Germline IgM predicts T cell immunity to Pneumocystis

Abstract

Pneumocystis is the most common fungal pulmonary infection in children under the age of 5 years. In children with primary immunodeficiency, Pneumocystis often presents at 3-6 months of age, a time period that coincides with the nadir of maternal IgG and when IgM is the dominant Ig isotype. Because B cells are the dominant antigen-presenting cells for Pneumocystis, we hypothesized the presence of fungal-specific IgMs in humans and mice and that these IgM specificities would predict T cell antigens. We detected fungal-specific IgMs in human and mouse sera and utilized immunoprecipitation to determine whether any antigens were similar across donors. We then assessed T cell responses to these antigens and found anti-Pneumocystis IgM in WT mice, Aicda-/- mice, and in human cord blood. Immunoprecipitation of Pneumocystis murina with human cord blood identified shared antigens among these donors. Using class II MHC binding prediction, we designed peptides with these antigens and identified robust peptide-specific lung T cell responses after P. murina infection. After mice were immunized with 2 of the antigens, adoptive transfer of vaccine-elicited CD4+ T cells showed effector activity, suggesting that these antigens contain protective Pneumocystis epitopes. These data support the notion that germline-encoded IgM B cell receptors are critical in antigen presentation and T cell priming in early Pneumocystis infection.

Keywords: Adaptive immunity; Fungal infections; Immunology; Infectious disease; Proteomics.

Figure 1. IgM responses to whole Pneumocystis antigen in Pneumocystis-naive mice.

(A) Anti-Pneumocystis IgM titers in WT C57BL/6 mice. (B) Anti-Pneumocystis IgM titers in Aicda^–/– and Rag2^–/– mice. In this assay, an OD of more than 0.1 was considered positive. The asterisk denotes significant differences between Aicda^–/– and Rag2^–/– mice (by Mann-Whitney).

Figure 2. IgM responses to Pneumocystis antigens in human cord blood.

(A) Anti-Pneumocystis IgM titers in human cord blood in response to whole Pneumocystis antigen. (B). Western blot analysis of human cord blood IgM in response to whole Pneumocystis antigen. (C). Venn diagram analysis of Pneumocystis proteins immunoprecipitated by human cord blood IgM. (D) Human cord blood IgM specificity in response to Pneumocystis-specific antigens PNEG_01454 and Meu10 (PNEG_01435). PNEG_02812 was identified in all 3 donors. Data are shown as mean ± SD.

Figure 3. Pneumocystis-specific IgG responses 6 weeks after PC infection in mice.

(A) P. murina Meu10/ PNEG_01435–specific IgG titers (n = 3 per group). Responses were only observed in PC-infected WT mice and not in naive C57BL/6 mice or Aicda^–/– mice. (B) P. murina PNEG_01454–specific IgG titers (n = 3 per group). Two-way ANOVA analysis showed that IgG titers for each Pneumocystis-specific antigen were significant in C57BL/6 mice. *P < 0.05. Data are shown as mean ± SD.

Figure 4. Pneumocystis-specific T cell responses after PC infection in mice.

(A) C57BL/6 and (B) Aicda^–/– mice were infected with 100 μL P. murina inoculum (2 × 10⁵ cysts) via oral pharyngeal administration. Two weeks later, single cells from mouse lungs were prepared for IFN-γ and IL-5 Elispot or LegendPlex assays (n = 4 per group). Uninfected naive lungs had a background of 0–1 spots in all stimulation conditions, so 1 representative naive lung sample is depicted as background in the assay. PC antigen and P1–P6 were significantly greater in lung samples from Aicda^–/– mice compared with those from naive lung controls (P < 0.05 by ANOVA or Mann-Whitney test). (C) C57BL/6 Aicda^–/– and CD4⁺ cre Stat3^fl/fl mice at 6 weeks. Both C57BL/6 and Aicda^–/– mice had significantly higher type I responses compared with CD4⁺ cre Stat3^fl/fl mice (P < 0.05, ANOVA). Data are shown as mean ± SD.

Figure 5. Effector activity of antigen-enriched CD4⁺ T cells.

(A) C57BL/6 mice were immunized with 10 μg Meu10 and 10 μg LTA1, 10 μg LTA1 only, or vehicle (naive) by oral pharyngeal aspiration and boosted once 3 weeks later. One week after boosting, 2 × 10⁵ purified CD4⁺ T cells were transferred into Rag2^–/– mice intravenously. Two weeks after cell adoptive transfer, all mice were infected with approximately 2 × 10⁵ asci of P. murina inoculum by oral pharyngeal aspiration. Four weeks later, right lung middle lobes were removed for RNA isolation and fungal burden assessment by RT-qPCR. Dunnett’s multiple-comparison test shows that Rag2^–/– mice that received CD4⁺ T cells from Meu10-primed C57BL/6 mice had significantly lower fungal burden in the lungs compared with all other groups. (B) The experiments described in A were repeated with whole PC Ag and PNEG_01454. CD4⁺ T cells from OTII-Rag2^–/– mice were used as an irrelevant antigen control. *P < 0.05, ***P < 0.001 by 1-way ANOVA followed by Dunn’s multiple comparisons test.