The fundamental premise of our application is to provide unbiased, non-hypothesis driven analyses of immune signatures (IMS) associated with two important human diseases, namely dengue virus (DENV) and Mycobacterium tuberculosis (Mtb). In the current cycle, we successfully accomplished the goals set forth in the original proposal and have characterized memory T cell IMS associated with DENV and Mtb in the context of: 1) natural immunity and/or control of infection, 2) active and severe disease, and 3) administration of licensed or experimental vaccines. Our group has studied DENV and Mtb for several years for the following reasons: 1) they are both current global health problems, 2) memory T cells are fundamentally associated with protective immunity for these diseases, 3) antigen-specific T cell responses are detected in sufficient numbers ex vivo, making them accessible for “omics” studies without the need for in vitro expansion, and 4) human specimens associated with natural infection/immunity and severe disease are easily accessible in large numbers. The HIPC study format was ideally suited to accomplish the proposed mission. Each project and core was and remains critically dependent on other elements of the program. In the context of HIPC, we were able to realize synergies, benefit from economies of scale, and bring groups of investigators with different expertise together. As a result, we developed a critical mass that is extensively leveraged in the proposed extension, based on: 1) an established team of experienced investigators, 2) clinical collaborations with leaders in the field, 3) an extensive collection of epitopes restricted by a variety of different HLA class I and II molecules to allow an analysis of memory T cells with unprecedented precision, 4) IMS associated with specific T cell subsets representing key players in immunity, and 5) established “omics” assays and analysis pipelines to generate IMS related to DENV and Mtb. In this proposed extension, we plan to build on the definition of the T cell IMS in DENV and Mtb accomplished in the current 5-year funding period by expanding the significance and the dimensionality of the observations in multiple, yet related and synergistic, directions. As mentioned above, the work performed in the current cycle identified IMS associated with specific T cell subsets in natural infection, vaccination, and acute/severe disease. It is also clear that these T cell subsets are themselves heterogeneous. Here, we will utilize single-cell sequencing to “drill down” and establish IMS of the specific cell types responsible for this heterogeneity. The second dimension will “drill up” by considering additional cell subsets. For DENV, this will be accomplished by determining the IMS of whole unfractionated PBMC stimulated by epitopes recognized by DENV memory T cells. For Mtb, we will extend our focus to unconventional CD4-CD8-double-negative T cells which we have found to be responsive to bacterial lys...