Infiltrating regulatory B cells control neuroinflammation following viral brain infection

Abstract

Previous studies have demonstrated the existence of a subset of B lymphocytes, regulatory B cells (Bregs), which modulate immune function. In this study, in vivo and in vitro experiments were undertaken to elucidate the role of these Bregs in controlling neuroinflammation following viral brain infection. We used multicolor flow cytometry to phenotype lymphocyte subpopulations infiltrating the brain, along with in vitro cocultures to assess their anti-inflammatory and immunoregulatory roles. This distinctive subset of CD19(+)CD1d(hi)CD5(+) B cells was found to infiltrate the brains of chronically infected animals, reaching highest levels at the latest time point tested (30 d postinfection). B cell-deficient Jh(-/-) mice were found to develop exacerbated neuroimmune responses as measured by enhanced accumulation and/or retention of CD8(+) T cells within the brain, as well as increased levels of microglial activation (MHC class II). Conversely, levels of Foxp3(+) regulatory T cells were found to be significantly lower in Jh(-/-) mice when compared with wild-type (Wt) animals. Further experiments showed that in vitro-generated IL-10-secreting Bregs (B10) were able to inhibit cytokine responses from microglia following stimulation with viral Ags. These in vitro-generated B10 cells were also found to promote proliferation of regulatory T cells in coculture studies. Finally, gain-of-function experiments demonstrated that reconstitution of Wt B cells into Jh(-/-) mice restored neuroimmune responses to levels exhibited by infected Wt mice. Taken together, these results demonstrate that Bregs modulate T lymphocyte as well as microglial cell responses within the infected brain and promote CD4(+)Foxp3(+) T cell proliferation in vitro.

Figure 1. B regulatory cells (Bregs) persist in the brain during chronic viral infection

Single cell suspensions of brain tissue obtained from MCMV-infected mice (3–5 animals) per time point were banded on a 70% Percoll cushion. Brain leukocytes were collected and labeled with PE-Cy5-conjugated Abs specific for CD45, AF700-labeled anti-CD11b Abs, and APC-Cy7 anti-CD19 Abs; and analyzed using flow cytometry. Breg cells were identified within the CD45^hi cell population by using a Breg staining kit (BioLegend, San Diego, CA). The data obtained were processed using FlowJo software. A. Representative flow cytometric plots show the percentages of CD45^hiCD19⁺CD1d^hiCD5⁺ Breg cells within the infiltrating CD45^hiCD3⁻ population obtained from infected brains at 7, 14, and 30 d p.i. B. CD19⁺ CD1d^hiCd5⁺ Abs were used to determine the total number of Breg cells within the infiltrating CD45^hiCD3⁻ population. Data shown are mean (±SEM) absolute number of infiltrating cells pooled from 3 independent experiments (n = 3–5). C. Percentage of Bregs within the infiltrating CD45^hiCD3⁻ cell population at indicated time points from three independent experiments are shown (n = 3–5). ^*P <0.05, 30 d p.i. versus 7 and 14 d p.i. D. Absolute numbers of CD19⁺ cells determined from the total CD45hi population including CD3⁺ cells. E. Absolute numbers of CD1dhiCD5⁺ cells determined within the infiltrating CD45hi population including CD3⁺ cells. *P <0.05, 30 d p.i. versus 7 and or 14 d p.i.

Figure 2. T-cell responses are altered within the brains of B-cell deficient mice

Wt and B-cell deficient (Jh^−/−) mice were infected with MCMV and brain-infiltrating leukocytes were collected at 30 d p.i. and labeled with PE-Cy5-conjugated Abs specific for CD45, AF700-labeled anti-CD11b, eFlour 450-CD4, and PE-Cy7-CD8; and analyzed using flow cytometry. A. Representative contour plots showing the percentages of CD4⁺ and CD8⁺ T-cells within the brain-infiltrating CD45^hi population at 30 d p.i. are shown. B. Specific fluorescent-tagged mAB were used to stain CD4⁺ and CD8⁺ cells and determine total T-cell numbers within the infiltrating CD45^hi population. Data shown are mean (±SEM) absolute number of infiltrating cells pooled from 3 independent experiments (n=5). C. The percentage of CD4⁺ and CD8⁺ T-cell subsets at 30 d p.i. from three independent experiments are shown (n=5). D. Representative contour plots from dLN of Wt and Jh^−/− mice infected with MCMV at 30 d p.i. E. The absolute number of CD4⁺ and CD8⁺ T-cells from the dLN of Wt and Jh^−/− animals was determined and presented as pooled data from 3 independent experiments (n=5). F. The percentage of CD4⁺ and CD8⁺ T-cell subsets in dLN from three independent experiments are shown (n=5). ^*P <0.05 versus infected Wt.

Figure 3. Absence of B-cells leads to decreased numbers of regulatory T-cells within the brain

Wt and Jh^−/− mice were infected with MCMV and brain tissue samples were obtained at 30 d p.i. Brain leukocytes were collected and labeled with PE-Cy5-conjugated Abs specific for CD45, AF700-labeled anti-CD11b, eFlour450-CD4, and FITC-Foxp3; and analyzed using flow cytometry. A. Representative contour plots show the percentages of CD4⁺Foxp3⁺ Tregs within the infiltrating CD45^hi population in the infected brains at 30 d p.i. Also, representative contour plots are shown from dLN of Wt and Jh−/− mice collected at 30 d p.i. B. FITC labeled anti-Foxp3 Abs were used to determine the total number of Tregs within the infiltrating CD45^hi population (n=5). Data shown are mean (±SEM) absolute number of infiltrating cells pooled from 3 independent experiments. C. The percentage of CD4⁺Foxp3⁺ Tregs within CD45^hi population from three independent experiments are also shown (n=5). D. The absolute number of CD4⁺Foxp3⁺ T-cells was also determined from the dLN of Wt and Jh^−/− animals and presented as pooled data from 3 independent experiments (n=5). E. The percentage of CD4⁺Foxp3⁺ T-cell subsets in dLN from three independent experiments are shown (n=5). ^*P <0.05 versus infected Wt.

Figure 4. B-cell deficient mice display increased levels of chronic microglial cell activation

Brain mononuclear cells isolated from animals at 30 d p.i. were stained using anti-CD45, anti-CD11b, and anti-MHC-II surface marker Abs. A. Up-regulation of MHC class II on the CD45^intCD11b⁺ resident microglial cells from Wt and Jh^−/− mice in response to viral brain infection was compared. An overlay of histograms from isotype (grey, shaded), Wt (red, solid line) mice and from Jh^−/− (blue, dotted line) animals is shown. B. Data presented show mean fluorescent intensity (MFI) of MHC-II binding to microglia from Wt versus Jh^−/− mice (n=5). C. Representative histogram overlays for CD40 and CD86 expression on microglial cells isolated from Wt and Jh^−/− mice are shown. The overlay of histograms from isotype (grey, shaded) and specific marker (solid line) are shown for the respective groups. D. Data presented in the bar graph shows percentage of CD40 and CD86 expression on microglia from Wt versus Jh^−/− mice (n=5). *p < 0.01 versus infected Wt for Fig 4B and *p < 0.05 versus infected Wt for Fig 4D. E. Mean fluorescent intensity (MFI) of CD40 and CD86 expression on microglia obtained from Wt versus Jh^−/− mice. *p < 0.01 versus infected Wt for Fig 4B; and *p < 0.05 versus infected Wt for Fig 4D and E.

Figure 5. Induced B10 cells modulate microglial cell activation

A. Splenic CD19⁺ B-cells from IL-10-GFP knock-in mice were cultured with agonistic CD40 mAB (1 μg/ml, eBioscience San Diego, CA) for 48 h. For the last 5 h, cells were treated with LPS (10 μg/ml, Sigma), PMA (50 ng/ml, eBioscience San Diego, CA), ionomycin (100 ng/ml, eBioscience San Diego, CA), and monensin (PIM) (eBioscience San Diego, CA) for 5 h to induce IL-10-producing B10 cells. B. Representative flow cytometry contour plots showing IL-10 induction in CD19⁺ cells that were stimulated with the above protocol. C. The induced B10 cells were then added to purified cultures of primary murine microglia (1:1 ratio) and IL-10 production in these co-cultures was assessed using ELISA. D. CD19⁺IL-10-GFP⁺ B10, as well as CD19⁺IL-10-GFP⁻, cells were enriched using FACS and then added to primary microglial cell cultures. Co-cultures were incubated for 5 h, followed by stimulation with MCMV (MOI=5), and assessment of TNF-α production (at 24h) using ELISA. *p < 0.05 CD19⁺GFP⁺ versus microglia alone; *p < 0.05 anti-IL-10 versus IgG isotype

Figure 6. Induced B10 cells promote proliferation of Foxp3⁺ Treg cells

Regulatory B10 cells were prepared from CD19⁺ cells as described and CD4⁺ T-cells were isolated from MHC-matched donors, Foxp3^EGFP+, using a negative selection kit. Induced B10 cells were added to the CD4⁺ T-cell cultures at a 1:1 ratio. The co-cultures were then incubated for 72 h and Foxp3^EGFP+ expression within the CD4⁺ T-cell population was assessed by flow cytometry for GFP expression to assess Treg phenotype. A. Flow cytometry histogram overlays show staining for isotype (grey, shaded) and intracellular IL-10 (solid line) levels in the induced B10 cells. B. B10 cells generated through this protocol were subjected to phenotyping to determine expression of various B-cell surface markers. Representative histogram overlays, gated from the CD19⁺IL-10⁺ population, which include isotype (grey, shaded) and specific markers (solid line) are shown. C. Representative flow cytometry contour plots of Foxp3^EGFP+ within the CD4⁺ T-cell population are shown: non-GFP, CD4⁺ T-cells only (CD4), total B-cells with CD4⁺ T-cells (CD19⁺:CD4⁺), and induced B10 cells with CD4⁺ T-cells (B10:CD4⁺); along with a standard protocol used to generate in vitro Treg cells as a positive control (iTregs). D. Representative flow cytometry analysis of Foxp3⁺Ki67⁺ expression within CD4⁺ T-cells co-cultured with CD4 only, CD4:B10 and positive control iTreg are shown. E. The percentages of CD4⁺Foxp3⁺ in the various treatment groups from three independent experiments are shown. ^*P <0.01 versus CD4⁺T-cells alone and total B-cells+CD4⁺ T-cells

Figure 7. B-cell replenishment restores T-cell levels within chronically-infected brains

MCMV-primed splenocytes and lymph node cells from donor BALB/c mice were enriched for CD19⁺ cells using negative selection. These B-cells were then adoptively transferred via tail vein injection into MHC-matched Jh^−/− recipients 1 d prior to the infection with MCMV. Wt and Jh^−/− mice served as appropriate controls and brain tissue samples were obtained from each group at 30 d p.i. Brain leukocytes were collected and labeled with PE-Cy5-conjugated Abs specific for PE-Cy5-labeled anti-CD45, AF700-labeled anti-CD11b, eFlour450-labeled anti-CD4, PE-Cy7-labeled anti-CD8, FITC-labeled anti-Foxp3 and APC-Cy7-labeled anti-MHC II and analyzed using flow cytometry. A. Representative contour plots showing the percentages of CD4⁺, CD8⁺ (upper panel), and CD4⁺Foxp3⁺ T-cells (lower panel) in the infiltrating CD45^hi population within the infected brains from each group. B. The absolute numbers of CD4⁺, CD8⁺ and CD4⁺Foxp3⁺ T-cells were also determined among the brain-infiltrating CD45^hi cells from Wt, Jh^−/−, and Jh^−/− with B-cell AT animals. Pooled data obtained from 3 independent experiments are presented (n=5 per group). *p < 0.05 Jh^−/−+AT versus Jh^−/− C. Histogram overlays from isotype (grey, shaded), Wt (red, solid), Jh^−/− (blue, dashed), and Jh^−/− mice that received CD19⁺ cells (green, dotted) are shown for MHC class II up-regulation on CD45^intCD11b⁺ resident microglia. D. Data presented show mean fluorescent intensity (MFI) of MHC-II binding from Wt, Jh^−/− and Jh^−/−+AT mice (n=5 per group).*p < 0.01 Jh^−/−+AT versus Jh^−/−