B cells mediate chronic allograft rejection independently of antibody production

Abstract

Chronic rejection is the primary cause of long-term failure of transplanted organs and is often viewed as an antibody-dependent process. Chronic rejection, however, is also observed in mice and humans with no detectable circulating alloantibodies, suggesting that antibody-independent pathways may also contribute to pathogenesis of transplant rejection. Here, we have provided direct evidence that chronic rejection of vascularized heart allografts occurs in the complete absence of antibodies, but requires the presence of B cells. Mice that were deficient for antibodies but not B cells experienced the same chronic allograft vasculopathy (CAV), which is a pathognomonic feature of chronic rejection, as WT mice; however, mice that were deficient for both B cells and antibodies were protected from CAV. B cells contributed to CAV by supporting splenic lymphoid architecture, T cell cytokine production, and infiltration of T cells into graft vessels. In chimeric mice, in which B cells were present but could not present antigen, both T cell responses and CAV were markedly reduced. These findings establish that chronic rejection can occur in the complete absence of antibodies and that B cells contribute to this process by supporting T cell responses through antigen presentation and maintenance of lymphoid architecture.

Figure 1. B cells are sufficient for CAV in the absence of antibodies.

WT, AID/μS KO, μMT, and μMT + B cells^{AID/μS KO} recipients were transplanted with BALB/c hearts and treated with costimulation blockade (0.25 mg, CTLA4Ig and anti-CD40L, on days 2, 4, 6, and 8). μMT + B cells^{AID/μS KO} recipients were μMT mice that received naive AID/μS KO B cells (2 × 10⁷, 2 to 4 days before transplantation). Allografts were beating and contracting at harvest (100–110 days). (A and B) Morphometric quantitation of vasculopathy in each recipient (A), and luminal occlusion in each vessel (B) (mean ± SEM; n = 26–64 vessels, 5–9 mice per group). (C) Morphometric quantitation of luminal occlusion in each vessel in Bm12 heart allografts from untreated recipients (at 90–100 days, mean ± SEM; n = 34–87 vessels, 5–10 mice per group). (D) Median fluorescence intensity of BALB/c-reactive antibodies in sera of BALB/c heart recipients at harvest (1:4 dilution, mean ± SD; n = 4–6 mice per group). *P < 0.05; **P < 0.005; ***P < 0.0005.

Figure 2. Alloreactive T cell responses are diminished in the absence of B cells.

(A) Spleen, LN, and BM cells were harvested from WT, AID/μS KO and μMT recipients of BALB/c hearts treated with costimulation blockade for assessment of T cell activation and cytokine production (at allograft harvest for CAV, 100–110 days after transplantation). Harvested cells were stimulated with BALB/c splenocytes for 6 hours and IFN-γ⁺ and TNF-α⁺ cells within the H-2^d negative recipient T cells were assessed by intracellular cytokine staining. Percentage and total numbers of IFN-γ⁺ and TNF-α⁺, CD4⁺ and CD8⁺ T cells in allograft recipients and naive mice are shown. Percentage of CD44^hi T cells, and CD62L^lo, CD25^hi, and CD69^hi cells within CD44^hi T cells in allograft recipients are shown. (B) Representative images of immunofluorescence staining in cryosections of heart allografts from recipients showing CD4⁺ (red), CD8⁺ (green), and DAPI (blue). Arrows point to CD4⁺ or CD8⁺ T cells. Original magnification, ×20. Scale bars: 150 μm. (C) Total T cell numbers were enumerated in spleen, LN, and BM cells from allograft recipients at the time of harvest. Data shown are representative of 2 independent experiments (mean ± SD; n = 4–6 mice per group). *P < 0.05; **P < 0.005; ***P < 0.0005.

Figure 3. Cognate and noncognate functions of B cells contribute to CAV.

All recipients were transplanted with BALB/c hearts, treated with costimulation blockade, and harvested between 90 and 100 days. Recipient spleen, LN, and BM cells were stimulated with BALB/c splenocytes for 24 hours, and the total number of IFN-γ⁺ and TNF-α⁺ cells within H-2^d-negative recipient T cells were enumerated. (A) Morphometric quantitation of CAV in heart allografts from Ig^HEL recipients (μMT and WT controls are the same as in Figure 1B). (B) Total IFN-γ⁺ and TNF-α⁺ T cells in Ig^HEL and μMT + B cells^{AID/μS KO} recipients. (C) Morphometric quantitation of CAV in heart allografts from chimeras. (D) Total IFN-γ⁺ and TNF-α⁺ T cells in chimera recipients. Results are mean ± SEM (n = 29–68 vessels and 4–9 mice per group) (A and C) and mean ± SD (n = 4–6 mice per group) (B and D). *P < 0.05; **P < 0.005; ***P < 0.0005.