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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 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. In cancer, neoantigen (NeoAg)-specific CD8 +  T cells capable of direct tumor recognition have been extensively studied but little is known of the role of NeoAg-specific CD4 +  T cells. Here Schoenberger and colleagues analyze an oligoclonal CD4 +  T cell response to a naturally arising murine tumor NeoAg at the level of TCR usage and functionality.

Therapeutic benefit to immune checkpoint blockade (ICB) is currently limited to the subset of cancers thought to possess a sufficient tumor mutational burden (TMB) to allow for the spontaneous recognition of neoantigens (NeoAg) by autologous T cells. We explored whether the response to ICB of an aggressive low-TMB squamous cell tumor could be improved through combination immunotherapy using functionally defined NeoAg as targets for endogenous CD4+ and CD8+ T cells. We found that, whereas vaccination with CD4+ or CD8+ NeoAg alone did not offer prophylactic or therapeutic immunity, vaccines containing NeoAg recognized by both subsets overcame ICB resistance and led to the eradication of large established tumors that contained a subset of PD-L1+ tumor-initiating cancer stem cells (tCSC), provided the relevant epitopes were physically linked. Therapeutic CD4+/CD8+ T cell NeoAg vaccination produced a modified tumor microenvironment (TME) with increased numbers of NeoAg-specific CD8+ T cells existing in progenitor and intermediate exhausted states enabled by combination ICB-mediated intermolecular epitope spreading. We believe that the concepts explored herein should be exploited for the development of more potent personalized cancer vaccines that can expand the range of tumors treatable with ICB.

BackgroundCD4+ T cells play a critical role in the positive and negative regulation of cellular immunity through the many functional subsets they comprise. The progressive growth of immunogenic tumors which nonetheless generate mutation-specific T cells suggests that effective immune control may be avoided or suppressed at the level of the neoantigen-specific CD4+ T-cell response. Despite their importance, little is known about the ontogeny, architecture, and development of the CD4+ NeoAg-specific repertoire induced by progressively growing tumor.MethodsWe used a tetramer specific for a validated neoantigen, CTLCH129>Q/I-Ak, to characterize the ontogeny of natural CD4+ T-cell responses to an aggressive and poorly immunogenic major histocompatibility complex class II-deficient tumor, squamous cell carcinoma VII (SCC VII), during progressive growth or following therapeutic peptide vaccination using a combination of flow cytometry, single-cell genomics, and T-cell receptor (TCR) gene engineering.ResultsWe find that the natural CD4+ T-cell response to a growing tumor is phenotypically and functionally diverse, with distinct subsets including type 1 helper, T follicular helper-like, and regulatory T cell (Treg) lineages appearing as early as 9 days after tumor implantation. Therapeutic vaccination using the CLTCH129>Q peptide in adjuvant plus α-programmed cell death protein-1 reduces the frequency of CLTCH129>Q-specific Treg in both tumor and tumor-draining lymph node. Single-cell transcriptomic analysis of CLTC-specific CD4+ T cells recapitulated and extended the diversity of the response, with TCRs of varying affinity found within each functional subset. The TCR affinity differences did not strictly correlate with function, however, as even the lowest affinity TCRs isolated from Treg can mediate therapeutic efficacy against established tumors in the setting of adoptive cellular therapy (ACT).ConclusionsThese findings offer unprecedented insight into the functional diversity of a natural neoantigen-specific CD4+ T-cell response and show how immunotherapeutic intervention influences the phenotype, magnitude, and efficacy of the antitumor immune response. This information could lead to new approaches to immune monitoring in the clinical setting of checkpoint blockade immunotherapy and cancer vaccines. Furthermore, we show that Treg can be a potent source of TCRs that can mediate therapeutic benefit in the setting of ACT.

CD4+ T cells play a critical role in antitumor immunity via recognition of peptide antigens presented on MHC class II (MHC-II). Although some solid cancers can be induced to express MHC-II, the extent to which this enables direct recognition by tumor-specific CD4+ T cells is unclear. We isolated and characterized T cell antigen receptors (TCRs) from naturally primed CD4+ T cells specific for 2 oncoproteins, HPV-16 E6 and the activating KRASG12V mutation, from patients with head and neck squamous cell carcinoma and pancreatic ductal adenocarcinoma, respectively, and determined their ability to recognize autologous or human leukocyte antigen-matched antigen-expressing tumor cells. We found in both cases that the TCRs were capable of recognizing peptide-loaded target cells expressing the relevant MHC-II or B cell antigen-presenting cells (APCs) when the antigens were endogenously expressed and directed to the endosomal pathway but failed to recognize tumor cells expressing the source protein even after induction of surface MHC-II expression by IFN-γ or transduction with CIITA. These results suggest that priming and functional recognition of both a nuclear (E6) and a membrane-associated (KRAS) oncoprotein are predominantly confined to crosspresenting APCs rather than via direct recognition of tumor cells induced to express MHC-II.

Therapeutic benefit to immune checkpoint blockade (ICB) is currently limited to the subset of cancers thought to possess a sufficient tumor mutational burden (TMB) to allow for the spontaneous recognition of neoantigens (NeoAg) by autologous T cells. We explored whether the response to ICB of an aggressive low-TMB squamous cell tumor could be improved through combination immunotherapy using functionally defined NeoAg as targets for endogenous [CD4.sup.+] and [CD8.sup.+] T cells. We found that, whereas vaccination with [CD4.sup.+] or [CD8.sup.+] NeoAg alone did not offer prophylactic or therapeutic immunity, vaccines containing NeoAg recognized by both subsets overcame ICB resistance and led to the eradication of large established tumors that contained a subset of PD-LV tumor-initiating cancer stem cells (tCSC), provided the relevant epitopes were physically linked. Therapeutic [CD4.sup.+]/[CD8.sup.+]T cell NeoAg vaccination produced a modified tumor microenvironment (TME) with increased numbers of NeoAg-specific [CD8.sup.+] T cells existing in progenitor and intermediate exhausted states enabled by combination ICB-mediated intermolecular epitope spreading. We believe that the concepts explored herein should be exploited forthe development of more potent personalized cancer vaccines that can expand the range of tumors treatable with ICB.

Therapeutic benefit to immune checkpoint blockade (ICB) is currently limited to the subset of cancers thought to possess a sufficient tumor mutational burden (TMB) to allow for the spontaneous recognition of neoantigens (NeoAg) by autologous T cells. We explored whether the response of an aggressive low TMB squamous cell tumor to ICB could be improved through combination immunotherapy using functionally defined NeoAg as targets for endogenous CD4 + and CD8 + T cells. We found that, whereas vaccination with CD4 + or CD8 + NeoAg alone did not offer prophylactic or therapeutic immunity, vaccines containing NeoAg recognized by both subsets overcame ICB resistance and led to the eradication of large established tumors that contained a subset of PD-L1 + tumor-initiating cancer stem cells (tCSC), provided the relevant epitopes were physically linked. Therapeutic CD4 + /CD8 + T cell NeoAg vaccination produced a modified tumor microenvironment (TME) with increased numbers of NeoAg-specific CD8 + T cells existing in progenitor and intermediate exhausted states enabled by combination ICB-mediated intermolecular epitope spreading. The concepts explored herein should be exploited for the development of more potent personalized cancer vaccines that can expand the range of tumors treatable with ICB.Therapeutic benefit to immune checkpoint blockade (ICB) is currently limited to the subset of cancers thought to possess a sufficient tumor mutational burden (TMB) to allow for the spontaneous recognition of neoantigens (NeoAg) by autologous T cells. We explored whether the response of an aggressive low TMB squamous cell tumor to ICB could be improved through combination immunotherapy using functionally defined NeoAg as targets for endogenous CD4 + and CD8 + T cells. We found that, whereas vaccination with CD4 + or CD8 + NeoAg alone did not offer prophylactic or therapeutic immunity, vaccines containing NeoAg recognized by both subsets overcame ICB resistance and led to the eradication of large established tumors that contained a subset of PD-L1 + tumor-initiating cancer stem cells (tCSC), provided the relevant epitopes were physically linked. Therapeutic CD4 + /CD8 + T cell NeoAg vaccination produced a modified tumor microenvironment (TME) with increased numbers of NeoAg-specific CD8 + T cells existing in progenitor and intermediate exhausted states enabled by combination ICB-mediated intermolecular epitope spreading. The concepts explored herein should be exploited for the development of more potent personalized cancer vaccines that can expand the range of tumors treatable with ICB.

CD4 T cells play critical roles in the positive and negative regulation of cellular immunity through the many functional subsets they comprise. The progressive growth of immunogenic tumors which nonetheless generate mutation-specific T cells suggests that effective immune control may be avoided or suppressed at the level of the neoantigen-specific CD4 T cell response. We used a tetramer specific for a validated neoantigen, CTLC /I-E , to characterize the ontogeny of natural CD4 T cell responses to an aggressive and poorly immunogenic Major Histocompatibility Complex Class II (MHCII)-deficient tumor, SCC VII, during progressive growth or following therapeutic peptide vaccination. We find that the natural CD4 T cell response to a growing tumor is phenotypically and functionally diverse, with distinct subsets including type 1 helper (T 1), T follicular helper (T )-like, and regulatory T cell (T ) lineages appearing as early as 9 days after tumor implantation. Therapeutic vaccination using the CLTC peptide in adjuvant plus α-PD-1 sharply reduces the frequency of CLTC -specific T frequency in both tumor and tumor-draining lymph node (tdLN). Single cell transcriptomic analysis of CLTC-specific CD4 T cells recapitulated and extended the diversity of the response, with TCRs of varying affinity found within each functional subset. The TCR affinity differences did not strictly correlate with function, however, as even the lowest affinity TCRs isolated from T can mediate therapeutic efficacy against established tumors in the setting of adoptive cellular therapy (ACT). These findings offer unprecedented insight into the functional diversity of a natural neoantigen-specific CD4 T cell response and show how immunotherapeutic intervention influences the phenotype, magnitude, and efficacy of the anti-tumor immune response.