Mixed chimerism is a promising approach to inducing allograft and xenograft tolerance. Mixed allogeneic and xenogeneic chimerism in mouse models induced specific tolerance and global hyporesponsiveness, respectively, of host mouse NK cells. In this study, we investigated whether pig/human mixed chimerism could tolerize human NK cells in a humanized mouse model. Our results showed no impact of induced human NK cell reconstitution on porcine chimerism. NK cells from most pig/human mixed chimeric mice showed either specifically decreased cytotoxicity to pig cells or global hyporesponsiveness in an vitro cytotoxicity assay. Mixed xenogeneic chimerism did not hamper the maturation of human NK cells, but was associated with an alteration in NK cell subset distribution and IFN-γ production in the bone marrow. In summary, we demonstrate that mixed xenogeneic chimerism induces human NK cell hyporesponsiveness to pig cells. Our results support the use of this approach to inducing xenogeneic tolerance in the clinical setting. However, additional approaches are required to improve the efficacy of tolerance induction while assuring adequate NK cell functions.

Introduction] Specific allodepletion is an attractive approach to preventing graft-vs-host disease or enhancing immune reconstitution of patients receiving bone marrow transplantation. However, current approaches to allodepletion are not satisfactory. It has been shown that methotrexate (MTX) induces apoptosis of activated lymphocytes and fludarabine (FLU) induces apoptosis of alloantigen-stimulated peripheral blood mononuclear cells in primary mixed lymphocyte reactions (MLR). However, it is unclear if these chemotherapeutic drugs can mediate efficient allodepletion. In this study, we investigated whether or not treatment of MLR cells with MTX and FLU could lead to effective allodepletion.[Methods] B6 (H-2 b ) splenocytes were stimulated with MHCmismatched BALB/c (H-2 d ) splenocytes in vitro in the presence of MTX and FLU for 5 days. These responder splenocytes were harvested and restimulated with BALB/c splenocytes in the presence of MTX and FLU for another 3 days. In vitro proliferative and cytotoxic responses to BALB/c alloantigens of these allodepleted MLR cells were then measured by H 3 incorporation and Cr 51 release assay.To determine the in vivo response of MLR cells allodepleted with MTX and FLU, the allodepleted MLR cells were co-infused with T cell-depleted (TCD) bone marrow cells (BMC) from B6 and BALB/c mice into lethally irradiated B6 recipients. Peripheral chimerism was followed. To determine the ability of MLR cells allodepleted with MTX and FLU to persist in vivo, the allodepleted CD45.2 B6 MLR cells were infused with TCD BMC from congenic CD45.1 B6 and BALB/c mice into lethally irradiated congenic CD45.1 B6 recipients. The number of allodepleted MLR cells in the spleen and lymph nodes were determined at various time points. To determine the response of the allodepleted MLR cells to 3rd party alloantigens, the allodepleted MLR cells were infused with TCD BMC from B6 and CBA (H-2 k ) mice into lethally irradiated B6 recipients. Peripheral chimerism was followed.[Results] MTX did not alter the proliferative or cytotoxic responses of B6 responders to BALB/c alloantigens. Treatment of MLR cells with FLU resulted in loss of proliferative but not cytotoxic responses. Two rounds of treatment of MLR cells with both MTX and FLU resulted in a marked decrease in proliferative and cytotoxic responses upon restimulation by BALB/c alloantigens in vitro. Infusion of MLR cells allodepleted with MTX and FLU with a mixture of B6 and BALB/c BMC to lethally irradiated B6 recipients allowed the persistence of mixed chimerism for up to 6 weeks. The allodepleted MLR cells were able to persist in vivo for up to 6 weeks post-transfer. However, these allodepleted MLR cells were unable to reject 3rd party allogeneic cells.[Conclusion] MTX and FLU act synergistically to result in non-specific allodepletion.