Mycobacterium tuberculosis-specific CD8+ T cells require perforin to kill target cells and provide protection in vivo

Abstract

Optimal immunity to Mycobacterium tuberculosis (Mtb) infection requires CD8(+) T cells, and several current Mtb vaccine candidates are being engineered to elicit enhanced CD8(+) T cell responses. However, the function of these T cells and the mechanism by which they provide protection is still unknown. We have previously shown that CD8(+) T cells specific for the mycobacterial Ags CFP10 and TB10.4 accumulate in the lungs of mice following Mtb infection and have cytolytic activity in vivo. In this study, we determine which cytolytic pathways are used by these CD8(+) T cells during Mtb infection. We find that Mtb-specific CD8(+) T cells lacking perforin have reduced cytolytic capacity in vivo. In the absence of perforin, the residual cytolytic activity is CD95 and TNFR dependent. This is particularly true in Mtb-infected lung tissue where disruption of both perforin and CD95 eliminates target cell lysis. Moreover, adoptive transfer of immune CD8(+) T cells isolated from wild-type, but not perforin-deficient mice, protect recipient mice from Mtb infection. We conclude that CD8(+) T cells elicited following Mtb infection use several cytolytic pathways in a hierarchical and compensatory manner dominated by perforin-mediated cytolysis. Finally, although several cytolytic pathways are available, adoptively transferred Mtb-specific CD8(+) T cells require perforin-mediated cytolysis to protect animals from infection. These data show that CD8(+) T cell-mediated protection during Mtb infection requires more than the secretion of IFN-gamma and specifically defines the CD8(+) cytolytic mechanisms utilized and required in vivo.

FIGURE 1

TB10.4_4–11-specific CD8⁺ T cells are cytolytic in vivo. A, Lung mononuclear cells isolated from uninfected (left panel) or Mtb-infected (right panel)B6 mice 6 wk after infection were stained with H-2 K^b:TB10.4_4–11 tetramer. The percentage of tetramer⁺CD8⁺ lymphocytes is shown in each panel. B, In vivo cytolytic activity of TB10.4_4–11-specific CD8⁺ T cells in the spleen and lung tissue was calculated as described in Materials and Methods. Bar, mean ± SEM of three mice per group.

FIGURE 2

Cytolytic mechanisms used by TB10.4_4–11-specific CD8⁺ T cells in vivo. A, The absolute number of TB10.4 _4–11 tetramer⁺CD8⁺ T cells per spleen was determined and plotted vs TB10.4_4–11-specific killing for individual Mtb-infected B6 (●) or Pfn^−/− (○) mice. Line, Nonlinear regression analysis for B6 (R² = 0.97) and Pfn^−/− (R² = 0.99). Each data set is best fit by two separate curves, p = 0.0003. B, The absolute number of CFP10_32–39 tetramer⁺CD8⁺ T cells per spleen was determined and plotted vs CFP10_32–39-specific killing for individual Mtb-infected B10.BR (●) and B10.BR.Pfn^−/− (○) mice. Line, Nonlinear regression analysis for B10.BR (R² = 0.59) and B10.BR.Pfn^−/− (R² = 0.76) is shown. Each data set is best fit by two separate curves, p = 0.0012. C, CMTMR (CFSE^neg) WT splenocytes, TB10.4_4–11-pulsed CFSE^low WT, CFSE^mid TNFR^−/−, and CFSE^high lpr splenocytes were mixed ~1:1:1:1 and injected into mice. Target cells recovered from the spleens of uninfected WT mice (top), WT (middle), and Pfn^−/− (bottom) Mtb-infected mice and were analyzed by flow cytometry. Representative histograms are shown. D, The percent TB10.4_4–11specific killing was calculated for five mice per group and normalized to the average specific killing of WT targets in infected WT mice to determine the percent maximal killing for each target population (as described in Materials and Methods). Bar, mean ± SEM. **, p < 0.01 and ***, p < 0.001 by one-way ANOVA vs WT targets within the same recipient group or as indicated by the bracket. Data are representative of three similar experiments.

FIGURE 3

Cytolytic mechanisms used by pulmonary TB10.4_4–11-specific CD8⁺ T cells in vivo. A, 1:1:1:1 mixture of unpulsed CMTMR-labeled WT, TB10.4_4–11-pulsed CFSE^low WT, unpulsed CFSE^mid TNFR^−/−, and TB10.4_4–11-pulsed CFSE^high. TNFR^−/− targets cells were injected into uninfected and Mtb-infected B6 and Pfn^−/− mice. The lungs were analyzed to determine the relative TB10.4_4–11-specific killing of TNFR targets vs WT targets. B, Same as in A, but with lpr targets instead of TNFR^−/− targets. Bar, mean ± SEM of four to five mice per group. ***, p < 0.001 by one-way ANOVA vs WT targets within the same recipient group or as indicated by the bracket.

FIGURE 4

Mtb-specific CD4⁺ T cell cytolytic activity in vivo. A, 1:1 mixture of unpulsed CFSE^high and CFP10_11–25-pulsed CFSE^low-labeled splenic B cells were injected into uninfected B10.BR mice (left) and Mtb-infected mice untreated (center) or pretreated with anti-CD4 Ab (right). The percent specific killing of CFP10_11–25-pulsed cells is indicated in B, Bar, mean ± SEM of four to five mice; ***, p < 0.0001 by Student’s t test. C, In vivo cytolytic assay of ESAT6_1–15-pulsed B cells was used to determine the percent ESAT6_1–15 specific killing in B6 mice infected with Mtb 5 wk before. Bar, mean ± SEM of five mice. Data are representative of three similar experiments. D, Unpulsed CMTMR-labeled WT, ESAT6_1–15-pulsed CFSE^low WT, CFSE^mid TNFR^−/−, and CFSE^high lpr splenic B cells were mixed 1:1:1:1 and injected into mice. Target cells recovered from the spleens of uninfected and Mtb-infected B6, Pfn^−/−, and gld mice were analyzed for specific killing. The percent maximal killing was calculated as described in Materials and Methods. Bar, mean ± SEM of three to five mice per group. Means were not significantly different when tested using a one-way ANOVA.

FIGURE 5

Protection by adoptively transferred immune CD8⁺ T cells requires Pfn. A, B6 (CD45.2) mice were irradiated and injected i.v. with highly purified immune CD8⁺ T cells isolated from congenic CD45.1 mice and then challenged with Mtb by the aerosol route within 24 h. Three weeks later, the CD45.1 and CD45.2 expression by pulmonary CD4⁺ and CD8⁺ T cells was analyzed. Data are representative of five individual mice. B, Splenic CFU from mice in A that received splenic CD8⁺ T cells from uninfected (naive) or infected (immune) mice or no cells (no transfer). Bar, mean. C and D, Three weeks after infection of mice that received no cells (No tx) or WT or Pfn^−/− CD8⁺ T cells, pulmonary cells were isolated, enumerated, and analyzed by flow cytometry. The total number of pulmonary CD4⁺ (□) and CD8⁺ (●) T cells (C) and the total number of TB10.4_4–11-specific (left panel) and Mtb32_93–102-specific (right panel) CD8⁺ T cells (D) was determined by tetramer staining for each individual animal. Bar, mean ± SEM of five mice per group; *, p < 0.05 by one-way ANOVA vs no transfer (E). Purified CD90⁺ T cells pooled from mice that received WT or Pfn^−/− CD8⁺ T cells (five mice per group) were stimulated with TB10.4_4–11 and IFN-γ-releasing CD8⁺ T cells were enumerated by ELISPOT. Bar, mean ± SEM of triplicate wells. Splenic (F) and pulmonary (G) Mtb bacterial burden was assessed 3 wk after no transfer or transfer of WT or Pfn^−/− CD8⁺ T cells and challenge with aerosolized Mtb. Bar, mean ± SEM of five mice per group; n.s., Not significant; *, p < 0.05 and **, p < 0.01 by one-way ANOVA vs no transfer or between groups as indicated by the brackets. The data are representative of three experiments.