ABSTRACT Developing an effective vaccine for tuberculosis (TB) remains challenging because we do not understand how to improve protective immunity to Mycobacterium tuberculosis (Mtb) infection. While CD4 T cells and IFN- g responses are critical for controlling Mtb infection, they are not sufficient for conferring protective immunity. Moreover, Mtb impairs DC functions, leading to antigen-specific CD4 T cells that are ineffective in controlling infection. Therefore we need to design efficacious vaccines that induce additional DC-T cell responses that confer better protection. There is accumulating evidence that IL-17 and Th17 responses are important for protective immunity to TB in mice and humans but the molecular mechanisms for Th17 generation in TB are not well defined. We previously showed that an avirulent hip1 mutant Mtb strain induces earlier and higher IL-17 responses and attenuated disease, suggesting that wild type Mtb restricts optimal Th17 responses in the lung. Here we present novel data that links the CD40-costimulatory capacity of DCs with Th17 generation during Mtb infection. We show that interaction between CD40 and CD40L is critical for generating antigen-specific IL-17 responses to Mtb and that exogenously engaging CD40 on DCs enhances antigen-specific lung Th17 responses. We propose to test the hypothesis that boosting the CD40-CD40L costimulatory pathway and enhancing Th17 responses will improve protection against TB in mice and that these pathways contribute to immune control in human TB. We will undertake 3 distinct yet complementary approaches consisting of mechanistic studies in the mouse model of TB and clinically relevant studies in human cells. Using a mucosal DC vaccination model, we will test whether boosting CD40-CD40L interactions improves protection and define the Mtb-specific T cell memory responses associated with protective immunity (Aim 1). We will use a BCGDhip1 strain, which induces higher CD40 expression and increased Th1 and Th17 responses, to assess its vaccine efficacy compared to BCG (Aim 2). To expand insights from mice to human TB, we will use ex vivo functional assays to determine whether the capacity of DCs to promote IL-17 is greater in individuals who latently control infection compared to those who develop active TB. (Aim 3). Our studies will provide important mechanistic insights into how Mtb modulates DC-T crosstalk in animal models and human cells and open new avenues to target host pathways that can boost protective immunity and improve vaccine efficacy.