The Interplay Between PD-1 Blockade and T cell Development in the Context of Thymic Regeneration in Prostate Cancer

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PROJECT SUMMARY Immunotherapies, such as PD-1 blockade, have shown therapeutic potential in prostate cancer; however, only a portion of patients respond, suggesting unidentified mechanisms of resistance. The current standard of care for prostate cancer is androgen deprivation therapy (ADT), which targets hormone-sensitive tumor cells. Interestingly, ADT also results in regeneration of the thymus and increased thymic output of newly developed T cells. We have shown that newly generated T cells resulting from ADT-induced thymic regeneration traffic to tumors where they can contribute to the tumor immune milieu. Further, PD-1 deficiency or blockade leads to an increase in the generation of thymic regulatory T cells (Tregs), suggesting that PD-1 regulates the selection of these cells in the thymus and, therefore, the Treg repertoire. It is unclear whether PD-1 limits Treg development by inhibiting the T cell receptor signaling pathway or the CD28 costimulatory pathway, but it has already been established that CD28 is critical for Treg development. Based upon these data, I hypothesize that inhibition of PD-1 could broaden the repertoire of Tregs available to traffic to prostate tumors and PD-1 limits Treg selection through inhibition of CD28 signaling. First, I will identify changes in tumor infiltrating Treg repertoire resulting from PD-1 blockade in the context of thymic regeneration. A castration model of ADT is used to induce thymic regeneration in a castration and immunotherapy resistant murine model of prostate cancer. Animals are treated with aPD-1 or control and the T cell repertoire is examined. Additionally, I will examine pre- and post- treatment samples collected as a part of an ongoing clinical trial where treatment-naïve men diagnosed with advanced prostate cancer receive ADT in combination with aPD-1, allowing us to perform real-time evaluation of the impact of PD-1 blockade during thymic regeneration on repertoire selection in patients . Second, I will determine the mechanism by which PD-1 limits Treg development. This will be accomplished through the use of the CRISPR-Cas9 system to mutate motifs in the CD28 cytoplasmic tail that are sites of PD-1 inhibition to determine dependence on CD28 signaling. Further, alterations of downstream signaling pathways resulting from PD-1 deficiency will be examined by transcriptomic and proteomic studies. The outcomes of this proposal are anticipated to reveal the how PD-1 blockade impacts the immune repertoire in the context of ADT-induced thymic regeneration to identify potential mechanisms of immunotherapy resistance in prostate cancer. These studies may also lead to new justifications for combination therapies in prostate cancer to increase the number of patients responding to checkpoint blockade.