Maintenance of peripheral T cell responses during Mycobacterium tuberculosis infection

Abstract

Fully functional T cells are necessary for the maintenance of protective immunity during chronic infections. However, activated T cells often undergo apoptosis or exhaustion upon chronic stimulation mediated by Ag or inflammation. T cell attrition can be compensated for by the production of thymus-derived T cells, although the new naive T cells must undergo T cell priming and differentiation under conditions different from those encountered during acute infection. We used a murine model of Mycobacterium tuberculosis infection to address how the activation and differentiation of new thymic emigrants is affected by chronic inflammation, as well as whether the newly developed effector T cells help to maintain peripheral T cell responses. Although new thymic emigrants contributed to the peripheral T cell response early during acute M. tuberculosis infection, the relative contribution of new effector T cells to the peripheral CD4 and CD8 T cell pools declined during chronic infection. The decline in new T cell recruitment was a consequence of quantitative and/or qualitative changes in Ag presentation, because during chronic infection both the priming and expansion of naive T cells were inefficient. Thus, although thymic tolerance is not a major factor that limits protective T cell responses, the chronic environment does not efficiently support naive T cell priming and accumulation during M. tuberculosis infection. These studies support our previous findings that long-term protective T cell responses can be maintained indefinitely in the periphery, but also suggest that the perturbation of homeostasis during chronic inflammatory responses may elicit immune pathology mediated by new T cells.

Figure 1. The thymus is a target of Mtb infection

Mice were infected via the low-dose aerosol route with Mtb (strain H37Rv), and bacterial titers and effector T cell responses were measured. (A) Colonization of the thymus, lung, MLN, spleen, and liver after aerosol Mtb infection. The data shown were accumulated from two experiments. (B) The flow cytometric gating strategy used to analyze ESAT6_4-17/A^b and Tb10.4_4-11/K^b antigen- specific T cells in the thymus, on day 11, 33, 68, 98 post-infection, is shown. (C) The data indicate the number of ESAT6_4-17/A^b and Tb10.4_4-11/K^b antigen-specific T cells detected in the thymus during Mtb infection. (D) Representative dot plots indicate the frequency of IFNγ̃ and TNFα-producing CD4 and CD8 single-positive thymic T cells detected on day 31, 67, and 100 post-infection. The data are representative of three experiments of similar design, wherein 5 mice were used per group.

Figure 2. RTEs contribute to the T cell response during Mtb infection

(A) A schematic illustrating the experimental protocol is shown. CD45.1/CD45.2 mice either remained uninfected, or were infected, via the low-dose aerosol route, with Mtb (strain H37Rv); 30 or 90 days later, the mice were administered busulfan (0.6 mg/mouse). One day later, 1.5×10⁷ T cell-depleted bone marrow cells from C57BL/6 mice were injected, and ESAT6_4-17/A^b -specific CD44^hi thymus-derived donor and host CD4 T cells were quantitated in the lungs 63 days later. (B) Analyses of donor and host T cells are shown. The numbers in the dot plots indicate the frequencies of ESAT6_4-17/A^b antigen-specific donor or host CD4 T cells after bone marrow reconstitution of uninfected, day 30, or day 90 Mtb-infected animals. Data are representative of two experiments, where five mice were analyzed in each experimental group.

Figure 3. Naïve T cell priming during Mtb infection

Naïve ESAT6 and SM1 transgenic T cells were isolated from mice by negative magnetic bead selection, and 1×10⁶ cells were injected, at a 1:1 ratio, into infected CD45- and Thy1-congenic recipient mice. Mice were analyzed 12 hours after cell transfer on each of the days indicated. (A) Representative flow cytometric analysis of CD69expression on ESAT6 or SM1 transgenic T cells from the spleen on day 25 post-infection. (B) Expression of CD69 on donor ESAT6 transgenic T cells in the MLN and spleen during infection. The datum represents individual mice; the dashed line indicates the mean CD69 expression of SM1 transgenic T cells in mice analyzed throughout the experiment. The data shown were accumulated from two experiments, and are representative of three independent experiments.

Figure 4. Effector T cells detect similar levels of antigen during Mtb infection

In vitro differentiated ESAT6 Th1 effectors (1×10⁶) were injected into congenic recipient mice, and mice were analyzed 12 hours after cell transfer on each of the days indicated. The percent of cells that expressed of CD69 on donor ESAT6 Th1 effector T cells in the spleen, MLN, and lung is shown. The datum represents individual mice; the dashed line indicates the mean CD69 expression detected on effector Th1 cells that were transferred into uninfected mice. The data shown were accumulated from three experiments.

Figure 5. Expansion of naïve transgenic T cells during acute and chronic Mtb infection

Naïve ESAT6 transgenic T cells (2 × 10³) were injected intravenously into CD45-congenic mice that had been aerosol-infected 11, 30, 60, or 90 days prior. The number of transgenic donor cells in the spleen, MLN, and lung, were determined on days 2, 7, and 10 post-transfer. The arrows in each plot indicate the day the transgenic T cells were transferred. The data are representative of two experiments of similar design, wherein 5 mice were used per group.

Figure 6. RTEs contribute to the peripheral CD4 T cell response during chronic infection

Infected CD45-congenic mice were administered 0.6 mg of busulfan on day 30, 60, or 90 post-infection. One day later, 1.5×10⁷ T cell-depleted bone marrow cells from C57BL/6 mice were injected, and ESAT6_4-17/A^b -specific CD4 T cells were quantitated in the lungs 49, 63, 84, and 140 days after reconstitution. (A) The number of ESAT6_4-17/A^b-specific host and donor CD4 T cells is shown. (B) Representative dot plots are shown of IFNγ̃ and TNFα-producing donor or host CD4 T cells and the frequencies of ESAT6_4-17/A^b antigen-specific CD4 T cells from busulfan-treated mice that were analyzed 140 days post-reconstitution. Intracellular and tetramer staining were preformed on two independent samples from the same animals. (C) The frequencies of ESAT6_4-17 / I-A^b tetramer-positive cells within the donor- and host-expressing populations were plotted against the frequency of ESAT6_1-20 / I-A^b cells that specifically produced IFNγ for each busulfan-treated mice within each group; the mice received busulfan on either day 30 or 90 post-infection, and were analyzed 140 days post-reconstitution. The best-fit line was generated using least squares regression analysis. No statistical difference was found upon comparison of the two slopes (p=0.83 day 30, p=0.87 day 90). The data are representative of two experiments of similar design, wherein five mice were used per group.

Figure 7. RTEs contribute to the peripheral CD8 T cell response during chronic infection

Infected mice were administered 0.6 mg of busulfan on the indicated days post-infection. One day later, 1.5×10⁷ T cell- depleted bone marrow cells from C57BL/6 mice were injected, and Tb10.4_4-11/K^b antigen-specific CD8 T cells were quantitated in the lungs. (A) Analyses of donor and host T cells are shown. The numbers in the dot plots indicate the frequencies of Tb10.4_4-11/K^b antigen-specific CD8 T cells after donor-bone marrow transfer in mice treated with busulfan on day 30, day 90 post-infection. (B) The number of Tb10.4_4-11/K^b -specific host and donor CD8 T cells after donor-bone marrow reconstitution is shown.