Diversity of the T-cell response to pulmonary Cryptococcus neoformans infection

Abstract

Cell-mediated immunity plays an important role in immunity to the pathogenic fungus Cryptococcus neoformans. However, the antigen specificity of the T-cell response to C. neoformans remains largely unknown. In this study, we used two approaches to determine the antigen specificity of the T-cell response to C. neoformans. We report here that a diverse T-cell receptor (TCR) Vbeta repertoire was maintained throughout the primary response to pulmonary C. neoformans infection in immunocompetent mice. CD4+ T-cell deficiency resulted in relative expansion of all CD8+ T-cell subsets. During a secondary immune response, preferential usage of a TCR Vbeta subset in CD4+ T cells occurred in single individuals, but the preferences were "private" and not shared between individuals. Both CD4+ and CD8+ T cells from the secondary lymphoid tissues of immunized mice proliferated in response to a variety of C. neoformans antigens, including heat-killed whole C. neoformans, culture filtrate antigen, C. neoformans lysate, and purified cryptococcal mannoprotein. CD4+ and CD8+ T cells from the secondary lymphoid tissues of mice undergoing a primary response to C. neoformans proliferated in response to C. neoformans lysate. In response to stimulation with C. neoformans lysate, lung CD4+ and CD8+ T cells produced the effector cytokines tumor necrosis factor alpha and gamma interferon. These results demonstrate that a diverse T-cell response is generated in response to pulmonary C. neoformans infection.

FIG. 1.

Vβ TCR repertoire of CD4⁺ and CD8⁺ T cells in uninfected CBA/J mice. The frequencies of splenic CD8⁺ (A) and CD4⁺ (B) T cells expressing each Vβ subset were determined as described in Materials and Methods by flow cytometric analysis. Each bar represents a single animal. The frequencies (C and D) and absolute numbers (E and F) of each Vβ subset in lung CD4⁺ and CD8⁺ T cells were determined by flow cytometric analysis of lymphocytes recovered from enzymatic digests of whole lungs, as described in Materials and Methods. Each bar represents a single animal. The numbers indicate the mean for each group.

FIG. 2.

Vβ TCR repertoire of lung T cells in CBA/J mice after C. neoformans infection. The absolute number (A, C, and E) of T cells expressing each Vβ subset and the increase compared to uninfected control lungs (B, D, and F) are shown. Mice were infected intratracheally with 10⁴ CFU of C. neoformans. CD4⁺ T cells were recovered from the lungs of infected mice at weeks 3 (A and B) and 5 (E and F) postinfection, and CD8 T cells were recovered from the lungs of infected mice at week 3 postinfection (C and D). Each bar represents a single animal, and the numbers indicate the mean for each group.

FIG. 3.

Effect of CD4⁺ T-cell deficiency on the CD8⁺ Vβ repertoire during pulmonary C. neoformans infection. The increases in the numbers of lung CD8 T cells at weeks 3 (A) and 5 (B) postinfection relative to the numbers in CD4⁺ T-cell-sufficient, uninfected control (UC) lungs are shown. CBA/J mice were made CD4⁺ T cell deficient throughout the infection by using anti-CD4 depleting monoclonal antibodies, as described in Materials and Methods. The TCR Vβ repertoire of lung CD8⁺ T cells in CBA/J mice was assayed as described above. Each bar represents a single animal, and the numbers indicate the mean for each group.

FIG. 4.

Relative expansion of Vβ TCR subsets in lung CD8⁺ (A) and CD4⁺ (B) T cells during the secondary response to C. neoformans. Mice were infected with 10³ CFU of C. neoformans and rested to allow resolution of the primary infection, as described in Materials and Methods. The mice were given a secondary infection consisting of 10⁴ CFU C. neoformans, and the Vβ TCR usage of CD4⁺ and CD8⁺ T cells in the lungs was assayed by flow cytometry at day 10 after the secondary infection. Each bar represents a single animal, and the numbers indicate the mean for each group. UC, uninfected control.

FIG. 5.

Proliferation of T cells from secondary lymphoid tissues of immune mice. Cells from LALN and spleens of immunized mice were stained with CFSE and cultured with (i) no restimulation (No Stim), (ii) anti-CD3/anti-CD28 (αCD3 αCD28), or (iii) C. neoformans lysate (C. neo Lysate). The histograms show the results for CD4⁺ or CD8⁺ gated populations after 2 and 4 days in culture.

FIG. 6.

Proliferation of (A) and effector cytokine production by (B and C) T cells from immunized mice in response to a variety of C. neoformans antigens. (A) Pooled LALN cells and splenocytes from immunized mice were cultured with the following stimuli, as described in Materials and Methods: anti-CD3/anti-CD28, no stimulus (No Stim), heat-killed C. neoformans (Cneo), C. neoformans lysate, C. neoformans culture filtrate antigen (CneF), and purified C. neoformans mannoprotein (MP). The data are the data from one of two experiments in which similar results were obtained. (B and C) Frequency of production of IFN-γ and TNF-α by CD4⁺ (B) and CD8⁺ (C) T cells from immunized mice in response to C. neoformans antigens. Pooled LALN cells and splenocytes from immunized mice were cultured with the various stimuli, as described in Materials and Methods. The frequency is the percentage of cytokine-positive T cells in the stimulated wells minus the percentage in unstimulated wells. The data are the data from one of two experiments in which similar results were obtained.

FIG. 7.

Proliferative responses of T cells from the LALN of mice infected with C. neoformans. Splenocytes from uninfected mice were used as a control, because there were insufficient numbers of T cells from the LALN of uninfected specific-pathogen-free mice for analysis. T cells were enriched by MACS, stained with CSFE, and cultured with adherent splenocytes from uninfected controls for 4 days in the presence of (i) no additional stimulus (No Stim), (ii) C. neoformans lysate (C. neo Lysate), or (iii) anti-CD3 antibodies (αCD3). The histograms show data for viable cells gated on CD4⁺ or CD8⁺ cells, and the percentages indicate the percentages of daughter cells (cells which had undergone ≥1 cell division). The data are the data from one of two experiments in which similar results were obtained.

FIG. 8.

Frequency of production of effector cytokines by T cells from the lungs and secondary lymphoid tissues. T cells from the lungs, LALN, and spleens of infected mice and from the lungs and spleens of uninfected mice were enriched by MACS or FACS and cocultured with adherent lung cells from uninfected mice, as described in Materials and Methods. Cultures received no additional stimulus or C. neoformans lysate. The bars indicate the percentage of cytokine-positive T cells in the stimulated wells minus the percentage of cytokine-positive cells in unstimulated wells. The data are the data from one of two experiments in which similar results were obtained.