Project Summary Tuberculosis remains a serious global epidemic and with the rise of multi-drug resistant strains, an efficacious vaccine solution is imperative. Synthetic subunit vaccines, which do not contain live bacteria, present numerous advantages over attenuated vaccines by offering a safer option for immunocompromised individuals and being amenable to rapid, scalable, and reproducible production. The subunit vaccines currently being developed for Mycobacterium tuberculosis (Mtb) use peptide or protein antigens, which target MHC-restricted conventional T cells, overlooking the potential of Mtb lipid antigens as vaccine candidates. Mycobacterial lipids are presented by group 1 CD1 (CD1a, b, c) and group 2 CD1d to cognate T cells. Group 1 CD1-restricted T cells can be identified in patients with TB and have been shown to contribute to protective immunity against Mtb infection. In order to study further study group 1 CD1-restricted Mtb response, we previously created CD1 expressing transgenic mice (hCD1Tg) and CD1b-restricted T cell receptor transgenic T cells (DN1Tg) specific for the dominant Mtb lipid antigen mycolic acid (MA). We have since showed that DN1 T cells can be activated in vivo by vaccination with nanoparticles (NP) loaded with MA. We immunized hCD1Tg-DN1 mice with MA-NP to generate memory DN1 T cells. Using transcriptome analysis, we then determined that memory DN1 T cells most closely resemble T follicular helper (TFH) cells. In this proposal, we will assess whether two key TFH cell molecules, Bcl6 and PD-1, are necessary for memory DN1 T cell differentiation. We will also validate findings in the polyclonal setting using single cell sequencing. Finally, we will test whether memory DN1 T cell can provide protection in Mtb infection conditions. Through this work, we will be able to understand unique determinants of memory group 1 CD1-restricted T cells differentiation and their role in Mtb infection bringing us a step closer to creating a lipid-based vaccine for Mtb.