INTERACTIONS BETWEEN HUMAN NATURAL KILLER CELLS AND B CELLS CAUSE AUGMENTATION OF IGG HUMORAL RESPONSES

Abstract

53 Background: Delayed vascular rejection of pig-to-primate xenografts involves NK cells, macrophages and deposition of induced IgG antibodies. Since we and others have shown previously that human NK cells bind to the same major porcine carbohydrate xenoantigen recognized by human xenoreactive antibodies, gal alpha 1,3-gal (α Gal), we investigated the role of human NK cells in augmentation of the human anti-porcine B cell antibody response. Methods and Results: Human B cells isolated by immunomagnetic beads to > 95% purity demonstrated minimal proliferation above background when exposed to porcine aortic endothelial cells (PAEC). Following addition of irradiated NK cells (> 90% purity), B cell proliferation increased by over 6-fold. This effect required NK cell pre-activation with IL-2 or IL-12, suggesting a role for inducible co-stimulatory molecules. Incubation with Mabs against either CD40 ligand (on activated NK cells) or CD40 (on B cells) reduced B cell proliferation by 52% and 44%, respectively, whereas isotype control antibody had no effect. In the absence of NK cells, stimulation of B cells with anti-Ig plus anti-CD40 had minimal effect on proliferation, whereas the addition of IFN-gamma or GM-CSF, but not other cytokines such as IL-10, increased proliferation to levels comparable following culture with activated NK cells. Pre-incubation of NK cells with IB4, a lectin with specific binding affinity for α Gal, reduced NK-driven B cell proliferation by 80-90%, whereas control had no effect. In an ELISA using porcine endothelial cell monolayers as targets, addition of NK cells to B cells induced anti-pig antibody isotype switching, with the ratio of anti-pig IgM/IgG decreasing from 1.6 for B cells alone to 0.67 for B cells co-cultured with NK cells. Conclusions: These results demonstrate that human NK cells augment B cell anti-α Gal IgG responses, that these effects involve CD40L/CD40 and specific cytokines, and that NK-B cell interactions may be critical to the delayed vascular process of pig-to-primate xenograft rejection.