Memory CD73+IgM+ B cells protect against Plasmodium yoelii infection and express Granzyme B

Abstract

To better understand anti-malaria protective immune responses, we examined the cellular mechanisms that govern protective immunity in a murine Plasmodium yoelii 17X NL (PyNL) re-infection model. Initially, we confirmed that immune B cells generated during a primary PyNL infection were largely responsible for protection from a second PyNL infection. Using the previously identified memory B cell markers CD80, PD-L2, and CD73, we found an increase in the frequency of CD80-PD-L2-CD73+ B cells up to 55 days after a primary PyNL infection and at 4-6 days following a second PyNL infection. Moreover, injection of enriched immune CD19+CD73+ B cells into nonimmune mice were significantly more protective against a PyNL infection than CD73- B cells. Interestingly, a substantial fraction of these CD73+ B cells also expressed IgM and granzyme B, a biomolecule that has been increasingly associated with protective responses against malaria.

Fig 1. B cells enriched from splenocytes 9 months post-PyNL clearance conferred protection against a secondary PyNL infection.

Results are expressed as percent parasitemia ± SEM following i.p. infection of C57BL/6 mice with 1 x 10⁶ PyNL erythrocytic stage parasites. Parasitemia levels from naive (nonimmune), PyNL Immune mice and mice adoptively transferred with B cells from naive mice were compared with mice receiving PyNL immune B cells. Parasitemia levels were evaluated by examining blood smears beginning at day 5 post-infection until parasitemia clearance. Mann-Whitney test was used for statistical evaluation; *p<0.05; n = 7 mice per group.

Fig 2. Kinetics of the expansion of CD19⁺ B cells expressing CD80, PD-L2 and CD73 markers post-primary PyNL infection.

(A) Percent parasitemia over the course of a PyNL infection measured by blood smear evaluation. Expansion of splenic (B) CD80^-PD-L2^-CD73⁺ and (C) CD80⁺PD-L2⁺CD73⁺ B cell populations (mean ± SEM) measured by flow cytometry over the course of a PyNL infection and at 1 month post-parasitemia clearance (1-M PC). (D) Representative dot plots show gating strategy and frequencies of splenic CD80^-PD-L2^-CD73⁺ B cells from mice at D-0 and at 1-M PC. Mann-Whitney test was used for statistical evaluation; *p<0.05; n = 5 mice per group. Results are expressed as per 1 x 10⁶ CD19⁺ B cells from one experiment out of two with similar results. *p<0.05; n = 5 mice per group.

Fig 3. PyNL immune CD73⁺ B cells confer protection against a primary PyNL infection.

CD73^- (3 x 10⁷) or CD73⁺ enriched B cells (9 x 10⁶ or 3 x 10⁷) obtained from immune mice 7 months post-PyNL clearance were transferred into nonimmune, naive mice. Two hours after the transfer of cells, mice were infected i.p. with 1 x 10⁶ PyNL erythrocytic stage parasites. Parasitemias were evaluated by blood smears starting at day 4 post-infection until parasitemia clearance. Results are expressed as the percent parasitemia ± SEM. A representative experiment out of two experiments is shown. Mann-Whitney test was used for statistical evaluation, p<0.05; n = 5 mice per group.

Fig 4. Comparison of CD19⁺ B cell frequencies expressing CD73 and/or Granzyme B at different time points following a primary PyNL infection and at one month post-parasitemia clearance.

Representative flow cytometry dot plots show frequencies of CD19^- or CD19⁺ splenocytes (T cells were excluded) expressing CD73 or frequencies of CD19⁺CD73⁺ B cells expressing Granzyme B (Granz B) at (A) Day 0 or (B) Day 13 post-PyNL infection. Alternatively, frequencies of CD19^- or CD19⁺ splenocytes expressing Granz B at (C) both time points or (D) frequencies of CD19⁺Granz B⁺ cells expressing CD73 at D-13 were determined. Also shown is the development of CD19⁺ B cells expressing (E) CD73, (F) Granzyme B (GrB), (G) both CD73 and GrB or (H) or CD73^- B cells expressing GrB following a primary PyNL infection beginning at Day 0 through Day 30 post-parasitemia clearance (PC). A representative experiment of two experiments is shown. Mann Whitney test was used for statistical evaluation. *p<0.05; n = 5 mice per group.

Fig 5. Comparison of CD19+ B cell frequencies expressing CD73 and GrB the day before and at Day 6 post-secondary infection with PyNL.

Splenic B cells were recovered from PyNL immune mice two months post-primary PyNL infection (Pre-SI) or six days post-secondary PyNL infection (D-6 post-SI) (secondary infection at 2 months post-primary infection). Frequencies of CD19⁺ B cells expressing (A, B) CD73, (C, D) GrB, (E, F) CD73 and GrB, (E, H) CD73, GrB, and IgM markers or (G, I) CD19⁺CD73^-, GrB⁺, IgM⁺ cells were measured by flow cytometry at these time points. Representative dot plots (A, C, E, G) show frequencies of these different populations at Pre-SI and D-6 post-SI. Representative experiment of two experiments is shown. Mann-Whitney test was used for statistical evaluation. Mean ± SEM; *p<0.05; n = 5 mice per group.

Fig 6. Comparison by flow cytometry of CD19⁺ B cell frequencies expressing CD73, IgM or Granzyme B (GrB) following an in vitro PyNL infection.

Splenic CD19⁺ B cells were recovered from noninfected mice (naive) or at 4 months post-PyNL clearance (PyNL Immune mice). Five million spleen cells were co-cultured with 5 x 10⁶ PyNL iRBC (Naive-PyNL; PyNL Immune-PyNL) or without PyNL iRBC (Naive; PyNL Immune). Representative flow cytometry dot plots from two independent experiments show frequencies of different B cell subsets from stimulated Naïve (left column) or Immune splenocytes (right column) (A, C), or after gating on CD19⁺CD73⁺ (E), or CD19⁺CD73^- cells (G). Frequencies of B cell subsets shown in B, D, F, H and I are expressed as the percentage of CD19⁺ cells and, thus, were obtained after gating on CD19⁺ B cells as shown on the y-axis (n = 5 mice per group). Mann-Whitney test was used for statistical evaluation. *p<0.05; n = 5 mice per group.

Fig 7. Flow Cytometry Imaging of CD73⁺GrB⁺ B cells.

Splenocytes from PyNL immune mice were co-incubated with PyNL infected erythrocytes for 4 days and then surface stained with fluorochrome-conjugated antibodies specific for CD19, CD73, and CD8 followed by intracellular staining using a monoclonal antibody specific for granzyme B (GrB). The cells were incubated with violet live-dead stain to differentiate viable from nonviable cells and then visualized using an ImageStream system via bright field (BF) or fluorescence mode at 60X magnification (CD8⁺ cells were excluded from the gates). Using fluorescence mode, CD73⁺ B cells expressing GrB were visualized by placing a gate on live CD19⁺ cells (red) followed by gating on CD73⁺ (purple), GrB⁺ (green) or CD19⁺CD73⁺GrB⁺ cells (purple and green).