CD40 Expression by B cells is Required for Optimal Immunity to Murine Pneumocystis Infection

Abstract

CD40-CD40L interactions are critical for controlling Pneumocystis infection. However, which CD40-expressing cell populations are important for this interaction have not been well-defined. We used a cohousing mouse model of Pneumocystis infection, combined with flow cytometry and qPCR, to examine the ability of different populations of cells from C57BL/6 mice to reconstitute immunity in CD40 knockout (KO) mice. Unfractionated splenocytes, as well as purified B cells, were able to control Pneumocystis infection, while B cell depleted splenocytes and unstimulated bone-marrow derived dendritic cells (BMDCs) were unable to control infection in CD40 KO mice. Pneumocystis antigen-pulsed BMDCs showed early, but limited, control of infection. Consistent with recent studies that have suggested a role for antigen presentation by B cells, using cells from immunized animals, B cells were able to present Pneumocystis antigens to induce proliferation of T cells. Thus, CD40 expression by B cells appears necessary for robust immunity to Pneumocystis.

Figure 1.

Flow cytometry analysis of lung and spleen cells following transfer of CD19− cells or CD19⁺ B cells; gating was on live cells, then lymphocytes, then CD19 and CD45.1. Representative results are shown for spleen and lung cells from CD40 KO mice exposed to a Pneumocystis-infected seeder for 65 days. The exposed mice were reconstituted at day 20 with PBS (control) or with CD19⁺ B cells (positive), or with CD19 depleted (negative) spleen cells. Transferred B cells (CD45.1⁺/CD19⁺) were detected in the spleen and lung of the mouse that received CD19+ B cells, while transferred non-B cells were detected in the mouse that received cells that had been depleted of CD19⁺ B cells. Neither population was detected in the mouse that had received PBS.

Figure 2.

Transfer of splenocytes controls P. murina infection in CD40 KO mice. CD40 KO mice were cohoused with a P. murina infected seeder beginning at day 0. At day 20 (Cage 1) or 24 (cage 2) mice were injected via tail-vein with either PBS or ~50 million splenocytes from C57BL/6 mice. Organism loads were deteremined by qPCR targeting the single copy dfhr gene, and are expressed as dhfr copies per mg lung tissue. Data represent the geometric mean ± SD for 2–4 mice per time-point for the splenocyte group, and 1 (day 50) to 2 mice per time-point for the PBS control group. P value is shown for the difference between the 2 groups, using Student’s unpaired t-test. *, p<0.05

Figure 3.

Transfer of B cells is sufficient to control P. murina infection in CD40 KO mice. A. CD40 KO mice were cohoused with a P. murina infected seeder beginning at day 0. At day 20 mice were injected via tail-vein with either PBS or ~28 million CD19⁺ B cells from C57BL/6 mice. For A and B, organism loads were deteremined by qPCR targeting the single copy dfhr gene, and are expressed as dhfr copies per mg lung tissue. Data represent the geometric mean ± SD for 2–3 mice per time-point for the splenocyte group, and 1 (days 25 and 65) to 2 (day 90) mice per time-point for the PBS control group. Student’s unpaired t-test was used to compare the groups at each time-point. *, p=0.02 B. CD40 KO mice cohoused as above received either CD19⁺ B cells (B cell⁺, ~50 million cells/mouse), splenocytes that had been depleted of CD19⁺ B cells (B cell-, ~15–25 million cells/mouse) or saline at day 20. For these studies the focus was on later time-points (days 65 and 90), when the differences were seen in the prior studies, to allow for greater numbers of mice at each of these time-points, given the restrictions on the number of mice permitted per cage. In one experiment, no flow data were available due to technical difficulties at the day 65 timepoint; all animals from that time-point were included in the analysis. Data represent the geometric mean ± SD; number of mice per time-point is as follows: day 35, n=1 for each group; day 65, 7 to 12 per group; day 90, 6 to 9 per group. Student’s unpaired t-test was used to compare the different groups at each time-point. *, p=0.02 for B cell⁺ vs B cell⁻; **, p=0.02 for B cell⁺ vs saline; ‡, p=0.005 for B cell⁺ vs B cell⁻; ‡ ‡, p=0.0002 for B cell⁺ vs saline. C. CD4⁺ (left) and CD8⁺ (right) T cells, as determined by flow cytometry and shown as a percent of live lymphocytes, for the 3 groups at days 65 and 90. Symbols represent mean, error bars represent standard deviation. No significant differences were seen among the groups at either time-poiint. D. Anti-Pneumocystis antibodies, as determined by ELISA, for the animals in B, demonstrated that mice receiving CD19⁺ B cells developed antibodies by day 65 with a further increase by day 90, while no antibodies developed in the other 2 groups. Data represent the mean ± SD. Student’s unpaired t-test was used to compare the different groups at each time-point. *, p=0.02 for B cell⁺ vs B cell⁻; **, p=0.03 for B cell⁺ vs saline; ‡, p=0.001 for B cell⁺ vs B cell⁻; ‡ ‡, p=0.0003 for B cell⁺ vs saline.

Figure 3.

Transfer of B cells is sufficient to control P. murina infection in CD40 KO mice. A. CD40 KO mice were cohoused with a P. murina infected seeder beginning at day 0. At day 20 mice were injected via tail-vein with either PBS or ~28 million CD19⁺ B cells from C57BL/6 mice. For A and B, organism loads were deteremined by qPCR targeting the single copy dfhr gene, and are expressed as dhfr copies per mg lung tissue. Data represent the geometric mean ± SD for 2–3 mice per time-point for the splenocyte group, and 1 (days 25 and 65) to 2 (day 90) mice per time-point for the PBS control group. Student’s unpaired t-test was used to compare the groups at each time-point. *, p=0.02 B. CD40 KO mice cohoused as above received either CD19⁺ B cells (B cell⁺, ~50 million cells/mouse), splenocytes that had been depleted of CD19⁺ B cells (B cell-, ~15–25 million cells/mouse) or saline at day 20. For these studies the focus was on later time-points (days 65 and 90), when the differences were seen in the prior studies, to allow for greater numbers of mice at each of these time-points, given the restrictions on the number of mice permitted per cage. In one experiment, no flow data were available due to technical difficulties at the day 65 timepoint; all animals from that time-point were included in the analysis. Data represent the geometric mean ± SD; number of mice per time-point is as follows: day 35, n=1 for each group; day 65, 7 to 12 per group; day 90, 6 to 9 per group. Student’s unpaired t-test was used to compare the different groups at each time-point. *, p=0.02 for B cell⁺ vs B cell⁻; **, p=0.02 for B cell⁺ vs saline; ‡, p=0.005 for B cell⁺ vs B cell⁻; ‡ ‡, p=0.0002 for B cell⁺ vs saline. C. CD4⁺ (left) and CD8⁺ (right) T cells, as determined by flow cytometry and shown as a percent of live lymphocytes, for the 3 groups at days 65 and 90. Symbols represent mean, error bars represent standard deviation. No significant differences were seen among the groups at either time-poiint. D. Anti-Pneumocystis antibodies, as determined by ELISA, for the animals in B, demonstrated that mice receiving CD19⁺ B cells developed antibodies by day 65 with a further increase by day 90, while no antibodies developed in the other 2 groups. Data represent the mean ± SD. Student’s unpaired t-test was used to compare the different groups at each time-point. *, p=0.02 for B cell⁺ vs B cell⁻; **, p=0.03 for B cell⁺ vs saline; ‡, p=0.001 for B cell⁺ vs B cell⁻; ‡ ‡, p=0.0003 for B cell⁺ vs saline.

Figure 3.

Transfer of B cells is sufficient to control P. murina infection in CD40 KO mice. A. CD40 KO mice were cohoused with a P. murina infected seeder beginning at day 0. At day 20 mice were injected via tail-vein with either PBS or ~28 million CD19⁺ B cells from C57BL/6 mice. For A and B, organism loads were deteremined by qPCR targeting the single copy dfhr gene, and are expressed as dhfr copies per mg lung tissue. Data represent the geometric mean ± SD for 2–3 mice per time-point for the splenocyte group, and 1 (days 25 and 65) to 2 (day 90) mice per time-point for the PBS control group. Student’s unpaired t-test was used to compare the groups at each time-point. *, p=0.02 B. CD40 KO mice cohoused as above received either CD19⁺ B cells (B cell⁺, ~50 million cells/mouse), splenocytes that had been depleted of CD19⁺ B cells (B cell-, ~15–25 million cells/mouse) or saline at day 20. For these studies the focus was on later time-points (days 65 and 90), when the differences were seen in the prior studies, to allow for greater numbers of mice at each of these time-points, given the restrictions on the number of mice permitted per cage. In one experiment, no flow data were available due to technical difficulties at the day 65 timepoint; all animals from that time-point were included in the analysis. Data represent the geometric mean ± SD; number of mice per time-point is as follows: day 35, n=1 for each group; day 65, 7 to 12 per group; day 90, 6 to 9 per group. Student’s unpaired t-test was used to compare the different groups at each time-point. *, p=0.02 for B cell⁺ vs B cell⁻; **, p=0.02 for B cell⁺ vs saline; ‡, p=0.005 for B cell⁺ vs B cell⁻; ‡ ‡, p=0.0002 for B cell⁺ vs saline. C. CD4⁺ (left) and CD8⁺ (right) T cells, as determined by flow cytometry and shown as a percent of live lymphocytes, for the 3 groups at days 65 and 90. Symbols represent mean, error bars represent standard deviation. No significant differences were seen among the groups at either time-poiint. D. Anti-Pneumocystis antibodies, as determined by ELISA, for the animals in B, demonstrated that mice receiving CD19⁺ B cells developed antibodies by day 65 with a further increase by day 90, while no antibodies developed in the other 2 groups. Data represent the mean ± SD. Student’s unpaired t-test was used to compare the different groups at each time-point. *, p=0.02 for B cell⁺ vs B cell⁻; **, p=0.03 for B cell⁺ vs saline; ‡, p=0.001 for B cell⁺ vs B cell⁻; ‡ ‡, p=0.0003 for B cell⁺ vs saline.

Figure 3.

Transfer of B cells is sufficient to control P. murina infection in CD40 KO mice. A. CD40 KO mice were cohoused with a P. murina infected seeder beginning at day 0. At day 20 mice were injected via tail-vein with either PBS or ~28 million CD19⁺ B cells from C57BL/6 mice. For A and B, organism loads were deteremined by qPCR targeting the single copy dfhr gene, and are expressed as dhfr copies per mg lung tissue. Data represent the geometric mean ± SD for 2–3 mice per time-point for the splenocyte group, and 1 (days 25 and 65) to 2 (day 90) mice per time-point for the PBS control group. Student’s unpaired t-test was used to compare the groups at each time-point. *, p=0.02 B. CD40 KO mice cohoused as above received either CD19⁺ B cells (B cell⁺, ~50 million cells/mouse), splenocytes that had been depleted of CD19⁺ B cells (B cell-, ~15–25 million cells/mouse) or saline at day 20. For these studies the focus was on later time-points (days 65 and 90), when the differences were seen in the prior studies, to allow for greater numbers of mice at each of these time-points, given the restrictions on the number of mice permitted per cage. In one experiment, no flow data were available due to technical difficulties at the day 65 timepoint; all animals from that time-point were included in the analysis. Data represent the geometric mean ± SD; number of mice per time-point is as follows: day 35, n=1 for each group; day 65, 7 to 12 per group; day 90, 6 to 9 per group. Student’s unpaired t-test was used to compare the different groups at each time-point. *, p=0.02 for B cell⁺ vs B cell⁻; **, p=0.02 for B cell⁺ vs saline; ‡, p=0.005 for B cell⁺ vs B cell⁻; ‡ ‡, p=0.0002 for B cell⁺ vs saline. C. CD4⁺ (left) and CD8⁺ (right) T cells, as determined by flow cytometry and shown as a percent of live lymphocytes, for the 3 groups at days 65 and 90. Symbols represent mean, error bars represent standard deviation. No significant differences were seen among the groups at either time-poiint. D. Anti-Pneumocystis antibodies, as determined by ELISA, for the animals in B, demonstrated that mice receiving CD19⁺ B cells developed antibodies by day 65 with a further increase by day 90, while no antibodies developed in the other 2 groups. Data represent the mean ± SD. Student’s unpaired t-test was used to compare the different groups at each time-point. *, p=0.02 for B cell⁺ vs B cell⁻; **, p=0.03 for B cell⁺ vs saline; ‡, p=0.001 for B cell⁺ vs B cell⁻; ‡ ‡, p=0.0003 for B cell⁺ vs saline.

Figure 4.

Transfer of BMDCs is insufficient to control P. murina infection in CD40 KO mice. A. CD40 KO mice were cohoused with a P. murina infected seeder beginning at day 0. At day 24–26 mice were injected via tail-vein with either PBS or ~2.5–12 million BMDCs from C57BL/6 mice. For A and B, organism loads were deteremined by qPCR targeting the single copy dfhr gene, and are expressed as dhfr copies per mg lung tissue. Data represent the geometric mean ± SD for 2–5 mice per time-point for the BMDC group, and 2–3 mice per time-point for the PBS control group. B. CD40 KO mice cohoused as above received either PBS or 10 million antigen-primed BMDCs at day 20 (5 cages) or 27 (1 cage); the BMDCs had been incubated with a crude P. murina antigen overnight prior to trasfer. For the latter cage one mouse from each group was harvested at day 45 rather than day 35 as the earliest time-point. For the other times, data represent the geometric mean ± SD for 4–15 mice per time-point for the BMDC group, and 2–11 mice per time-point for the PBS control group. Student’s unpaired t-test was used to compare the groups at each time-point. †, p=0.01; ‡, p=0.001

Figure 4.

Transfer of BMDCs is insufficient to control P. murina infection in CD40 KO mice. A. CD40 KO mice were cohoused with a P. murina infected seeder beginning at day 0. At day 24–26 mice were injected via tail-vein with either PBS or ~2.5–12 million BMDCs from C57BL/6 mice. For A and B, organism loads were deteremined by qPCR targeting the single copy dfhr gene, and are expressed as dhfr copies per mg lung tissue. Data represent the geometric mean ± SD for 2–5 mice per time-point for the BMDC group, and 2–3 mice per time-point for the PBS control group. B. CD40 KO mice cohoused as above received either PBS or 10 million antigen-primed BMDCs at day 20 (5 cages) or 27 (1 cage); the BMDCs had been incubated with a crude P. murina antigen overnight prior to trasfer. For the latter cage one mouse from each group was harvested at day 45 rather than day 35 as the earliest time-point. For the other times, data represent the geometric mean ± SD for 4–15 mice per time-point for the BMDC group, and 2–11 mice per time-point for the PBS control group. Student’s unpaired t-test was used to compare the groups at each time-point. †, p=0.01; ‡, p=0.001

Figure 5.

B cells can present P. murina antigens in vitro. A cell proliferation assay was used to determine if Pneumocystis antigens, presented by B cells or dendritic cells plus monocytes, could induce CD4⁺ T cells to proliferate. Spleen cells from 3–4 C57BL/6 mice that had been previously immunized with 20 μg crude P. murina antigen or adjuvant alone were combined and purified by cell sorting to provide populations of CD4⁺ T cells, CD19⁺ B cells, and CD11c⁺/CD11b⁺ DCs and monocytes. Purified CD4+ T cells (44,000/well) were cultured for 5 days either alone or co-incubated with B cells (50,000/well) or DCs/monocytes (5,000 cells/well) incubated with P. murina antigen (20 μg/ml), purified Msg (10 μg/ml) or media alone; results are shown as the stimulation index. CD4⁺ T cells or B cells cultured alone and unpurified splenocytes served as controls. Results are shown as mean stimulation index of 3 replicates for each condition, and are shown for one of 2 experiments with similar results. P values are shown for the difference between the RLU of the antigen incubated wells vs. no antigen control wells, using Student’s unpaired t-test. *, p<0.05; †, p<0.01; ‡, p<0.001.