CD154 blockade and donor-specific transfusions in DLA-identical marrow transplantation in dogs conditioned with 1-Gy total body irradiation

Abstract

Stable mixed donor/host chimerism has been reliably established in dogs given a sublethal dose (2 Gy) of total body irradiation (TBI) before and immunosuppression with mycophenolate mofetil (MMF) or rapamycin combined with cyclosporine (CSP) after marrow transplantation from dog leukocyte antigen (DLA)-identical littermates (hematopoietic cell transplantation [HCT]). When TBI was reduced to 1 Gy, only transient engraftment was observed. Here we investigated whether stable engraftment after 1-Gy TBI could be accomplished by reducing host-versus-donor immune responsiveness through preceding CD154 blockade and infusion of donor peripheral blood mononuclear cells (PBMCs). We found that the anti-human CD154 antibody, 5c8, cross-reacted with canine lymphocytes and blocked alloimmune responses in vitro. Based on pharmacokinetic studies, 6 dogs received a single intravenous injection of 5 mg/kg anti-CD154 antibody (on day -5), followed 1 day later by donor PBMCs. On day 0, the dogs were given 1 Gy of TBI and underwent DLA-identical marrow grafts. Postgraft immunosuppression consisted of MMF and CSP. All 6 dogs demonstrated initial engraftment; 3 dogs sustained the engraftment for >26 weeks, whereas 3 dogs rejected their grafts, after 9, 22, and 24 weeks, and survived with autologous recovery. Graft survival was significantly improved over that in 11 historical controls conditioned with 1-Gy TBI and given either MMF or rapamycin with CSP after HCT, all of which rejected their grafts between 3 and 12 weeks (P = .03). Preceding donor PBMC infusion and CD154 blockade improved survival of DLA-identical marrow grafts after 1-Gy TBI.

Figure 1

Distribution of PMA and ionomycin- activated canine lymphocytes stained with FITC-labeled anti-CD154 antibody 5c8 (solid line) and a mouse isotype control (dotted line). Events are gated for living lymphocytes (vital stain: propidium iodide).

Figure 2

MLR results. Shown are averages of 6 experiments, with each individual experiment done in triplicate. Anti-CD154 mAb 5c8, irrelevant mAb 31A (negative control), and CTLA4-Ig (positive control) were added at concentrations of 10 μg/mL each. The maximum ³H-thymidine uptake of the medium control in each experiment was set at 100%. Error bars represent 1 standard deviation. P values were calculated using the paired Student t test. Medium versus irrelevant mAb 31A, P = .14; medium versus 5c8, P < .0001; medium versus CTLA4Ig, P < .0001; mAb 5c8 versus CTLA4Ig, P = .04.

Figure 3

(A) Serum concentrations of anti-CD154 mAb 5c8 in 3 dogs given 1, 5, and 10 mg mAb/kg. Concentrations were calculated against a standard of purified mAb 5c8. (B) Antibody responses to anti-CD154 mAb 5c8 in 3 dogs given 1, 5, and 10 mg mAb/kg. IgG and IgM responses were assessed by isotype-specific secondary antibodies.

Figure 4

(A) Serum concentrations of mAb 5c8 in the 6 dogs that underwent transplantation. Concentrations were calculated against a standard of purified mAb 5c8. (B) IgG antibody responses against mAb 5c8, assessed by isotype-specific secondary antibodies.

Figure 5

Hematologic changes in 6 dogs receiving DLA-identical littermate marrow grafts. (A) Median neutrophil, lymphocyte, and platelet counts. (B) Donor granulocyte chimerism levels. (C) Donor mononuclear cell chimerism levels.

Figure 6

Kaplan-Meyer estimates of graft survival in the 6 dogs pretreated with anti-CD154 mAb 5c8 and donor PBMCs and 11 control dogs that did not receive pretransplantation treatment. All dogs were conditioned with 1-Gy TBI, received marrow grafts from DLA-identical littermates, and were given MMF or rapamycin for 28 days and CSP for 35 days posttransplantation.