Figure 2\n  Figure 2F:\n• PD-1 and TIM-3 aren’t sufficient markers to determine differences in exhaustion between single and combined T cell treatment. Suggest looking at transcriptional markers of exhaustion, i.e. TOX.\n• The most definitive method to test for exhaustion is to conduct an ex vivo stimulation and assess functional activity.\n  Are there differences in the frequencies of Tregs in the CD4 only vs combined CD4+CD8 treatments\no Figure 3\n  Figure E:\n• Authors claim enhanced proliferation just by obtaining the T cell counts at the end of all of their stimulations but this method leaves too much room for misinterpretation.\n  In figure 3B, it looks like there is less p/HLA multimer staining cells than the other CCR groups. Please clarify how you normalize for this because it doesn’t look like they stain for the multimer after the stimulation. Does this mean the CD8 αβ only T cells would have similar cytokine expression if TCR expression was equal across groups in Figure 3C. The data shown actually look like the trend in IFNγ+ cells correlates pretty well with TCR expression.\no Figure 4\n  Figure 4A, please provide a rationale as to T cells are injected 6 days earlier here than they do for the rest of their experiments? At this time point, the tumors do not look like they would be at a palpable size.\n  Figure 4H—again, consider expanding the markers you use to characterize exhaustion. At this point the tumors are so small that leads one to wonder if the reduced TIM3 phenotype they’re seeing is because the cells don’t have anything to interact with anymore yet. The authors mention differences in tumor burden in the results section for figure 5 as a reason for why the CD8αβ groups have more proliferation associated genes than the CD8/CD28. Repeat some of these studies but euthanizing mice when the tumors are varying sizes might shed some light on this matter.\n  Authors claim improved infiltration but only show that there are more cells in the tumor. The increased numbers could be due to increased proliferation and/or Survival but not necessarily infiltration.\n  Figure 4G captions claim that the heightened CD4 to CD8 ratio is indicative of CD4 proliferation. This could be the result of many other mechanisms. CD8 death, for one, in addition to the other concerns I’ve mentioned about proliferation.\no Figure 5\n  They mention that the CD8 T cells from the CD8/28 group has more stem-like gene expression, but that is only in reference to the CD8αβ combo control and not the CD8αβ control. Not sure if that is a fair comparison\n  If exhaustion is being averted then it is unclear why a cluster of exhausted T cells from this sequencing data does not show up.\no Figure 6\n  The manuscript would have benefited from a strong ending (Figures 6 and 7). The lack of data showing that the mutant had any beneficial effects in vivo and characterized it with an in vitro repeat stim/sequencing assay. They show upregulation in proliferation and exhaustion related genes but follow this with flow data that only looks at PD1 and TIM3 again for exhaustion, which also had very little differences between the mutant and wildtype cd8/cd28 groups. Furthermore they saw no differences in cytokine expression after restimulation. Did not show any functional proof of differences in proliferation again.\n  In figures 6 and 7 they did not show any signaling differences for why their mutant might be affecting the T cells differently than their wildtype version of the CD8/CD28. They mention in their discussion possible signaling molecules that are involved but didn’t look at a single one.\nFinally, please citing other relevant work demonstrating the use of CD4 or CD8a molecules modified to express costimulatory domains."}, "author_response": "Point by point answers to reviewers:\nReviewer #1:\n1-1. The Introduction section lacks sufficient clarity regarding the well-established limitations of T cells\ngenetically modified with T cell receptors in the treatment of solid tumors. The authors should briefly\noutline the key mechanisms underlying this limited efficacy, such as tumor antigen heterogeneity, immune\nsuppressive microenvironments, and poor T cell infiltration, and then use this background to clearly\narticulate their central hypothesis and the rationale for their proposed strategy to overcome these\nbarriers. Providing this context would greatly enhance the coherence and scientific relevance of the\nstudy.\nWe appreciate the reviewer’s suggestion and have edited the introduction accordingly.\nPage 3, line 57 now reads:\n“Multiple barriers have been shown to contribute to this lack of efficacy: In addition to tumor\nheterogeneity limiting antigenic encounter and the presence of immunosuppressive populations the solid\ntumor microenvironment (TME) is typically rich in inhibitory cytokines, suppressive checkpoint ligands\nand the general lack of positive co-stimulatory signals necessary for full T cell activation. Together, these\nobstacles underscore the imminent need to improve T cell trafficking and function within the solid tumor\nTME.”\nPage 4, line 79 now reads:\n“Using a vector backbone including a TCR that recognizes the MAGE A1 cancer testes antigen\n(T ) with co-expression of the CD8ab co-receptor, we sought to provide co-stimulation within the\nTCR-MA1\nTCR-associated complex for robust T cell activation independent of the presence/absence of TME-derived\nco-stimulatory ligands while also directing CD4+ T cells to the tumor”\n1-2. The presentation of the experimental findings in the Results section lacks coherence and narrative\nflow. In some places, the transitions between paragraphs are abrupt, and the rationale linking each\nexperimental step is insufficiently developed. As a result, it is difficult for the reader to follow the logical\nprogression of the study or to understand why specific experiments were performed at each stage.\nWe have extensively revised the manuscript to clarify the rationales leading to each successive\nexperimental step so that the logical progression is built on the previous finding. For example:\nPage 5, line 106 now reads:\n“To determine the impact of CD4+ and CD8+ T both alone and combined, in vitro\nTCR-MA1-CD8αβ\nsurvival of the MAGE-A1 and HLA-A*0201-expressing ME275 melanoma cell line was assessed in a\nreal-time killing assay where T cells were repeatedly challenged with tumor”\nPage 6, line 116-117 now reads:\n\"To assess the proliferative capacity, CD4+ and CD8+ T were exposed once to ME275\nTCR-MA1-CD8αβ\nand their divisions assessed 7 days later (Extended Fig. 3B-G).”\nPage 6, line 128 now reads:\n“To examine this in vivo, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were engrafted with 1 x 106\nME275 cells (Fig. 1E).”\nPage 8, line 180 now reads:\n“To achieve ligand-independent signaling, we tethered positive intracellular costimulatory\ndomains to the requisite CD8αβ co-receptor chains, based on findings that TCR chain modifications\n4\nimpaired function (Extended Fig. 6A-C) and prior evidence that CD8α chain modifications can transmit\nactivation signals.”\nPage 9, line 200 now reads:\n“To standardize TCR expression, constructs were first titrated in CD4+ T cells, selected for their\nlack of endogenous CD8αβ expression thus forcing the use of CCRs for class I-restricted responses,\nensuring comparable transduction efficiency and transgene expression (Fig. 3B, first 3 panels to the left).”\nPage 12, line 276 now reads:\n“Based on studies demonstrating that modifications CARs co-stimulatory motifs can enhance function\nwe generated T constructs with distinct CD28 intracellular domain (ICD) motif mutation\nTCR-MA1-CD8/28\ncombinations.”\n1-3. The Discussion section largely reiterates the results without offering a focused or integrative\nanalysis of their broader implications. It would benefit from a more critical interpretation of the findings\nin the context of existing literature, highlighting how this work advances the field. Moreover, key\nlimitations, such as the reliance on a single clinical case, limited T cell persistence, and the use of\nimmunodeficient mouse models, are not adequately addressed. A more balanced discussion\nacknowledging these weaknesses, along with suggestions for future directions, would strengthen the\nmanuscript considerably.\nWe appreciate the reviewer’s suggestions and have revised the Discussion to acknowledge the use of\nimmunodeficient mouse models. Regarding the single patient reported, we have clarified that the rationale\nto undertake the task of increasing efficacy does not rely on the patient case, rather the patient case adds a\ndimension, but the lack of efficacy was also evident in mouse models.\nWe have now included a discussion regarding that limitation and revised other parts of discussion to\naddress broader implications.\nThe revised Discussion, pages 15&16, starting on lines 347 and 385 now reads:\n“CD4+ T cells can be transduced with a Class I TCR by co-expressing the CD8αβ co-receptor thus\nenhancing the persistence and function of engineered CD8+and CD4+ T cells that recognize the same tumor\ntarget when class II expression is often limited. However, both our experience in one patient-case - although\nhampered by limited interpretability - and stringent murine models, point to the insufficiency of this\nintervention alone in consistently controlling solid tumors.”\n“This study relies on immunodeficient NSG models, which lack critical components of the human\nimmune system and do not fully recapitulate the complexity of the TME. Furthermore, insufficient number\nof patients received T which impedes drawing meaningful conclusions of the effect of co-\nTCR-MA1-CD8ab\nengaging CD4+ T cells towards the same Class I target. While human studies further assessing whether\nthe T and T mediate similar effects in patients are being implemented,\nTCR-MA1-CD8/CD28 TCR-MA1-CD8/CD28mut\nhumanized mouse models may provide a reflective platform, as they replicate key constraints of the solid\ntumor microenvironment such as stromal barriers, immunosuppressive cells and cytokines that are likely\nto prevent infiltration and negatively impact the efficacy of transferred T cells.”\n1-4. The authors engineered TCR-transduced CD4+ and CD8+ T cells targeting MAGE-A1/HLA-A2 and\nshowed enhanced cytokine production and tumor cell killing in vitro, as well as antitumor activity in vivo.\nBased on these findings, they treated a patient with MAGE-A1＋ NSCLC, demonstrating safety but no\nclinical efficacy. Although the authors explain the reason for the evaluation of only one case, a single-\n5\npatient case limits interpretability, and additional cases are needed to draw meaningful conclusions\nregarding therapeutic potential.\nWe appreciate the reviewer’s comment regarding the limitations of a single-patient clinical case.\nThe trial was ended after 1 patient received cells due to sponsorship withdrawal as a result of a change in\ncorporate strategy. We are acutely aware of the limitations to drawing firm conclusions about either safety\nand have therefore revised our claims to clarify these points to the reader. See response to point 1-3 above\nregarding the changes made to the Discussion.\nAdditionally, we have modified the text in the Results section.\nResults Section Title change. Page 7, line 137 now reads:\n“CD4+ and CD8+ T are safely infused in one patient, require potency enhancement in\nTCR-MA1-CD8αβ\nstringent murine models”\nIn the Results section, Page 7, line 147 now reads:\n“Given the limitation of a single patient, this result cannot be conclusive but suggests additional\nmeasures may be needed to improve efficacy.”\n1-5. Fig. 1D: What is the explanation for the higher proliferative responses observed in the CD4+/CD8+\nTTCR-MAI-CD8αβ group? Does the \"coordinated response\" contribute to enhanced cytokine expression\n(what cytokines?), thereby promoting proliferation?\nRelevant to this point and in line with Reviewer 3 Comment 5 & 6’s suggestion, we have updated the\nmanuscript to characterize the increased T cell numbers observed at the conclusion of the live killing\nassays while intentionally avoiding attribution to either enhanced survival or increased proliferation.\nTo address cytokine expression of CD4 alone, CD8 alone and in combination, we have now referenced\nour previously published manuscript1 (Supplementary Figure S15 shown below for this Reviewer’s ease):\nFor WT1-specific T cells (also an over-expressed self-antigen), wells in which CD4 alone (T\nTCR37-45\ndpCD4+ - same design as T where CD4 T cells co-express CD8αβ) were exposed to supra-\nTCR-MA1-CD8αβ\nphysiological levels of cognate peptide (10 µg/ml) had overall increased cytokine concentrations\nincluding IL-2 and cytokines associated with helper function compared to wells in which CD8 alone were\nexposed to peptide (eCD8+ - same as CD8 T in this manuscript).\nTCR-MA1-CD8αβ\n6\n+ + + +\nFig. S15. Cytokine profiles of peptide stimulated eCD8 , dpCD4 and eCD8 /dpCD4 TTCR37-45 T cells.\nCytokine production by TTCR37-45 T cells was measured after 4, 24, and 48 hours of stimulation with T2 cells\nexogenously loaded with 10μg peptide. Cytokines are found at significantly higher concentrations in cultures of\n+ + + + + +\ndpCD4 cells compared to eCD8 cells and the mixture of eCD8 /dpCD4 . The mixture of eCD8 /dpCD4\n+\nproduced significantly more cytokine than eCD8 cells alone. Data are presented as mean+ standard deviation (SD).\nDifferences between groups were calculated using a student t test.\nFor this manuscript and the experiment shown in Fig. 1D, to address cell proliferation for CD4 alone,\nCD8 alone or each subset in combination with the other, we have now included a new Extended Fig. 3\nshown below.\nThe revised Result Section, Page 6, line 116 now reads:\n“To assess the proliferative capacity, CD4+ and CD8+ T were exposed once to ME275\nTCR-MA1-CD8αβ\nand their divisions assessed 7 days later (Extended Fig. 3B-G). CD8+ T divided more in the\nTCR-MA1-CD8αβ\npresence of CD4+ T (Extended Fig. 3B-D), while CD4+ T divided less when\nTCR-MA1-CD8αβ TCR-MA1-CD8αβ\ncombined with CD8+ T (Extended Fig. 3E-G). Despite the increased ability of TCR-transgenic\nTCR-MA1-CD8αβ\nCD4+ T cells co-transduced with CD8ab to secrete helper cytokines when exposed to supraphysiological\nlevels of peptide1 and proliferate after tumor exposure (Extended Fig. 3E), they showed reduced tumor\nkilling upon repeated challenge (Fig. 1C). In contrast, CD8+ T cells divided less after single tumor exposure\nbut were more effective at repeated killing (Extended Fig. 3B, 3E, Fig. 1C). Together, CD4+ and CD8+ T\ncells sustained tumor control (Extended Fig. 3C), likely through CD4+ -derived cytokine support that\nenhances CD8+ T cells proliferation and cytotoxicity. The reduced proliferation of CD4⁺ T cells in co-\nculture (Extended Fig. 3F), may reflect rapid tumor clearance by CD8⁺ T cells and/or sequestration of\nCD4+ T cell -derived cytokines.”\nTCR-MA1-CD8αβ TCR-MA1-CD8αβ\n(A) Growth kinetics for the ME275 cell line in a live tumor visualization assay (Incucyte S3) in the absence\n(black line) or presence of CD8+ T (red line) or CD4++ CD8+ T (purple line). The\nTCR-MA1-CD8αβ TCR-MA1-CD8αβ\nE:T ratio was 2:1, and arrows indicate the addition of tumor cells to the culture. The mean and standard\ndeviation (SD) of triplicate wells is shown. (n=3 wells/group). (B-G) Co-culture of ME275 with CD4+ T\nTCR-\n(blue), CD8+ T (red), or CD4+ + CD8+ T (purple)at E:T ratio of 5:1 for 7\nMA1-CD8αβ TCR-MA1-CD8αβ TCR-MA1-CD8αβ\ndays. (B-C) Histogram representing cell division between CD8+ T alone without tumor\nTCR-MA1-CD8αβ\nstimulation(gray), CD8+ T alone(red) or CD8+ T with CD4+ T (purple)\nTCR-MA1-CD8αβ TCR-MA1-CD8αβ TCR-MA1-CD8αβ\nwith tumor stimulation identified by CFSE proliferation dye. (D) Percentage divided (left panel) and mean\nMFI of divided cells(right) of CD8+ T alone(red) compared with CD8+ T (purple)\nTCR-MA1-CD8αβ TCR-MA1-CD8αβ\nfrom CD4+/CD8+ T combination group. The mean and standard deviation (SD) of duplicated\nTCR-MA1-CD8αβ\nwells from two experiment is shown(n=4). p value determined by unpaired two-tailed t-test. (n=4 wells)\n(E-F) Histogram representing cell division between CD4+ T alone without tumor stimulation\nTCR-MA1-CD8αβ\n(gray), CD4+ T alone(red) or CD4+ T with CD8+ T (purple) with tumor\nTCR-MA1-CD8αβ TCR-MA1-CD8αβ TCR-MA1-CD8αβ\nstimulation identified by CFSE proliferation dye. (n=4 wells/group). (G) Percentage divided (left panel)\nand mean MFI of divided cells(right) of CD4+ T alone(red) compared with CD4+ T\nTCR-MA1-CD8αβ TCR-MA1-\n(purple) from CD4+/CD8+ T combination group. The mean and standard deviation (SD)\nCD8αβ TCR-MA1-CD8αβ\nof duplicated wells from two experiment is shown. p value determined by unpaired two-tailed t-test. (n=4\nwells/group).\nLahman, M. C. et al. Targeting an alternate Wilms' tumor antigen 1 peptide bypasses\nimmunoproteasome dependency. Sci Transl Med 14, eabg8070 (2022).\nhttps://doi.org:10.1126/scitranslmed.abg8070\n1-6. Page 7, line 120: Consider replacing “lacks tumor control” with “fails to control tumor growth” for\n8\nimproved clarity and precision.\nWe have revised the manuscript and replaced “lacks tumor control” with “fails to control tumor growth”\nthroughout.\nPage 7, line 120 now reads:\n“Indeed, mice infused with half doses of CD4+ plus CD8+ T (5 x 106 vs. 107 total\nTCR-MA1-CD8αβ\ncells) (Fig. 2D) failed to control tumor growth (Fig. 2E).”\n1-7. Fig. 2H–J: It is unclear whether the reported scRNA-seq data are representative of samples from\nmultiple patients and whether the findings are reproducible. Have the authors conducted similar analyses\nusing samples from their own patient cohort to validate the findings?\nWe investigated the tumor heterogeneity expression of the costimulatory ligands by analyzing publicly\navailable single-cell RNA-seq datasets taken from patients with NSCLC (n= 42 patients) and acral or\ncutaneous melanoma (n=8 patients). We do not have additional patient samples from our own cohort to\nperform additional scRNA-seq analysis. We have now clarified the number of patients from which the\nscRNAseq analysis was performed.\nPage 8, line 166 now reads:\n“To examine the expression of both positive and negative co-stimulatory ligands across human\nTMEs, in silico analysis of previous published single-cell RNA sequencing (scRNA-seq) datasets from\nNSCLC (n=42 patients) and melanoma patients (n=8 patients)2,3 was performed.”\n1-8. Page 7, lines 128–129: The statement “Patient tumors have limited and inconsistent expression of\nboth positive and negative co-stimulatory binders” refers to a well-recognized limitation of CAR T cell\ntherapy in solid tumors, unlike in hematologic malignancies, where co-stimulatory ligands are more\nconsistently present (PMID: 24667962). For clarity, the authors should first provide a brief background\non this issue to orient readers before presenting the scRNAseq findings.\nWe appreciate the reviewer for this helpful suggestion and have integrated a brief background well as the\nsuggested reference.\nPage-9, line 162 now reads:\n“T cells can receive co-stimulation either naturally through ligands in the TME or synthetically\nvia immunomodulatory fusion proteins (IFPs) that couple inhibitory receptor ectodomains to\ncostimulatory endodomains4. The CD28 co-stimulatory molecule was naturally expressed on T\nTCR-MA1-\ninfused in a patient (Fig. 2A) and in our in vivo murine models5 (Extended Fig. 5A), indicating their\nCD8αβ\ncapacity to respond to positive co-stimulation. Unlike in hematologic malignancies, costimulatory ligands\nare less consistently present in solid tumors6. ”\nKakarla, S. & Gottschalk, S. CAR T cells for solid tumors: armed and ready to go? Cancer J 20,\n151-155 (2014). https://doi.org:10.1097/PPO.0000000000000032\n1-9. Importantly, the study lacks translational evidence demonstrating the effectiveness of TCR-\nengineered T cells incorporating co-stimulatory signaling via a chimeric CD8/CD28 receptor in human\npatients with solid tumors. Including such data would greatly enhance the clinical relevance of the\n9\nfindings.\nIncorporating the chimeric CD8/CD28 coreceptor in future clinical trials is the planification stages\n(typically from the planification stages to completion of a clinical trial with associated immune\nmonitoring at our institution can take 3-5 years) of our group and outside of the scope of this manuscript.\nWe have now addressed this point in the Discussion.\nPage 16, line 388 now reads:\n“While human studies further assessing whether the T and T\nTCR-MA1-CD8/28 TCR-MA1-CD8/28mut\nmediate similar effects in patients are being implemented, humanized mouse models may provide a\nreflective platform, as they replicate key constraints of the solid tumor microenvironment such as stromal\nbarriers, immunosuppressive cells and cytokines that are likely to prevent infiltration and negatively\nimpact the efficacy of transferred T cells”\nReviewer #2:\n2-1. The authors should clarify the proportion of engineered CD4+ and CD8+ T cells used in the in vivo\nexperiments.\nIn all experiments equal total T cell numbers were infused across groups. We have clarified the\nproportion of engineered CD4⁺ and CD8⁺ T cells used in the in vivo experiments updating the relevant\nfigures and figure legends (updated Figures 1E, 1F, 2D) and explicitly stating “equal total number of T\ncell across groups” on the Figures and the proportions in the text. Updated text, figures and figure\nlegends for Figures 1E, F and 2D shown below:\nPages 6, Line 128 now reads:\n“To examine this in vivo, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were engrafted with 1 x 106\nME275 cells (Fig. 1E). CD4+, CD8+, or combined CD4+/CD8+ T (1:1 ratio, 1 x 107 total)\nTCR-MA1-CD8αβ\nmediated tumor control,…”\nPages 6, Line 132 now reads:\n“In this model, CD4+ or CD8+ T alone slowed but did not prevent tumor growth while\nTCR-MA1-CD8αβ\nthe CD4+/CD8+ combination(1:1 ratio, 1 x 107 total) had significantly reduced tumor volumes compared\nto controls (Fig. 1F and G),…”\nsubcutaneously into NSG mice with subsequent transfer of 1 x 107 equal numbers of total irrelevant T cells\n(T ) , CD4+ T alone, CD8+ T alone, or a 1:1 ratio of combined CD4+ and\nIrr.-CD8αβ TCR-MA1-CD8αβ TCR-MA1-CD8αβ\nCD8+ T 22 days after engraftment. The tumors were allowed to reach ~60mm3 in size before\nTCR-MA1-CD8αβ,\nT cell infusion (n=3 mice/group, 2 tumors/mouse). (F) Schematic (left) and representative growth kinetics\n(right) of 1 x 105 A375F tumors engrafted into NSG mice with subsequent transfer of 1 x 107 equal numbers\nof total T cells (T , CD4+ T alone, CD8+ T alone, or a 1:1 ratio of combined\nIrr.-CD8αβ TCR-MA1-CD8αβ TCR-MA1-CD8αβ\nCD4+ and CD8+ T ) 12 days after engraftment. The tumors were allowed to reach ~60mm3 in\nTCR-MA1-CD8αβ\nsize before T cell infusion (n=3-4 mice/group, 2 tumors/mouse).\nPage 7, line 149 now reads:\n“Indeed, mice infused with half doses of CD4+ plus CD8+ T (5 x 106 vs. 1 x 107 total\nTCR-MA1-CD8αβ\ncells) (Fig. 2D) failed to control tumor growth (Fig. 2E).”\nFigure 2 (D) Schematic (left) and growth kinetics (right) of 1 x 105 A375F tumors engrafted into NSG mice\nwith subsequent transfer of 5x 106 total T cells (T , CD4+ T alone, CD8+ T\nIrr.-CD8αβ TCR-MA1-CD8αβ TCR-MA1-CD8αβ\nalone, or a 1:1 ratio of combined CD4+ and CD8+ T ), 12 days after engraftment. (n=3\nTCR-MA1-CD8αβ\nmice/group, 2 tumors/mouse, data shown are representative of three independent experiments.)\n2-2. In Figure 4B, the tumor growth curves for mice treated with irrelevant TCR or MAGE-A1 TCR plus\nCD8αβ appear identical to those shown in Figure 2. The authors should either consolidate this data into\na single figure or present an independent experiment with a new control group.\nWe appreciate the reviewer for pointing this out. To address the concern, we have updated Figure 2D to\npresent data from a new, independent experiment. This ensures that the tumor growth curves shown in\nstate as below.\nFigure 2 (D) Schematic (left) and growth kinetics (right) of 1 x 105 A375F tumors engrafted into NSG mice\nwith subsequent transfer of 5x 106 total T cells (T , CD4+ T alone, CD8+ T\nIrr.-CD8αβ TCR-MA1-CD8αβ TCR-MA1-CD8αβ\nalone, or a 1:1 ratio of combined CD4+ and CD8+ T ), 12 days after engraftment. (n=3\nTCR-MA1-CD8αβ\nmice/group, 2 tumors/mouse, data shown are representative of three independent experiments.)\n2-3. An in vivo comparison of tumor growth in mice treated with MAGE-A1 TCR-engineered T cells co-\nexpressing CD8β/CD28WT versus CD8β/CD28Mut is essential to validate the enhanced efficacy\nobserved in vitro.\nWe agree with the review’s comment. To validate the enhanced efficacy observed in vitro, we conducted\na new in vivo comparison of CD8β/CD28WT versus CD8β/CD28Mut using the A375F tumor model.\nWhen administering a reduced number of T cells (2.5 × 10⁶ instead of 5 x 106 as shown in Fig. 6C) 12\ndays post tumor engraftment, this approach effectively revealed differences in antitumor activity between\nMAGE-A1 TCR-engineered T cells co-expressing CD8β/CD28 WT versus CD8β/CD28mut. These\nresults are now included in the revised manuscript (new Fig. 6D).\nThe Results Section, Page 13, line 291 now reads:\n“However, when the total infused dose was halved (2.5 x 106 T cells), T achieved\nTCR-MA1-CD8/28mut\nmore robust tumor burden reduction relative to controls (Fig. 6D-E).”\nCD4+ and CD8+ T cells (T T and T ) at a ratio of 1:1 on day 12\nTCR-Irr.-CD8/28, TCR-MA1-CD8/28, TCR-MA1-CD8/28mut\npost engraftment. (n=3mice/group, 2 tumors/mouse, data shown are representative of two independent\nexperiments.).(E) A375F tumor volume assessed at sacrifice 9 days after T cell infusions for each indicated\ncondition same as (D). p value determined by one-way ANOVA with Tukey’s multiple comparison\ntest. (n=4-5 mice/group, data shown are representative of two independent experiments).\n2-4. It would also be important to assess in vivo persistence of the transferred T cells and the overall\nsurvival of treated mice.\nWe thank the reviewer for this suggestion. To assess in vivo persistence we have now added a new\nExtended Data Figure 11B which shows T cell percentage obtained from the single cell suspension of\nspleens and tumors at the time of sacrifice from the experiment shown in Figure 6C/6D.\nThe revised manuscript Page 13, line 294 now reads:\n“Cells preferentially localized to tumors while remaining detectable in concurrent spleens\n(Extended Fig. 11B).\nExtended Fig. 11 (B) Schematic (left) and T cell counts in spleens and tumors (right) of A375F tumors\nengrafted NSG mice with subsequent transfer of 5 x 106 or 2.5 x 106 equal numbers of T cells (T ,\nTCR-Irr.-CD8/28\nT , T 1:1 ratio of combined CD4+ and CD8+) 12 days after implantation. Spleens\nTCR-MA1-CD8/28 TCR-MA1-CD8/28mut\nand tumors were collected at 8 days after T cell infusion and T cells were identified in flow cytometry\n13\nanalysis. The mean and standard error of mean (SEM), p values determined with unpaired two-tailed t-test.\n(n=3 mice per group).\nTo address survival of the treated we added a new Figure 6G showing a murine model with the SK-\nMEL37 melanoma tumor cell line, which also addresses reviewer #2 Comment 3 regarding\nCD8β/CD28WT versus CD8β/CD28Mut in vivo. In this model, CD8αβ/CD28Mut presented superior\ntumor control compared to CD8αβ/CD28WT in vivo 60 days after T cell transfer (new Figure 6G):\nThe revised manuscript Page 13, line 296 now reads:\n“T also suppressed growth of the cell death-resistant HLA-A2+ MAGE-A1+ SK-MEL-37\nTCR-MA1-CD8/28mut\nmelanoma cell line in vitro (Extended Fig. 2, Fig. 6F),7 and mediated superior tumor reduction in vivo at\n60 days compared to T (Fig 6G). Thus, T conferred modest but consistent\nTCR-MA1-CD8/28 TCR-MA1-CD8/28mut\nimprovements in both short- (10 days) and long-term (60 days) tumor control, potentially due to an\nincreased proliferative capacity.”\nFigure 6 (G) Non-humanized MISTRG mice were engrafted with 1 x 106 SK-MEL-37 (left) and subsequently\ninfused with total 2 x 107 CD4+ and CD8+ T cells (T T and T ) at\nTCR-Irr.-CD8/28, TCR-MA1-CD8/28, TCR-MA1-CD8/28mut\na ratio of 1:1 on day 21 post engraftment (n=4 mice, 2 tumors per mouse). SK-MEL-37 tumor volume\n(right) assessed at sacrifice 62 days after T cell infusions for each indicated condition. p value determined\nby two-tailed unpaired t-test. (n=4 mice/group, 2 tumors/mouse averaged).\nReviewer #3:\n3-1. Abstract: The abstract was not clearly written and led to some confusion especially as it relates to\ndescribing CD4 T cells. A clearer and more concise description of describing their strategy to engineer\nCD8αβ into CD4 T cells to improve TCR-T cell would be helpful.\nWe appreciate the reviewer’s suggestion and have re-written the abstract for conciseness and to clarify the\nCD4 strategy.\nThe revised abstract (Page 2, lines 35-50) now reads:\n“Adoptive transfer of T cells engineered with tumor-specific T cell receptors (TCR-T) has shown\nlimited efficacy in solid tumors, hindered by insufficient persistence, tumor trafficking, and dependence on\ntumor-associated co-stimulatory ligands. In one clinical case and NSG murine models, infusion of CD4⁺\nand CD8⁺ T cells co-expressing a class I MAGE-A1–specific TCR and CD8αβ failed to control tumor\n14\nprogression. To enhance function downstream of TCR signaling, we explored the adaptability of\nintracellular TCR components to synthetic modification. By leveraging the obligate co-expression of\nCD8αβ required for CD4+ T cells engagement, the CD8β chain provided a tractable site for engineering\nwithout loss of function. In vitro screening of CD4+ T cells identified CD28 intracellular tail as the most\neffective co-stimulatory signal. The synthetic chimeric co-receptor (CD8/CD28) enhanced cytokine\nproduction, persistence, and tumor control in immunodeficient murine models with preservation of stem-\nlike transcriptional programs. Rational modification of CD28 cytoplasmic motifs further augmented in vivo\ntumor control associated with increased intratumoral T cell proliferation and reduced expression of\nexhaustion-associated genes. These benefits extended in vitro to PRAME- and WT1-specific TCRs,\nsuggesting generalizability. Our findings establish a framework for integrating synthetic co-stimulation\ndirectly into the TCR complex independent of co-stimulatory ligand expression.”\n3-2. Introduction: A point for clarity. The authors state that “modifying the intracellular TCR chain\ndomains decreased TCR function, likely by blocking the initiation of TCR signaling”. However, the\npublication they cited used a single-chain TCR that wouldn’t engage TCR signaling in the first place as\nsuch their rationale seems unfounded.\nWe thank the reviewer for this insightful comment. We have removed the reference and the related\nstatement in the introduction and have now supplemented the manuscript with new experimental data\n(new Extended Fig. 6A-C) showing that in the case of a TCR specific a similarly over-expressed self-\nantigen (WT1), modification of the intracellular TCR signaling domain directly impairs TCR function.\nThese findings are now included in the revised Results section.\nThe Results section (Page 8, lines 180-183) now reads:\n“To achieve ligand-independent signaling, we tethered positive intracellular costimulatory\ndomains to the requisite CD8αβ co-receptor chains, based on findings that TCR chain modifications\nimpaired function (Extended Fig. 6A-C) and prior evidence that CD8α chain modifications can transmit\nactivation signal.”\ndesign;(B) Chimeric TCRs are expressed on the cell surface identified by p/HLA multimer. CD8+ T cells were\ntransduced with the modified TCR constructs and p/HLA multimer binding was analyzed by flow cytometry. (C)\nC4\nTCR transduced CD8+ T cells were stimulated with cognate peptide at 1uM and IL-2 and IFNγ levels were analyzed\nby flow cytometry.\n3-3. Figure 1: What is the significance of the K278T mutation in MAGE-A1 peptide for selecting TCR 5\nas TCR of interest? How does this relate to the modifications made to the T cell. Is this a higher\nMA1\naffinity version? If so, would one want to use a lower affinity version to demonstrate the ability to alter\nthe TCR activation threshold with said modifications?\nWe appreciate the reviewer’s request for clarification regarding the significance of the K278T mutation in\nthe MAGE-A1 peptide and the rationale for selecting TCR 5. First, we noted that the legend to\nMA1\nExtended Figure 1D was erroneous and led to believe that TCR modifications were made (not the case\nin this figure) rather than the TCR recognizing a variant of the MAGE peptide. This is now corrected in\nthe manuscript and shown below.\nThe revised manuscript Page 5, line 95 now reads:\n“TCR 5 had a similar functional avidity towards the wild-type peptide compared to TCR 2, 3 and 4\nMA1 MA1\n(range 48.1-85.8nM) (Extended Fig. 1C), and also recognized the previously described K278T mutated\nMAGE-A1 peptide (Extended Fig. 1D). Based on these characteristics, TCR 5 was selected for\n278-286 MA1\nfuture studies.”\n16\n“Extended Data Fig. 1. (D) Recognition of wildtype MAGE-A1 peptide and MAGE-A1 K278T\n278-86 278-86\nmutant peptide [loaded on TAP-deficient T2 B lymphoblastoid cell lines (T2-BLCL)] by the TCR-deficient\nNur-77 reporter Jurkat cells transduced TCR 1-5. Histograms represent GFP expression downstream\nMA1\nof Nur77-GFP triggered when the TCR is engaged”.\nA new section was added in the Methods: Page 18 line 438 now reads:\n“Selection of the MA1-specific TCR for clinical translation\n“While other somatic mutations exist within the MAGE-A1 gene, only the K278T missense mutation\nfalls within the targeted epitope (MAGE-A1₍₂₇₈₋₂₈₆₎). The K278T mutation has been identified in publicly\navailable datasets and is including in the breast cancer cell line HCC10088. Of the 5 isolated TCRs and to\nchoose one for clinical translation, we proceeded by first excluding the least avid TCR 1 (mean EC50 of\nMA1\n134 nM). The remaining 4 TCRs had very similar functional avidity (ranging from 48.1-85.8 nM). TCR 5\nMA1\nwas chosen based on its ability to recognize the wild-type and mutated peptide, thus broadening the targeted\nepitope to include the K278T mutation. All 5 TCRs (TCR 1-5) were isolated from healthy HLA-A2+\nMA1\ndonors. To avoid safety concerns, none were modified or affinity enhanced.”\n3-4. Figure 1D: It is not entirely clear whether the proliferation was determined via incucyte or a\nseparate stimulation. It will be helpful offer a clear description of how they enumerated the CD4 vs CD8\nT cells.\nCD4⁺ and CD8⁺ T cell counts shown in Figure 1D were analyzed from the live tumor killing assay shown\nin Figure 1C at the 144-hour timepoint. Cell counts were determined by counting beads via flow\ncytometry. The Results and Methods were revised to clarify this point.\nThe Results Section Page 5, line 109 now reads:\n“Although CD8+ T remained superior at repeated tumor killing compared to CD4+ T\nTCR-MA1-CD8αβ TCR-MA1-\n, the 1:1 combination of CD4+ and CD8+ T (with each subset contributing half of the\nCD8αβ TCR-MA1-CD8αβ\ntotal cell number) significantly outperformed an equal total number of CD8+-only or CD4+-only T\nTCR-MA1-\nat 144 hours and 192 hours (Fig. 1C, right panel). At 144 hours, CD4+ and CD8+ T\nCD8αβ TCR-MA1-CD8αβ\nremaining in wells within the combination group were ~30x more abundant whereas CD4+ and CD8+\nT alone were ~3x more abundant (Fig. 1D).”\nTCR-MA1-CD8αβ\nThe Methods Section Page 21, line 509 now reads:\n“Analysis of T cell counts in Real-time killing assays: At indicated timepoints remaining T cells in the wells\nwere counted via flow cytometry with count bright counting beads (Invitrogen).”\n3-5. Figure 1D: Were only T cells seeded the same on their own as combined or reduced to half for their\n17\n1:1 (or are they doubling the total number of T cells in the combination group)? Could this affect fold-\nchange numbers when determining expansion? One could assume reduced numbers but as written this is\npoint is ambiguous.\nWe thank the reviewer for this clarification request which was also a concern for Reviewer 2. We\nconfirm that throughout the manuscript for each experiment where the combination of CD8 and CD4 is\ncompared to each subset alone, the total cell remains the same. We have revised the relevant figures to\navoid any ambiguity. Additionally, the measure of CD4 and CD8 fold change in alone or combined\nconditions compared to input is now clarified in Figure 1D.\nThe Results Section Page 5, line 109 now reads:\n“Although CD8+ T remained superior at repeated tumor killing compared to CD4+\nTCR-MA1-CD8αβ\nT , the 1:1 combination of CD4+ and CD8+ T (with each subset contributing half of\nTCR-MA1-CD8αβ TCR-MA1-CD8αβ\nthe total cell number) significantly outperformed an equal total number of CD8+-only or CD4+-only T\nTCR-\nat 144 hours and 192 hours (Fig. 1C, right panel). At 144 hours, CD4+ and CD8+ T\nMA1-CD8αβ TCR-MA1-CD8αβ\nwithin the combination group were ~30x more abundant whereas CD4+ and CD8+ T alone were\nTCR-MA1-CD8αβ\n~3x more abundant (Fig. 1D).”\nFigure 1 (C) Growth kinetics (left panel) for the ME275 cell line in a live tumor visualization assay\n(Incucyte S3) in the absence (gray line) or presence of CD4+ T (blue line), CD8+ T\nTCR-MA1-CD8αβ TCR-MA1-CD8αβ\n(red line), or CD4+ + CD8+ T (purple line). The E:T ratio was 2:1, and arrows indicate the\nTCR-MA1-CD8αβ\naddition of tumor cells to the culture. Final tumor integrated intensity (right panel) for the same experiment\nat 144 and 192 hours. The mean and standard deviation (SD) of triplicate wells is shown. Data were\nanalyzed using a one-way ANOVA with Tukey’s multiple comparison test. (n=3 wells/group). (D) Counts\nof survived T cells were measured at 144 hours post tumor simulation from Incucyte S3 live tumor assay\n(C). Change in absolute numbers of CD4+ T (left panel) and CD8+ T (right panel)\nTCR-MA1-CD8αβ TCR-MA1-CD8αβ\nin either alone (CD4 - blue bars, CD8 - red bars) or combined (purple bars) conditions compared to seeded\ncells. The mean ± SD from triplicated wells is shown (n=3 wells/group). p value determined by unpaired\ntwo-tailed t-test.\n3-6. Figure 1D Measuring T cell numbers over 6 days seems too brief. I suggest extending this time to a\ncouple of weeks (at least). How was survival impacted? Please consider using a proliferation dye to\nvisualize the extent of cell division (vs increased numbers due to cell survival).\nWe have modified/added 3 additional figures. 1) The measurements for intensity of tumor for the live\nkilling assay shown in Figure 1C, left panel was extended from 144 hours (6 days) to 192 hours (~8\ndays) and statistical analysis for both the 144 and 192 hours timepoints added to Figure 1C, right panel.\n2) We added a new Extended Figure 3A with the combination group continuing to show tumor control at\n13 days. 3) We added a new Extended Figure 3B showing proliferation (using a proliferation dye) of\nspecifics).\nThe revised Results Section Page 5, line 110 now reads:\n“……the 1:1 combination of CD4+ and CD8+ T (with each subset contributing half of\nTCR-MA1-CD8αβ\nthe total cell number) significantly outperformed an equal total number of CD8+-only or CD4+-only T\nTCR-\nat 144 hours and 192 hours (Fig. 1C, right panel). At 144 hours, CD4+ and CD8+ T\nMA1-CD8αβ TCR-MA1-CD8αβ\nwithin the combination group were ~30x more abundant whereas CD4+ and CD8+ T alone were\nTCR-MA1-CD8αβ\n~3x more abundant (Fig. 1D). CD4+ and CD8+ T combination group continued to control tumor\nTCR-MA1-CD8αβ\nfor 13 days (Extended Fig. 3A).”\nFigure 1. (C) Growth kinetics (left panel) for the ME275 cell line in a live tumor visualization assay\n(Incucyte S3) in the absence (gray line) or presence of CD4+ T (blue line), CD8+ T\nTCR-MA1-CD8αβ TCR-MA1-CD8αβ\n(red line), or CD4+ + CD8+ T (purple line). The E:T ratio was 2:1, and arrows indicate the\nTCR-MA1-CD8αβ\naddition of tumor cells to the culture. Final tumor integrated intensity (right panel) for the same experiment\nat 144 and 192 hours. The mean and standard deviation (SD) of triplicate wells is shown. Data were\nanalyzed using a one-way ANOVA with Tukey’s multiple comparison test. (n=3 wells/group).\nExtended Fig. 3. (A) Growth kinetics for the ME275 cell line in a live tumor visualization assay for 312\nhours(13 days) (Incucyte S3) in the absence (black line) or presence of equal total numbers of seeded CD8+\nTCR-MA1-CD8αβ TCR-MA1-CD8αβ\nindicate the addition of tumor cells to the culture. The mean and standard deviation (SD) of triplicate wells\nis shown. (n=3 wells/group).\n3-7. Figure 1E: The numbers for the CD4 only and CD8 only treatments vs combined is ambiguous\nhere too.\nWe thank the reviewer for pointing this out and have clarified the total cell infused into mice throughout\nthe manuscript. Please see answer to Reviewer 2 Comment 1.\n3-8. Figure 2F:\n• PD-1 and TIM-3 aren’t sufficient markers to determine differences in exhaustion between single and\ncombined T cell treatment. Suggest looking at transcriptional markers of exhaustion, i.e. TOX.\n• The most definitive method to test for exhaustion is to conduct an ex vivo stimulation and assess\nfunctional activity.\nWe appreciate the reviewer’s suggestion regarding a more comprehensive assessment of T cell\nexhaustion. 1) To provide a more nuanced view of the expression of markers associated with an\nexhausted phenotype, we have added the expression of CD39 and 2B4 alongside PD-1 and TIM-3 in the\nnew Figures 2F and Extended Fig. 4B. 2) Intratumoral T from CD4+ alone, CD8+ alone and\nTCR-MA1-CD8αβ\nCD4+& CD8+ combined groups were further ex vivo stimulated with peptide and their function assessed.\nThe results are shown in a new Figure 2G. Of note, similar revisions were performed in new Figure 4 G\nand H addressed in Reviewer 3 Comment 13.\nThe revised Results Section Page 7, line 149 now reads:\n“Indeed, mice infused with half doses of CD4+ plus CD8+ T (5 x 106 vs. 1 x 107 total\nTCR-MA1-CD8αβ\ncells) (Fig. 2D) failed to control tumor growth (Fig. 2E). In the combined infusion group, only\nintratumoral CD4+ exhibited reduced expression of markers exhaustion-associated markers (Tim3, PD-1,\nCD39, and 2B4), compared to either subset infused alone (Extended Fig. 4B, Fig. 2F), yet all T\nTCR-MA1-\nsubsets - either alone or combined - produced <10% median IFNg and TNFa upon cognate peptide\nCD8αβ\nexposure (Fig. 2G).\ngroups from (D) in CD4+ (top panel) and CD8+ (bottom panel) T for the indicated conditions.\nTCR-MA1-CD8αβ\nGraph represents mean ± SD. p values determined by unpaired two-tailed t-test. (n=3 mice, 6 tumors/group,\n2 tumors/mouse). (G) Quantification of IFNγ + /TNFα+ binding after 1μM cognate peptide stimulation for\n18 hours ex vivo across the same experimental groups from (D) in CD4+ (left) and CD8+ (right) T\nTCR-MA1-\nfor the indicated conditions. Single cell suspensions were obtained at sacrifice, 6 days after T cell\nCD8αβ\ninfusion. Graph represents mean ± SD. p values determined by unpaired two-tailed t-test. (n=3 mice/group,\n2 tumors/mouse).\nExtended Data Fig.4 (B) Co-expression of PD-1 (x-axis) and Tim-3 (y-axis) in CD4+ (left 3 panels) and\nCD8+ (right 3 panels) T cells identified in spleen (top panels) or tumor (second row panels) for the indicated\nconditions. Co-expression of CD39+ (x-axis) and Tim-3+ (y-axis) (third row panels) and expression of 2B4+\n(fourth row panels) in CD4+ T (left 3 panels) and CD8+ T (right 3 panels). Single\nTCR-MA1-CD8αβ TCR-MA1-CD8αβ\ncell suspensions were obtained at sacrifice, 8 days after T cell infusion from (Fig. 2D).\n3-9. Figure 2: Are there differences in the frequencies of Tregs in the CD4 only vs combined CD4+CD8\ntreatments\nWe have now included this analysis in Extended Fig. 4C. Using immunohistochemistry on formalin-\nfixed, paraffin-embedded tumors from the in vivo experiment shown in Fig. 2D, we assessed FOXP3\nexpression in CD4⁺ T cells. Although studies have found infused CAR T cells become\nTCR-MA1-CD8αβ\nTregs9,10, we found no evidence infused CD4 T cells became T-regs (Extended Fig. 4C).\nThe revised the manuscript page 7, line 154 now reads:\n“There were no Treg populations (FOXP3+) found in CD4+ T alone and CD4+ /\nTCR-MA1-CD8αβ\nCD8+ combined group (Extended Fig 4C).”\nTCR-\nbetween the CD4+ alone and the CD4+ + CD8+ combination (1:1 ratio) groups from (Fig. 2D).\nMA1-CD8αβ\nThe mean and ±SD of 6 tumor is shown(n=6). p value determined by unpaired two-tailed t-test. (n=3\nmice, 2 tumors/mouse).\n3-10: Figure 3E: Authors claim enhanced proliferation just by obtaining the T cell counts at the end of all\nof their stimulations, but this method leaves too much room for misinterpretation.\nRelevant to this point and in line with Reviewer 1 Comment 5’s suggestion and Reviewer 3 Comment\n6, we have updated language throughout the manuscript to only characterize the increased T cell numbers\ncompared to input observed at the conclusion of the live killing assays while intentionally avoiding\nattribution to either enhanced survival or increased proliferation.\nThe Results section Page 9, line 208 now reads:\n“Among these, the CD8/CD28 co-receptor (CD8/28) most effectively restrained ME275 tumor\ngrowth in repeated killing assays (Fig. 3E, left and middle) and yielded greater live total remaining T\ncells in wells at 384 hours (16 days) (Fig. 3E, right).”\n3-11: Figure 3B: it looks like there is less p/HLA multimer staining cells than the other CCR groups.\nPlease clarify how you normalize for this because it doesn’t look like they stain for the multimer after the\nstimulation. Does this mean the CD8 αβ only T cells would have similar cytokine expression if TCR\nexpression was equal across groups in Figure 3C. The data shown actually look like the trend in IFNγ+\ncells correlates pretty well with TCR expression.\nWe thank the reviewer for the opportunity to clarify this point. T cells were transduced using titrated\nlentiviral constructs to ensure similar transduction efficiency and vector copy numbers. After transduction\nand to standardize TCR expression across groups, transduced T cells were further FACS sorted on\np/HLA+ cells and expanded using a rapid expansion protocol (REP). We have now revised Figure 3B\n(shown below) to show the MFI of p/HLA multimer binding across groups after FACS sorting and\nexpansion.\nThe revised Results Section Page 9 199 now reads:\n“To standardize TCR expression, constructs were first titrated in CD4+ T cells, selected for their\nlack of endogenous CD8αβ expression thus forcing the use of CCRs for class I-restricted responses,\nensuring comparable transduction efficiency and transgene expression (Fig. 3B, first 3 panels to the left).\nTransduced T cells were then sorted on similar p/HLA+ binding and expanded (Fig. 3B, right panel).”\nCD4+ transduced to express T with the indicated CCRs after transduction for equal transduction\nTCR-MA1\ncopy numbers and post-REP (rapid expansion protocol) p/HLA multimer binding.\n3-12. Figure 4A: please provide a rationale as to T cells are injected 6 days earlier here than they do for\nthe rest of their experiments? At this time point, the tumors do not look like they would be at a palpable\nsize.\nThe experiment in now revised Figure 4A (injection of T cells at day 12 or 13) allowed the assessment of\nthe interaction between tumor and T cells but only produced a modest prolongation of murine overall\nsurvival (new Extended Figure 8D). However, when the T cell injections were administered 6 days after\ninoculation (Figure 4I, previously Figure 4A, with tumor sizes at inoculation now shown in the new\nExtended Figure 8E, the differences between CD8ab and CD8/28 were evident.\nThe revised Results Section Page 10, lines 229 now reads:\n“While the survival of all mice infused with T on day 13 after A375F engraftment was\nTCR-MA1-CD8/28\nonly modestly prolonged compared to controls (T ) (Extended Fig. 8D), when administered on\nTCR-Irr-CD8/28\nday 6 into mice with measurable tumors (Extended Fig. 8E), T significantly extended survival\nTCR-MA1-CD8/28\ncompared with T (Fig. 4I).”\nTCR-MA1-CD8αβ\ninfused with total 5 x 106 CD4+ and CD8+ T CCR cells (T T T\nTCR-MA1-CD8αβ TCR-Irr.-CD8/28, TCR-MA1-CD8αβ, TCR-MA1-\n) at a E to T ratio of 1:1 13 days post engraftment. Survival analysis (right) was carried out when\nCD8/CD28\ntumor burden reached 500mm3, and assessed tumor progression occurring 37 days vs. 32 days vs. 25 days\npost T cell transfer, respectively; Kaplan-Meier test for analysis of survival applied (left). p values were\ndetermined using a log-rank test. (n=4-6 mice/group).\nExtended Fig. 8 (E) Schematic (left) and tumor volume (right) of 1 x 105 A375F tumors engrafted into NSG\nmice before subsequent transfer 5 x 106 CD4+ and CD8+ T CCR cells equal numbers of T cells\nTCR-MA1-CD8αβ\n(T , T , T )1:1 ratio of combined CD4+ and CD8+ T ) 6 days after\nIrr.-CD8/28 TCR-MA1-CD8αβ TCR-MA1-CD8/28 TCR-MA1-CD8αβ\nengraftment (n=6 mice/group).\nFigure 4 (I) NSG mice were engrafted with 1 x 105 cells A375F and subsequently infused with total 5 x 106\nCD4+ and CD8+ T CCR cells (T T T ) at a E to T ratio\nTCR-MA1-CD8αβ TCR-Irr.-CD8/28, TCR-MA1-CD8αβ, TCR-MA1-CD8/28\nof 1:1 6 days post engraftment. Survival analysis was carried out when tumor burden reached 500 mm3 and\nassessed tumor progression occurring 44 days vs. 23 days vs. 16 days post T cell infusion, respectively;\nKaplan-Meier test for analysis of survival applied. p values determined by a log-rank test. (n=6\nmice/group).\n3-13. Figure 4H: point 1 - again, consider expanding the markers you use to characterize exhaustion.\nSimilar to the changes made to Figure 2D, we expanded the characterization of T cells in Figures 4G and\nExtended Figure 8B to include the co-expression of additional markers associated with exhaustion\n(CD39 with TIM-3, and PD-1 with TIGIT) and added a new Figure 4H assessing the function of\nintratumoral CD4+ and CD8+ T compared toT .\nTCR-MA1-CD8/28 TCR-MA1-CD8αβ\nThe revised Results section Page 10, Line 222 now reads:\n“T cells showed reduced expression of surface markers associated with exhaustion\nTCR-MA1-CD8/28\n(PD1+/Tim3+, CD39+/Tim3+, and PD1+/TIGIT+) (Fig. 4G, Extended Fig. 8C) and produced significantly\nmore cytokines upon cognate peptide exposure ex vivo (Fig. 4H).”\nFigure 4 (G) Bar graph showing PD-1+/Tim3+, CD39+/Tim3+, and PD-1+/TIGIT+ frequency of CD4+ and\nCD8+ TILs between T and T . Single cell suspensions were obtained at sacrifice,\nTCR-MA1-CD8αβ TCR-MA1-CD8/28\n8 days after T cell infusion from (same experiment design as A). p value determined by unpaired two-tailed\nt-test. (n=3 mice, 6 tumors/group, 2 tumors/mouse).\n(H) Quantification of IFNγ+/TNFα+ populations after 1μM cognate peptide stimulation for 18 hours ex vivo\nacross the same experimental groups from (A) in CD4+ (left panel) and CD8+ (right panel) T\nTCR-MA1-CD8αβ\nfor the indicated conditions. Single cell suspensions were obtained at sacrifice, 6 days after T cell infusion.\nThe mean ± SD of 6 tumors per group is shown. Unpaired two-tailed t-test. (n=3 mice, 5-6 tumors/group,\n2 tumors/mouse).\naxis) (top row panels), co-expression of CD39 (x-axis) and Tim-3 (y-axis) (middle row panels), and co-\nexpression of TIGIT (x-axis) and PD-1 (y-axis) (bottom row panels) in CD4+ (left 2 panels) and CD8+\n(right 2 panels) TILs identified in tumor between T and T . Single cell suspensions\nTCR-MA1-CD8αβ TCR-MA1-CD8/28\nwere obtained at sacrifice, 8 days after T cell infusion from (Fig. 4A).\n3-13. Figure 4H: point 2: At this point the tumors are so small that leads one to wonder if the reduced\nTIM3 phenotype they’re seeing is because the cells don’t have anything to interact with anymore yet. The\nauthors mention differences in tumor burden in the results section for figure 5 as a reason for why the\nCD8αβ groups have more proliferation associated genes than the CD8/CD28. Repeat some of these\nstudies but euthanizing mice when the tumors are varying sizes might shed some light on this matter.\nTo address this question, we have updated / added the following figures.\n1) Figure 4A: Additional data points added showing an average tumor volume of 37mm3 for the\nCD8/28 group at the time of mice euthanasia.\n2) New Figure 4G (also see answer to Reviewer 3 Comment 13 part 1): T cell in the CD8/28\ngroup had reduced expression of exhaustion-associated surface markers.\n3) New Figure 4H (also see answer to Reviewer 3 Comment 13 part 1): T cells in the CD8/28\ngroup produced more cytokines ex vivo compared to the CD8αβ group.\nPage 10, line 217 now reads:\n“Mice in the T group markedly reduced tumor size 9 days post-transfer (average tumor\nTCR-MA1-CD8/28\nvolume at euthanasia 37mm3) (Fig. 4A). Tumors from T -treated mice contained significantly\nTCR-MA1-CD8/28\nmore CD4+ and CD8+ T cells per mm2 (Fig. 4B, quantified in Extended Fig. 8B), with higher T cell-to-\ntumor ratios overall (Fig. 4C), and within CD4+ (Fig. 4 D) and CD8+ (Fig. 4E) T cell subsets. The\nCD4+:CD8+ ratio in this group approached 1 (mean 0.872) versus 0.381 in the T group where\nTCR-MA1-CD8αβ\nCD8+ T cells predominated (Fig. 4F). T cells showed reduced expression of surface markers\nTCR-MA1-CD8/28\nassociated with exhaustion (PD1+/Tim3+, CD39+/Tim3+, and PD1+/TIGIT+) (Fig. 4G, Extended Fig. 8C)\nand produced significantly more cytokines upon cognate peptide exposure ex vivo (Fig. 4H). Collectively\n26\nthis data show that T were detected at higher frequency in tumors, potentially due to either\nTCR-MA1-CD8/28\npreferential tumor accumulation or an earlier burst of intra-tumoral expansion. Notably, the remaining\ncells displayed fewer exhaustion-associated features, and exhibited enhanced function compared to T\nTCR-\n.”\nMA1-CD8αβ\nTo link the findings in Figures 4 and 5, the last paragraph associated with Figure 5 was updated.\nPage 12, line 269 now reads:\n“Taken together these observations suggest that combining CD8+ and CD4+ T cells enhances\nproliferation of both subsets, while CD28 co-stimulation promotes cytotoxic signatures, consistent with\ntheir increased ability to produce cytokines (Fig. 4H) and preserves early memory programs, consistent\nwith their reduced expression of exhaustion-associated surface markers (Fig. 4G).\n3-14: Figure 4: Authors claim improved infiltration but only show that there are more cells in the tumor.\nThe increased numbers could be due to increased proliferation and/or Survival but not necessarily\ninfiltration.\nTo avoid over- or misinterpretation, we have revised the manuscript and now refer to these findings more\naccurately as an increase in “The number of CD4+ and CD8+ per mm2 of tumor” rather than “improved\ninfiltration.”\nThe revised manuscript Page 10, lines 219 now reads:\n“Tumors from T -treated mice contained significantly more CD4+ and CD8+ T cells\nTCR-MA1-CD8/28\nper mm2 (Fig. 4B, quantified in Extended Fig. 8B), with higher T cell-to-tumor ratios overall (Fig. 4C),\nand within CD4+ (Fig. 4 D) and CD8+ (Fig. 4E) T cell subsets.”\n3-15. Figure 4G: captions claim that the heightened CD4 to CD8 ratio is indicative of CD4 proliferation.\nThis could be the result of many other mechanisms. CD8 death, for one, in addition to the other concerns\nI’ve mentioned about proliferation.X\nWe have removed language referencing proliferation or speculation on the mechanisms leading to\nincreased T cell numbers/mm2 to avoid over-interpretation. The text in reference to Figure 4G (now\nFigure 4F) and the corresponding Figure Legend have been revised.\nThe Results Section Page 10, line 221 now reads:\n“The CD4+:CD8+ ratio in this group approached 1 (mean 0.872) versus 0.381 in the T group\nTCR-MA1-CD8αβ\nwhere CD8+ T cells predominated (Fig. 4F).”\ntwo-tailed t-test. Analysis performed using HALO Link software on IHC slides. (n=3 mice, 6\ntumors/group, 2 tumors/mouse).\n3-16: Figure 5: They mention that the CD8 T cells from the CD8/28 group has more stem-like gene\nexpression, but that is only in reference to the CD8αβ combo control and not the CD8αβ control. Not sure\nif that is a fair comparison\nWe appreciate the comment. In our scRNA-seq data, CD8/28 T cells display an intermediate phenotype:\nthey up-regulate cytotoxic programs while retaining features consistent with early memory relative to the\nCD8αβ combo control. This suggests that adding CD28 co-stimulation can enhance cytotoxicity yet\npreserve elements of an early memory program. We have now clarified the text by showing first pairwise\ncomparisons between the different CD8+ T cell groups (Fig. 5D and F) with corresponding tumor volumes\n(Fig. 5 E and G) and then the comparison regarding the 3 groups (Extended data Fig. 9C). We note that\nCD4⁺ T cells in the combo conditions likely sustain proliferation and provide help, whereas the single\nCD8αβ control, which lacks CD4⁺ cells, is less proliferative and may therefore maintain some early memory\nfeatures, which could explain why the contrast between CD8/28 and pure CD8αβ is not significant. These\nmechanistic interpretations are hypotheses and will require further study – this is now clearly stated in the\ntext.\nThe Results Section Page 11 line 255 now reads:\n“Relative to CD8+-only cells, CD8+ T cells in the combo CD8αβ group showed enriched cytotoxic\nand proliferative but reduced stem-like signatures (Fig. 5D), aligning with their failure to achieve tumor\nvolume reduction (Fig. 5E). In contrast, CD8+ T cells in the combo CD8/28 group showed enriched\ncytotoxic and stem-like but reduced proliferative signatures (Fig. 5F), correlating with tumor volume\nreduction (Fig. 5G). Although speculative, the lower proliferative signature may reflect diminished antigen\navailability after tumor shrinkage, favoring the acquisition of self-renewing programs. Comparisons across\ngroups suggest that CD8+-only and CD8+ combo CD8/28proliferate and differentiate less than CD8+\ncombo CD8αβ cells, thereby maintaining stem-like features. For CD8+-only, this may reflect absence of\nCD4⁺ help (less proliferative stimulation), whereas for CD8+ combo CD8/28this may be linked to tumor\nburden reduction (Extended Fig. 9C). Cell proportions (Extended Fig. 9D) and absolute numbers\n(Extended Fig. 9E) further support this model: CD8+ in the combo CD8/28 group showed a predominance\nof cytotoxic and stem-like rather than proliferative signatures (tumor burden reduction), whereas the CD8+\nin the combo CD8αβ group displayed cytotoxicity and proliferative rather than stem-like signatures (no\ntumor burden reduction compared to control).\nTaken together these observations suggest that combining CD8+ and CD4+ T cells enhances\nproliferation of both subsets, while CD28 co-stimulation promotes cytotoxic signatures, consistent with\ntheir increased ability to produce cytokines (Fig. 4H) and preserves early memory programs, consistent\nwith their reduced expression of exhaustion-associated surface markers (Fig. 4G).\nLEF1, NELL2), proliferation (MKI67, TOP2A, PCNA), and cytotoxicity (KLRG1, KLRD1, NKG7, GZMB,\nGNLY, PRF1, GZMA, GZMM, CSTA, CSTW) markers across CD8+ T-cell transcriptional states between\nCD8+-alone and CD8+ in T combo(x-axis). Statistical significance was assessed using the\nTCR-MA1-CD8αβ\nWilcoxon rank sum test. Asterisks indicate the following thresholds of significance: *p < 0.05, **p < 0.01,\n***p < 0.001, ****p < 0.0001.\n(E) Tumor burden measurements for NSG mice engrafted with 1 x 105 cells A375F and subsequently\ninfused with total 5 x 106 T cells of CD4+ and CD8+ (1:1 ratio combo) T , CD8+ only T\nTCR-Irr.-CD8αβ TCR-MA1-\n, CD4+ and CD8+ (1:1 ratio combo) T 12 days post engraftment. (n=3 mice, 2\nCD8αβ TCR-MA1-CD8αβ\ntumors/group averaged). p value determined by unpaired two-tailed t-test.(F) Violin plots displaying the\nexpression levels (y-axis) of stem-like (TCF7, SELL, IL7R, LTB, LEF1, NELL2), proliferation (MKI67,\nTOP2A, PCNA), and cytotoxicity (KLRG1, KLRD1, NKG7, GZMB, GNLY, PRF1, GZMA, GZMM, CSTA,\nCSTW) markers across CD8+ T-cell transcriptional states between T and T (x-\nTCR-MA1-CD8αβ TCR-MA1-CD8/28\naxis). Statistical significance was assessed using the Wilcoxon rank sum test. Asterisks indicate the\nfollowing thresholds of significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.\n(G) Tumor burden measurements for NSG mice engrafted with 1 x 105 cells A375F and subsequently\ninfused with total 5 x 106 T cells of CD4+ and CD8+ (1:1 ratio combo) T and CD4+ and CD8+\nTCR-MA1-CD8αβ ,\n(1:1 ratio combo) T and CD4+ and CD8+ (1:1 ratio combo) T 12 days post\nTCR-MA1-CD8/28 TCR-Irr.-CD8/28\nengraftment. (n=3 mice, 2 tumors/group averaged). p value determined by unpaired two-tailed t-test.\n3-17 Figure 6: If exhaustion is being averted then it is unclear why a cluster of exhausted T cells from this\nsequencing data does not show up.\nactivated or cytotoxic T cells, a difficulty noted by others in the field. In our cytotoxic cluster, we\nobserved expression of some exhaustion-associated markers (e.g., TIGIT, DUSP2); however, a distinct\nexhausted T-cell cluster was not clearly separated from the cytotoxic population in our dataset.\nWe have modified the manuscript to acknowledge this point and added relevant references. Page 11, line\n252 now reads:\n“A distinct “exhausted” cluster was not identified, consistent with prior studies showing that\nexhaustion markers can also be expressed in activated T cells, complicating their distinction in scRNA-seq\ndata-sets11-13.”\nGros, A. et al. PD-1 identifies the patient-specific CD8+ tumor-reactive repertoire infiltrating\nhuman tumors. Journal of Clinical Investigation 124, 2246-2259 (2014).\nhttps://doi.org:10.1172/JCI73639\nInozume, T. et al. Selection of CD8+PD-1+ lymphocytes in fresh human melanomas enriches for\ntumor-reactive T cells. J Immunother 33, 956-964 (2010).\nhttps://doi.org:10.1097/CJI.0b013e3181fad2b0\nMietz, J. et al. Human effector CD8(+) T cells with an activated and exhausted-like phenotype\ncontrol tumour growth in vivo in a humanized tumour model. EBioMedicine 106, 105240 (2024).\nhttps://doi.org:10.1016/j.ebiom.2024.105240\n3-18: Figure 6: The lack of in vivo antitumor activity using the TCR-PRAME-CD8/28 mut is critical to\ninclude but missing from these data sets.\nFor this manuscript, we prioritized maintaining the focus on the in vivo validation of the TCR-MAGE\nCD8/28 mut and have included two additional in vivo murine models detailed in the responses to\nReviewer 2, Comments 3 and 4. To show the potential applicability of this approach to other TCRs, we\nhave added the effect of TCR-PRAME CD8/28 mut in vitro targeting 2 different PRAME-expressing\ntumors as well as TCR-WT1 CD8/28mut and moved these to Extended Data Figure 12 (shown below).\nWe have also clarified the title and the text of the paragraph to reflect this point.\nThe Results Section Page 12, line 274 (title) now reads:\n“CD8-CD28 CCR tail mutations enhance anti-tumor activity in MAGE tumor models”\nThe Results Section Page 13, line 301 now reads:\n“To test generalizability, we applied the CD28-mut strategy to PRAME- and WT1-specific TCRs\n(Extended Fig. 12). T displayed slightly improved functional avidity (EC IFNγ 86.78 vs\nTCR-PRAME-CD8/28mut 50\n91.79; TNFα 24.2 vs 38.37nM) compared with T (Extended Fig. 12A & B), while T\nTCR-PRAME-CD8/28 TCR-WT1-\nshowed similar functional avidity to T (EC IFNγ 2.25 vs 0.57; TNFα 1.92 vs 0.26 nM)\nCD8/28mut TCR-WT1-CD8/28 50\n(Extended Fig. 12C &D). However, both T and T achieved superior tumor\nTCR-PRAME-CD8/28mut TCR-WT1-CD8/28mut\ncontrol after repeated tumor challenge in vitro against HLA A2+ PRAME+ PANC1 (pancreatic ductal\nadenocarcinoma), HLA-A2+ PRAME+ A375F and HLA-A2+ WT1+ SW480 (colon adenocarcinoma) lines\ncompared with their non-mutated counterparts (Extended Fig. 12E-G). These findings indicate that CD28\ntail mutations may enhance efficacy beyond TCR across to other TCR therapies.”\nMA1,\n(A) Dose response curves showing IFNγ and TNFα levels in CD4+ T T T\nTCR-PRAME-CD8αα, TCR-PRAME-CD8αβ, TCR-\nand T cells incubated with decreasing concentrations of cognate peptide\nPRAME-CD8/28, TCR-PRAME-CD8/28mut\novernight. (B) Mean EC of IFNγ and TNFα cytokine expression for CD4+ T T\nT and T cells from previous dose response curve (A).\nTCR-PRAME-CD8/28, TCR-PRAME-CD8/28mut\n(C) Dose response curve showing IFNγ and TNFα levels in CD4+ T T T\nTCR-WT1-CD8αα, TCR-WT1-CD8αβ, TCR-WT1-\nand T cells incubated with decreasing concentrations of cognate peptide overnight.\nCD8/28, TCR-WT1-CD8/28mut\n(D) Mean EC of IFNγ and TNFα cytokine expression for CD4+ T T T\nand T .\nCD8/28, TCR- WT1-CD8/28mut\n(E) (left) Growth kinetics of the PANC1 cell line in the presence of CD4+ with the indicated CCR constructs\n(T T T and T ) at a ratio of E:T 5:1. (right)\nTCR-PRAME-CD8αα, TCR-PRAME-CD8αβ, TCR-PRAME-CD8/28, TCR-PRAME-CD8/28mut\nFinal tumor integrated intensity of Incucyte killing assay was analyzed. p values were determined with one-\nway ANOVA with Tukey’s multiple comparison test. (n=3 wells/group).\n(F) (left) Growth kinetics of the A375F cell line in the presence of CD4+ with the indicated CCR constructs\n(T T T and T ) at a ratio of E:T 10:1. (right)\nTCR-PRAME-CD8αα, TCR-PRAME-CD8αβ, TCR-PRAME-CD8/28, TCR-PRAME-CD8/28mut\nFinal tumor integrated intensity of Incucyte killing assay was analyzed. p values were determined by one-\nway ANOVA with Tukey’s multiple comparison test. (n=3 wells/group).\n(G) (left) Growth kinetics of SW480 cell line in the presence of CD4+ with the indicated CCR constructs\n(T T T and T ) at a ratio of E:T 5:1. Final tumor\nTCR-WT1-CD8αα, TCR- WT1-CD8αβ, TCR- WT1-CD8/28, TCR- WT1-CD8/28mut\n31\nintegrated intensity of Incucyte killing assay was analyzed. p values were determined by one-way ANOVA\nwith Tukey’s multiple comparison test. (n=3 wells/group).\n3-19: Figure 7: The manuscript would have benefited from a strong ending (Figures 6 and 7). The lack\nof data showing that the mutant had any beneficial effects in vivo and characterized it with an in vitro\nrepeat stim/sequencing assay. They show upregulation in proliferation and exhaustion related genes but\nfollow this with flow data that only looks at PD1 and TIM3 again for exhaustion, which also had very\nlittle differences between the mutant and wildtype cd8/cd28 groups. Furthermore, they saw no differences\nin cytokine expression after restimulation. Did not show any functional proof of differences in\nproliferation again.\nWe agree with the reviewer’s comments, and we have included two additional in vivo murine models\ndetailed in the responses to Reviewer 2, Comments 3 and 4 regarding the effects on CD8/28mut\ncompared to CD8/28wt. We have also included a new Extended Figure 11C showing Ki-67 expression\non CD4+ and CD8+ T vs T from in vivo tumor IHC analysis.\nTCR-MA1-CD8αβ TCR-MA1-CD8/28\nThe result Section Page 13, line 294 now reads:\n“CD4+ and CD8+ T had higher expression of the Ki67 proliferation marker than\nTCR-MA1-CD8/28mut\nT by IHC (Extended Fig. 11C).”\nTCR-MA1-CD8/28\nExtended Figure 11(C) Immunohistochemistry analysis of Ki-67 expression in CD4+ or CD8+ T cells\nbetween T , T and T groups from (Fig. 6D). p value determined with\nTCR-Irr.-CD8/28 TCR-MA1-CD8/28 TCR-MA1-CD8/28mut\nBrown-Forsythe and Welch ANOVA test with Dunnett’s multiple comparison test. (n=3 mice/group, 2\ntumors per mouse).\n3-20: – point 1: In figures 6 and 7 they did not show any signaling differences for why their mutant might\nbe affecting the T cells differently than their wildtype version of the CD8/CD28. They mention in their\ndiscussion possible signaling molecules that are involved but didn’t look at a single one.\nWe have now included a new figure (Extended Fig. 10B) that examines signaling patterns between the\nwildtype and mutant CD8/CD28 constructs. Specifically, we analyzed phosphorylation of both AKT (p-\nAKT) and ERK\n32\n(p-ERK) as key downstream effectors of CD28-mediated costimulatory signaling and TCR-driven\nactivation14. This approach allows for distinguishing the signaling pathways downstream of the CD28\ndomain in the canonical TCR signaling pathway.\nThe Results Section Page 14, lines 317 now reads:\n“Exposure to cognate peptide-loaded TAP-deficient T2 B lymphoblastoid cell lines (T2-BLCL)\nrevealed higher phosphorylated downstream TCR and CD28 signaling molecules (p)-ERK and (p)-AKT in\nT than T at 5 minutes, with both exceeding T (Extended Fig.\nTCR-MA1-CD8/CD28mut TCR-MA1-CD8/CD28 TCR-MA1-CD8αβ\n13A), consistent with more pronounced early CD28-mediated costimulatory signaling and TCR-driven\nsignaling14.”\nAdachi, K. & Davis, M. M. T-cell receptor ligation induces distinct signaling pathways in naive vs.\nantigen-experienced T cells. Proc Natl Acad Sci U S A 108, 1549-1554 (2011).\nhttps://doi.org/10.1073/pnas.1017340108\nExtended Data Fig. 13 (A) Intracellular expression of (p)-ERK(top) and (p)-AKT(bottom) across CD4+ T\ncells constructs (T , T , and T ) stimulated with 1uM of cognate\nTCR-MA1-CD8αβ TCR-MA1-CD8/28 TCR-MA1-CD8/CD28mut\npeptide at three different time point. Time course (left) and fold change (right) compared to CD8αβ . The\nmean and standard deviation (SD) of triplicate wells from 2 independent experiment is shown. p value\ndetermined by one-way ANOVA with Tukey’s multiple comparison test. n= 6 wells/group, from 2 separate\nexperiments).\n3-20: – point 2: Finally, please citing other relevant work demonstrating the use of CD4 or CD8a\nmolecules modified to express costimulatory domains.\nWe have now referenced three relevant studies that inspired our chimeric co-receptor (CCR) design:\nModification of the CD8α intracellular domain to incorporate MyD88 resulting in enhanced CD8 T-cell\nfunction and anti-tumor activity in vivo, and CD8α homodimers fused with either CD3ζ or CD28\nintracellular domain shown to transmit activation signals upon crosslinking.\nThe Result Section Page 8, line 179 now reads:\n33\n“To achieve ligand-independent signaling, we tethered positive intracellular costimulatory\ndomains to the requisite CD8αβ co-receptor chains, based on findings that TCR chain modifications\nimpaired function (Extended Fig. 6A-C) and prior evidence that CD8α chain modifications can transmit\nactivation signals15-17.\n34"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have satisfactorily addressed all of my concerns, and the revised manuscript has been substantially improved.", "reviewer_2": "The authors have thoroughly addressed the reviewer’s comments, and the revised manuscript demonstrates substantial improvement. I consider it suitable for publication in Nature Communications", "reviewer_3": "The author's have more than adequately addressed each of my critiques. The additional studies outlined in their response, both experimentally and within the body of the manuscript, make for a clearer and more complete report."}, "author_response": ""}]

Decision letter