A Novel Approach to Determine the Protective Value of ESAT-6-specific T Cells in Tuberculosis

Abstract RATIONALE: Vaccination is the most efficient way to limit transmission, lower mortality, and achieve tuberculosis eradication. The discovery that CD4 T cells coordinate elimination of cells infected with M. tuberculosis (Mtb) in humans led to the identification of several T cell antigens that underlie the current vaccine pipeline. However, the antigens that confer the most protective T cell response against Mtb remain unknown. Multiple candidate vaccines in clinical trials include the Mtb protein “early secreted antigenic target of 6 kDa” (ESAT-6), based on the high proportion of T cells in Mtb-infected humans and animals that recognize this antigen and modest protection in vaccinated animals. Despite this immunogenicity, Mtb-exposed humans with an ESAT-6-specific T cell response are not protected from developing tuberculosis. To determine the value of ESAT-6-specifc T cells in the host response to Mtb, we developed an ESAT-6-tolerant mouse (termed “6T”), bypassing the need to use attenuated, ESAT-6-deficient Mtb strains. METHODS: The murine Rosa26 locus was edited to introduce a sequence encoding an N-terminal ESAT-6 peptide only in cells expressing Foxn1-Cre. 6T mice were infected with Mtb via aerosol, and lung and spleen cells were interrogated by flow cytometry, T cell receptor sequencing, and ELISA. Mice previously infected with intradermal Mtb for 4 weeks were also re-challenged with aerosolized Mtb. RESULTS: Whereas ∼9% of all lung CD4 T cells in Mtb-infected wildtype mice are specific for ESAT-6, none are present in infected 6T mice. Lung Mtb burden was assessed at 4 and 8 weeks post-infection. At 4 weeks, Mtb colony-forming units and bulk Mtb-specific T cell responses were unchanged between wildtype and 6T mice. T cell receptor sequencing demonstrated that 6T mice exhibited more unique and diverse clonotypes, some of which cluster into predicted antigen specificity groups that were not observed among T cells from wildtype mice. Stimulation of spleen T cells from 6T mice with a panel of peptide pools spanning 60 individual Mtb antigens revealed both repression and induction of distinct T cell specificities by interferon gamma production, relative to the distribution in wildtype T cells. CONCLUSIONS: The 6T mouse model eliminates ESAT-6-specfic T cells without altering the broader naïve repertoire and demonstrates that ESAT-6-specific T cells are dispensable for Mtb control at 4 weeks post-infection. In the absence of ESAT-6-specific T cells, the global Mtb-specific T cell response is dramatically altered, highlighting the critical importance of antigen selection for tuberculosis vaccine design.