Antigen-Presenting Cell Control of CD8+ T Cell Exhaustion in Cancer - Project Summary Tumor-reactive cytotoxic T lymphocytes (CTLs) often progress to dysfunction defined as T cell exhaustion. Marked by expression of the programmed cell death protein 1 (PD-1), the exhausted T (Tex) cell lineage is a developmental continuum wherein PD-1low Tex progenitors give rise to terminally dysfunctional PD-1high Tex cells. Notably, the immune checkpoint blockade therapy revives Tex progenitors, but not terminal Tex cells, calling for exploration of their differentiation mechanisms and means of therapeutic intervention. In a murine cancer model, tumor development induces differentiation of tumor-associated macrophages (TAMs) in association with generation of Tex cells. Transcriptome analysis revealed that TAMs exhibit shared characteristics with type 1 dendritic cells (DC1s) including expression of the transcription factor interferon regulatory factor-8 (IRF8). IRF8 promotes TAM presentation of cancer cell antigens to CD8+ T cells similar to DC1, but TAMs differ from DC1s in promoting high PD-1 expression. Importantly, macrophage-specific deletion of IRF8 attenuates Tex cell differentiation, and suppresses tumor growth. Furthermore, human TAMs express IRF8, and a TAM IRF8 gene signature tracks with a Tex cell gene signature in multiple cancer types. Based on these findings, we hypothesize that terminal Tex cell differentiation is driven by IRF8-expressing TAMs with a tolerogenic antigen-presenting cell (APC) function in the tumor tissue, and such a TAM-Tex cell regulation axis can be targeted for novel cancer immunotherapy. To test this hypothesis, we will first determine how IRF8 is induced in TAMs, and how it promotes TAM APC function. By performing chromatin profiling experiments and using genetic mouse models, we will assess whether the TAM-enriched transcription factor Batf2 enables IRF8 autoactivation via the +32kb Irf8 enhancer element. IRF8-deficient TAMs are defective in acquiring cancer cell antigens. Using mouse strains with macrophage- or cancer cell-specific deletion of the B2m gene, we will investigate whether IRF8 promotes TAM acquisition of antigens through cross-presentation or cross-dressing. Secondly, we will define how the tolerogenic function of TAMs is specified, and how it can be reprogrammed for therapy. Compared to DC1s, TAMs express lower levels of interleukin-15 (IL-15), but exhibit heightened transforming growth factor-b (TGF-b) signaling. By generating macrophage-specific gain- or loss-of-function mouse models, we will explore whether blockage of TGF-b signaling reverses the tolerogenic APC function of TAMs in an IL-15-dependent manner, and whether overexpression of IL-15 in macrophages is sufficient to induce T cell-stimulatory TAMs in genetic models and in a cell therapy setting. Compared to DC1s, TAMs have a smaller cell size. We will investigate whether and how activation of the metabolic regulator mammalian target of rapamycin complex 1 (mTORC1) reprogram TAMs to be immunostimulatory APCs. Successful completion of this project will not only generate mechanistic insights into APC control of Tex cell differentiation in cancer, but also guide the targeting of the TAM-Tex cell regulation axis for therapy of a wide range of malignancies.
