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. 2016 May 15;196(10):4100-9.
doi: 10.4049/jimmunol.1502678. Epub 2016 Apr 13.

Regulatory IgDhi B Cells Suppress T Cell Function via IL-10 and PD-L1 during Progressive Visceral Leishmaniasis

Affiliations

Regulatory IgDhi B Cells Suppress T Cell Function via IL-10 and PD-L1 during Progressive Visceral Leishmaniasis

Robert G Schaut et al. J Immunol. .

Abstract

During visceral leishmaniasis (VL), Th1-based inflammation is induced to control intracellular parasites. Inflammation-based pathology was shown to be dampened by IL-10 and eventual programmed death 1-mediated T cell exhaustion. Cell type(s) responsible for the initiation of T cell-produced IL-10 during VL are unknown. CD19(+), CD5(-), CD1d(-), IgD(hi) regulatory B cells from healthy controls produced IL-10 in the absence of infection or stimulation, in contrast to IgD(lo/neg) B cells. IgD(hi) B cells may have a de novo versus induced regulatory program. The population of IgD(hi) B cells increased 3-fold as VL progressed. B cells from VL dogs were necessary and sufficient to suppress Th1 cell effector function. IgD(hi) B cells induced IL-10 production by T cells and IgD(lo) B cells. Blockage of B cell-specific PD-L1 restored Th1 responses. IgD(hi) regulatory B cells represent a novel regulatory B cell that may precipitate T cell exhaustion during VL.

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Conflict of interest statement

Competing Financial Interests

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
L. infantum infected dogs from Brazil display high levels of immunoglobulin D on the surface of their B cells suggesting the occurrence of a naïve-like B cell during chronic VL. (A) Representative flow cytometry plot of IgD expression on CD19+ B cells isolated from clinically symptomatic, L. infantum infected Brazilian dogs. (B) Quantification of CD19+, IgDhi populations in individuals. Endemic controls (EC), or Brazilian symptomatic (BR-SY) dogs. N=5, 1 experiment. Significance determined via one-way ANOVA ±SEM **p<0.01,
Figure 2
Figure 2
Immunoglobulin IgD significantly increased on the surface of B cells during visceral leishmaniasis. (A) Histogram of isotype (dashed), endemic control (open-solid), asymptomatic (grey) or symptomatic (black) magnetically selected B cells. Percentages of CD19 (left), IgM (center) or IgDhi (right). Histograms representative of n=7 per group and 3 experiments. (B) Quantification of CD19+, IgDhi populations in individuals. Endemic controls (EC), asymptomatic (AS), symptomatic (SY) dogs. N=13 per group from 5 experiments, except SY-BR where N=5, 1 experiment. Significance determined via one-way ANOVA ±SEM **p<0.01, ***p<0.001. (C) Relative expression of IgD and IgM in healthy endemic control dog. Representative of n=7 and 3 experiments. (D) Relative surface expression of IgM and IgD positive peripheral blood B cell populations. N=12 per group from 3 experiments. Statistical significance determined via one-way ANOVA with multiple comparisons was used for statistical analysis. Bars represent ±SEM *p<0.05, **p<0.01.
Figure 3
Figure 3
All IgDhi B cells have significantly increased IL-10 production. (A) CD19+ B cells gated for IgDhi and IL-10 via flow cytometry. Left panel (a) represents IgDhi gating scheme of CD19+ cells. Following panels are representative of IgDhi intracellular IL-10 from progressive clinical groups. Representative of n=7 per group and 5 experiments. (B) Percentage of CD19+, IgDhi, IL-10 positive cells per clinical group as represented in A. EC= endemic controls, AS= asymptomatic, SY= symptomatic. (C) Total CD19+ B cells gated for intracellular IL-10 via flow cytometry. Left represents CD19+ gate and following panels show representative IL-10 production in CD19+ cells from progressive clinical groups. (D) Quantification of CD19+, IL-10 percent positive cells per clinical group. Representative of n=7 per group and 5 experiments. (E) CD19+ cells analyzed for IgD and intracellular IL-4 via flow cytometry. Representative of n=7 per group and 2 experiments. (F) Quantification of CD19+, IgDhi, IL-4 percent positive cells per clinical group. Graph is representative of n=7 per group and 2 experiments. (G) Representative plot of IgD, IL-10 expression of splenic B cells from VL symptomatic dogs. (H) Graphical representation of percentage of IgDhi B cells within the spleen of L. infantum-infected symptomatic dogs. (I) IgD distribution across all CD19+ cells in the three clinical groups (top) and intracellular IL-10 production in IgD and B cell groups. All bars (B,D,F) represent ± SEM. One-way ANOVA with multiple comparisons was used for all statistical analysis. *p<0.05, ***p<0.001.
Figure 4
Figure 4
B cells isolated from VL symptomatic dogs sufficient and necessary to suppress CD4+ T cell TH1 function. (A) CD4+ T cell proliferation, IFN-γ production or IL-10 production in response to freeze-thawed (FT) L. infantum antigen when co-cultured with B cells (left) or co-cultured with B cell negative- PBMCs (right panel set). Histogram of responses by endemic control (open-solid line), asymptomatic (grey) or symptomatic (black) VL ex vivo CD4+ T cells. Representative of n=7 per group. (B,C,D) Percent CD4+ T cell population containing (B) EdU (proliferation), (C) IFNγ, and (D) IL-10 after co-culture with B cells (+) ×ばつ105 cells/well] or co-cultured with B cell negative PBMCs ×ばつ105 cells/well]. Graphs representative of n=7 per group. Percentages are normalized to isotype control and values subtracted from endemic control baseline. Statistical analysis performed utilizing one way-ANOVA with multiple comparison of the means. *p<0.05, **p<0.01, ****p<0.0001. EC = endemic controls, AS = asymptomatic and SY = symptomatic groups. All B cell co-culture was performed at 1:1 B/T cell ratios. Error bars represent ±SEM.
Figure 5
Figure 5
IgDhi B cells induced IgDlo B cells to produce IL-10. (A) Schema of magnetic IgDhi/IgDlo B cell separation. (B) IL-10 production in IgDhi (red line), IgDlo (black dots) and IgDlo-MHCII, with IgDhi B cell depleted co-culture (grey dots with dotted line). B cells were selected using CD19+ positive selection magnetic beads followed by labeling with anti-human-IgD magnetic beads. MHCII cells were isolated via adherence from total CD19−, cells. Dots represent results from individual dogs. (C) IL-10 secretion as measured by ELISA in IgDhi and co-cultured IgDhi/lo cells compared to only IgDlo cells after 7 days of culture. Dots represent results from individual asymptomatic VL dogs. Bars are ±SEM. N=18 per group. (D) CD5 vs. IgD expression, CD1d+ cells significantly increased IL-10 expression during progressive VL. ** p<0.01, *** p<0.001, **** p<0.0001 One-way ANOVA with multiple comparisons was used to for statistical analysis. Cells were incubated in a ratio of 10:1 (IgDlo:IgDhi) or (IgDlo:MHCII).
Figure 6
Figure 6
Inhibitory receptor PD-L1 and IL-10 drove B cell suppression of T cell function in ex vivo cells from VL symptomatic dogs. (A) mRNA expression of PDL1 via qPCR in B cells (left). MFI surface expression of PD-L1 on CD19+, IgDhi B cells (right) (B) Representative contour plots of IFN-γ vs. proliferation in non-stimulated, positive-control ConA or L. infantum antigen stimulated T cells after B cell co-culture. B cells treated with isotype IgG, anti-PD-L1 (αPD-L1), or anti-IL-10 (αIL-10) specific antibodies prior to incubation with indicated T cell populations. (C) Intracellular IL-10 expression in non-stimulated CD3+ T cells isolated from symptomatic VL group. Histograms represent intracellular IL-10 percent positive cells. (D) CD3+T cell responses to co-culture with anti-PDL1 or IL-10 antibody-treated B cells from symptomatic animals. Percentages normalized to isotype control. N=7 per group. (E) Population data of CD3+ T cells producing IL-10 from symptomatic dogs co-cultured with B cells. Percentages normalized to isotype control subtracted from endemic control. N=7 per group. One-way ANOVA with multiple comparisons ± SEM. p<0.05, **p<0.01, ***p<0.001.

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