Marginal zone B cells (MZB) participate in the early immune response to several pathogens. In this study, we show that in μMT mice infected with Leishmania donovani, CD8 T cells displayed a greater cytotoxic potential and generated more effector memory cells compared with infected wild type mice. The frequency of parasite-specific, IFN-γ+ CD4 T cells was also increased in μMT mice. B cells were able to capture parasites, which was associated with upregulation of surface IgM and MyD88-dependent IL-10 production. Moreover, MZB presented parasite Ags to CD4 T cells in vitro. Depletion of MZB also enhanced T cell responses and led to a decrease in the parasite burden but did not alter the generation of effector memory T cells. Thus, MZB appear to suppress protective T cell responses during the early stages of L. donovani infection.

B cells are mainly known for their role in the production of Abs aimed at facilitating pathogen and/or other Ag clearance. However, an increasing body of literature has now shown that they may also regulate adaptive T cell responses by various Ab-independent mechanisms, such as cytokine production, costimulation, and Ag presentation (14). Recent studies in various infectious disease models have demonstrated that B cells can enhance Th2 responses (57). Conversely, B cells were also shown to support Th1 responses (6). In contrast, IL-10–producing B cells can suppress CD4 T cell responses (8) and prevent the induction of autoimmune diseases in several mouse models (9, 10). These reports suggest that depending on the disease model, B cells can both enhance and/or suppress CD4 T cell responses.

The role of B cells in regulating CD8 T cell responses is less clear, but also seems to depend on the disease model. In some models, B cells are not critical for the priming of CD8 T cells (1113) or for memory generation (14). In contrast, in LCMV- (13, 15) and L. monocytogenes- (12) infected mice, the absence of B cells seems to affect memory generation. In addition, TGF-β–secreting B cells can induce anergy in CD8 T cells (16). Moreover, B cells were shown to inhibit CD4 and CD8 T cells in tumor-induced immunity by a yet unknown mechanism (17).

Various studies have now demonstrated that B cells play a negative role in several experimental models of leishmaniasis (5, 1820). B cell depletion was shown to enhance resistance to Leishmania tropica and Leishmania mexicana in BALB/c mice (18), and cotransfer of B cells converts T cell-reconstituted, Leishmania major-resistant, C.B-17 scid mice into a susceptible phenotype (21). Furthermore, C57BL/6 B cell-deficient mice are highly resistant to Leishmania donovani infection (20). The mechanism by which B cells exacerbate Leishmania infections is yet unknown. A recent study has suggested that IL-10 derived from L. major-induced regulatory B cells (Bregs) skews the balance toward unprotective Th2 responses (5). In contrast, Deak et al. (19) recently proposed that IgM production and polyclonal B cell activation, which requires activation of complement, are the main cause of disease exacerbation in Leishmania infantum-infected mice rather than IL-10 production by B cells.

In this study, we investigated the role of B cells in the regulation of Ag-specific CD8 T cell responses in a murine model of visceral leishmaniasis (VL). CD8 T cells are very important effector cells in the immune response to the protozoan parasite L. donovani, a causative agent of VL (22, 23). However, we have previously reported that mice infected with L. donovani developed deficient parasite-specific CD8 T cell responses, characterized by limited clonal expansion and functional exhaustion during chronic disease (23). In this study, we demonstrate that marginal zone B cells (MZB) suppress Ag-specific CD8 and CD4 T cell responses during the early stages of VL. Suppression of NK1.1+ cell functions appears to be, in part, mediated by MyD88-dependent IL-10 production. Moreover, B cells inhibit the generation of effector memory CD8 T cells after L. donovani infection.

All experiments were approved by and conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Animal Care and Use Committee of The Johns Hopkins University School of Medicine (Protocol No. MO08M501).

Six- to 8-wk-old C57BL6N/Cr and CD45.1-C57BL6 female mice were obtained from National Cancer Institute (Frederick, MD). C57BL/6-Il10−/−, B6.129S7-Rag1tm1Mom/J, and C57BL/6-Tg(OT-I)-RAG1tm1Mom mice were obtained from The Jackson Laboratory. μMT mice were provided by Dr. S. Desiderio (The Johns Hopkins University, Baltimore, MD). C57BL/6-MyD88−/− mice were a gift from Dr. F. Zavala (The Johns Hopkins University). Ly5.1-OT-I mice were generated by crossing CD45.1-C57BL6 with C57BL/6-Tg(OT-I)-RAG1tm1Mom mice. Mice were housed at the animal facility of The Johns Hopkins University under specific pathogen-free conditions. All experiments were approved and performed in compliance to regulations set by the Animal Care and Use Committee of The Johns Hopkins University School of Medicine. Transgenic L. donovani parasites expressing OVA (PINK) were a gift from Drs. P. Kaye and D. F. Smith (University of York, York, U.K.). Wild type (WT) L. donovani (strain LV9) and PINK were maintained by serial passage in B6.129S7-Rag1tm1Mom/J mice. Mice were infected by injecting 2 × 107 amastigotes i.v. into the lateral tail vein. Splenic parasite burden was determined by limiting dilution (23).

CD45.1-OT-I transgenic mice, expressing an MHC class I-restricted TCR specific for chicken OVA257–264, were used for adoptive transfers. CD8 T cells were enriched from the spleen of naive OT-I/RAG1 mice using MACS following manufacturer’s instructions (Miltenyi Biotech). Naive CD8 T cells were then sorted to >98% purity using FACSVantage (Becton Dickinson) based on the CD62L and CD44 expression. A total of 2 × 104 sorted cells were then injected via the lateral tail vein of mice.

For the IL-10 blockade, mice were treated biweekly with 200 μg anti–IL-10R Ab (clone IBI.3A) or with isotype control. MZB were depleted using the mixture of Abs described previously (24, 25). In brief, mice were injected with a single dose of 100 μg of either isotype or anti–LFA-1α (clone M17/4; eBioscience) and anti-CD49d Ab (clone R1-2; eBioscience). Mice were infected 1 wk after the administration of anti–LFA-1α and anti-CD49d. To deplete B cells, we injected mice with 250 μg MB20-11 Ab (26), which was a kind gift from Dr. Thomas Tedder (Duke University). Mice were infected 6 d after B cell depletion.

WT mice were infected with PINK. Sera from infected mice were collected between days 12 and 14 p.i. and pooled. μMT mice were administered with 200 μl of the pooled sera the day before infection. Control μMT mice received naive sera.

L. donovani parasites were stained with PKH67 (Sigma) following manufacturer’s instructions. Mice received 5 × 107 PKH67-labeled parasites. Spleens from naive and infected mice were harvested 20 h later and surface stained for flow cytometric analysis.

Naive B cells were purified using the naive B cell isolation kit (Miltenyi Biotech) according to manufacturer’s protocol. Purified B cells (purity 90–92%) were then incubated either alone, with different ratios of parasites, or with 1 μg/ml CpG (InvivoGen). For cross-linking of IgM and C3dg receptor (CD21), latex beads were incubated either alone, with anti-IgM (5 μg/ml), or with anti-CD21 (5 μg/ml) for 2 h at room temperature (RT). The beads were washed twice with PBS and then incubated with B cells at 37°C to remove unbound Abs. IL-10 production by B cells was assessed at 24 h using the IL-10 secretion kit from Miltenyi Biotech following the manufacturer’s protocol.

Adoptively transferred OT-I T cells were identified by staining the splenocytes with biotinylated anti-CD45.2 and/or CD45.1 Ab followed by streptavidin-PerCp, or with anti–CD45.1-FITC. Other Abs used to further characterize CD8 T cells were CD8-allophycocyanin, CD8-Pacific blue, CD69-PE, CD44-allophycocyanin, CD44-biotin, CD62L-PE, CD62L-allophycocyanin, CD122-PE, CD127-PE, and KLRG1-allophycocyanin. All Abs unless specified were from BD Pharmingen. For intracellular staining, splenocytes were stimulated with the SIINFEKL peptide (Genosphere Biotechnologies) and IL-2 (Amgen) in the presence of brefeldin A (BD Pharmingen) for 4 h at 37°C followed by surface staining for anti-CD45.2/1 and -CD8 (23). The cells were then fixed, permeabilized, and stained with either isotypes or anti-granzyme B–PE (Caltag), anti–IFN-γ–allophycocyanin, anti–IL-2–PE (BD Pharmingen), and anti–TNF-α–PE–Cy7 (eBioscience). More than 1 million cells per sample were acquired with an LSRII (Beckon Dickinson), and analysis was done using FACSDiva software.

Endogenous CD4 T cell responses were analyzed as follows. Bone marrow-derived dendritic cells (BMDCs) were pulsed with fixed parasites for 24 h at 37°C. Splenocytes were added to BMDCs and incubated for 2 h at 37°C. Brefeldin A (BD Pharmingen) was then added for a further 4 h. Cells were then stained with biotinylated anti-CD3 followed by PerCP-conjugated streptavidin, FITC-conjugated anti-CD4, and allophycocyanin-conjugated anti–IFN-γ (all BD Bioscience). For B cell analysis, samples were surface stained with anti–CD19-allophycocyanin-Cy7 (1D3), anti–CD5-FITC (53-7.3), anti-CD21 PE (7G6; BD Pharmingen), anti–CD1d-PE (1B1), and anti–CD23-PE-Cy7 (B3B4; eBioscience). Flow cytometric analysis was performed with an LSRII flow cytometer (Becton Dickinson). A total of 350,000 cells per sample were acquired and analyzed with the FACSDiva software.

The expression of costimulatory molecules by B cells 24 h after incubation with parasites was assessed with the following Abs (BD Pharmingen): anti–CD19-allophycocyanin-Cy7, anti–CD86-PE (GL1), anti–CD80-PerCP-Cy5.5 (16-10A1), anti–MHC class II-FITC (2G9), anti–CD40-biotin (323), and anti–SA-PE-Cy7.

L. donovani parasites were stained with PKH67 (Sigma) following manufacturer’s instructions. Naive B cells were purified using the naive B cell isolation kit (Miltenyi Biotech) according to manufacturer’s protocol. The purity of the B cells was 90–92%. One million purified B cells were then incubated either alone or with 1:5 or 1:10 ratios of parasites in a 48-well plate for 24 h. B cells in culture were cytospin on glass slides, allowed to air-dry, and then fixed with cold acetone for 10 min. The slides were then washed with PBS and stained as follows: the fixed B cells were incubated for 35 min at RT with 100 μl PBS with naive mouse serum (diluted 1:500) and then washed with PBS. The slides were then incubated with anti-IgM (BD Pharmingen) at 37°C for 1 h, washed, and incubated with rabbit anti-rat AF594 for 35 min at RT. The slides were subsequently washed and mounted with mounting media. Images were taken at 600× with an Olympus BX51 microscope.

Cytospins of parasites were prepared and fixed with cold acetone. The parasites were stained with anti-C3 (Cedarlane) or with anti-mouse IgG2a and conjugated with rabbit anti-rat AF594 (Invitrogen). The slides were washed and mounted with mounting media. Images were taken at 1000× with an Olympus BX51 microscope.

L. donovani amastigotes were incubated with supernatants collected from the B cell in vitro studies (naive B cells, infected B cells, CpG stimulated B cells) for 20 min on ice. They were then washed, incubated with biotinylated anti-IgM for 20 min on ice, and subsequently with streptavidin allophycocyanin (BD Pharmingen). Samples were fixed with 2% paraformaldehyde and analyzed by flow cytometry.

Glass-bottomed petri dishes (Mattek, Ashland, MA) were coated with 10 μg/ml fibronectin for 3 h and washed once with PBS. Isolated naive B cells were seeded on the dish at a density of 25,000 cells/ml and allowed to settle for 30 min before adding parasites at a final density of 100,000 cells/ml. Imaging was started within 1 min of addition of the parasites to the medium with Zeiss Axiovert 2000 microscope using a 40× apochromatic lens (numerical aperture = 1.5). Images were acquired at 37°C, 5% CO2 every 30 s for 4 h using Slidebook 4.2 (Intelligent Image Innovations, Denver, CO). Movies were created using Slidebook and ImageJ (National Institutes of Health, Bethesda, MD).

KZO and B3Z hybridomas were kind gifts from Dr. N. Shastri (University of California, Berkeley, CA). KZO is a CD4 T cell hybridoma that recognizes OVA246–264 in the context of I-Ab. B3Z is a CD8 T cell hybridoma specific for the OVA257–264 epitope in the context of H-2Kb. Both hybridomas have been transfected with an NFAT-lacZ reporter construct (27). MZB were sorted from a starting population of naive splenic B cells (enriched as described earlier) to >98% purity using FACSVantage (Becton Dickinson) based on the expression of CD21 and CD23. A total of 105 sorted MZB were incubated at 37°C with PINK and/or LV9 (multiplicity of infection 1:5) and 105 KZO and/or B3Z. OVA (for KZO) or SIINFEKL (for B3Z) were added to the culture for the positive control groups. Eighteen hours later, the cells were loaded with fluorescein di-β-D-galactopyranoside (Invitrogen) and analyzed for lacZ expression on a flow cytometer as described by Karttunen and Shastri (28). MZB were gated out of the analysis based on the relative small size compared with T cell hybridomas.

WT and μMT mice were infected with 2 × 107 PINK amastigotes. Spleens were harvested on days 1, 3, and 6 p.i. Spleens were collagenase digested followed by isolation of CD11c+ dendritic cells (DCs) using anti-CD11c beads (Miltenyi Biotec) as previously described (29). RNA was extracted using RNeasy mini kit (Qiagen) as per manufacturer’s instruction. Reverse transcription was performed using iScript cDNA synthesis kit (Bio-Rad) using manufacturer’s protocol. Real-time PCR analysis was performed on cDNA using iQ SYBR Green supermix kit (Bio-Rad). IL-12p35, IL-12/IL-23 p40, and actin were amplified using primers described previously (29). All PCRs were carried out with an iCycler (Bio-Rad).

C57BL/6 mice were irradiated with two doses of 5.5 Gy within a 2-h interval. Irradiated mice were reconstituted with bone marrow mixed from μMT, IL-10−/−, and C57BL/6. The bone marrow was mixed at a ratio of 3:1 for the following combinations: 75% μMT with 25% Il10−/− and 75% C57BL/6 with 25% Il10−/−. Irradiated mice were reconstituted with 107 bone marrow cells from the earlier combinations. Mice were provided with antibiotics in drinking water for 4 wk. Engraftment was allowed to proceed for 6 wk. A group of mice was checked for reconstitution 6 wk after bone marrow transfer.

Results were analyzed using unpaired Student t test. A p value <0.05 was considered significant. Real-time PCR results were analyzed using an unpaired Student t test. All experiments were repeated at least twice.

It is known that B cells exacerbate Leishmania infections (5, 1820). B cell-deficient (μMT) mice are indeed more resistant than WT mice to infection with L. donovani (see Supplemental Fig. 1A) (20). However, the mechanisms by which B cells help to exacerbate disease have not yet been clarified. Hence we first wanted to determine whether B cells interfere with the priming and development of protective, Ag-specific CD8 T cells responses in vivo. Thus, OT-I T cells, which recognize an H-2Kb–restricted CD8 T cell epitope of OVA257–264, were adoptively transferred into μMT mice. These mice were subsequently infected with OVA-transgenic L. donovani amastigotes. Between days 3 and 7 p.i., OT-I T cells undergo clonal expansion during L. donovani infection. This is typically followed by clonal contraction (days 9–14) (23). We first compared the number of OT-I T cells that engrafted after the adoptive transfer in both types of host and found similar numbers of OT-I cell in the spleen of both mouse strains 7 d after adoptive transfer (1240 ± 549 cells in the spleen of naive μMT mice, and 1213 ± 528 in WT mice). Next, we compared the expansion of OT-I T cells in μMT and WT mice. B cells did not seem to affect the proliferative capacity of OT-I T cells. Indeed, the number of OT-I T cells present in the spleen of WT mice was similar to that in μMT mice (Fig. 1A). Interestingly, a greater percentage of OT-I T cells expressed granzyme B in μMT mice compared with WT mice (Fig. 1B, Supplemental Fig. 1B). A similar increase in granzyme B expression was observed in the endogenous CD8 T cell population (Fig. 1C, Supplemental Fig. 1B) and the NK1.1+ population in μMT mice (Fig. 1D, Supplemental Fig. 1B). The IFN-γ, IFN-γ/TNF-α, and IFN-γ/IL-2 production by OT-I T cells was similar in both μMT and WT mice (Fig. 1E–G). In contrast, the frequency of endogenous CD4 T cells producing IFN-γ was significantly increased in the absence of B cells (Fig. 1H, Supplemental Fig. 1C), suggesting that B cells are not only interfering with CD8 T cell responses during VL, but also with CD4.

FIGURE 1.

Enhanced CD8 and CD4 T cell responses in μMT mice infected with L. donovani. (A) A total of 2 × 104 OT-I T cells were adoptively transferred before infection with PINK amastigotes. OT-I T cells were identified by gating on CD8+ Ly5.1+ cells. Graphs represent the average number ± SE of OT-I T cells in the spleen of individual mice on days 7 and 13 p.i. (BG) Splenocytes from infected WT and μMT mice were restimulated and stained for granzyme B, IFN-γ, TNF-α, and IL-2. (B) Graph represents the percentage of OT-I T cells producing granzyme B, (C) granzyme B-producing endogenous CD8 T cells, (D) NK1.1+ granzyme B-producing cells, (E) IFN-γ–producing OT-I T cells, (F) TNF-α– and IFN-γ–producing OT-I T cells, and (G) IFN-γ– and IL-2–producing OT-I cells. (H) For endogenous CD4 T cell responses, infected WT and μMT splenocytes were incubated with BMDCs pulsed with fixed parasites for 2 h and then an additional 4 h with brefeldin. Graph represents the percentage of IFN-γ–producing CD4 T cells. Data represent mean percentages ± SE, representative of four independent experiments, n = 3–5. *p < 0.05, **p < 0.01.

FIGURE 1.

Enhanced CD8 and CD4 T cell responses in μMT mice infected with L. donovani. (A) A total of 2 × 104 OT-I T cells were adoptively transferred before infection with PINK amastigotes. OT-I T cells were identified by gating on CD8+ Ly5.1+ cells. Graphs represent the average number ± SE of OT-I T cells in the spleen of individual mice on days 7 and 13 p.i. (BG) Splenocytes from infected WT and μMT mice were restimulated and stained for granzyme B, IFN-γ, TNF-α, and IL-2. (B) Graph represents the percentage of OT-I T cells producing granzyme B, (C) granzyme B-producing endogenous CD8 T cells, (D) NK1.1+ granzyme B-producing cells, (E) IFN-γ–producing OT-I T cells, (F) TNF-α– and IFN-γ–producing OT-I T cells, and (G) IFN-γ– and IL-2–producing OT-I cells. (H) For endogenous CD4 T cell responses, infected WT and μMT splenocytes were incubated with BMDCs pulsed with fixed parasites for 2 h and then an additional 4 h with brefeldin. Graph represents the percentage of IFN-γ–producing CD4 T cells. Data represent mean percentages ± SE, representative of four independent experiments, n = 3–5. *p < 0.05, **p < 0.01.

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Because IgM is thought to contribute to disease exacerbation in BALB/c mice infected with L. infantum (19), we reconstituted μMT mice with sera from days 12–14 infected WT mice before adoptive transfer of OT-I T cells, and infected them with L. donovani. μMT mice reconstituted with sera had a similar parasite burden when compared with unreconstituted μMT mice (see Supplemental Fig. 2A). Furthermore, no difference was observed in the number or effector function of OT-I T cells present in the spleen of both groups of mice (data not shown). Taken together, these results show that B cells appear to be involved in the regulation of CD8 and CD4 T cell responses during the early stages of VL.

In a previous study, we reported that CD8 T cells fail to generate effector memory cells (TEM) during VL. Further, we reported that at the end of contraction, ∼70–80% of the OT-I T cells displayed a central memory cell (TCM)-like phenotype and 20–30% were effector cells (23). Hence we next characterized the phenotype of adoptively transferred OT-I T cells in μMT mice at two different time points: during clonal expansion (day 7) and at the end of contraction (day 13). Phenotypes that were considered were: effector cells (CD62LloCD127), TEM (CD62LloCD127+), and TCM (CD62LhiCD127+). No difference in the percentage of OT-I T cells expressing an effector phenotype (CD62LloCD127neg) was observed at day 7 p.i. (Fig. 2A, 2B, Supplemental Fig. 2B). In contrast, when we looked at the OT-I T cell responses at day 13 p.i., we noticed a significant increase in the frequency of CD62Llo OT-I T cells present in the spleen of μMT mice compared with WT mice (Fig. 2A). Nevertheless, the percentages of CD127neg OT-I T cells at day 13 p.i. were similar in both groups of mice (Fig. 2B). Moreover, there was no difference in the percentages of CD44+ OT-I T cells between both groups (data not shown). Hence 40–50% of the adoptively transferred OT-I T cells that survived contraction in μMT mice was CD44+CD62loCD127+, a phenotype typically associated with TEM. OT-I T cells did not generate TEM in infected WT mice. We also looked at the KLRG1 expression at days 7 and 13 p.i., which is a marker for short-lived effector cells (30). In WT mice, only 10–14% of the OT-I T cells expressed KLRG1 at both time points; in contrast, ∼25% at day 7 and 35% of the OT-I T cells at day 13 were positive for KLRG1 in μMT mice (Fig. 2C, Supplemental Fig. 2B). A recent study demonstrated that the generation of KLRG1+ effector CD8 T cells is governed by IL-12 (31). Thus, we assessed the IL-12 p35 and IL-12/IL-23 p40 mRNA expression by splenic CD11chi DCs at days 1, 3, and 6 after L. donovani infection. L. donovani induces a moderate and transient increase in IL-12p35 mRNA levels in DCs, which peaks at 5 h p.i (32). As shown in Fig. 2D, IL-12p35 mRNA levels gradually decreased from day 1 to 6 p.i. in DC isolated from infected WT mice. In contrast, in infected μMT mice, IL-12 p35 expression was induced at greater levels compared with WT mice and sustained over the first 6 days of infection (Fig. 2D). The expression levels for the IL-12/IL-23 p40 mRNA were comparable in DCs isolated from infected WT and μMT mice, with the exception of day 3, when p40 was expressed at greater levels in infected WT mice (Fig. 2E).

FIGURE 2.

TEM are generated in μMT mice but not in WT mice after infection with L. donovani. OT-I T cells were identified by gating on CD8+ Ly5.1+ cells. Graphs represent the percentage ± SE of gated cells that were (A) CD62Llo, (B) CD127, and (C) KLRG1+ on days 7 and 13 p.i. (D and E) Splenic DCs were MACS enriched after infection with parasite on days 1, 3, and 6. Fold gene induction was calculated after normalization to the housekeeping gene actin. Expression of (D) IL-12p35 and (E) IL-12p40 in naive WT and naive μMT was taken as 1 and compared with the respective infected WT and μMT mice. (FK) B cells were depleted in naive C57BL/6 mice with the anti-CD20 Ab. OT-I T cells were then adoptively transferred into depleted and control undepleted mice. Animals were infected a day after transfer with L. donovani. Graphs represent (F) the splenic parasite burden, (G) granzyme B-producing endogenous CD8 T cells, (H) NK1.1+ granzyme B-producing cells, and (I) IFN-γ production by CD4 T cells. Graphs represent the percentage of OT-I T cells that were (J) KLRG1+, (K) CD62Llo CD127+, CD62LhiCD127+, and CD62Llo CD127. Data represent mean percentages ± SE, representative of two to four independent experiments, n = 3–5. *p < 0.05, **p < 0.01.

FIGURE 2.

TEM are generated in μMT mice but not in WT mice after infection with L. donovani. OT-I T cells were identified by gating on CD8+ Ly5.1+ cells. Graphs represent the percentage ± SE of gated cells that were (A) CD62Llo, (B) CD127, and (C) KLRG1+ on days 7 and 13 p.i. (D and E) Splenic DCs were MACS enriched after infection with parasite on days 1, 3, and 6. Fold gene induction was calculated after normalization to the housekeeping gene actin. Expression of (D) IL-12p35 and (E) IL-12p40 in naive WT and naive μMT was taken as 1 and compared with the respective infected WT and μMT mice. (FK) B cells were depleted in naive C57BL/6 mice with the anti-CD20 Ab. OT-I T cells were then adoptively transferred into depleted and control undepleted mice. Animals were infected a day after transfer with L. donovani. Graphs represent (F) the splenic parasite burden, (G) granzyme B-producing endogenous CD8 T cells, (H) NK1.1+ granzyme B-producing cells, and (I) IFN-γ production by CD4 T cells. Graphs represent the percentage of OT-I T cells that were (J) KLRG1+, (K) CD62Llo CD127+, CD62LhiCD127+, and CD62Llo CD127. Data represent mean percentages ± SE, representative of two to four independent experiments, n = 3–5. *p < 0.05, **p < 0.01.

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To test whether Abs were involved in suppressing the generation of TEM, we reconstituted a group of μMT mice with serum collected at days 12–14 p.i. from infected WT mice. TEM OT-I T cells were generated in infected, serum-reconstituted μMT mice at a similar frequency to that observed in μMT (Supplemental Fig. 2C, 2D). Adoptively transferred OT-I T cells in these mice also had similar KLRG1 expression at days 7 and 12 p.i. (Supplemental Fig. 2E). This implies that Abs alone may not play a role in the regulation of TEM responses; nevertheless, we cannot exclude the participation of Abs in the suppressive effects mediated by B cells.

To exclude the fact that our observations were mainly due to differences in the splenic architecture, cellular composition, and physiology between μMT and C57BL/6 mice rather than to the lack of B cells, we depleted B cells in C57BL/6 mice using a depleting Ab directed against CD20 (Supplemental Fig. 2F). We then infected these mice and control undepleted C57BL/6 mice with L. donovani. Like μMT mice, B cell-depleted C57BL/6 mice had a similar parasite burden to the undepleted group at day 6 p.i. By day 12 p.i., though, B cell-depleted mice showed a superior capacity in controlling parasite growth and had nearly cleared infection (Fig. 2F). B cell depletion also resulted in an increase in granzyme B expression in CD8 T cells (Fig. 2G) and NK1.1+ cells (Fig. 2H) at day 6 p.i. No difference in granzyme B production by both cell populations was observed at day 12 p.i. Similarly, B cell-depleted mice had a significantly higher frequency of IFN-γ+ CD4 T cells at day 6 p.i. compared with the control group (Fig. 2I); no differences were detected at day 12 p.i.

As already observed in L. donovani-infected μMT mice, the frequency of KLRG1+ OT-I T cells was significantly greater in B cell-depleted mice compared with the undepleted group (Fig. 2J). Moreover, OT-I T cells generated TEM in anti-CD20–treated mice at day 12 p.i., but not in the control group (Fig. 2K, Supplemental Fig. 2G).

To understand how B cells may interfere with the development of CD8 T cell responses, we monitored the interaction between naive splenic B cells and L. donovani amastigotes over 24 h. One to 2 h after exposure to the parasites, some B cells already had amastigotes attached to their surface and were projecting protrusions (see Supplemental Video). This was a transient phenomenon that was only observed at the early time points after exposure, suggesting that B cells were activated (33). Moreover, some B cells had formed clusters within 3–5 h after exposure to the parasite. At 48 h, most of the B cells that had clustered were dead (data not shown).

To better understand the B cell–parasite interaction, we exposed B cells to fluorescently labeled L. donovani. After 24 h, B cells were labeled with an anti-IgM Ab and analyzed by immunofluorescence. As shown in Fig. 3A (1–3), most of the B cells carrying parasites had formed clusters after 24 h in culture. Interestingly, the IgM staining seemed to form pockets that partially surrounded the parasite aggregates. This suggests that IgM was not only increasingly expressed on the surface of B cells but probably also secreted. We next investigated whether the interaction between B cells and parasites resulted in B cell activation. Hence we incubated parasites with naive B cells and monitored the surface modulation of the costimulatory molecules CD86, CD80, and CD40 24 h after exposure with L. donovani. The B cell–Leishmania interaction resulted in the upregulation of CD86 (Fig. 3B). The majority of these cells also expressed high levels of MHC class II and CD80 (Fig. 3B). CD40 was not upregulated after coculture with L. donovani (data not shown).

FIGURE 3.

B cells form clusters and upregulate IgM and CD86 on exposure to L. donovani. (A) Naive splenic B cells were isolated and incubated with (1–3) or without (4) PKH67-labeled LV9 (WT L. donovani) for 24 h. B cell cytospins were stained with anti-IgM followed by secondary conjugation to AF594. Immunofluorescence staining; original magnification ×600. (B) CD86 and CD80 expression on CD19+MHC class II+B220+ B cells after incubation with different ratios of B cells to parasite (1:2 and 1:5) for 24 h. Numbers indicate percentage of cells expressing the respective markers, as measured by flow cytometry. (C) PKH67-labeled parasites were transferred to mice and spleens were harvested 20 h later. Splenocytes were stained for CD19, CD5, CD21, and CD23. Cells gated on CD19+CD5+ and PKH67+ are shown. Data are representative of two to three independent experiments.

FIGURE 3.

B cells form clusters and upregulate IgM and CD86 on exposure to L. donovani. (A) Naive splenic B cells were isolated and incubated with (1–3) or without (4) PKH67-labeled LV9 (WT L. donovani) for 24 h. B cell cytospins were stained with anti-IgM followed by secondary conjugation to AF594. Immunofluorescence staining; original magnification ×600. (B) CD86 and CD80 expression on CD19+MHC class II+B220+ B cells after incubation with different ratios of B cells to parasite (1:2 and 1:5) for 24 h. Numbers indicate percentage of cells expressing the respective markers, as measured by flow cytometry. (C) PKH67-labeled parasites were transferred to mice and spleens were harvested 20 h later. Splenocytes were stained for CD19, CD5, CD21, and CD23. Cells gated on CD19+CD5+ and PKH67+ are shown. Data are representative of two to three independent experiments.

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To determine whether B cells were also capturing parasites in vivo, we infected C57BL/6 mice i.v. with PKH67-labeled L. donovani and sacrificed them 20 h later. As expected from the results obtained in vitro, a small percentage (0.2–0.3%) of splenic CD19+ cells was indeed PKH67+ (Fig. 3C). These cells were mostly CD21hi, CD19+, and CD23lo/int.

Recent literature has demonstrated that some Leishmania species activate B cells and induce IL-10 production (5, 19). Thus, we investigated whether B cells also produce IL-10 after L. donovani infection. Purified naive splenic B cells were incubated with L. donovani amastigotes for 24 h and IL-10 production was assessed by FACS. As expected, B cells stimulated with CpG produced IL-10 (34) (Fig. 4A); the majority of the CpG-stimulated cells producing IL-10 were CD1d+CD5+CD23hi, a phenotype that has been associated to Bregs. Of the B cells exposed to L. donovani amastigotes, 2.3% also produced IL-10. However, the IL-10 producers were divided into two populations, both of which were CD21+ (data not shown): CD19+CD21+CD1d+/−CD5+CD23lo cells, a phenotype similar to MZB, and CD19+CD21+CD1d+CD5+CD23hi cells, which have been described as Bregs (4).

FIGURE 4.

CD5+CD1+CD23hi B cells and MZB produce IL-10 on exposure to L. donovani. (AD) IL-10 production was assessed by the IL-10 secretion assay kit. IL-10 producers were gated on forward scatter (FSC) and then identified using surface markers CD23, CD1d, and CD5. (A) Naive splenic B cells were isolated and incubated with either parasite, CpG, or medium alone for 24 h. (B) Naive B cells were isolated from MZB-depleted spleens and incubated with either parasite, CpG, or medium alone. (C) Naive splenic B cells were incubated with either latex beads alone, anti-IgM–coated latex beads, or anti-CD21–coated latex beads for 24 h. (D) Naive splenic B cells from Myd88−/− mice were incubated with either parasite, CpG, or medium alone for 24 h.

FIGURE 4.

CD5+CD1+CD23hi B cells and MZB produce IL-10 on exposure to L. donovani. (AD) IL-10 production was assessed by the IL-10 secretion assay kit. IL-10 producers were gated on forward scatter (FSC) and then identified using surface markers CD23, CD1d, and CD5. (A) Naive splenic B cells were isolated and incubated with either parasite, CpG, or medium alone for 24 h. (B) Naive B cells were isolated from MZB-depleted spleens and incubated with either parasite, CpG, or medium alone. (C) Naive splenic B cells were incubated with either latex beads alone, anti-IgM–coated latex beads, or anti-CD21–coated latex beads for 24 h. (D) Naive splenic B cells from Myd88−/− mice were incubated with either parasite, CpG, or medium alone for 24 h.

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Next, we investigated the role of MZB in the production of IL-10. Thus, we depleted MZB (25) in naive mice, purified naive splenic B cells, and exposed them to L. donovani in vitro. Strikingly, the IL-10 production was reduced by ∼60% after incubation with the parasite (Fig. 4B) and most of the cells that were still producing IL-10 in the MZB-depleted group were CD1d+CD5+CD23hi cells (Fig. 4B). The IL-10 production by B cells after exposure with CpG was also severely reduced after depletion of MZB (Fig. 4B).

Given the quick kinetics of IL-10 production by B cells exposed to L. donovani, we investigated the mechanism that leads to IL-10 secretion. We have previously reported that HASPB1, a surface protein of L. donovani, is recognized by natural Abs and complement (35); and in Fig. 3A, we have shown that B cells dramatically upregulate IgM expression after coincubation with parasites. Thus, we first coated beads with anti-IgM Abs to cross-link IgM on B cells. Of the B cells incubated with anti-IgM–coated latex beads, 3.7% produced IL-10, and the majority of these cells showed a phenotype similar to MZB (Fig. 4C). Because amastigotes purified from Rag1−/− mice were coated with complement C3 (see Supplemental Fig. 3A), we next assessed whether cross-linking of CD21 (complement receptor 2), would also induce IL-10 production. Hence we incubated B cells with anti-CD21–coated latex beads for 24 h and monitored IL-10 production. Only 1.8% of cells produced IL-10, the majority of which displayed a MZB-like phenotype. These results suggest that Leishmania may be inducing IL-10 production by cells with an MZB phenotype via cross-linking of surface IgM and/or CD21. However, neither mechanism induced IL-10–producing B cells expressing CD1d+CD5+CD23hi, implying that there may be an additional pathway of IL-10 induction. Thus, we investigated whether the adaptor protein MyD88 was involved in the induction of IL-10 secretion by B cells after exposure with L. donovani. Naive B cells were purified from Myd88−/− mice and coincubated with L. donovani or CpG. As expected, no IL-10 production was detected in culture treated with CpG in Myd88−/− B cells (Fig. 4D). Similarly, IL-10 was also nearly completely abrogated after incubation of Myd88−/− B cells with L. donovani (compare Fig. 4A and 4D).

Because L. donovani was captured by cells with an MZB-like phenotype (Fig. 3C) and this interaction resulted in the upregulation of CD86 (Fig. 3B), we next investigated whether MZB were able to present parasite-derived Ags to T cells. Hence we coincubated sorted MZB and PINK parasites with the KZO (ova-specific CD4 T cell hybridoma) and/or B3Z (ova-specific CD8 T cell hybridoma) (27, 28, 36) overnight. As shown in Fig. 5A, presentation of the SIIKFEKL peptide by MZB led to the activation of the B3Z cells, as measured by the lacZ expression. However, when MZB were incubated with PINK, we did not detect any activation in the B3Z cell population, suggesting that MZB were not able to present parasite-derived ova to B3Z (Fig. 5A). We also assessed whether MZB were capable of presenting parasite-derived OVA to ova-specific CD4 T cell hybridoma (KZO). As a positive control, we coincubated MZB and OVA with KZO, which showed increased lacZ expression (Fig. 5B). Interestingly, when we coincubated MZB with PINK, we also noticed a significant increase in T cell activation compared with the negative controls (Fig. 5B). We next investigated whether follicular B cells (FoB) were able to present parasite-derived OVA to KZO and B3Z cells. Neither B3Z (Fig. 5C) nor KZO (Fig. 5D) were activated on coincubation with FoB and PINK, suggesting that FoB are not capable of presenting parasite-derived OVA to B3Z or KZO.

FIGURE 5.

MZB present parasite-derived OVA to CD4 T cell hybridoma. MZB and FoB were purified from the spleen of C57BL/6 mice and incubated with KZO and/or B3Z, and PINK, OVA, or the SIINFEKL peptide at 37°C. Eighteen hours later, KZO and B3Z were assessed for their lacZ expression by flow cytometry. (A) FACS profiles of lacZ expression by B3Z cells incubated with MZB from naive C57BL/6 mice. (B) FACS profiles of lacZ expression by KZO cells incubated with MZB from naive mice. (C) FACS profiles of lacZ expression for B3Z cells incubated with FoB from naive mice. (D) FACS profiles of lacZ expression for KZO cells incubated with FoB from naive mice. Data are representative of three independent experiments.

FIGURE 5.

MZB present parasite-derived OVA to CD4 T cell hybridoma. MZB and FoB were purified from the spleen of C57BL/6 mice and incubated with KZO and/or B3Z, and PINK, OVA, or the SIINFEKL peptide at 37°C. Eighteen hours later, KZO and B3Z were assessed for their lacZ expression by flow cytometry. (A) FACS profiles of lacZ expression by B3Z cells incubated with MZB from naive C57BL/6 mice. (B) FACS profiles of lacZ expression by KZO cells incubated with MZB from naive mice. (C) FACS profiles of lacZ expression for B3Z cells incubated with FoB from naive mice. (D) FACS profiles of lacZ expression for KZO cells incubated with FoB from naive mice. Data are representative of three independent experiments.

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Next, we evaluated whether IL-10 could mediate the suppressive effects observed on the T cell responses in infected mice. Hence we treated C57BL/6 mice with an anti–IL-10R Ab and monitored the development of adoptively transferred OT-I T cells. In vivo IL-10R blockade resulted in a 3- to 4-fold increased expansion of OT-I cells at day 6 p.i. (Fig. 6A). Interestingly, though, despite the increased expansion at day 6, OT-I T cells in mice treated with the anti–IL-10R Ab contracted to about the same number at day 9 p.i. IL-10R blockade did not improve IFN-γ production by CD8 OT-I T cells (data not shown); however, we noticed an increase in granzyme B production by OT-I T cells (Fig. 6B, Supplemental Fig. 3B) and in the frequency of endogenous CD4 T cells producing IFN-γ (Fig 6C, Supplemental Fig. 3B) at day 13 p.i. in infected mice treated with the anti–IL-10R Ab. However, IL-10R blockade failed to restore the generation of TEM in L. donovani-infected mice (see Supplemental Fig. 3C, 3D), but we observed a significant increase in the frequency of KLRG1+ cells in anti–IL-10R–treated, infected mice (Fig. 6D). Taken together, these data show that IL-10R blockade enhanced the effector functions of CD8 and CD4 T cells, and KLRG1 expression by CD8 T cells; however, it did not induce TEM, suggesting that the pathway leading to the generation of TEM is not governed by IL-10.

FIGURE 6.

IL-10 suppresses expansion and function of OT-I T cells. Mice were administered either isotype or anti–IL-10R Ab before transfer of OT-I T cells and infection with PINK. (A) Absolute numbers of OT-I T cells on days 6 and 13 p.i. were identified by gating on CD8+ Ly5.1+ cells. (B) Granzyme B production by OT-I T cells upon restimulation on days 6 and 13 p.i. (C) IFN-γ production by endogenous CD4 T cells. (D) Percentage of OT-I T cells expressing KLRG1+. Data represent mean percentages ± SE, representative of four independent experiments, n = 3–5. *p < 0.05, **p < 0.01.

FIGURE 6.

IL-10 suppresses expansion and function of OT-I T cells. Mice were administered either isotype or anti–IL-10R Ab before transfer of OT-I T cells and infection with PINK. (A) Absolute numbers of OT-I T cells on days 6 and 13 p.i. were identified by gating on CD8+ Ly5.1+ cells. (B) Granzyme B production by OT-I T cells upon restimulation on days 6 and 13 p.i. (C) IFN-γ production by endogenous CD4 T cells. (D) Percentage of OT-I T cells expressing KLRG1+. Data represent mean percentages ± SE, representative of four independent experiments, n = 3–5. *p < 0.05, **p < 0.01.

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We then wanted to determine which B cell population was involved in the regulation of T cell responses during the early stages of L. donovani infection. Thus, we proceeded to deplete MZB before infection with L. donovani (see Supplemental Fig. 4A). MZB depletion did not affect OT-I T cell expansion in infected mice (Fig. 7A) and did not promote the generation of TEM at day 14 p.i. (Fig. 7B, 7C, Supplemental Fig. 4B). However, the frequency of KLRG1+ OT-I T cells was significantly increased in mice depleted of MZB compared with undepleted mice (Fig. 7D, Supplemental Fig. 4B). Moreover, the percentage of granzyme B producing OT-I T cells was also increased at day 7 p.i. in mice depleted of MZB compared with the control group (Fig. 7E, Supplemental Fig. 4C). No differences were observed at day 14 p.i. Endogenous CD8 T cells also expressed more granzyme B in infected MZB-depleted mice at day 7 p.i. (Fig. 7F, Supplemental Fig. 4C) and the frequency of IFN-γ–producing CD4 T cells was also slightly greater in these mice compared with undepleted mice (Fig. 7G, Supplemental Fig. 4C). More importantly, depletion of MZB not only improved T cell responses, but also resulted in a significantly lower parasite burden (Fig. 7H), suggesting that MZB contribute to the establishment of chronic L. donovani infection in mice.

FIGURE 7.

MZB depletion enhances CD8 and CD4 T cell responses. Mice were treated with a single dose of 100 μg anti-CD11a and anti-CD49d 1 wk before transfer of OT-I cells followed by infection with PINK. (A) Absolute numbers of OT-I T cells on days 7 and 14 p.i. were identified by gating on CD8+ Ly5.1+ cells. OT-I T cells were analyzed for expression of (B) CD62Llo, (C) CD127, and (D) KLRG1+ at days 7 and 14 p.i. (E and F) Splenocytes were restimulated and intracellularly stained for granzyme B. (E) Granzyme B production by OT-I T cells and (F) endogenous CD8 T cells. (G) IFN-γ production by endogenous CD4 T cells. Data represent mean percentages ± SE, representative of two independent experiments, n = 3–5. (H) Splenic parasite burden was determined by limiting dilutions. Data represent mean numbers ± SE, representative of two independent experiment, n = 3–5. *p < 0.05, **p < 0.01.

FIGURE 7.

MZB depletion enhances CD8 and CD4 T cell responses. Mice were treated with a single dose of 100 μg anti-CD11a and anti-CD49d 1 wk before transfer of OT-I cells followed by infection with PINK. (A) Absolute numbers of OT-I T cells on days 7 and 14 p.i. were identified by gating on CD8+ Ly5.1+ cells. OT-I T cells were analyzed for expression of (B) CD62Llo, (C) CD127, and (D) KLRG1+ at days 7 and 14 p.i. (E and F) Splenocytes were restimulated and intracellularly stained for granzyme B. (E) Granzyme B production by OT-I T cells and (F) endogenous CD8 T cells. (G) IFN-γ production by endogenous CD4 T cells. Data represent mean percentages ± SE, representative of two independent experiments, n = 3–5. (H) Splenic parasite burden was determined by limiting dilutions. Data represent mean numbers ± SE, representative of two independent experiment, n = 3–5. *p < 0.05, **p < 0.01.

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Because the enhanced T cell effector functions observed after IL-10 blockade were similar to those observed in μMT mice, we finally wanted to investigate the role of B cell-derived IL-10 in the suppression of T cell responses. Thus, we generated mix bone marrow chimera using μMT and Il10−/− mice as donors and irradiated C57BL/6 as recipients. Because 25% of non-B cells will also be IL-10 deficient and this could compromise the course of infection, we generated C57BL/6 and Il10−/− chimeric mice as control group. Twenty-five percent of the cells in these mice are also IL-10 deficient. First, we determined whether both groups of mice had comparable frequencies of B and T cells in the spleen. The percentage of B cells (Supplemental Fig. 4D), CD4 T cells (Supplemental Fig. 4E), and CD8 T cells (Supplemental Fig. 4F) was not significantly different in both groups of mice. We then proceeded to analyze the effect of B cell-derived IL-10 on Ag-specific CD8 T cell responses during L. donovani infection. As shown in Fig. 8A, the absence of IL-10 production by B cells did not have any effect on the expansion of OT-I T cells. Likewise, no differences were observed in the surface modulation of CD62L (Supplemental Fig. 4G) and CD127 (Supplemental Fig. 4H). IFN-γ (Fig. 8B) and granzyme B (Fig. 8C) production by OT-I T cells were also comparable in both groups of mice. Granzyme B production by endogenous CD8 T cells was also similar in both groups at day 6 p.i., but slightly increased in the WT/Il10−/− control group at day 13 p.i. (Fig. 8D). These results imply that B cell-derived IL-10 does not affect CD8 T cell responses during VL. In contrast, when we analyzed granzyme B expression in NK1.1+ cells, we noticed a significant increase in granzyme B+ NK1.1+ cells in the μMT/Il10−/− group at day 6 p.i. (Fig. 8E). As observed in infected mice depleted of MZB (Fig. 7G), the frequency of IFN-γ+ CD4 T cells was also slightly greater in the μMT/Il10−/− group at day 6 p.i. (Fig. 8F). By day 13 p.i., though, both groups of mice had comparable frequencies of IFN-γ+ CD4 T cells. Interestingly, μMT/Il10−/− mice had a significantly lower parasite burden at day 12 p.i. compared with the WT/Il10−/− group (Fig. 8G). Taken together, our data suggest that IL-10 production by B cells does not interfere with the development of CD8 T cell responses; however, it significantly reduces the cytotoxic capacity of NK and/or NKT cells, and slightly contributes to delay the onset of Th1 responses and to exacerbate disease during the early stages of infection.

FIGURE 8.

B cell-derived IL-10 contributes to disease exacerbation. Mix bone marrow chimeras were generated. One group of irradiated mice was reconstituted with a mix of bone marrow cells consisting of 75% μMT and 25% Il10−/− mice; the control group received bone marrow cells from 75% C57BL/6 and 25% Il10−/− mice. Six weeks after engraftment, OT-I T cells were adoptively transferred into both groups of mice. The animals were infected the day after with PINK. (A) Absolute numbers of OT-I T cells on days 6 and 13 p.i. were identified by gating on CD8+ Ly5.1+ cells. (B and C) Splenocytes from infected mice were restimulated and stained for IFN-γ and granzyme B. Graph represents the percentage of OT-I T cells producing IFN-γ (B) and granzyme B (C). (D) Granzyme B production by endogenous CD8 T cells. (E) Percentage of NK1.1+ cells producing granzyme B. (F) IFN-γ production by endogenous CD4 T cells. Data represent mean percentages ± SE, representative of two independent experiments, n = 3. (G) Splenic parasite burden was determined by limiting dilutions. Data represent mean numbers ± SE, representative of two independent experiments, n = 3. *p < 0.05, **p < 0.01.

FIGURE 8.

B cell-derived IL-10 contributes to disease exacerbation. Mix bone marrow chimeras were generated. One group of irradiated mice was reconstituted with a mix of bone marrow cells consisting of 75% μMT and 25% Il10−/− mice; the control group received bone marrow cells from 75% C57BL/6 and 25% Il10−/− mice. Six weeks after engraftment, OT-I T cells were adoptively transferred into both groups of mice. The animals were infected the day after with PINK. (A) Absolute numbers of OT-I T cells on days 6 and 13 p.i. were identified by gating on CD8+ Ly5.1+ cells. (B and C) Splenocytes from infected mice were restimulated and stained for IFN-γ and granzyme B. Graph represents the percentage of OT-I T cells producing IFN-γ (B) and granzyme B (C). (D) Granzyme B production by endogenous CD8 T cells. (E) Percentage of NK1.1+ cells producing granzyme B. (F) IFN-γ production by endogenous CD4 T cells. Data represent mean percentages ± SE, representative of two independent experiments, n = 3. (G) Splenic parasite burden was determined by limiting dilutions. Data represent mean numbers ± SE, representative of two independent experiments, n = 3. *p < 0.05, **p < 0.01.

Close modal

In this study, we show that MZB are involved in the suppression of CD8 and CD4 effector functions during the early stages of L. donovani infection, and that this suppression contributes to disease exacerbation. Moreover, we demonstrate that B cells also prevent the generation of TEM by a yet unidentified mechanism.

We have previously shown that L. donovani induces defective CD8 T cell responses with limited expansion capacity (23). Interestingly, although most of the CD8 T cells during peak expansion are CD62Llo, they do not express other markers typically associated with effector cells, such as PD-1 (23), Fas (data not shown), or KLRG1, and the majority of the CD62Llo effectors are uncharacteristically highly positive for Bcl2 (data not shown). This phenotype is not typically associated with effector CD8 T cells. Moreover, 70–80% of the CD8 surviving contraction are TCM-like cells and TEM are not generated (23). Thus, it seems that there is a bias toward the development of TCM-like cells rather than effectors during the early stages of L. donovani infection. The transition of effector to memory CD8 T cells is affected by extracellular stimuli such as costimulation, the strength and timing of TCR–Ag interactions, and inflammatory cytokines (3739). In the experimental model for VL, Ag may only be available in low quantities during the first few days of infection, because parasites are quickly segregated into macrophages. Moreover, the parasite actively suppresses IL-12 production in macrophages (40, 41), and DCs only transiently express this cytokine (42). Hence parasite-specific CD8 T cells are primed in a low-Ag, IL-12–poor environment. This could explain why only a small percentage of effector CD8 T cells expresses KLRG1 and 70–80% of the CD8 surviving contraction are TCM-like cells (31).

However, in B cell-deficient mice, IL-12 production by DCs was sustained during the first week of infection. Consequently, in agreement with the literature (31), a larger percentage of effector CD8 T cells expressed KLRG1, and CD8 TEM were generated in those mice. L. donovani infection in μMT mice also resulted in a stronger Th1 response compared with WT mice. CD4 T cells are known to provide help during CD8 T cell priming, among others by secreting IL-2. A recent study has shown that priming of CD8 T cells in an IL-2–deficient environment results in decreased KLRG1 and granzyme B expression by effector CD8 T cells and in premature upregulation of CD127 and CD62L (43). Hence Leishmania-specific CD8 T cells in WT mice may be primed in an environment in which both IL-12 and IL-2 are present only at very low levels.

The mechanism by which B cells interfere with the development of T cell responses is still not clear and is only partly mediated by IL-10, which is known to suppress IL-12 production by DCs and also Th1 effector functions (44). MZB depletion and IL-10R blockade resulted in increased KLRG1 and granzyme B expression by CD8 T cells and in a higher frequency of IFN-γ–producing CD4 T cells. A similar outcome was observed after depletion of regulatory CD4 T cells (data not shown), which are also thought to express IL-10 during the early stages of L. donovani infection (32). Among B cells, IL-10 is mainly produced by cells with an MZB phenotype and by Bregs after in vitro exposure to L. donovani. Nevertheless, B cell-derived IL-10 had only transient inhibitory effects on CD4 T cells and on NK1.1+ cells but did not affect CD8 T cell responses. This suggests that MZB do not directly suppress CD8 effector function via IL-10 secretion, but may operate via other pathways and/or could be inducing IL-10 production by other cell populations and suppress the function of APCs. Further studies aimed at investigating whether MZB suppress APC functions and/or induce IL-10 production by other cells are currently under way.

The IL-10–rich environment present during the first week of infection may explain the partial suppression on T cell functions but does not explain why CD8 TEM are not generated after L. donovani infection. Several transcription factors are involved in the induction of memory and/or effector CD8 T cells (30, 4549). T-bet deficiency, the lack of Blimp-1, inactivation of the transcription factor Id2, or forced expression of Bcl-6 all induce CD8 T cells that developed into memory precursor cells and more rapidly acquired TCM characteristics (4851). Whether these transcription factors also play a role in governing the generation (or the lack) of TCM-like and TEM CD8 T cells during L. donovani infection and the role of B cells in inducing/inactivating these factors still remains to be determined.

MZB are able to bind immune complexes and migrate toward the splenic T/B cell border (52, 53). The migration into the white pulp is induced by ligation of CD21 (54). In this study, we demonstrate that B cells have the capacity to bind L. donovani. Hence it is possible that after L. donovani injection, MZB capture C3-coated parasites via CD21 (complement receptor 2) and/or via surface IgM. This interaction could result in the activation of MZB and consequently in the upregulation of CD86 and surface IgM, and secretion of IgM. In vivo, the majority of the CD19+ cells that were positive for PKH67-labeled parasites were CD21+ CD23lo/int, a phenotype that is also shared by MZB. This suggests that possibly, after capturing the parasite, MZB migrate to the white pulp, where they might interact with FoBs, follicular DCs, and/or follicular Th cells. This interaction could result in the overactivation of FoBs, which further contribute to the suppression of protective T cell responses by a yet unknown mechanism. After interaction with the parasite, cells with a MZB phenotype also secrete IL-10 in an MyD88-dependent way. The steps upstream of MyD88 activation in B cells are as yet unknown. Definition of these steps will require a deeper understanding of how MZB interact with the parasite. For instance, one needs to clarify how MZB recognize L. donovani and which pathways are triggered during these early recognition events. We also need to determine whether L. donovani is merely bound at the cell surface of MZB or these cells are actually able to internalize the parasite. Although B cells from early vertebrates are able to phagocytose 2-μm particles (55), phagocytosis by murine MZB has not yet been reported.

How MyD88 activation in B cells interferes with the regulation of T cell responses is not yet clear. Our findings suggest that the suppressive effects of B cells during Leishmania infection are only in part mediated by IL-10. It is tempting to speculate that MZB may prime CD4 T cells with a regulatory phenotype during the very early stages of infection, which then contribute to suppress protective Th1 responses. A recent study has shown that signaling via MyD88 in B cells suppresses NK cells and T cell responses in Salmonella typhimurium-infected mice (56). In this study, IL-10 was an essential mediator of the B cell inhibitory effects on NK and T cells, and MyD88 was crucial for mediating these effects. Inhibition of T cell functions by B cells has also been reported in tumor immunity (17). A recent vaccination study that used CpG as an adjuvant has shown that B cells suppressed CD8 T cell responses (57).

In conclusion, we have identified a novel inhibitory function for MZB, which contribute to suppress NK1.1+ cells, CD8, and CD4 T cell responses during the early stages of L. donovani infection. Suppression of NK1.1+ cells and CD4 T cells is only partly mediated by MyD88-dependent production of IL-10 by B cells. Furthermore, depletion of MZB results in increased resistance to infection.

Hence the early recognition of some pathogens by MZB contributes to shape the development of adaptive T cell responses and may help the establishment of chronic infections.

We thank Dr. Thomas Tedder for the MB20-11 Ab, Dr. Fidel Zavala for Myd88−/− mice, Dr. Nilabh Shastri for the B3Z and KZO hybridomas, Lee Blosser for sorting cells, and Dr. Stephen Desiderio for the μMT mice and for critical reading of the manuscript.

This work was supported by start-up packages from The Johns Hopkins School of Medicine and from the Institut National de la Recherché Scientifique–Institut Armand Frappier (to S.S.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

BMDC

bone marrow-derived dendritic cell

Breg

regulatory B cell

DC

dendritic cell

FoB

follicular B cell

MZB

marginal zone B cell

RT

room temperature

TCM

central memory T cell

TEM

effector memory T cell

VL

visceral leishmaniasis

WT

wild type.

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The authors have no financial conflicts of interest.