Blockade of T-Cell Homeostasis by Anti-Interleukin-7 Receptor Monoclonal Antibody Allows Indefinite Islet and Long-Term Skin Allograft Survival

Abstract

Background: T cell depletion is followed by T cell homeostasis with increase in memory T cells which are more potent in mediating rejection. We demonstrate that IL-7R blockade with or without prior T cell depletion inhibits T cell homeostasis and prolongs graft survival. Methods: Tail skin from C57BL/6 was transplanted to Balb/c mice with 3 treatment protocols: protocol 1 (no treatment, n=10), 2 (T cell depletion by combining 2 depleting mAbs: anti-CD4 and anti-CD8 on day -3 and -1, n=10), and 3 (same as protocol 2, followed by a blocking anti-IL-7Rα mAb qod, n=11). Islets from C57BL/6 were transplanted to streptozotocin-induced diabetic Balb/c mice, divided into 3 groups: group A (no treatment, n=10), B (anti-IL-7Rα mAb qod from day 0 to rejection, n=5), and C (anti-IL-7Rα mAb qod from day -21 to PTD90, n=6). Results: Median skin graft survival with protocol 1, 2, and 3 was 9.5, 30, and 58 days, respectively (protocol 3 vs 2, p< 0.0001). IL-7R blockade following T cell depletion profoundly inhibited lymphocyte reconstitution. At PTD35, the mean absolute numbers of CD3+, CD3+CD4+, CD3+CD8+, and CD19+ cells in the spleen were 6.7, 4.1, 0.8, and 119 in protocol 3 vs 57.5, 43.6, 7.3, and 276 ×105 cells in protocol 2, respectively (all p< 0.05). Importantly, the mean absolute number of CD44hiCD62Llomemory T cells was 4-fold lower in protocol 3 vs protocol 2: 3.8 vs 14.3 ×105 cells, and the percentage of CD4+CD25+FOXP3+ Treg to total CD4+ T cells was slightly increased: 13 vs 10% (all p< 0.05). Similar results were found in the lymph node and in the blood. Moreover, thymic cellularity was markedly reduced in protocol 3 vs 2: 0.7 vs 142 ×105 cells, p< 0.05. IL-7R blockade abrogated cellular immune responses as shown by direct IFNγ ELISPOT (41.3 vs 193.5 spots/105 T cells) and by MLR-3H thymidine (238 vs 10510 CPM) and inhibited humoral immune responses as shown by a reduction in DSA (310 vs 996 MFI) (protocol 3 vs 2, all p< 0.05). Histology revealed abundant subcutaneous and dermal leukocyte infiltrates at PTD21 in protocol 2, whereas few infiltrates were seen in protocol 3 at PTD21 and PTD35. The addition of low-dose tacrolimus 0.5 mg/kg/d to protocol 2 did not improve median graft survival (32.5 days, n=6, p=NS), whereas the same dose of tacrolimus added to protocol 3 further prolonged skin graft survival to >90 days in 4 of 6 recipients (p< 0.05). Median islet graft survival was not significantly prolonged when anti-IL-7Rα was given from day 0 (group B vs A: 29 vs 21 days, p=NS). However, when treatment was started 21 days before islet graft, 5 of 6 recipients had indefinite graft survival >180 days (group C vs A, p< 0.005), associated with an abrogation of DSA (group C vs A: 260 vs 976 MFI, p< 0.01). Conclusion: IL-7R blockade following with T cell depletion is a potent immunosuppressive regimen which abrogates both cellular and humoral allo-immune responses and may be used synergistically with low-dose tacrolimus to avoid nephrotoxity.