The C-Type Lectin Receptor MCL Mediates Vaccine-Induced Immunity against Infection with Blastomyces dermatitidis

Abstract

C-type lectin receptors (CLRs) are essential in shaping the immune response to fungal pathogens. Vaccine-induced resistance requires Dectin-2 to promote differentiation of antifungal Th1 and Th17 cells. Since Dectin-2 and MCL heterodimerize and both CLRs use FcRγ as the signaling adaptor, we investigated the role of MCL in vaccine immunity to the fungal pathogen Blastomyces dermatitidis. MCL(-/-) mice showed impaired vaccine resistance against B. dermatitidis infection compared to that of wild-type animals. The lack of resistance correlated with the reduced recruitment of Th17 cells to the lung upon recall following experimental challenge and impaired interleukin-17 (IL-17) production by vaccine antigen-stimulated splenocytes in vitro. Soluble MCL fusion protein recognized and bound a water-soluble ligand from the cell wall of vaccine yeast, but the addition of soluble Dectin-2 fusion protein did not augment ligand recognition by MCL. Taken together, our data indicate that MCL regulates the development of vaccine-induced Th17 cells and protective immunity against lethal experimental infection with B. dermatitidis.

FIG 1

Vaccine-induced immunity against B. dermatitidis requires MCL signaling. MCL^−/− mice and wild-type littermates were vaccinated with 10⁶ live vaccine yeast twice over the course of 4 weeks. Two weeks after the boost, vaccinated and unvaccinated mice were challenged with 2 × 10³ wild-type yeast. At 2 weeks postinfection (A), when unvaccinated mice were moribund, or 4 days postinfection (B), during the peak influx of antigen-specific CD4 T cells into the lung, lung CFU were assessed. Data are the means ± standard errors of the means (SEM) (n = 8 to 12 mice/group); data are from a single experiment representative of 3 independent experiments. *, P < 0.05 versus vaccine-induced reduction in lung CFU in wild-type mice. Numbers indicate the n-fold difference in lung CFU versus that of unvaccinated controls.

FIG 2

Number of activated T cells migrating to the lung on recall is reduced in MCL^−/− mice. MCL^−/− mice and wild-type littermates received an adoptive transfer of 10⁶ CD4⁺ purified, naive 1807 Tg cells and were vaccinated with 10⁶ live vaccine yeast or left unvaccinated. At day 4 postinfection, the number of activated (CD44⁺) transferred 1807 T cells was enumerated from the lung by flow cytometry. (A and B) Dot plots show concatenated samples of 4 to 6 mice/group. The numbers indicate the means ± SEM of activated (CD44^hi) 1807 Tg (Thy1.1⁺) T cells. Data are expressed as means ± SEM (n = 4 to 6 mice/group). Data are means ± SEM (n = 4 to 6 mice/group) from single experiments representative of 3 independent experiments. *, P < 0.05 versus the number of 1807 T cells in vaccinated wild-type mice.

FIG 3

Number of IL-17-producing T cells migrating to the lung on recall is reduced in MCL^−/− mice. MCL^−/− mice and wild-type littermates received an adoptive transfer of 10⁶ CD4⁺ purified, naive 1807 Tg cells and were vaccinated with 10⁶ live vaccine yeast cells or left unvaccinated. At day 4 postinfection, the number and frequency of cytokines producing 1807 T cells were enumerated from the lung by FACS. (A to C) Dot plots show concatenated samples of 4 to 6 mice/group. The numbers inside the dot plots indicate the mean number ± SEM of IFN-γ- and IL-17-producing 1807 Tg (Thy1.1⁺) T cells. The numbers outside the dot plots indicate the mean frequencies ± SEM of IFN-γ- and IL-17-producing 1807 Tg (Thy1.1⁺) T cells. Data are expressed as the means ± SEM (n = 4 to 6 mice/group). *, P < 0.05 versus cytokine production by T cells from wild-type mice. (D) At day 4 postinfection, the splenocytes were stimulated with CW/M Ag and IFN-γ and IL-17 production was determined by ELISA. Data are the means ± SEM (n = 4 to 6 mice/group) from single experiments representative of 3 independent experiments. *, P < 0.05 versus the number of lung CFU from vaccinated wild-type mice.

FIG 4

MCL and Dectin-2 regulate T-cell expansion and Th17 cell differentiation. C57BL6 wild-type mice (Dectin-2^+/+), Dectin-2^−/− mice, and MCL^−/− mice and wild-type littermates received an adoptive transfer of 10⁶ CD4⁺ purified, naive, CFSE-labeled 1807 Tg cells and were vaccinated with 10⁶ live vaccine yeast or left unvaccinated. At day 8 postvaccination, the number of activated (CD44⁺) (A) and cytokine-producing (B) 1807 T cells were enumerated from the lung by FACS. Dot plots show concatenated samples of 4 to 6 mice/group. The numbers inside the dot plots indicate the mean number ± SEM of activated (CD44⁺) 1807 Tg (Thy1.1⁺) T cells. Data are expressed as the means ± SEM (n = 4 to 6 mice/group). *, P < 0.05 versus activated 1807 T cells from wild-type mice. SP, spleen. (C) T cells from the sdLN and spleen were stimulated with CW/M antigen for 3 days, and cytokines were measured in the cell culture supernatant. *, P < 0.05 versus cytokines from wild-type control mice.

FIG 5

Recognition of B. dermatitidis ligands by Dectin-2 and MCL. (A and B) B3Z cells expressing FcRγ, Dectin-2 and FcRγ, MCL and FcRγ, or Dectin-2, MCL, and FcRγ were stimulated with heat-killed B. dermatitidis (A) or plate-coated cell wall extract (CWE) isolated from live and heat-killed B. dermatitidis (B). After 18 h, lacZ activity was measured using a colorimetric assay and expressed as OD₅₆₀/OD₆₂₀ values. Data are the means ± standard deviations from duplicate wells. (C) Recombinant human IgG1 Fc, Dectin-2-Fc, MCL-Fc, and a mixture (1:1) of Dectin-2-Fc and MCL-Fc were incubated with the indicated amount of plate-coated cell wall extract isolated from live or heat-killed B. dermatitidis. Bound proteins were detected with anti-hIgG-HRP and expressed as OD₄₅₀ values. (D) Fc protein staining of B. dermatitis. Heat-killed and live yeast cells were incubated with Fc fragment alone, Dectin-2-Fc, MCL-Fc, or a mixture of Dectin-2-Fc and MCL-Fc, as indicated by different colors, followed by staining with PE-conjugated anti-human Fc Ab and analysis by FACS. As a negative control, Ab-stained fungal cells without any Fc proteins are indicated by gray filled histograms.