Distinct compartmentalization of CD4+ T-cell effector function versus proliferative capacity during pulmonary cryptococcosis

Abstract

The activation and expansion of T cells and their acquisition of effector function are key steps in the development of the adaptive immune response. Most infections are predominantly outside of the lymphoid tissues, and it is unclear at what point developmentally and anatomically T cells acquire effector function in vivo. In these studies, we compared the activation and polarization of T cells during murine pulmonary Cryptococcus neoformans infection in the secondary lymphoid tissues and at the site of primary infection. Few CD4(+) and CD8(+) T cells expressed an activated phenotype (CD44(hi,) CD25(+), CD69(+), CD62L(lo), CD45RB(lo)) at the sites of clonal expansion (lymph nodes, spleen, and blood). In contrast, a high percentage of T cells expressed activation markers at the site of primary infection, the lungs. Additionally, the polarization of CD4(+) T cells to interferon-gamma-producing effector cells occurred at the site of infection, the lungs. CD4(+) and CD8(+) T cells from secondary lymphoid organs responded to TCR restimulation by proliferating, whereas T cells from the lungs proliferated poorly. This report demonstrates for the first time that T-cell activation and effector function in secondary lymphoid tissues during fungal infection is characteristically different from that at the site of primary infection.

Figure 1

Kinetics of the host response to pulmonary C. neoformans infection at the time of infection and weeks 1, 2, and 4 after infection. Pulmonary fungal burden (A), lung T-cell (B), and LALN T-cell (C) numbers were assessed as described in the Materials and Methods. Each time point represents the mean ± SEM of six animals. Data are from two independent experiments. Note that the y axis in A is in log₁₀ scale. **In C, T-cell numbers in the LALNs of uninfected, specific pathogen-free mice are less than 10⁵ cells. *P < 0.05, compared to CD4⁺ or CD8⁺ T-cell numbers at week 0 (uninfected controls).

Figure 2

Activation profiles of T cells isolated from the spleens, LALNs, and lungs of uninfected mice and mice 1, 2, and 4 weeks after infection. The expressions of CD44, CD25, CD69, CD62L, and CD45RB were determined by flow cytometry on freshly isolated cells, as described in the Materials and Methods. Data points are the means of pooled samples from three mice per time point. Similar results were obtained in three independent experiments.

Figure 3

Production of IFN-γ in stimulated T cells from the lymph nodes and lungs of C. neoformans-infected mice. Leukocytes were cultured overnight in the presence or absence of anti-CD3 cross-linking antibodies, as described in the Materials and Methods, stained for the presence of IFN-γ and analyzed by intracellular flow cytometry. In A, representative dot plots from week 4 after infection are shown. In B, the percentage of IFN-γ-positive CD4⁺ and CD8⁺ T cells in lungs or lymph nodes after restimulation with anti-CD3 and anti-CD28 at weeks 1, 2, and 4 after infection. The percentage of IFN-γ-positive cells was multiplied by the absolute number of each T-cell population to determine the absolute number of IFN-γ-positive T cells per LALN and per lung. Data points represent pooled samples of three mice per time point. Similar results were obtained in three independent experiments.

Figure 4

IFN-γ production by T cells from the lungs and secondary lymphoid tissues. T cells from the lungs, LALNs, and spleens of infected mice and the lungs and spleens of uninfected (UC) mice were enriched via MACS or FACS and co-cultured with adherent lung cells from uninfected mice, as described in Materials and Methods. Cultures received no additional stimulus, C. neoformans lysate, or anti-CD3/anti-CD28. Bars represent the percentage of IFN-γ⁺ T cells in the stimulated wells minus IFN-γ⁺ T cells in unstimulated wells. Data points represent pooled samples of three mice per time point and are from one of two experiments with similar results.

Figure 5

Effect of anti-CD3 stimulation on lymphoblast formation by T cells obtained from the LALNs and lungs of mice 2 weeks after C. neoformans infection. T cells were cultured overnight with or without anti-CD3 antibodies and forward scatter was assessed by flow cytometry. Data are representative of three independent experiments consisting of three mice per time point.

Figure 6

Effect of anti-CD3 stimulation on proliferation of T cells obtained from the spleen, LALNs, and lungs of mice after C. neoformans infection. Cells from each of the organs were labeled with CFSE and cultured for 3 days in the presence or absence of anti-CD3 antibodies. The intensity of CFSE staining on CD4⁺ and CD8⁺ T cells was assessed by flow cytometry. Data are representative of two independent experiments consisting of three mice per time point.