Project Summary/Abstract Aging is associated with dramatically increased susceptibility to infectious diseases like influenza. Although vaccination is the primary strategy for minimizing flu deaths, older adults have consistently impaired vaccine responses. Preliminary analyses of tonsil T cells from older vs younger adults reveal that CXCL13 is significantly decreased in old follicular helper T cells as compared to young. Further preliminary experiments in tonsil organoids reveal a role for CXCL13 in memory CD4+ T cell homeostasis. While CXCL13 is expressed by follicular helper T cells in humans, mouse T cells lack CXCL13 expression entirely. The Research Strategy proposed here will leverage human immune organoid technology, developed in sponsor Dr. Mark Davis’ lab, to define the role of CXCL13 as an aging-associated defect in memory T cell homeostasis. This work will reveal the mechanism of CXCL13-mediated maintenance of memory T cells (Aim 1) and define the identity of the T cell pool that is maintained by CXCL13 (Aim 2). In Aim 1, the applicant Dr. Casey Beppler will use CRISPR- engineered CXCL13-KO tonsil organoids, specific cell type depletions, and single cell RNA sequencing to reveal the cellular and molecular mediators of CXCL13-mediated T cell homeostasis. In Aim 2, Dr. Beppler will integrate cellular phenotyping of human tonsil and spleen T cells across adult lifespan with spheromer staining of antigen-specific T cells (also developed in the Davis Lab) in CXCL13-KO tonsil organoids to determine the specific T cell population that is maintained by CXCL13. Although work proposed here will focus on the role of CXCL13 in secondary lymphoid organs, these findings are likely to reveal mechanisms at play in tertiary lymphoid structures across disease states from cancer to autoimmunity. The research and career development training plans are tailored to enable Dr. Beppler to gain new skills in modeling human lymphoid tissues with immune organoids and single cell RNA sequencing, as well as professional development skills in mentoring, oral presentation, and grant writing. Sponsor Dr. Mark Davis is a long-standing expert in the field of antigen- specific T cell responses and a leader in human systems immunology. Advisors will contribute with their complementary expertise: Dr. Purvesh Khatri (bioinformatics), Dr. Le Cong (CRISPR and gene editing), and Dr. Bali Pulendran (multi-omics analysis of human immune responses). The Stanford Institute for Immunity, Transplantation & Infection directed by Dr. Davis is an excellent environment for collaborative and cutting-edge research, supported by outstanding infrastructure (Stanford Center for Genomics, Human Immune Monitoring Center). In summary, the strong mentorship and training plan will prepare Dr. Beppler for her future independent career studying human T cell immunology. This project will improve our understanding of an aging-associated defect in human lymphoid tissues, thus providing the potential for future therapies to promote memory homeostasis and limiting the number of deaths to vaccine-preventable diseases.

Project Narrative 79% of flu-related deaths between the 2010/11-2019/20 seasons were of people 65 years or older, and although vaccinations are strongly encouraged in this group, elderly vaccine responses are consistently impaired. This study will define the role of CXCL13 as an aging-associated defect in memory T cell homeostasis using CRISPR-engineered tonsil organoids. Results obtained here will improve our understanding of aging immune memory, thus providing the potential for future therapies directed at this axis and limiting the number of deaths to vaccine-preventable diseases.