CD28 is required for optimal induction, but not maintenance, of vaccine-induced immunity to Blastomyces dermatitidis

Abstract

Cellular immunity mediated by T lymphocytes, in particular CD4+ and CD8+ type 1 cells, is the main defense against pathogenic fungi. Here, CD28-deficient (CD28-/-) mice were used to study the role of costimulation for the generation and maintenance of T-cell-mediated, type 1 cytokine-dependent mechanisms of vaccine immunity to Blastomyces dermatitidis infection. Disruption of CD28 costimulation reduced the number of type 1 CD4 and CD8 cells generated and impaired resistance to infection. Type 1 T-cell subsets generated in vaccinated CD28-/- mice were durable and protected mice for at least 3 months after vaccination. Our findings suggest that CD28 is required for the induction of optimal, protective T-cell responses to B. dermatitidis infection but may be dispensable for the maintenance of T-cell memory.

FIG. 1.

T-cell priming and production of IFN-γ, TNF-α, and GM-CSF transcript and protein by lung T cells after B. dermatitidis infection in vaccinated and unvaccinated wild-type and CD28^−/− mice. (Top panel) Priming of lung T cells as measured by surface expression of CD44 on cells analyzed 4 days postinfection. The data are an average of six to nine mice/group. Mice were infected with 2 × 10³ yeast. *, P < 0.05; **, P < 0.1 (versus vaccinated wild-type controls). Controls (C) represent vaccinated mice treated with rat IgG. (Middle panel) n-fold changes in lung cytokine transcripts in vaccinated wild-type and CD28^−/− mice versus unvaccinated controls as measured by real-time PCR. The data represent an average of three to five mice/group. Mice were infected with 2 × 10³ yeast and analyzed 2 and 3 days after infection. *, P < 0.05 (versus unvaccinated controls). Cytokine transcripts did not differ significantly in vaccinated CD28^−/− mice versus wild-type controls, except for a marginal difference in GM-CSF transcript at day 2. **, P = 0.06. (Bottom panel) Intracellular cytokine production by CD44⁺ CD4⁺ and CD44⁺ CD8⁺ cells. The data represent an average of six to nine mice per group. Mice were infected with 2 × 10³ yeast and analyzed 4 days after infection. *, P < 0.008 (versus corresponding unvaccinated mice); **, P ≤ 0.02; ***, P < 0.07 (versus corresponding vaccinated wild-type controls). Controls (C) represent vaccinated mice treated with rat IgG.

FIG. 2.

Role of CD28 costimulation for the generation of protective CD4 and CD8 T-cell responses. Mice were vaccinated subcutaneously twice with 10⁴ live yeast at each site of injection. To induce protective CD8 T-cell immunity, CD28^−/− and wild-type mice were depleted by anti-CD4 MAb throughout the experiment. Mice were infected with 2 × 10³ wild-type yeast and analyzed for lung CFU 14 to 16 days after infection. The data are geometric mean CFU ± the standard error of the mean of two independent experiments; n = 8 to 12 mice/group. *, P < 0.001 (versus unvaccinated controls); **, P < 0.001 (versus corresponding vaccinated CD28^−/− mice); ***, P < 0.005 (versus corresponding CD4-depleted mice).

FIG. 3.

Histological appearance of skin tissue at the site of vaccination of vaccinated CD28^−/− and wild-type mice. Tissue was analyzed 3 weeks postvaccination when CD28^−/− mice were harvested to determine lung CFU. Skin tissue was stained with Gomori's methenamine silver; all images are at ×40 magnification. Yeast cells were 10-fold more plentiful in CD4-depleted CD28^−/− mice than in nondepleted CD28^−/− mice and were concentrated mainly along the periphery of the lesions.

FIG. 4.

Maintenance of vaccine induced resistance by CD4 and CD8 cells in the absence of CD28. Mice were vaccinated subcutaneously twice with 10⁶ live yeast at each site of injection. After a resting period of 3 months, mice were infected intratracheally with 2 × 10³ wild-type yeast. The data are geometric mean CFU ± the standard error of the mean; n = six to nine mice/group. *, P < 0.001 (versus unvaccinated control mice).