IL-10 from marginal zone precursor B cells controls the differentiation of Th17, Tfh and Tfr cells in transplantation tolerance

Abstract

B cells are known to control CD4T cell differentiation in secondary lymphoid tissues. We hypothesized that IL-10 expression by marginal zone precursor (MZP) regulatory B cells controls the differentiation and positioning of effector and regulatory T cells during tolerization. Costimulatory blockade with donor-specific transfusion (DST) and anti-CD40L mAb in C57BL/6 mice induced tolerance to allogeneic cardiac allograft. B cell depletion or IL-10 deficiency in B cells prevented tolerance, resulting in decreased follicular regulatory CD4(+) T cells (Tfr) and increased IL-21 expression by T follicular helper (Tfh) cells in the B cell and T cell zones. IL-21 acted with IL-6 to induce CCR6(+) Th17 that caused rejection. Deficiency or blockade of IL-6, IL-21, IL-21R, or CCR6 prevented B cell depletion-induced acute cellular rejection; while agonistic mCCL20-Ig induced rejection. Adoptive transfer of IL-10(+/+) MZP in tolerogen treated CD19-Cre(+/-):IL-10(fl/fl) mice rescued the localization of Tfh and Tfr cells in the B cell follicle and prevented allograft rejection. MZP B cell IL-10 is necessary for tolerance and controls the differentiation and position of Th17, Tfh and Tfr cells in secondary lymphoid tissues. This has implications for understanding tolerance induction and how B cell depletion may prevent tolerance.

Keywords: B cells; Breg; Costimulatory blockade; Tfh; Th17; Transplantation tolerance.

Figure 1. B cell depletion alters Th17 differentiation

(A) C57BL/6 recipient mice given tolerogen (DST + anti-CD40L mAb), BALB/c cardiac allografts on d0, and anti-mCD20 mAb or control IgG on d+1. Expression of intracellular IL-21 in CD4 T cells analyzed in LN, spleen and graft and d5. Allograft infiltrating cells were pooled from 3–4 mice/group. Data representative of one of two independent experiments. (B) CD4⁺CD45⁺ cells from recipient spleen and donor hearts purified by flow cytometry sorting, and expression of mRNA analyzed by qRT-PCR on d5. Data are cumulative of three independent experiments. In each experiments cells pooled from 3–4 mice. Mean and s.e.m shown.

Figure 2. B cell depletion induces alloantigen specific Th17 cells. (A and B)

C57BL/6 recipients given tolerogen, anti-mCD20 mAb or control IgG, and CFSE-labeled TEa CD4⁺ T cells adoptively transferred on d+1. After 5 days, CD4⁺CFSE⁺ T cells from (A) spleen and (B) LN purified using flow cytometry, and mRNA expression analyzed by qRT-PCR. Purified cells pooled for RNA isolation from 3–4 mice/group. Data are cumulative of three independent experiments. Mean and s.e.m shown.

Figure 3. B cell depletion alters the Tfh cell location and expression of IL-21, CCR6, and CCR7

(A) C57BL/6 mice given tolerogen and anti-CD20 mAb, and after 5 days percentages of CD4⁺CD44⁺CXCR5⁺ Tfh cells in spleen analyzed by gating on CD4⁺ cells (left). Mean percentage of CD4⁺CD44⁺CXCR5⁺ cells plotted (right). Mean and s.e.m shown. 5–6 mice/group. (B) C57BL/6 recipients given tolerogen plus anti-mCD20 mAb or control IgG, and CFSE-labeled TEa CD4⁺ T cells adoptively transferred on d+1. After 5 days, CD4⁺CFSE⁺ T cells from LN and spleen purified and BCL6 mRNA expression analyzed by qRT-PCR. Purified cells pooled for RNA isolation from 3–4 mice/group. (C) C57BL/6 mice treated as indicated. After 5 days, percentages of CD44⁺CXCR5⁺ Tfh cells in spleen analyzed by gating on CD4⁺ cells. 4 mice/group. (D) C57BL/6 mice given tolerogen and anti-CD20 mAb. After 5 days, CD4⁺CD44⁺CXCR5⁺ cells purified from spleen, and IL-21, CCR6 and CCR7 mRNA expression analyzed by qRT-PCR. Purified cells pooled for RNA isolation from 3–4 mice/group. Error bars are standard deviation. (E) C57BL/6 mice given tolerogen, anti-CD20 mAb, and purified CFSE labeled CD4⁺CD44⁺CXCR5⁺ Tfh cells adoptively transferred. After 5 days, spleens harvested, frozen tissue sections stained for CD4 and B220, and location of CFSE⁺ Tfh cells in spleen analyzed (top). Magnification 400x. Quantitative analysis of CFSE⁺ Tfh cells in T cell and B cell zones (bottom). 3 mice/group, 3–5 sections/spleen, 4–5 follicles/section. (F) C57BL/6 mice given tolerogen and anti-CD20 mAb. After 5 days, spleens harvested, 8µM frozen tissue sections stained for CD4, CXCR5, B220 and Foxp3, and location of CD4⁺CXCR5⁺Foxp3⁺ Tfr cells in the B cell follicle analyzed. Tfr cells quantitated and plotted. 3 mice/group, 3–5 sections/spleen, 3–4 follicles/section.

Figure 4. B cell IL-10 deficiency alters Tfh and Tfr location

(A, B) Naïve wild type or naïve B-IL-10^−/− mice, or B-IL-10^−/− recipients given tolerogen and BALB/c allografts. On the day of transplant, IL-10 sufficient MZP B cells adoptively transferred into recipients. Mice euthanized on day 40, and spleens harvested and frozen. (A) Spleen sections (8µM) stained for CD4, B220, CXCR5 and Foxp3. Representative images shown from B-IL-10^−/− + tolerogen + allograft or B-IL-10^−/− + tolerogen + allograft + WT MZP B cell treated mice (upper). Mean numbers of Foxp3⁺CD4⁺ cells in the follicles (CXCR5⁺ zone) of spleen (lower). 3 mice/group, 3–4 follicles/section. Magnification 200X. (B) Spleen sections (8µM) stained for CD4, B220, CXCR5 and Foxp3, and analyzed. Representative images of naïve littermate control or B-IL-10^−/− spleen (upper). Mean CD4⁺CXCR5⁺Foxp3⁻ Tfh cells per follicle (lower). Error bars are standard deviation. 3 mice/group, 3–4 follicles/section. Magnification 200X.

Figure 5. Neutralization of IL-6 or IL-21, or deficiency of CCR6, prevents B cell depletion induced allograft rejection

(A) C57BL/6 recipients given tolerogen, anti-mCD20 mAb, anti-IL-21 mAb (10 mg/kg i.v. on d −1 and +5), or IL-21R–Ig fusion protein (200 µg/mouse i.v. on d −1, +3 and +5), and allograft survival monitored. Anti-CD20 mAb vs. anti-CD20 mAb + anti-IL-21 mAb (p=0.0017); anti-CD20 mAb vs. anti-CD20 mAb + IL-21R–Ig (p=0.0045); (B) C57BL/6 recipients given tolerogen, and anti-mCD20 mAb with or without anti-IL-6 mAb (1 mg/mouse i.p. on d +1, +3 and +5). Allograft survival monitored. Tolerogen + anti-CD20 mAb vs. tolerogen + anti-CD20 mAb + anti-IL-6 (p=0.0003). (C) B-IL-10^−/− recipients given tolerogen, transplanted with allografts, and given anti-IL-6 mAb (1 mg/mouse i.p. on d +1, +3 and +5) or anti-IL-21 mAb (10 mg/kg i.v. on d −1 and +5). Allograft survival monitored (left) and parenchymal rejection scored (right). B-IL-10^−/− vs. B-IL-10^−/− + anti-IL-21 mAb (p=0.03); B-IL-10^−/− vs. B-IL-10^−/− + anti-IL-6 mAb (p=0.05) (D) C57BL/6 recipients given tolerogen, and anti-mCD20 mAb or control IgG, and allograft survival monitored. Donor grafts and native heart harvested on d5 and expression of CCL20 assayed by immunofluorescence microscopy. Magnification 200X (left). Mean number of CCL20⁺ cells/section, 3 grafts/group, 3–4 sections/graft (right) (E) Wild type C57BL/6 or CCR6^−/− recipients given tolerogen, and anti-mCD20 mAb, and graft survival monitored. WT + anti-CD20 mAb vs. CCR6−/− with or without anti-CD20 mAb (p<0.001). (F) C57BL/6 recipients given tolerogen, anti-mCD20 mAb, and either control IgG or mCCL20-Ig fusion protein (200 µg/mouse i.v., d +1). Graft survival monitored. DST + anti-CD40L mAb vs. DST + anti-CD40L + mCCL20-Ig (p<0.0001); DST + anti-CD40L mAb + anti-CD20 mAb vs. DST + anti-CD40L + anti-CD20 mAb + mCCL20-Ig (p, ns).

Figure 6. Role of IL-10 producing MZP B cells in tolerance and rejection

Schematic representation of interaction of effector B cells, MZP B cells, Tfh, Teff and Tfr in the secondary lymphoid tissue during co-stimulatory blockade induced tolerance. During tolerance, IL-10 produced by MZP B cells induces the differentiation of Tfr cells which migrate into the T cell zone and control the immune response in the secondary lymphoid tissues. Treg also migrate to the site of inflammation and control the alloimmune response. In the absence of IL-10 producing MZP B cells, Tfh cells down-regulate CXCR5 and up regulate CCR7, leading to migration into the T cell zone. Tfh also produce IL-21 which together with other cytokines in the microenvironment such as IL-6 drives the differentiation of Th17. Th17 cells express CCR6 and migrate to the site of inflammation.