From T Cells to Dendritic Cells: Mechanistic Dissection of CTLA4 and TIM3 in Cancer Immunity - PROJECT SUMMARY Immunotherapy targeting the immune checkpoint receptors (ICRs) PD1, CTLA4 and Lag3 – molecular brakes of our immune system - has revolutionized oncology, but it only benefits a small subset of patients. The limited mechanistic understanding of checkpoint signaling has become a significant barrier to expanding the clinical benefits of immunotherapy. In addition to the adaptive arm of anti-tumor immunity, particularly T cells where these receptors are predominantly active, the critical role of understudied innate anti-tumor immunity has been increasingly recognized. Notably, ICRs such as PD1, VISTA and TIM3 are also expressed in innate immune cells and play essential roles in anti-tumor immunity. Thus, an in-depth understanding of checkpoint signaling in both the adaptive and innate immunity will be pivotal for harnessing the full potential of immunotherapy. This project investigates the mechanism of two central ICRs, CTLA4 and TIM3, in adaptive and innate anti-tumor immunity respectively, to provide the foundation for novel therapeutic strategies. During the F99 phase of this project, I will investigate the molecular mechanism of CTLA4 in cytotoxic CD8+ T cells. CTLA4 research has historically centered on CD4+ T cells, where it maintains peripheral tolerance by restraining the activity of CD28, a costimulatory receptor known to be triggered by B7 (CD80/CD86) on antigen-presenting cells (APCs). However, recent studies and my preliminary data (see Progress Report) highlight the importance of CD8+ T cell-intrinsic CTLA4 in anti-tumor immunity, although the mechanism is unclear. Notably, CD8+ T cells can also express B7, either endogenously (B7endo) or acquired from APCs via trogocytosis (B7trogo), to trigger CD28 from the same T cell in cis in APC-sparse peripheral tissues. Based on these studies and the strong endocytic feature of CTLA4, I hypothesize that CTLA4 in CD8+ T cells act as a cell- intrinsic “molecular sink” to deplete B7 in cis. In the F99 phase, I will test this hypothesis both in vitro and in mouse tumor models, with the potential of addressing the long-standing mechanism of CTLA4 in CD8+ T cells. In the K00 portion, I plan to dissect the molecular mechanism of ICRs in innate immune cells, focusing on TIM3, another important yet poorly understood ICR. I plan to study TIM3 in dendritic cells (DCs), a type of myeloid innate immune cells critical for antigen presentation in the cancer immunity cycle. While TIM3 is primarily known as a marker for T cell terminal exhaustion, its function in T cells remains ambiguous with both stimulatory and inhibitory roles reported. Recent studies, however, highlight TIM3’s significance in intratumoral DCs in both human cancer patients and mouse tumor models. These exciting data urge a shift of focus of TIM3 biology from T cells to myeloid cells. In the K00 phase, I plan to leverage a unique combination of membrane reconstitution, cellular imaging, CRIPPR-editing, and mouse tumor models to elucidate the biochemical and molecular mechanism of TIM3 in DCs, including its physiological ligands, downstream effectors and its role in the development and function of DC subsets in cancers.
