Project Summary Cancer immunotherapy’s signature achievement is extension of overall survival with long-term durable remissions in a minority of patients. However, the question of how memory CD8 T cells form in cancer patients is poorly understood. Our long-term goal is to understand the relationship between cell cycle regulation and CD8 T cell memory fate. We examined CDK4/6 inhibition in mouse and human CD8 T cells during T cell priming and found a dramatic skewing of CD8 T cells toward a memory fate. Antigen-specific CD8 T cells treated ex vivo with CDK4/6i displayed increased long-lived memory responses upon adoptive transfer into mice. We further used single cell transcriptional profiling to evaluate recently activated human CD8 T cells from blood of breast cancer patients pre- and on-treatment with the CDK4/6i palbociclib or abemaciclib. TCR clonotype tracking and RNA velocity analysis revealed that CDK4/6 inhibition increased the ratio of central memory to effector CD8 T cell precursors. Analysis of paired samples showed that MYC targets were downregulated by CDK4/6 inhibitor therapy in humans, consistent with an increase in the transcriptional repressor MXD4. Our central hypothesis is that CDK4/6 inhibition in CD8 T cells in both mice and humans augments long-term protective immunity. To test this, we will use state-of-the-art technologies including: 1) TRP1 transnuclear mice developed by our lab to study antigen-specific TCRs with a physiologic range of affinities for an endogenous antigen; 2) scRNAseq and TCR clonotype tracking to analyze a large bank of longitudinal blood and matched tissue samples from patients on a Phase II trial; 3) ChIPseq, and co-immunoprecipitations to determine the molecular basis linking CDK4/6 inhibition to memory cell fate in CD8 T cells. In Aim 1 we will identify the molecular mechanism linking CDK4 and/or CDK6 to induction of memory cell fate. Slowing the cell cycle using other cell cycle inhibitors or by taking advantage of natural heterogeneity in cell division rates does not potentiate memory formation in CD8 T cells, implying a unique role for CDK4 and/or CDK6 in conferring a memory cell fate. We will identify the transcriptional regulators affected by CDK4/6 inhibition, with a focus on MYC and RUNX3. Combination of CDK4/6 inhibitors with PD-1 blockade in the clinic results in unexpected treatment-limiting toxicity. In Aim 2 we propose that a short course of CDK4/6 inhibition would successfully potentiate CD8 T cell memory, thus allowing cessation of the CDK4/6 inhibitor to minimize or obviate overlap with PD-1 blockade. We will test this hypothesis in mice using our TRP1 CD8 T cell adoptive transfer model, and in humans using 29 paired blood and tissue samples from a Phase I trial of CDK4/6 inhibition with PD-1 blockade. We will use high- resolution single-cell transcriptional profiling of recently activated CD8 T cells to track individual TCR clonotypes and their transcriptional profiles in CDK4/6 inh...