Immunologic tolerance to islets has been achieved in various rodent models using antibodies directed at CD45RB and Tim-1. We have shown that this tolerance mechanism is B lymphocyte dependent, presumably requiring immune regulatory function. In contrast to i.e autoimmunity, the regulatory B cells (Breg) in our model do not depend on IL-10 and may therefore represent a yet to be characterized subpopulation of Bregs. In an effort to elucidate the mechanism of B cell induced tolerance we investigated the requirement of NK and NKT cells in vitro and in vivo. Murine islets (200-300 Islets/recipient), isolated from male Balb/c mice, were transplanted under the kidney capsule of STZ treated (200 mg/kg) C57bl/6 (B6), CD1d^-/- and Lyst^bg/J (beige) mice. Tolerance was induced by intraperitoneal injection of 100 [mu]g anti-mouse CD45RB (Bio X cell) on days 0, 1, 3, 5, and 7 following transplantation and 500 [mu]g anti-mouse TIM-1 (Bio X cell, RMT1–10) i.p. on the day before transplant, and 300 [mu]g on days 0 and 5 post-transplant. Blood glucose levels were monitored for a minimum of 2 weeks up to the time of rejection or 100 days post-transplant. One group of B6 recipients received 3 doses of NK1.1 Antibody i.p. (200 [mu]g, days -7, 1 and 8) to deplete NK and NKT cells. This NK/NKT cell depletion resulted in the near absolute abrogation of the tolerizing effect of dual antibody treatment (anti CD45RB/Tim-1) as only 3/14 islet recipients treated with Nk1.1 antibody remained normoglycemic after 100 days. In the control group, 10/11 B6 recipients remained normoglycemic 100 days post islet transplant. In comparison, recipients that are deficient in NKT cells (CD1d^-/-) demonstrated comparable long term graft survival rates at 100 days (4/5 CD1d^-/- vs. 4/4 wt B6). Islet grafts were rejected between post-transplant day 14 and 20 in 4/4 beige recipient mice, treated with dual Antibody. In vitro assays of T cell stimulation have shown that NK cells are potent suppressors of T cell proliferation independent of B lymphocytes. There is a slight synergism in suppression between NK cells and Bregs.In summary, these result point towards a crucial role of NK cells but not NKT cells in the maintenance of immune transplant tolerance induced by combined anti-CD45RB/anti-Tim-1 Antibody treatment. The mechanism of NK cell mediated tolerance and the potential interaction with Bregs, is under current investigation.

CITATION INFORMATION: Schuetz C, Lee K, Kojima L, Washburn L, Liu L, Liu W.-H, Kim J, Markmann J. NK Cells Are Required in B-Cell Dependent, Antibody Mediated Islet Transplant Tolerance. Am J Transplant. 2016;16 (suppl 3).

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