Effect of infusion of monoclonal antibodies to tumour necrosis factor‐receptor super family 25 on graft rejection in allo‐immune mice receiving autologous marrow transplantation

Abstract

Significant barriers to transplantation exist for individuals who are pre-sensitized to donor antigen and have high titres of donor-reactive antibody. We report the effect of autologous bone marrow transplantation (BMTx) after myeloablation in pre-sensitized mice along with the use of monoclonal antibodies (mAbs) to tumour necrosis factor-receptor super family 25 (TNFRSF25), expressed on regulatory T (Treg) cells. C57BL/6 mice, which had been sensitized earlier with BALB/c skin allografts, received secondary BALB/c grafts after the primary grafts had been rejected. Subsequently, recipient mice underwent myeloablation with cyclophosphamide and busulphan and were injected with T-cell-depleted bone marrow from CD45.1 congenic donors (BMTx). Recipient mice underwent immunosuppressive treatment with rapamycin. A subgroup of mice was also treated with mAbs to TNFRSF25. Control mice were pre-sensitized mice that received cyclophosphamide and busulphan followed by rapamycin. BMTx-treated mice had significantly prolonged skin graft survival versus control mice. These mice also showed attenuated donor-specific mixed lymphocyte co-culture responses relative to controls, increased splenic Treg cells and markedly diminished serum anti-donor IgG. Infusion of anti-TNFRSF25 mAbs further augmented graft survival and increased graft-infiltrating Treg cells. These mAbs also expanded murine and human Treg cells in vitro with the capacity to attenuate mixed lymphocyte co-cultures using fresh peripheral blood mononuclear cells. Overall, this study delineates the roles of autologous BMTx and anti-TNFRSF25 mAbs in expanding Treg cells and attenuating alloimmune responses in pre-sensitized mice.

Importance of B cells to development of regulatory T cells and prolongation of tissue allograft survival in recipients receiving autologous bone marrow transplantation

“…In our previous studies, skin allograft survival was markedly improved in both naive and immune mice after myeloablation using a short course of busulphan/cyclophosphamide, followed by autologous marrow transplantation and ongoing rapamycin treatment, with no such prolongation seen using rapamycin with busulphan/cyclophosphamide in the absence of marrow transplantation. Data in Fig.…”

“…BALB/c skin grafts to BL/6 naive or immune mice (i.e. having previously rejected BALB/c grafts) were performed as described in previous manuscripts – immune mice were those which had previously received and rejected (≥ 21 days earlier) a skin graft from the same donor as used in subsequent graft studies . All experimental naive/pre‐sensitized transplanted mice subsequently received rapamycin (Wyeth, St.Laurent, QC, Canada; 1 mg/kg at 36 hr intervals) post transplantation.…”

“…The CTL killing assays were performed as previously described: 1·0 × 10 6 responder splenocytes from graft recipients were stimulated with equal numbers of irradiated donor (BALB/c) or third‐party (C3H) stimulators in the presence of IL‐2 (BioLegend). Cells from replicate wells were pooled at 5 days and titrated at different effector : target ratios for killing of 1 × 10 3 51 Cr‐labelled P815 tumour target cells, or 72 hr Concanavalin‐A‐activated C3H splenocyte blasts.…”

“…Although transplantation has become the preferred treatment for patients with end‐stage organ failure, immune rejection, immunosuppressive drug‐related toxicity, opportunistic infection and lympho‐proliferative disorders are currently the major limitations to this treatment . We previously showed that in either naïve animals or in animals immune to graft tissue, transplanted with skin allografts, myeloablation followed by congenic bone marrow rescue (BMTx), improved allograft survival with nearly 40% of recipients retaining their grafts long‐term, in the absence of immunosuppression . Long‐term graft survival was associated with increased numbers of CD4 + Foxp3 + regulatory T (Treg) cells in recipients, which could actively suppress anti‐graft reactivity.…”

“…However, we acknowledged that we had not eliminated an important effect of Breg cells in vivo at earlier times during induction of the tolerant state, or indeed in the induction of Treg cells themselves. The fact that Treg cell functional activity was only demonstrable at ~40 days post transplantation, but grafts were still not rejected at earlier times, might imply the existence of alternative regulatory cell populations at early times post transplantation.…”