Interleukin-10 From Marginal Zone Precursor B-Cell Subset Is Required for Costimulatory Blockade-Induced Transplantation Tolerance

Abstract

Background: Blocking CD40-CD40L costimulatory signals induces transplantation tolerance. Although B-cell depletion prevents alloantibody formation, nonhumoral functions of B cells in tolerance have not been well characterized. We investigated whether specific subsets of B cell or B cell-derived interleukin (IL)-10 contribute to tolerance.

Methods: Wild type C57BL/6, or B cell-specific interleukin (IL)-10 (CD19-Cre::IL-10) mice, received vascularized BALB/c cardiac allografts. BALB/c donor-specific splenocyte transfusion and anti-CD40L monoclonal antibody were used as tolerogen. B cells were depleted with antimouse CD20 monoclonal antibody. Various B-cell subsets were purified and characterized by flow cytometry, reverse transcription polymerase chain reaction, and adoptive transfer.

Results: B-cell depletion prevented costimulatory blockade-induced allogeneic tolerance. Costimulatory blockade increased IL-10 in marginal zone precursor (MZP) B cells, but not other subsets. In particular, costimulatory blockade did not change other previously defined regulatory B-cell subsets (Breg), including CD5CD1d Breg or expression of TIM1 or TIM4 on these Breg or other Breg cell subsets. Costimulatory blockade also induced IL-21R expression in MZP B cells, and IL-21R MZP B cells expressed even more IL-10. B-cell depletion or IL-10 deficiency in B cells prevented tolerance in a cardiac allograft model, resulting in rapid acute cardiac allograft rejection. Adoptive transfer of wild type MZP B cells but not other subsets to B cell-specific IL-10 deficient mice prevented graft rejection.

Conclusions: CD40 costimulatory blockade induces MZP B cell IL-10 which is necessary for tolerance. These observations have implications for understanding tolerance induction and how B cell depletion may prevent tolerance.

Figure 1. B cell depletion prevents tolerance

(A) C57BL/6 mice i.v. injected with anti-mCD20 mAb (100μg/mouse) or isotype control IgG. After 3 days, CD19⁺B220⁺ cells in the spleen, LN and thymus analyzed by flow cytometry. In bone marrow, IgM⁺IgD⁺ cells analyzed by gating on CD19⁺ cells. Based on FSC-A vs. SSC-A profile, all the cells gated on the lymphocytic population. (B) C57BL/6 mice i.v. injected with anti-mCD20 mAb (100μg/mouse). CD19⁺B220⁺ cells as a percentage of total lymphocytes in spleen, LN and thymus analyzed by flow cytometry at different time points after gating on lymphocytic population. (C) C57BL/6 recipient mice given tolerogen (DST, 1 X 10⁷ cells/mouse d−7 relative to transplantation; and anti-CD40L mAb 250 μg/mouse d−7, −4, 0 and +4) and received BALB/c cardiac allografts or C57BL/6 syngeneic grafts on d0. Recipients given anti-mCD20 mAb (100μg/mouse) d+1. Graft survival monitored (left). After 5 days, grafts harvested, sections made and stained with hematoxylin and eosin (H & E), and parenchymal rejection scores calculated (right). H & E staining of graft (bottom). (D) C57BL/6 recipients given tolerogen and BALB/c allografts as described above, and various doses of anti-mCD20 mAb on d +1. Graft survival plotted (left). After 5 days, grafts harvested, sections stained with H & E, and parenchymal rejection scores calculated (right). (E) Human CD20 transgenic (H-2^d) mice given tolerogen and C57BL/6 (H-2^b) cardiac allografts. Anti-human CD20 mAb (100μg/mouse) given i.v. on d+1. Graft survival (left), Masson’s trichrome staining and parenchymal rejection score calculated (middle), and H & E staining on d+5 (right).

Figure 2. B cell depletion does not cause serum cytokine storm or alloantibody responses

(A) C57BL/6 recipients given tolerogen and BALB/c allografts or C57BL/6 syngeneic grafts on d0, and anti-mCD20 mAb (100μg/mouse) d+1. 5 days after transplantation, sera collected and cytokine levels quantitated by Luminex bead assay. (B) Sera from animals in Figure 1C collected 5 days after transplantation and alloantibody responses measured by flow cytometry. Representative histogram (left) and mean fluorescence intensity (MFI) (right). n= 3–5 mice/group.

Figure 3. B cell depletion causes acute cellular rejection with increased infiltration in the allograft

(A) C57BL/6 recipients given tolerogen, BALB/c allografts and anti-mCD20 mAb as in Figure 1C. Grafts harvested 5 days after transplantation. Immunohistochemistry for graft infiltrating cells. Magnification 200× (left). Quantitative data (right). (B) Immunohistochemistry and quantitation of graft infiltrating CD4⁺ and Foxp3⁺ cells. Magnification 400×. 3 grafts/group, 3 sections/graft, 3–4 fields/section.

Figure 4. Costimulatory blockade does not increase the CD19⁺CD5⁺CD1d^hi Breg

(A) C57BL/6 given no treatment (naïve), DST, anti-CD40L mAb, or DST + anti-CD40L mAb; and after 5 days frequency of CD5⁺CD1d^hi B cells among all lymphocytes in the spleen analyzed after gating on B220⁺CD19⁺ cells (upper). Mean percentage of CD5⁺CD1d^hi cells among all CD19⁺ cells (lower). 5 mice/group. (B) C57BL/6 mice given tolerogen, and after 5 days, B220⁺CD19⁺CD5⁺CD1d⁺ Breg and B220⁺CD19⁺CD5⁺CD1d⁻ non-Breg cells purified from spleen, and IL-10 mRNA expression assessed by qRT-PCR. (C) Splenocytes were stimulated with PMA and ionomycin, intracellular IL-10 stained, and analyzed by gating on CD19⁺CD5⁺CD1d^hi cells. 5 mice/group. (D and E) C57BL/6 mice given tolerogen, and after 5 days TIM-1 and TIM-4 expression analyzed on CD19⁺CD5⁺CD1d⁺ Breg or CD19⁺CD5⁺CD1d⁻ non-Breg in spleen. 4 mice/group.

Figure 5. Costimulatory blockade increases MZP B cells and their IL-10 expression

C57BL/6 mice given no treatment (naïve), DST, anti-CD40L or DST + anti-CD40L (tolerogen), and after 5 days different subsets of B cells from spleen were analyzed. (A) Contour plots show the changes in MZP and MZ cells (left). B cell subset percentages (right) calculated based on gating scheme in Supplementary Figure S1A. 4 mice/group. (B) IL-10 mRNA expression assessed by qRT-PCR. Error bar is standard deviation. (C) Splenocytes (2 X 10⁶ cells/well) stimulated with PMA and ionomycin, intracellular IL-10 stained, and flow gated on various B cell subsets. 5 mice/group. (D) IL-21R expression analyzed on MZP and other B cell subsets. Representative histogram of IL-21R expression on MZP B cells (left). MFI of IL-21R expression on various subsets (right). 4 mice/group. (E) IL-21R⁺ and IL-21R⁻ B cells purified from control or tolerized mice and IL-10 mRNA analyzed by qRT-PCR. Error bar is standard deviation.

Figure 6. B cell specific IL-10 is required for tolerance

(A) MZP, MZ and CD19⁺CD5⁺CD1d^hi+ B cells characterized from B-IL-10^−/− or littermates by flow cytometry. IL-10 mRNA expression monitored by qRT-PCR. (B) Splenic B cell subsets (left), CD4⁺ and CD8⁺ cells subsets (upper right), and CD25 and Foxp3 subsets gated on CD4⁺ cells (lower right) analyzed. (C) B-IL-10^−/− and littermate control recipients given tolerogen and BALB/c grafts. MZP B cells or follicular (Fol) B cells (4 X 10⁵ cells/mouse) from wild-type or B-IL-10^−/− naïve mice adoptively transferred on the day of transplant. Graft survival (upper) and parenchymal rejection score (lower). Littermate control or B-IL-10^−/− + WT MZP B cells vs B-IL-10^−/− mice (p<0.05); B-IL-10^−/− vs B-IL-10^−/− + WT Fol B cells or B-IL-10^−/− + B-IL-10^−/− MZP B cells (p ns). (D) Alloantibody responses of allograft recipients with the indicated genotypes and treatments measured 14 days after transplantation.