Studies of Human T Regulatory Cells

National Institute Of Allergy And Infectious Diseases

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Abstract

Natural Killer (NK) cell-mediated innate immunity has evolved as a rapid, first line of defense against multiple pathogens and tumors and plays a critical role in the activation and regulation of T cell-mediated adaptive immunity. NK cells recognize MHC-I (HLA-A, -B, -C in humans or H2-K, -D, -L in the mouse) via differentially expressed inhibitory and activating receptors such as members of the human killer cell immunoglobulin-like receptor (KIR) and leukocyte Ig-like receptor (LILR) or of the murine Ly49 families. A dynamic balance of inhibitory and stimulatory signals thus maintains immune homeostasis and regulates NK cell activation. Complete loss of self-MHC-I expression on tumor cells (“missing self”) abrogates KIR/LILR or Ly49 dependent inhibition resulting in immediate NK cell activation and lysis of tumor cells. Structural studies have demonstrated that the murine Ly49 molecules interact with amino acid residues of the membrane proximal (alpha2 and alpha3) domains of the H2 heavy chain as well as with the beta2-microglobulin (b2m) light chain but do not interfere with the TCR binding site. We previously demonstrated that global inhibition of Ly49/MHC-I interactions by administration of an anti-pan-MHC-I mAb, M1/42, to unmanipulated mice resulted in marked activation and proliferation of NK cells, myeloid cells, and memory phenotype (MP) CD4+ and CD8+ T cells. Production of Interferon-gamma by the activated NK cells in turn stimulated antigen presenting cells (APC) to produce IL-15 that drove the activation of MP CD4+ and CD8+ T cells and the further activation of NK cells via a feed-forward loop. M1/42 failed to inhibit antigen-specific T cell activation, consistent with the MHC-I binding site of M1/42 being distinct from that of the TCR binding site. The biologic effects of M1/42 in vivo appeared to be solely the result of blocking H2/Ly49 interactions as they were independent of the Fc portion of the mAb. More importantly, treatment with M1/42 dramatically enhanced the expansion and activation of tumor antigen specific CD8+ T cells resulting in augmented anti-tumor immunity against the checkpoint inhibitor resistant B16F10 melanoma. The dramatic in vivo effects of blocking H2/Ly49 interactions raised the possibility of translating these findings to humans by blocking HLA/KIR or HLA/LILR interactions. Crystal structures of HLA/KIR complexes reveal that the binding sites of human KIR on HLA antigens overlap with the TCR binding site and anti-HLA mAbs would therefore likely inhibit CD8+ T cell activation. Leukocyte-Ig-like-receptors (LILRs) represent immunomodulatory receptors on cells of both the human innate and adaptive immune systems and the LILRs interact with conserved regions of the MHC-I membrane proximal alpha3 domain and the nonpolymorphic b2m subunit and are therefore unlikely to interfere directly with T cell recognition. We demonstrate that two previously well characterized anti-pan-HLA mAbs, W6/32 and DX17, blocked the binding of multiple LILR multimers to HLA. The addition of Fab fragments of W6/32 or DX17 or engineered Fc-silenced mAbs to cultures of human peripheral blood mononuclear cells (hPBMC), in the absence of any other treatment, resulted in enhanced NK cell proliferation, and the production of IFN-gamma and IL-15. Treatment of immune-deficient mice that had been reconstituted with hPBMC resulted in activation of NK cells and MP CD4+ and CD8+ T cells. Most importantly, treatment of immunodeficient mice expressing a human IL-15 transgene with Fc-silenced DX17 alone, resulted in inhibition of the growth of a human pancreatic adenocarcinoma-derived cell line. This therapeutic effect of the mAb was greatly enhanced by co-administration of human NK cells. Overall, these results demonstrate that HLA/LILR interactions represent both an important pathway regulating innate immune system homeostasis and a potential target for check-point inhibition for the treatment of cancers in humans.