Neoantigen-specific stem cell memory-like CD4+ T cells mediate CD8+ T cell-dependent immunotherapy of MHC class II-negative solid tumors

CD4+ T cells play key roles in a range of immune responses, either as direct effectors or through accessory cells, including CD8+ T lymphocytes. In cancer, neoantigen (NeoAg)-specific CD8+ T cells capable of direct tumor recognition have been extensively studied, whereas the role of NeoAg-specific CD4+ T cells is less well understood. We have characterized the murine CD4+ T cell response against a validated NeoAg (CLTCH129>Q) expressed by the MHC-II-deficient squamous cell carcinoma tumor model (SCC VII) at the level of single T cell receptor (TCR) clonotypes and in the setting of adoptive immunotherapy. We find that the natural CLTCH129>Q-specific repertoire is diverse and contains TCRs with distinct avidities as measured by tetramer-binding assays and CD4 dependence. Despite these differences, CD4+ T cells expressing high or moderate avidity TCRs undergo comparable in vivo proliferation to cross-presented antigen from growing tumors and drive similar levels of therapeutic immunity that is dependent on CD8+ T cells and CD40L signaling. Adoptive cellular therapy (ACT) with NeoAg-specific CD4+ T cells is most effective when TCR-engineered cells are differentiated ex vivo with IL-7 and IL-15 rather than IL-2 and this was associated with both increased expansion as well as the acquisition and stable maintenance of a T stem cell memory (TSCM)-like phenotype in tumor-draining lymph nodes (tdLNs). ACT with TSCM-like CD4+ T cells results in lower PD-1 expression by CD8+ T cells in the tumor microenvironment and an increased frequency of PD-1+CD8+ T cells in tdLNs. These findings illuminate the role of NeoAg-specific CD4+ T cells in mediating antitumor immunity via providing help to CD8+ T cells and highlight their therapeutic potential in ACT.


Neoantigen-specific stem cell memory-like CD4 + T cells mediate CD8 + T cell-dependent immunotherapy of MHC class II-negative solid tumors
CD4 + T cells play key roles in a range of immune responses, either as direct effectors or through accessory cells, including CD8 + T lymphocytes. In cancer, neoantigen (NeoAg)-specific CD8 + T cells capable of direct tumor recognition have been extensively studied, whereas the role of NeoAg-specific CD4 + T cells is less well understood. We have characterized the murine CD4 + T cell response against a validated NeoAg (CLTC H129>Q ) expressed by the MHC-II-deficient squamous cell carcinoma tumor model (SCC VII) at the level of single T cell receptor (TCR) clonotypes and in the setting of adoptive immunotherapy. We find that the natural CLTC H129>Q -specific repertoire is diverse and contains TCRs with distinct avidities as measured by tetramer-binding assays and CD4 dependence. Despite these differences, CD4 + T cells expressing high or moderate avidity TCRs undergo comparable in vivo proliferation to cross-presented antigen from growing tumors and drive similar levels of therapeutic immunity that is dependent on CD8 + T cells and CD40L signaling. Adoptive cellular therapy (ACT) with NeoAg-specific CD4 + T cells is most effective when TCR-engineered cells are differentiated ex vivo with IL-7 and IL-15 rather than IL-2 and this was associated with both increased expansion as well as the acquisition and stable maintenance of a T stem cell memory (T SCM )-like phenotype in tumor-draining lymph nodes (tdLNs). ACT with T SCM -like CD4 + T cells results in lower PD-1 expression by CD8 + T cells in the t umor m ic roenvironment and an increased frequency of PD-1 + CD8 + T cells in tdLNs. These findings illuminate the role of NeoAg-specific CD4 + T cells in mediating antitumor immunity via providing help to CD8 + T cells and highlight their therapeutic potential in ACT.
Neoantigen (NeoAg)-specific T cells are frequently observed in the tumor microenvironment (TME) and periphery of human patients with cancer before and during treatment with immunotherapies such as immune checkpoint blockade (ICB) and personalized cancer vaccines [1][2][3][4][5][6][7][8] . While it is known that NeoAg-specific CD8 + T cells are capable of directly recognizing and destroying tumor cells, clinical responses have also been observed in patients receiving adoptive cellular therapy (ACT) with autologous tumor-infiltrating lymphocytes Article https://doi.org/10.1038/s41590-023-01543-9 memory (T SCM )-like phenotype in TCR-engineered CLTC H129>Q -specific CD4 + T cells, enabling these cells to effectively control tumors in the therapeutic setting, highlighting the clinical relevance of TCR-engineered CD4 + T cells recognizing tumor-derived NeoAg.

Expansion of CLTC H129>Q -specific CD4 + T cells correlates with protective whole-cell vaccination
We have previously demonstrated that vaccination with irradiated SCC VII cells and adjuvant polyI:C is protective against subsequent live tumor challenge. SCC VII-immune mice generate CD4 + and CD8 + NeoAg-specific T cell responses, which includes recognition of a mutated clathrin heavy chain epitope (CLTC H129>Q ) by CD4 + T cells 16 . To identify TCRs from CLTC H129>Q -specific CD4 + T cells, C3H/HeJ mice were immunized with irradiated SCC VII tumor cells admixed with polyI:C and challenged 14 d later with live SCC VII cells. At 14 d after tumor challenge, splenocytes from immune mice were isolated and stained with a CLTC H129>Q /I-A K tetramer ( Fig. 1a and Extended Data Fig. 1a). Consistent with our previous ELISpot results, tetramer-positive CD4 + (TILs) containing NeoAg-specific CD4 + T cells, suggesting that CD4 + T cells also play a crucial role in directing tumor immune responses [9][10][11] . In human melanoma, infiltration of NeoAg-specific CD4 + T cells is associated with antitumor effector phenotypes of macrophages, B cells and CD8 + T cells 12 . Several mechanisms of antitumor immunity mediated by CD4 + T cells have been proposed by studies in mouse models, including direct cytotoxicity dependent on recognition of MHC-II + tumor cells, local secretion of effector cytokines in the TME and providing T cell help for CD8 + T cells [13][14][15] ; however, how key characteristics such as TCR avidity and cellular differentiation states impact CD4 + T cell-mediated antitumor immunity remains unknown.
In the present study, we identified four distinct TCR clonotypes recognizing an epitope derived from a mutated clathrin heavy chain gene (CLTC H129>Q ) in the SCC VII tumor model. We found that CLTC H129>Q -specific T cell receptors (TCRs) differed in their avidity for antigen but were nonetheless similarly able to undergo antigen-dependent expansion in vivo and provide CD8 + T cell-and CD40L-dependent protection from tumor challenge. Furthermore, treatment with interleukin (IL)-7 and IL-15 induced a durable T stem cell d, TCR β-chain diversity of tetramer-sorted T cells. e, CDR3 sequences for α-and β-chains of clonally expanded T cells. f, Concentration-response curves of IFN-γ production by primary CD4 + T cells retrovirally transduced with each CLTC H129>Qspecific TCR stimulated with splenocytes pulsed with either mutant or wild-type peptides. Data are representative of three independent experiments.

Identification of CLTC H129>Q -specific CD4 + T cell clonotypes
To isolate CLTC H129>Q -specific CD4 + T cells, single tetramer-positive cells from challenged and protected mice were sorted into 96-well plates and the CDR3 regions of both TCR α/β-chains were amplified by PCR as previously described 17 . Sequencing of complementary DNA libraries revealed three expanded TCR β clonotypes represented among tetramer-sorted cells (Fig. 1d). These three TCR β-chains paired with four distinct α-chains, corresponding to four unique expanded T cell clones (Fig. 1e). Of note, TCR 1 and TCR 2 shared the same TCR β-chain and nearly identical α-chains, which differ at a single alanine to proline substitution within the CDR region. To confirm TCR surface assembly and specificity, each α/β receptor was expressed via retroviral transduction of naive primary C3H CD4 + T cells and tested for recognition of wild-type versus H129 > Q forms of the CLTC 119-133 peptide. CD4 + T cells expressing each of the four TCRs produced interferon (IFN)-γ when stimulated with splenocytes pulsed with the H129 > Q peptide ( Fig. 1f) but produced less or no detectable IFN-γ in response to the wild-type epitope.

CLTC H129>Q -specific TCRs differ in avidity
Next, we set out to compare the functional characteristics of the CLTC H129>Q -specific TCRs. For all four populations of transduced primary CD4 + T cells, >79% of cells expressed the introduced TCR as evidenced by staining for the associated TCR β-chain variable regions (TRBVs) (Fig. 2a).
Gating on the TRBV-expressing cells revealed a consistent difference in tetramer binding, with both a greater frequency and magnitude of tetramer binding observed for cells expressing TCRs 1 and 2 than those expressing TCRs 3 and 4 (Fig. 2a). Indeed, the median fluorescence intensity (MFI) of tetramer binding for cells expressing TCR 1 was significantly greater than that of cells expressing either TCR 3 or TCR 4 (22.8× and 12.1× greater, respectively) ( Fig. 2b and Extended Data Fig. 1b). To further investigate the avidity differences between TCRs, we incubated transduced primary T cells with a titration of tetramer concentrations and measured the percent of maximal fluorescence at each concentration (Fig. 2c). These experiments were consistent with our initial observations, indicating that both TCRs 1 and 2 had significantly lower tetramer half-maximum effective concentration (EC 50 ) values than TCRs 3 and 4 ( Fig. 2d). To study the TCR-binding properties in the absence of CD4, we transduced the TCR-deficient CD8 + T cell hybridoma line 58α − β − with each TCR. The 58α − β − cells expressing TCRs 1 and 2 were able to bind tetramer independently of the CD4 co-receptor, whereas cells expressing TCRs 3 and 4 did not, despite comparable levels of TCR expression (Fig. 2e). In conclusion, the CLTC H129>Q -specific CD4 + T cell pool contains T cell clones expressing both high and moderate avidity antigen receptors.

Differences in TCR avidity do not correlate with differences in proximal TCR signaling in vitro
Given the differences in TCR avidity observed, we investigated whether these correlate with proximal TCR signaling, as has been demonstrated in studies of tumor-specific CD8 + T cells 18 . To measure levels of TCR signaling, we stained permeabilized TCR-expressing primary CD4 + T cells for phosphorylated ERK1/2 (pERK1/2) after a brief stimulation period with peptide-pulsed splenocytes. While all four groups of TCR-expressing CD4 + T cells expressed higher levels of pERK1/2 after stimulation with peptide-pulsed splenocytes compared to splenocytes without added peptide, there were no significant differences in the percentage of activated cells between different TCRs (Fig. 2f). These results suggest that differences in CLTC H129>Q -specific TCR avidity do not correlate with differences in proximal TCR signaling.

Expansion and activation of CLTC H129>Q -specific CD4 + T cells in vivo is TCR avidity independent
To investigate how differences in TCR avidity may impact antigen-specific responses in vivo, we transferred equal numbers of CellTrace Violet (CTV)-labeled CD4 + T cells expressing either TCR 1 or TCR 3 into either naive mice or mice that were subsequently challenged with SCC VII. In this experimental system we were able to differentiate between cells expressing TCR 1 or TCR 3 as either CD90.1 + TRBV8.3 + or CD90.1 + TRBV8.3 − , respectively (Fig. 3a,b). Antigen-specific expansion of adoptively transferred cells was apparent within 3 d, as the frequency of total CD90.1 + cells in tumor-draining lymph nodes (tdLNs) increased significantly in mice challenged with live SCC VII cells compared to naive animals (Fig. 3c). We found that cells expressing TCR 1 or TCR 3 proliferated to a similar extent in the context of antigen derived from live tumor cells, with no significant differences in the number of expanded CTV low cells in mice challenged with SCC VII (Fig. 3d). Neither CD4 + T cell population proliferated significantly in naive mice, suggesting that TCR 1 does not recognize the wild-type CLTC epitope in vivo despite producing IFN-γ in response to splenocytes pulsed with high concentrations of the corresponding peptide (Figs. 3d and 1f). Consistent with these results, the relative frequency of cells expressing either TCR 1 or TCR 3 did not change significantly in either naive mice or mice challenged with SCC VII compared to their starting frequencies (Fig. 3e). In addition, both TCR 1-and TCR 3-expressing cells upregulated similar levels of the acute activation marker CD69 in an antigen-specific manner (Extended Data Fig. 2a,b). Altogether, these results suggest that NeoAg-specific CD4 + T cells with distinct TCRs behave similarly in vivo independent of TCR avidity. Next, we investigated the impact of TCR avidity on T cell activation in the TME. tdLNs and tumors were collected from mice 10 d after SCC VII challenge to assess the relative frequency and activation phenotype of cells expressing either TCR 1 or TCR 3 (Fig. 3f). Consistent with our results with cells collected after 3 d, there was no significant difference in the relative frequencies of cells expressing each TCR in the tdLN or TME after 10 d (Fig. 3g). Expression of co-inhibitory markers such as PD-1 is a hallmark of both tumor-reactive CD4 + and CD8 + T cells in the TME. We determined that cells expressing either TCR 1 or TCR 3 expressed similar levels of PD-1 in the TME and both expressed significantly more PD-1 than CD90.1 − host CD4 + T cells ( Fig. 3h and Extended Data Fig. 2c). These results suggest that the observed differences in TCR avidity do not influence CD4 + T cell persistence or activation in the TME. Given that PD-1 upregulation occurs downstream of TCR signaling, these results also suggest that CLTC H129>Q -specific CD4 + T cells are recognizing antigen in the TME as well as the tdLNs. Data represent four independent experiments, **P = 0.0070 TCR 1 versus TCR 3, 0.0034 TCR 1 versus TCR 4 one-way ANOVA with Tukey correction for multiple comparisons. e, Flow cytometry plots demonstrating TCR expression and tetramer binding of CD8 + CD4 − 58α − β − cells expressing each TCR. Data are representative of three independent experiments. f, Representative flow cytometry plots of pERK1/2 staining of T cells expressing TCR 1 stimulated with naive splenocytes pulsed with dimethylsulfoxide (DMSO) or 1 µg ml −1 CLTC H129>Q peptide (left). Quantification of pERK1/2 staining for each TCR 5 min after stimulation with naive splenocytes pulsed with DMSO or 1 µg ml −1 CLTC H129>Q peptide. Data represent three independent experiments. All data represent mean ± s.e.m. Article https://doi.org/10.1038/s41590-023-01543-9 CLTC H129>Q -specific CD4 + T cell protect from live tumor challenge in a CD40L and CD8 + T cell-dependent manner We sought to determine whether activated CLTC H129>Q -specific CD4 + T cells could protect from live tumor challenge. Notably, SCC VII tumor cells do not express MHC-II and in vitro treatment with IFN-γ did not induce MHC-II expression, suggesting that CLTC H129>Q -specific CD4 + T cells are unable to directly recognize tumor cells even under inflammatory conditions in vivo (Fig. 4a). Notably, transfer of 3 × 10 6
This protection was antigen-specific, as mice receiving 3 × 10 6    of durable immune memory. Protection by CLTC H129>Q -specific CD4 + T cells was also dependent on the frequency of these cells at the time of tumor challenge, as mice receiving either 1 × 10 6 or 3.3 × 10 5 cells before challenge had decreased rates of survival after inoculation of SCC VII-GFP/Luc cells (Fig. 4e). Given that SCC VII tumor cells do not express MHC-II (Fig. 4a), we reasoned that CLTC H129>Q -specific CD4 + T cell-mediated protection may be dependent on their role as helper cells for CD8 + T cells 19 . Consistent with this, depletion of CD8 + T cells before the transfer of CD4 + T cells and tumor inoculation prevented tumor rejection (Fig. 4f). Recent work has demonstrated that CD4 + T cells provide help to CD8 + T cells by CD40L-dependent licensing of cDC1s during the primary tumor response 20 . Upon stimulation with CLTC H129>Q peptide-pulsed splenocytes in vitro, CD4 + T cells expressing TCR 1 upregulated CD40L, suggesting that this mechanism may be responsible for protection from tumor challenge (Extended Data Fig. 3). To interrogate the role of this pathway in our model, we administered anti-CD40L-blocking antibodies on the day of tumor implantation and 2 d later. Mice receiving 3 × 10 6 activated CLTC H129>Q -specific CD4 + T cells along with anti-CD40L were no longer protected from tumor challenge (Fig. 4g). Together, these data demonstrate that NeoAg-specific CD4 + T cells mediate antitumor immunity by helping endogenous CD8 + T cells via CD40L signaling.
These findings prompted us to further probe the efficacy of CD4 + T cells expressing either TCR 1 or TCR 3. As antitumor efficacy was dependent on CD40L help for CD8 + T cells, we used the SCC VII parental cell line for all future experiments to rule out the contribution of CD8 + T cells specific for epitopes contained within the GFP or Luc reporter proteins. While transfer of 3 × 10 6 cells expressing TCR 1 was no longer sufficient for complete tumor rejection against the less-immunogenic parental cell line, tumor growth was still significantly delayed compared to untreated mice or mice receiving 3 × 10 6 polyclonal activated CD4 + T cells (Fig. 4h). There was no significant difference in the rate of tumor growth for mice receiving CD4 + T cells expressing TCR 1 or TCR 3, however, suggesting that clones with differences in TCR avidity can mediate similar CD40L-and CD8-dependent antitumor functions in vivo.

TCR-engineered CLTC H129>Q -specific CD4 + T cells mediate therapeutic immunity
Given the observation that CLTC H129>Q -specific CD4 + T cells could contribute to primary tumor immunity, we investigated the potential of TCR-engineered T cells to limit tumor burden in the context of therapeutic ACT against large established tumors. In preliminary experiments, we found that ACT with CD4 + T cells expressing TCR 1 did not significantly improve survival after tumor challenge compared to mice receiving non-transduced T cells, despite evidence that adoptively transferred cells were able to proliferate in the tdLNs and infiltrate tumors (Extended Data Fig. 4). We therefore sought to improve the function and survival of the engineered cells. Studies suggest that CD8 + T cells primed under conditions that preferentially generate less-differentiated stem cell memory T (T SCM ) cells have a greater capacity for persistence and tumor destruction when adoptively transferred into tumor-bearing animals 21 . The common γ-chain cytokines IL-7 and IL-15 have been demonstrated to preferentially generate and expand T SCM -like cells from ex vivo-stimulated, naive human CD8 + T cells 22 . We therefore sought to determine whether culturing our TCR-engineered CD4 + T cells in IL-7/IL-15, rather than IL-2, could generate T SCM -like cells for ACT (Fig. 5a). CD4 + T cells cultured in IL-7/IL-15 after TCR transduction demonstrated a significant increase in the frequency of CD44 − CD62 + Sca-1 hi cells, consistent with a T SCM -like phenotype ( Fig. 5b and Extended Data Fig. 5a-d). Cells cultured in IL-7/IL-15 were also less proliferative in vitro compared to cells cultured in IL-2 (Extended Data Fig. 5e). Despite these differences in in vitro phenotype, the TCR transduction efficiency was comparable between both treatment groups as determined by CD90.1 expression (Extended Data Fig. 5f).
To compare the in vivo expansion and therapeutic efficacy of IL-7/IL-15-treated T SCM -like cells with those cultured in IL-2, we transferred equal numbers (3 × 10 6 ) of TCR-engineered cells from either culture condition into mice with established SCC VII tumors 1 d following treatment with the lymphodepleting chemotherapy agent cyclophosphamide (Cy) (Fig. 5c). Notably, as soon as 4 d after transfer there was a roughly tenfold increase in the frequency of CD90.1 + cells in the peripheral blood of mice receiving the IL-7/ IL-15-treated cells, with this population peaking in size 9 d following adoptive transfer (Fig. 5d,e). Furthermore, animals receiving the T SCM -like CD4 + T cells had significantly delayed tumor growth compared to mice receiving the same number of T cells expanded in IL-2 (Fig. 5f). ACT with CD4 + T cells cultured in IL-7/IL-15, but not IL-2, provided a significant increase in survival beyond the direct effects of the Cy chemotherapy (Fig. 5g). Consistent with our previous results, therapeutic ACT with T SCM -like CD4 + T cells expressing TCR 3 delayed tumor growth similarly to ACT with cells expressing TCR 1 (Fig. 5h,i). These results suggest that TCR-engineered NeoAg-specific CD4 + T cells differentiated in IL-7/IL-15 can be effective as a therapeutic ACT treatment.

T SCM -like CLTC H129>Q -specific CD4 + T cells are maintained in the tdLNs
To further investigate the cellular programs of therapeutic CLTC H129>Q -specific T SCM -like cells in vivo, CD90.1 + CD4 + T cells were sorted from tdLNs and TILs 9 d after adoptive transfer of CD4 + T cells expressing TCR 1 differentiated in either IL-2 or IL-7/IL-15. A significant increase in the frequency of CD90.1 + CD4 + T cells was observed in both the tdLNs and TILs of mice receiving IL-7/IL-15-conditioned T cells compared to those cultured in IL-2, consistent with trends in the peripheral blood (Fig. 6a,b). Next, we performed bulk RNA sequencing (RNA-seq) to compare the transcriptomic features of cells treated with IL-2 or IL-7/IL-15 in these two tissue compartments. Principal-component analysis (PCA) revealed clustering of tdLN samples and TIL samples together, suggesting that the microenvironment promotes consistent transcriptomic features independent of cytokine treatment (Fig. 6c). To assess tissue-specific genetic signatures, we investigated differentially expressed genes between all TIL and tdLN samples regardless of cytokine treatment. Among genes differentially expressed in the tdLNs were tcf7 and sell, markers of memory T cells, whereas cells from TILs expressed genes associated with effector functions, including type 1 helper T (T H 1) cytokines (tnf), TCR-signaling pathway components (fos) and cytotoxicity genes (prf1, gzmc, gzme and gzmf) (Fig. 6d). Therefore, we decided to further investigate the memory phenotypes of adoptively transferred cells in the tdLNs and TILs by flow cytometry. Consistent with our RNA-seq data, we identified memory subpopulations corresponding to effector memory (T EM ) cells (CD44 + CD62L − ), central memory T (T CM ) cells (CD44 + CD62L + ) and T SCM cells (CD44 − CD62L + ) within the tdLNs (Fig. 6e). Notably, we identified a population of CD90.1 + T SCM cells within the lymph nodes of mice receiving cells treated with IL-7/ IL-15 that was almost entirely absent in mice receiving IL-2-treated cells (Fig. 6f). In both groups, CD90.1 + cells within the tumor were almost entirely of the T EM phenotype, with little to no apparent expression of CD62L (Fig. 6g,h). These data suggest that adoptively transferred in vitro-generated T SCM cells are selectively maintained as a reservoir in the tdLNs, where they give rise to more differentiated subsets capable of trafficking to the TME.

ACT with CLTC H129>Q -specific CD4 + T cells alters host CD8 + T cell phenotypes
To determine whether therapeutically transferred CLTC H129>Q -specific CD4 + T cells are modulating CD8 + T cell immunity, we investigated the phenotypes of CD8 + T cells in the TILs and tdLNs of mice receiving therapeutic ACT. While Cy is lymphodepleting, other studies using a similar dose of the drug demonstrate that full lymphocyte recovery Article https://doi.org/10.1038/s41590-023-01543-9 is apparent between 5-10 d following treatment 23,24 , suggesting that endogenous host lymphocytes are available for interactions with adoptively transferred CD4 + T cells. In the TME, there were no significant differences in the percentage of CD8 + T cells expressing PD-1 (Fig. 7a,b). Given that higher levels of PD-1 expression correlate with terminally exhausted cell states 25 , we also investigated the level of PD-1 expression by analyzing the MFI of PD-1 + CD8 + T cells. We found that despite the similar absolute frequency of PD-1 + cells, CD8 + TILs from mice treated with T SCM -like CD4 + T cells cultured in IL-7/IL-15 expressed significantly lower levels of PD-1 compared to CD8 + T cells from mice treated with Cy alone, suggesting that these CD8 + T cells may be less terminally exhausted (Fig. 7c). Given that CD4 + T cell help for the priming of antigen-specific CD8 + T cells is believed to occur in the local lymph nodes, we also investigated CD8 + T cell phenotypes in the tdLNs of mice receiving therapy. Specifically, we again looked at PD-1 expression, which is known to correlate with stem-like, tumor-specific T cells in the tdLNs 26 . Notably, we found a significant increase in the frequency of PD-1 + CD8 + T cells in the tdLNs of mice receiving adoptively transferred cells cultured in IL-7/IL-15 as compared to mice receiving cells cultured in IL-2 (Fig. 7d). Furthermore, there was a significant positive correlation between the frequency of CD90.1 + adoptively transferred CD4 + T cells and the frequency of PD-1 + CD8 + T cells in lymph nodes from both IL-2 and IL-7/IL-15-treated mice (Fig. 7e). Overall, these data suggest that adoptively transferred T SCM -like CD4 + T cells recognizing CLTC H129>Q are actively involved in the priming of tumor-specific CD8 + T cells in the tdLNs.
To confirm the role of host CD8 + T cells in therapeutic ACT experiments, we treated mice with IL-7/IL-15-treated CD4 + T cells expressing TCR 1 with or without concurrent antibody-mediated depletion of CD8 + T cells. While initially, following treatment, CD8-depleted mice exhibited delayed tumor growth compared to mice treated with Cy alone, ultimately CD8-depleted mice had significantly increased tumor burden relative to treated mice without depletion (Fig. 7f,g). These results confirm that endogenous host CD8 + T cells are required for therapeutic efficacy of ACT with CLTC H129Q -specific CD4 + T cells.

Discussion
In this study we have analyzed an oligoclonal CD4 + T cell response to a naturally arising tumor NeoAg at the level of TCR usage and functionality. Although there has been a greater emphasis on CD8 + T cell responses in this context, perhaps due to the fact that they can directly recognize most tumors and the comparative ease in identifying the target NeoAgs presented by MHC-I versus MHC-II, the fact that CD4 + T cells are crucial for the priming and regulation of CD8 + T cells suggests that a deeper understanding of their response to cancer could significantly improve existing immunotherapies, including ACT. In this study, we investigated how differences in TCR characteristics and T cell functional states impact the efficacy of ACT with NeoAg-specific CD4 + T cells.
Cloning multiple CLTC H129>Q -specific TCRs from SCC VII-immune mice allowed us to investigate how TCR-binding kinetics may impact T cell-intrinsic and extrinsic factors contributing to the antitumor immune response. We demonstrate that while CLTC H129>Q -specific TCR 3 has a comparatively lower avidity for peptide-MHC complexes than TCR 1, CD4 + T cells expressing either TCR are similarly able to transduce TCR signaling, proliferate in vivo, contribute to CD8 + T cell-and CD40L-dependent primary tumor immunity and provide therapeutic tumor control. These data suggest that across the range investigated in this study, TCR avidity does not significantly affect cell-intrinsic TCR signaling or cell-extrinsic interactions providing CD40L stimulation to accessory antigen-presenting cells (APCs), as is likely required for effective licensing of dendritic cells and subsequent priming of CD8 + T cells in our model. These results are consistent with studies in chronic infection models suggesting that tetramer-binding avidity may not correlate with CD4 + T cell function or fate. Specifically, a study of lymphocytic choriomeningitis virus infection in mice found that tetramer-negative low affinity CD4 + T cells exist at a similar frequency to tetramer-positive cells and contribute inflammatory cytokines during the effector phase 27 . Other studies suggest that the off-rate (K off ) of TCR interactions with peptide-MHC complexes may play a role in the commitment of individual clonotypes to distinct T H cell and memory lineages, whereas TCR avidity as measured by tetramer-binding studies alone did not correlate with either 28,29 . Notably, recent studies of MHC-I-restricted TCRs recognizing tumor antigens with a similar range of tetramer-binding capacity to those in our study did observe a correlation between TCR avidity and T cell functions in vitro and in vivo, suggesting that the possibility that CD4 + and CD8 + T cell subsets and their respective functions have different TCR avidity requirements 18,30 .
One limitation of our study is the requirement for T cell activation before TCR expression by retroviral transduction; we were therefore unable to determine whether TCR avidity differentially regulates T cell activation and priming of naive T cells during primary tumor immunity. Furthermore, the TCRs identified in this study were isolated from polyclonal CD4 + T cells following tumor rejection, likely reflecting a memory population; TCRs collected from the effector phase during tumor growth may have more diverse functional properties. It is notable that SCC VII tumor cells do not express MHC-II, even after treatment with IFN-γ. While transcriptomic signatures corresponding to cytotoxic CD4 + T cells have been identified in patients with melanoma and bladder cancer 31,32 and direct tumor recognition by CD4 + T cells has been observed 33,34 , studies also suggest that only a small subset of melanomas harbor any MHC-II + cells 35 . Even among MHC-II + tumor cells, endogenous antigens are selectively presented by MHC-II, which may limit direct recognition of critical tumor NeoAg 36 . A recent study from Rosenberg and colleagues demonstrated that among 20 confirmed NeoAg-specific TCRs isolated from CD4 + TILs found in human tumors, none was able to directly recognize autologous tumor cells 37 . Despite the inability of SCC VII cells to express MHC-II, cells expressing TCR 1 within the TME were enriched for transcripts associated with cytotoxicity (prf1, gzmc, gzme and gzmf), suggesting that this transcriptomic signature may be broadly associated with local CD4 + effector T cell differentiation even in cases where such cells cannot directly engage tumor cells. Overall, MHC-II − solid tumor models such as SCC VII may more closely model human disease and are an important alternative to murine cancer cells with inducible or constitutive MHC-II expression.
While SCC VII cells do not express MHC-II, ACT with T SCM -like TCR-engineered CD4 + T cells generates effective therapeutic immunity in the context of established solid tumors that is dependent on CD8 + T cells and likely mediated by APC activation ('licensing') via CD40/ CD40L interactions 38 . Several studies now implicate migratory cDC1 as the recipient of CD40L stimulation from CD4 + T cells in humans and mice 20,39,40 , suggesting that these are the relevant APC in our study. Consistent with our hypothesis of improved CD8 + T cell priming, we observed a significant increase in the frequency of PD-1 + CD8 + T cells in the tdLNs during effective therapeutic ACT. In addition, CD8 + TILs in mice treated with T SCM -like CD4 + T cells expressed lower levels of PD-1 in the TME, consistent with previously published results suggesting that CD8 + T cells primed in the absence of help express higher levels of co-inhibitory receptors 41 . While our results implicate a role for T cell help in the tdLNs, we cannot rule out additional cooperative functions locally within the TME between newly primed CD8 + T cells and NeoAg-specific CD4 + T cells, such as CD4 + T cell-derived IL-2 and IFN-γ, which serve to support CD8 + T cell survival in and recruitment to tumors, respectively 42 . Indeed, our data suggest that adoptively transferred CD4 + T cells are also capable of recognizing antigen in the TME, where they express a transcriptomic signature associated with effector function and differentiation. In addition, given that CD8 + T cell depletion does not completely abrogate antitumor efficacy in our therapeutic models, it is likely that additional mechanisms beyond APC licensing, such as local effector cytokine secretion, are required.
To improve the persistence of adoptively transferred CD4 + T cells, we administered Cy as a lymphodepletion regimen before ACT. In addition to depleting inhibitory lymphocytes and increasing homeostatic cytokine levels, lymphodepleting regimens have been reported to promote the release of innate immune ligands such as lipopolysaccharide (LPS) from gut microbiota, which potentiates host dendritic cell activation 43 . Notably, C3H/HeJ mice have a missense mutation in TLR4, which likely reduces the impact of this signaling axis following lymphodepletion and may therefore underestimate the efficacy of Cy in our model.
The superior efficacy of IL-7/IL-15-treated CD4 + T cells merits further investigation. While previous studies have demonstrated an enhanced capacity for expansion and therapeutic efficacy of CD4 + T cells primed in the presence of IL-7 or expressing a constitutively active mutant of STAT5 (refs. 44,45), our study extends these findings by demonstrating that IL-7/IL-15-treated CD4 + T cells adopt a unique surface phenotype in vitro associated with T SCM -like cells, which has mainly described in the context of tumor-specific CD8 + T cells. Other cytokines and treatments, such as IL-21, IL-9 and Wnt pathway inhibitors, have been described as inducers of T SCM -like CD8 + T cells and may similarly promote this phenotype in CD4 + T cells 21,46,47 . While IL-7 and IL-15 were used at a single concentration in our study, it is possible that altering the concentration of these cytokines may improve the yield of T SCM -like cells in this context. Our data demonstrate that following adoptive transfer, TCR-engineered T SCM -like cells generated in culture with IL-7/IL-15 maintain a reservoir in the tdLNs accounting for nearly a quarter of all CD90.1 + cells, whereas tumor-infiltrating cells express a differentiated effector memory phenotype. Several recent studies have highlighted the tdLNs as a crucial site for the maintenance of CD8 + tumor-specific T cells expressing memory-associated features, which seem critical for responses to ICB 26,48,49 . Our results suggest that these tdLN-resident memory cell populations may be effectively installed via ACT in patients lacking a sufficient tumor-specific memory cell reservoir and highlight the potential for combination therapies with ICB.
ACT with NeoAg-specific CD4 + T cells may have important advantages over ACT with CD8 + T cells. First, studies suggest that in both murine models and human cancers, NeoAg-specific CD4 + T cells may be more abundant than NeoAg-specific CD8 + T cells. In preclinical and clinical studies of personalized cancer vaccines, epitopes selected for binding to MHC-I perhaps surprisingly predominantly gave rise to CD4 + T cell responses 6,50 . In our own functional NeoAg screening approach applied to SCC VII, which does not leverage bioinformatic MHC binding predictions, we identified four MHC-II-restricted NeoAg and only one MHC-I-restricted NeoAg following vaccination with irradiated tumor cells 16 . This suggests that there may be a relative abundance of MHC-II-restricted TCRs available for immunotherapy, including previously identified TCRs specific for shared oncogenic driver mutations such as BRAF V600E 11 , KRAS G12V 36,51 and G12D 4 and IDH1 R132H 52 . In addition, by operating independently of direct tumor recognition, such as by marshaling a polyclonal CD8 + T cell response, ACT with CD4 + T cells may circumvent immune escape mechanisms associated with monoclonal CD8 + T cell ACT 53,54 . Overall, our study demonstrates the Article https://doi.org/10.1038/s41590-023-01543-9 efficacy of ACT with NeoAg-specific CD4 + T cells in a physiologically relevant tumor model and brings new insights to the use of similar approaches for adoptive immunotherapy of human cancer to empower more diverse, potent and durable antitumor immune responses.

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Animals
Female C3H/HeJ mice (The Jackson Laboratory) were used in these experiments. Animals were 8-12 weeks of age and maintained/ bred in The La Jolla Institute for Immunology vivarium under specific-pathogen-free conditions in accordance with guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International and animal studies were approved by The La Jolla Institute for Immunology Institutional Animal Care and Use Committee.

Cell culture
The squamous cell carcinoma VII San Francisco line (SCC VII) spontaneously arose from the abdominal wall of a C3H mouse in the laboratory of Herman Suit (Harvard University) and was adapted for partial in vitro growth by K.K. Fu and K.N. Lam (University of California). SCC VII was maintained for a maximum of three passages in vitro in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and penicillinstreptomycin (100 U ml −1 each, Gibco). To regenerate SCC VII P 0 cells, C3H/HeJ mice were subcutaneously (s.c.) injected with 5 × 10 5 cells in 1× HBSS and tumors were collected 14 d after inoculation. Tumor tissue was dissociated with a mouse Tumor Dissociation kit (Miltenyi Biotec) followed by passage through a 70-µm cell strainer (Fisher Scientific) and homogenized cells were re-seeded in vitro. For generation of SCC VII expressing luciferase and copepod-derived GFP (SCC VII-Luc/ GFP), cells were transduced with the BVLIV713VA-1 HIV lentiviral vector (System Biosciences) under 10 µg ml −1 puromycin selection and further purified using GFP + FACS-sorting. The 58α − β − hybridoma cell line was cultured in RPMI medium supplemented with l-glutamine and HEPES (10 mM, Gibco), 10% FBS, 1 mM sodium pyruvate (Gibco), 1× MEM Non-Essential Amino Acids (Gibco) and penicillin-streptomycin (100 U ml −1 each, Gibco).

Whole-cell vaccination and tumor challenge
Whole tumor cell vaccination experiments were conducted via s.c. injection of 10 × 10 6 50 Gy-irradiated SCC VII cells in 1× HBSS with 50 µg polyI:C (Thermo Fisher). Immunized mice were challenged 14 d later by s.c. injection of 5 × 10 5 live SCC VII cells.

Preparation of single-cell suspensions
Spleens, inguinal lymph nodes and tumors were surgically removed at experiment end points. Spleens were dissociated manually and cell suspensions were passed through a 70-µm strainer. Before use as APCs in in vitro assays, red blood cells were lysed with ACK lysis buffer (Thermo Fisher). Inguinal lymph nodes and subcutaneous tumors were minced into small (<2 mm) pieces with dissection scissors. Tissue fragments were enzymatically dissociated in 20 µg ml −1 Liberase (Roche) and 20 µg ml −1 DNase I (Roche) at 37 °C for 30 min. Single cells were passed through a 70-µm strainer.

TCR sequencing
Single tetramer-positive CD4 + T cells from the spleens of SCC VII-immune mice 14 d after challenge were sorted into 96-well plates using an FACS Fusion (BD). Multiplexed PCR amplification of the TCR α and α variable regions was performed as previously described 17 . cDNA libraries were sequenced by Sanger sequencing (ETON). Full-length TCR sequences were reconstructed from cDNA fragments using the IMGT database to identify corresponding V and J gene usage 55 . IMGT nomenclature for TCR V and J genes is used throughout the manuscript for consistency.

TCR cloning and expression
TCR nucleotide sequences were synthesized and cloned into MSGV1 retroviral expression backbones using a BioXP (Codex DNA). TCR β and α-chains were separated by a P2A ribosomal skipping element. For in vivo studies, constructs were synthesized encoding the TCR β and α-chains as described above, followed by an additional P2A sequence and the coding sequence of CD90.1. Spleens from naive C3H/Hej mice were dissociated manually and cell suspensions passed through a 70-µm filter. CD4 + T cells were isolated by magnetic negative selection (StemCell). CD4 + T cells were stimulated with anti-CD3/CD28 Dynabeads (Gibco) for 24 h. TCR retroviral supernatants were generated by co-transfection of Platinum-Eco cells with the TCR containing retroviral vectors and pcL-ECo plasmid. Retroviral supernatants were collected at 48 and 72 h after transfection and either used fresh or frozen at −80 °C. Transductions were performed on RetroNectin-coated plates (Takara) as previously described. Murine T cells were maintained in RPMI 1640 supplemented with 10% FBS, 50 µM β-mercaptoethanol, 1× penicillin-streptomycin and HEPES supplemented with 100 IU ml −1 human IL-2 (Roche) or 5 ng ml −1 human IL-7 and IL-15 (StemCell) in the presence of anti-CD3/ CD28 Dynabeads (Thermo Fisher) for expansion and used for experiments between 10-14 d after isolation. Dynabeads were magnetically removed before use in in vitro assays or adoptive transfer experiments.

Flow cytometry
Splenocytes and transduced T cells were stained with the indicated concentration I-A K (VALVTDNAVYQWSME)-PE tetramer for 1 h at 37 degrees.

In vitro T cell functional assays
For splenocyte co-culture assays, 2 × 10 4 transduced T cells were co-cultured with 2 × 10 5 splenocytes pulsed overnight with indicated concentrations of CLTC H192>Q (SLNTVALVTDNAVYQWSMEG) or wild-type CLTC (SLNTVALVTDNAVYHWSMEG) peptide in 96-well U-bottom plates. Supernatants were collected after 18-24 h and IFN-γ was measured by ELISA (BD Bioscience). For measuring levels of proximal TCR signaling, 5 × 10 4 transduced T cells were co-cultured with 1 × 10 5 splenocytes pulsed overnight with 1 µg ml −1 CLTC H129>Q peptide in 96-well U-bottom plates. Cells were centrifuged for 10 s at 400g to initiate contact between T cells and APCs. After incubation for 5 min at 37 °C, the reaction was stopped on ice for 30 s. The plate was then centrifuged at 311g for 2 min, supernatants were discarded and wells were vortexed to resuspend cells in remaining volume. Cells were immediately fixed with ice-cold 4% paraformaldehyde for 15 min on ice, followed by two washes with FACS buffer. Cells were then permeabilized with ice-cold 90% methanol for 15 min, followed by an additional two washes with FACS buffer. Cells were then stained intracellularly for phosphorylated ERK1/2 for 30 min at room temperature, before washing twice with FACS buffer and analyzing levels of phosphorylated ERK1/2 by flow cytometry.

Adoptive transfers and in vivo treatments
Naive or tumor-bearing C3H/HeJ mice were injected i.v. via the tail vein with the indicated number of CD4 + T cells in 200 µl 1× HBSS at the Article https://doi.org/10.1038/s41590-023-01543-9 indicated time points. Where indicated, CD4 + T cells were first labeled with CTV (Thermo Fisher) according to manufacturer's instructions. Depletion of CD8 + T cells was achieved by intraperitoneal (i.p.) injection of 200 µg anti-CD8 (116-13.1, BE0118, BioXCELL) or IgG2a (C1.18.4, BE0085, BioXCELL) isotype control at D4 and D0 relative to tumor cell injection for primary tumor immunity experiments. To deplete CD8 + T cells during therapeutic experiments, 200 µg anti-CD8 was injected i.p. immediately following adoptive transfer of CD4 + T cells and every 7 d for the duration of the experiment. CD40L was blocked in vivo by i.p. administration of 200 µg anti-CD40L (MR1, BE0017-1, BioXCELL) compared to Armenian hamster IgG (PIP, BE0260, BioXCELL) isotype control on D0 and D2 for primary tumor immunity experiments. Where indicated, tumor-bearing mice were treated with 150 mg kg −1 cyclophosphamide monohydrate (Sigma) dissolved in 1× PBS i.p. 1 d before T cell transfer. Tumor volume was calculated by the equation V = (l × w 2 ) / 2 where l and w correspond to the longer and shorter perpendicular diameters respectively. For therapeutic experiments, mice were treated once tumor volumes reached 100-250 mm 3 . Mice were randomized before initiating treatment.

RNA sequencing and bioinformatic analyses
RNA paired-end sequencing reads were obtained using Illumina's NovaSeq 6000 system. FastQC (v.0.11.9) and Trimmomatic (v.0.32) were used to run quality control and trim low-quality control reads. The paired ends that passed Illumina filters were filtered for reads aligning to tRNA, rRNA, adaptor sequences and spike-in controls. The reads were then aligned to the GRCm38 reference genome and Gencode v.M9 annotations using STAR (v.2.6.1c) 56 . DUST scores were calculated with PRINSEQ Lite (v.0.20.3) 57 and low-complexity reads (DUST > 4) were removed from the BAM files. The alignment results were parsed via SAMtools 58 to generate SAM files. Read counts to each genomic feature were obtained with the featureCounts program (v.1.6.5) 59 . After removing absent features (zero counts in all samples), the raw counts were then imported to R/Bioconductor package DESeq2 (ref. 60) to identify differentially expressed genes among samples. P values for differential expression were calculated using the Wald test for differences between the base means of two conditions. These P values were then adjusted for multiple test correction using the Benjamini-Hochberg algorithm 61 to control the false discovery rate. We considered genes differentially expressed between two groups of samples when the DESeq2 analysis resulted in an adjusted P value of <0.05 and the absolute value of the log fold change in gene expression was >1. Variance stabilizing transformation (DESeq2 v.1.24.0) was applied to the read counts for all samples. As there were samples from multiple mapping runs, adjustment of batch effect with outcome of interest as disease state was performed using ComBat (sva v.3.32.1).

Statistics
Statistical analyses were performed with Prism 9 (GraphPad Software). Statistical tests and significance are indicated in the figure legends.

Study approval
All animal studies were approved by The La Jolla Institute for Immunology Institutional Animal Care and Use Committee Animal Protocol AP00001026.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
Bulk RNA-seq data have been uploaded to the NCBI Gene Expression Omnibus and are accessible under accession no. GSE229221. The mouse reference genome GRCm38 is accessible through GenBank under accession no. GCA_000001635.2. Source data are provided with this paper.

Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.

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The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy Data Availability Statement: Bulk RNA-seq data has been uploaded to NCBI GEO and is accessible under accession number GSE229221. Source data are provided with this article.

Animals and other research organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research, and Sex and Gender in Research Laboratory animals [8][9][10][11][12] week old female C3H/Hej mice were used in these studies

Wild animals
This study did not involve wild animals

Reporting on sex
Female mice were used because the tumor cell line (SCC VII) was derived from a female mouse Field-collected samples This study did not involve field collected samples

Ethics oversight
The animal study protocol was approved by the La Jolla Institute for Immunology Institutional Animal Care and Use Committee (IACUC) Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation Samples were prepared as described in the materials and methods. Mouse spleens, tumors, and inguinal lymph nodes were surgically removed. Spleens were dissociated manually and cell suspensions were passed through a 70 uM strainer. Prior to use as antigen presenting cells in T cell in vitro assays, red blood cells were lysed with ACK lysis buffer. Lymph nodes and subcutaneous tumors were minced into small (<2mm) pieces with dissection scissors. Tissue fragments were enzymatically dissociated in 20 ug/mL Liberase (Roche) and 20 ug/mL DNase I (Roche) at 37 degrees C for 30 minutes. Single cells were then passed through a 70 uM strainer.
Instrument BD LSR-II and BD FACS Celesta were used for data aquisition Software Flow cytometry data was collected using BD FACSDiva software and anlyzed with FlowJo v10.8.2 Cell population abundance For tetramer sorting experiments, given the low total number of cells, no post-sort purity assessment was made and all single cells were used for TCR sequencing experiments.