H. Deirawan1, W. Liu1, L. Liu1, R. Stott1, K. M. Lee1, J. I. Kim1, J. F. Markmann1 1Massachusetts General Hospital,Division Of Transplant Surgery,Boston, MA, USA
Introduction:
CD1d presents lipid antigen to a subset of NKT cells and is widely used as a marker of regulatory B cells. NK1.1+ cells, which includes natural killer (NK) and Natural Killer T(NKT) cells, play a critical role in graft rejection. Depletion of NK1.1+ cells has been demonstrated to abrogate graft survival of islet, corneal, and cardiac allografts. We explored the possibility of a B cell-NK/NKT interaction, with the goal of determining whether NK/NKT cells played a role in a mouse model of regulatory B cell-dependent tolerance.
Methods:
Diabetes was induced in C57BL/6 mice with streptozotocin. In some experiments, CD1d-/- mice, and Beige mice were used. Four to five hundred islets were transplanted into the renal sub-capsular space of diabetic recipients. Recipient mice received 100 µg anti-mouse CD45RB i.p. on days 0, 1, 3, 5, and 7 following transplantation and 500 µg anti-mouse TIM-1 i.p. on day -1, and 300 µg on days 0 and 5 following transplantation. Recipients may also receive 200 mg anti-NK1.1 antibody on days -7, +1, and +8. Regulatory B cells were purified from long-term graft survival animals (>100 days) for adoptive transfer. Adoptive transfer was performed by intravenous injection of 5×10^6 B cells.
Graft survival between experimental groups was compared using Kaplan-Meier survival curves and Wilcoxon statistics. P values less than 0.05 were considered statistically significant.
Results:
Anti-CD45RB/anti-TIM-1 antibody treatment results in nearly 100% percent long-term graft survival, and reduces the percentage of NK cells but increases the percentage of NKT cells. The additional administration of anti-NK1.1 abrogates tolerance induction, but did not alter the percentage of TIM-1+ B cells nor the percentage of Foxp3+ T cells. To examine whether anti-NK1.1 antibody interferes with the development of Bregs vs. their effector function, we took advantage of the fact that untreated B cell deficient mice reject islet allografts, but the addition of B cells from tolerant recipients induces tolerance without any antibody treatment to the secondary recipients. In this model, anti-NK1.1 antibody abrogates tolerance induction by established Bregs.
To further delineate the subset of cells necessary for Bregs function. NKT cells deficient mice (CD1d-/-), and Beige mice (functional NK cells deficiency) were transplanted, dual antibody treated and monitored for outcome. In contrast to wild type mice, all Beige mice acutely reject their grafts. Only 30% of NKT deficient mice fail to accept their graft long term.
Conclusion:
Our adoptive transfer study demonstrates that the presence of recipient NK1.1+ cells is necessary for graft survival mediated by Bregs. Rejection as a result of the loss of NK1.1+ cells does not result from a loss of Bregs or of Tregs. We continue to explore the interaction between subsets of NK1.1+ cells and Bregs that may be necessary for transplant tolerance induction.