Abstract

Immune checkpoint therapies (ICT) have shown promise in treating solid tumors, providing cures and durable protection in some cases. However, only a subset of patients benefits from ICT, indicating a need for a deeper understanding of the requirements for successful therapy. In both mice and humans, αβ T cells are key mediators of spontaneous and ICT-induced anti-tumor responses. While the anti-tumor roles of CD8+ T cells are well-studied, the role of CD4+ T cells, particularly in ICT for MHC-II negative tumors, is less understood. New insights into how CD4+ T cells modulate CD8+ T cells during ICT may lead to new therapeutic avenues for enhancing CD8+ T cell responses in patients for whom ICT is currently ineffective. Thus, the primary aim of this thesis was to define the key cellular and molecular aspects governing CD4+ T cell-driven enhancement of tumor-specific CD8+ T cells within the tumor microenvironment (TME) during successful ICT. We focused primarily on αPD-1/αCTLA-4 ICT using our well-characterized MHC-II negative T3 sarcoma model. In the first part of the study, we demonstrated that CD4+ T cell help is crucial both for priming and during the effector phase within the TME for successful ICT. We discovered that while the CD40/CD40L pathway is necessary for priming CD8+ T cells, it is dispensable during the effector stage, where cytokines IFN-γ and IL-2 play critical roles. Effector-phase CD4+ T cell help mediated three major ICT-induced changes in CD8+ tumor-infiltrating lymphocytes (TILs): (i) downregulation of inhibitory receptors, (ii) enhancement of pro-inflammatory cytokine production, and (iii) increased cytotoxic potential. Additionally, effector-phase CD4+ T cell help indirectly enhanced CD8+ TILs by upregulating MHC-I on tumor cells and facilitating the elimination of immunosuppressive macrophages, thus fostering a tumor milieu conducive for their cytolytic activity. The second part of this thesis explored the dynamics of MHC-II neoantigen presentation by host antigen-presenting cells, crucial for activating CD4+ T cells in MHC-II negative tumors. We generated a TCR-mimic monoclonal antibody (1G10) specific for the dominant T3 CD4+ T cell neoepitope (mItgb1) bound to MHC-II (I-Ab). Time course flow cytometry staining of tumors and tumor-draining lymph nodes (TDLNs) from T3 tumor-bearing mice revealed that 1G10 predominantly stained macrophages and cDC2 cells in both untreated and ICT-treated mice. These findings suggest that macrophages and cDC2s comprise a major fraction of the cellular interaction network associated with CD4+ T cell activation in T3 tumors. However, the contribution of cDC1 cells could not be formally ruled out due to the detection limit of flow cytometry. Furthermore, in RAG2-/- T3 tumor-bearing mice, the 1G10 mAb inhibited the capacity of adoptively transferred neoantigen vaccine-elicited CD4+ and CD8+ T cells to drive tumor rejection, indicating that the effector phase CD4+ T cell help program depends on continuous MHC-II neoantigen presentation beyond the initial priming phase. The specific identity of the necessary antigen-presenting cells in this context warrants further investigation. Collectively, our findings reveal the dynamic complexities of helper CD4+ T cell-driven anti-tumor immunity and underscore the necessity of CD4+ T cell assistance for effective CD8+ T cell anti-tumor responses in immunotherapy. The insights gained advocate for the inclusion of CD4+ T cell-modulating agents in immunotherapy modalities that aim to maximally unleash the cytolytic potential of tumor-specific CD8+ T cells, such as ICT and neoantigen vaccines.

Committee Chair

Robert Schreiber

Committee Members

Carl DeSelm; Kenneth Murphy; Marco Colonna; Maxim Artyomov; Sheila Stewart

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Immunology)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

8-7-2024

Language

English (en)

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