An unexpected counter-regulatory role of IL-10 in B-lymphocyte-mediated transplantation tolerance

Abstract

Monoclonal antibody against the CD45RB protein induces stable transplantation tolerance to multiple types of allograft. We have previously established that this tolerance protocol relies on the regulatory function of B lymphocytes for its effect. B lymphocytes have also been reported to participate in immune regulation in several other settings. In most of these systems, the regulatory function of B lymphocytes depends on the production of IL-10. Therefore, we investigated the role of IL-10 in the anti-CD45RB model of B-cell-mediated transplantation tolerance. Surprisingly, using antibody-mediated neutralization of IL-10, IL-10-deficient recipients and adoptive transfer of IL-10-deficient B lymphocytes, we found that IL-10 actually counter-regulates tolerance induced by anti-CD45RB. Furthermore, neutralization of IL-10 reduced the development of chronic allograft vasculopathy compared to anti-CD45RB alone and reduced the production of graft reactive alloantibodies. These data suggest that the participation of regulatory B lymphocytes in transplantation tolerance may be distinct from how they operate in other systems. Identifying the specific B lymphocytes that mediate transplantation tolerance and defining their mechanism of action may yield new insights into the complex cellular network through which antigen-specific tolerance is established and maintained.

Figure 1. Effect of neutralizing IL-10 on anti-CD45RB-induced tolerance

C57BL/6 animals received C3H heart allografts in the presence of anti-CD45RB. Treatment with anti-CD45RB-induced long-term survival in about 55% of recipients. Addition of neutralizing antibody against IL-10 improved outcomes such that 80% of recipients showed long-term survival (p = 0.436).

Figure 2. Insufficiency of IL-10 in B cells enhances anti-CD45RB-induced tolerance

(A) IL-10-deficient animals received C3H cardiac allografts in the presence or absence of anti-CD45RB. IL-10 deficiency enhanced induction of long-term allograft acceptance induced by anti-CD45RB as compared to IL-10 sufficient controls (p = 0.037). (B) B-cell-deficient animals were reconstituted with 5 million B lymphocytes from IL-10-deficient or -sufficient donors. In the absence of B cells, tolerance is not induced. IL-10-sufficient and -deficient cells were equally competent in their ability to induce tolerance.

Figure 3. Inhibition of IL-10 improves chronic allograft vasculopathy (CAV) over anti-CD45RB alone

Histology of CAV formation and morphometric analysis of CAV lesions in C3H cardiac allografts transplanted in naïve C57BL/6 recipients of anti-CD45RB treatment alone and combined treatment of anti-CD45RB and anti-IL-10, and in IL-10^−/− recipients of anti-CD45RB treatment are shown. Panels A, C and E demonstrate coronary artery emerging from the aorta, ×200; Panels B, D and F show the coronary artery near the middle of grafts, ×400. Elastic tissue staining shows apparent intimal thickening of the coronary artery in a recipient treated with anti-CD45RB alone on postoperative day +137 (A and B); the grafts in the recipients treated with a combination of anti-CD45RB and anti-IL-10 show diminished neointimal proliferation (C and D). Moreover, scant evidence of CAV lesions is seen in grafts from IL-10^−/− recipients treated with anti-CD45RB (E and F). Morphometric analysis of the coronary artery emerging from the aorta demonstrates that the severity of CAV lesions in anti-CD45RB-treated animals was significantly greater than in anti-CD45RB- and anti-IL-10-treated animals, or in anti-CD45RB-treated IL-10^−/− mice (G). Neointimal indices from the coronary artery near the middle of graft show CAV lesion in anti-CD45RB-treated IL-10^−/− mice is much less serious than that in the animals of other two groups (H).

Figure 4. Neutralization of IL-10 further reduces alloantibody production compared to anti-CD45RB alone

Sera were collected from animals on day 14 following allograft placement and anti-CD45RB treatment in the presence or absence of neutralizing anti-IL-10 antibody. Levels of alloreactive IgM and IgG were assessed. IgM levels were decreased by anti-CD45RB alone with no further reduction on the addition of anti-IL-10 (p = 0.001) (A). Alloreactive IgG levels were decreased by anti-CD45RB as well, although they remained elevated above naïve levels (p = 0.038); addition of anti-IL-10 led to a further reduction such that there was no difference from baseline, untransplanted animals (B).