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.
© 2023. The Author(s).

CD4(+)Foxp3(+) regulatory T cells (Treg cells) are largely autoreactive yet escape clonal deletion in the thymus. We demonstrate here that CD27-CD70 co-stimulation in the thymus rescues developing Treg cells from apoptosis and thereby promotes Treg cell generation. Genetic ablation of CD27 or its ligand CD70 reduced Treg cell numbers in the thymus and peripheral lymphoid organs, whereas it did not alter conventional CD4(+)Foxp3(-) T cell numbers. The CD27-CD70 pathway was not required for pre-Treg cell generation, Foxp3 induction, or mature Treg cell function. Rather, CD27 signaling enhanced positive selection of Treg cells within the thymus in a cell-intrinsic manner. CD27 signals promoted the survival of thymic Treg cells by inhibiting the mitochondrial apoptosis pathway. CD70 was expressed on Aire(-) and Aire(+) medullary thymic epithelial cells (mTECs) and on dendritic cells (DCs) in the thymic medulla. CD70 on both mTECs and DCs contributed to Treg cell development as shown in BM chimera experiments with CD70-deficient mice. In vitro experiments indicated that CD70 on the CD8α(+) subset of thymic DCs promoted Treg cell development. Our data suggest that mTECs and DCs form dedicated niches in the thymic medulla, in which CD27-CD70 co-stimulation rescues developing Treg cells from apoptosis, subsequent to Foxp3 induction by TCR and CD28 signals.

Type 1 diabetes is a CD4 cell-dependent disease that results from destruction of insulin-producing beta cells in pancreatic islets. An ideal therapy would reverse diabetes shortly after onset when islet function in not yet fully ablated, and also prevent re-emergence of disease through the generation of memory cells that control the autoimmune response. In this study, we show that adaptive/induced polyclonal regulatory (TR) cells, which contain islet-reactive cells, fulfill these criteria in the NOD mouse model. CD4 cells induced to express FoxP3, IL-10, and TGF-beta1 in response to TCR signaling and TGF-beta1 can reverse diabetes with clinical restoration of prediabetic serum levels of IL-10. Unlike naturally occurring TR cells, these adaptive TR cells persist indefinitely (>1 year) as FoxP3(+), CD25(-) memory cells that self-renew. Establishment of memory is accompanied by narrowing of the T cell repertoire to usage of a single TCR beta-chain, Vbeta11, implying selection by Ag. With islet-specific adaptive TR cells, we show that memory is functionally stable and transferable. Therefore, adaptive TR cells, which can be readily generated from normal CD4 populations and become focused by Ag with induction of memory, may provide a treatment and a vaccine for the long-term cure of diabetes making them attractive as immunotherapeutic agents.

Secondary exposure to respiratory syncytial virus (RSV) can lead to immunopathology and enhanced disease in vaccinated individuals. Vaccination with individual RSV proteins influences the type of secondary RSV-specific immune response that develops upon challenge RSV infection, as well as the extent of immunopathology. RSV-specific memory CD4 T cells can directly contribute to immunopathology through their cytokine production. Immunization of BALB/c mice with a recombinant vaccinia virus (vv) expressing the attachment (G) protein of RSV results in pulmonary eosinophilia upon RSV challenge, whereas immunization of mice with a vv expressing the fusion (F) protein does not. We analyzed the CD4 T-cell response to an I-E(d)-restricted CD4 T-cell epitope within the F protein of RSV corresponding to amino acids 51 to 66 in an effort to better understand the similarities and differences in the immune response elicited by the G versus the F protein. Vaccination with the G protein induces a mixture of RSV G-specific Th1 and Th2 cells with a restricted T-cell receptor repertoire. In contrast, we demonstrate here that immunization with the F protein elicits a broad repertoire of RSV F-specific CD4 T cells that predominantly exhibit a Th1 phenotype. However, in the absence of gamma interferon (IFN-gamma), RSV F(51-66)-specific CD4 T cells secreted interleukin-5, and mice developed pulmonary eosinophilia after RSV challenge. IFN-gamma-deficient mice exhibited decreased weight loss compared to wild-type controls, suggesting that IFN-gamma exacerbates systemic disease. These data demonstrate that IFN-gamma can have both beneficial and detrimental effects during a secondary RSV infection.

IL-15 is known to be critical in the homeostasis of Ag-specific memory CD8(+) T cells following acute viral infection. However, little is known about the homeostatic requirements of memory CD8(+) T cells during a latent viral infection. We have used the murine gammaherpesvirus-68 (MHV-68) model system to investigate whether IL-15 is necessary for the maintenance of memory CD8(+) T cells during a latent viral infection. IL-15 is not essential either for the initial control of MHV-68 infection or for the maintenance of MHV-68-specific memory CD8(+) T cells. Even at 140 days postinfection, the proportion of CD8(+) T cells recognizing the MHV-68 epitopes were the same as in control mice. The maintenance of these memory CD8(+) T cells was attributable to their ability to turn over in vivo, probably in response to the presence of low levels of Ag. IL-15(-/-) mice had a significantly higher turnover rate within the virus-specific memory CD8(+) T cell population, which was the result of increased levels of viral gene expression rather than an increase in viral load. These cells did not accumulate in the spleens of the IL-15(-/-) mice due to an increased sensitivity to apoptosis as a result of decreased Bcl-2 levels. Intriguingly, memory CD8(+) T cells from latently infected mice failed to undergo homeostatic proliferation in a naive secondary host. These data highlight fundamental differences between memory CD8(+) T cells engaged in active immune surveillance of latent viral infections vs memory CD8(+) T cells found after acute viral infections.