Bacterial protein secretion is required for priming of CD8+ T cells specific for the Mycobacterium tuberculosis antigen CFP10

Abstract

Mycobacterium tuberculosis infection elicits antigen-specific CD8(+) T cells that are required to control disease. It is unknown how the major histocompatibility complex class I (MHC-I) pathway samples mycobacterial antigens. CFP10 and ESAT6 are important virulence factors secreted by M. tuberculosis, and they are immunodominant targets of the human and murine T-cell response. Here, we test the hypothesis that CFP10 secretion by M. tuberculosis is required for the priming of CD8(+) T cells in vivo. Our results reveal an explicit dependence upon the bacterial secretion of the CFP10 antigen for the induction of antigen-specific CD8(+) T cells in vivo. By using well-defined M. tuberculosis mutants and carefully controlling for virulence, we show that ESX-1 function is required for the priming of CD8(+) T cells specific for CFP10. CD4(+) and CD8(+) T-cell responses to mycobacterial antigens secreted independently of ESX-1 were unaffected, suggesting that ESX-1-dependent phagosomal escape is not required for CD8(+) T-cell priming during infection. We propose that the overrepresentation of secreted proteins as dominant targets of the CD8(+) T-cell response during M. tuberculosis infection is a consequence of their preferential sampling by the MHC-I pathway. The implications of these findings should be considered in all models of antigen presentation during M. tuberculosis infection and in vaccine development.

FIG. 1.

Priming of CFP10- and ESAT6-specific CD8⁺ and CD4⁺ T cells requires intact ESX-1 genes. (A) Splenic CD90⁺ T cells isolated from mice (n = 3, pooled) 3 weeks postinfection with Rv0573::Tn, Rv3870::Tn, or Rv3874::Tn were restimulated in vitro with peptide or H37Rv sonicate. Bars indicate means ± standard deviations. ***, P < 0.001 compared to results for medium alone. (B) Numbers of splenic CFU from the C3H mice used for panel A. The line indicates the median value. The means were not significantly different. (C) Pooled CD90⁺ T cells isolated from mice (n = 3) 3 weeks postinfection with the indicated M. tuberculosis strain were analyzed by flow cytometry using the H2-K^k/CFP10_32-39 tetramer. Percentages indicate the proportion of CD8⁺ T cells positive for tetramer binding. (D) H2-K^k/CFP10_32-39 tetramer staining of purified CD90⁺ T cells pooled from mice (n = 3) 3 to 4 weeks postinfection compiled from three experiments from two independent infections. Bars indicate means ± standard errors of the means. *, P < 0.05 compared to results for Rv0573::Tn.

FIG. 2.

Priming of CFP10- and ESAT6-specific CD8⁺ and CD4⁺ T cells requires an intact espA gene. (A) Pooled CD90⁺ splenic T cells isolated from H37Rv- or ΔespA-infected C3H mice (n = 3) 3 weeks postinfection were restimulated in vitro with peptide or H37Rv sonicate. Bars indicate means ± standard deviations. * and ***, P < 0.05 and P < 0.001, respectively, compared to results for medium alone by one-way analysis of variance. (B) Pooled CD90⁺ splenic T cells isolated from C3H mice (n = 3) 3 weeks postinfection were stained with the H2-K^k/CFP10_32-39 tetramer. Percentages indicate the proportion of CD8⁺ T cells positive for tetramer binding. (C) H2-K^k/CFP10_32-39 tetramer staining of purified CD90⁺ splenic T cells pooled from mice (n = 3) 3 to 5 weeks postinfection with the indicated M. tuberculosis strain compiled from three experiments from two independent infections. Bars indicate means ± standard errors of the means. The dotted line represents nonspecific staining based on tetramer staining from Rv3874::Tn-infected animals analyzed in parallel. *, P < 0.05 compared to results for H37Rv. (D and E) Spleen (D) or lung (E) CD90⁺ T cells pooled and isolated from C3H mice (n = 3) infected with ΔespA-pJEB (uncomplemented; open bars) or ΔespA-pEspA (espA plasmid complemented; filled bars) 5 weeks postinfection were restimulated in vitro with peptide or H37Rv sonicate. Bars indicate means ± standard deviations. ***, P < 0.001 compared to results for medium alone. Note that the data presented in panels A to C and panels D and E are from separate experiments.

FIG. 3.

Priming of T cells to non-RD1 antigens is independent of CFP10/ESAT6 secretion. (A and B) Pooled (n = 3 to 4 mice) CD90⁺ splenic T cells isolated from (C3H × BALB/c)F₁ H37Rv-infected (filled bars) or empty-plasmid-transformed ΔespA-infected (open bars) mice 5 weeks postinfection were restimulated in vitro with peptide epitopes from RD1-encoded (A) or non-RD1-encoded (B) antigens or H37Rv sonicate (B). Bars indicate means ± standard deviations. ***, P < 0.001 compared to results for medium alone. The dotted line indicates the average amount of IFN-γ released by T cells cultured with APC but without antigen. (C) Pooled (n = 3 to 4 mice) CD90⁺ splenic T cells isolated from (C3H × BALB/c)F₁ mice 5 weeks postinfection with H37Rv or ΔespA were analyzed by flow cytometry after being stained with the H2-K^d/TB10.4_20-28 tetramer (left, center) or SA-PE (right). Percentages indicate the proportions of CD8⁺ T cells positive for tetramer/SA binding. (D) Splenocytes from individual (C3H × BALB/c)F₁ mice 3 to 6 weeks postinfection were stained with the H2-K^d/TB10.4_20-28 tetramer. Mice were infected with H37Rv (left column) or ΔespA or ΔespA-pJEB (right column). Data points represent the percentages of H-2K^d/TB10.4_20-28 tetramer-positive or CD8⁺ cells for each subject. Data are compiled from three time points from two independent experiments. To establish the background value, each sample was stained with SA-PE. The dotted line represents the background means plus five standard deviations; n.s., not significant.