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MHC class I family members serve multiple functions beyond antigen presentation. We provide insight into the structure, expression and function of the Mill subfamily. This family includes two surface glycoproteins, Mill1 and Mill2. Protein sequences for Mill1 and Mill2 are most highly related to the NKG2D ligands, MICA and MICB, but neither of them bound to NKG2D. Computer-based protein modelling indicated that hereditary haemochromatosis protein (HFE), a molecule involved in iron uptake, was most similar. Mill1 and Mill2 were observed on cycling thymocytes, proliferating smooth muscle cells and fibroblasts. Using soluble Mill proteins, we found evidence for a soluble ligand in serum. Like HFE, the Mill family may be involved in nutrient metabolism. Skin was one of the only three organs found to express transcripts for both Mill1 and Mill2. Addition of antibodies specific for Mill2 to wounded skin enhanced healing. Our results suggest a role for the Mill proteins in cellular metabolism, with possible therapeutic significance.

ULBPs are human ligands for NKG2D, an activating receptor expressed on natural killer (NK) cells, NK1.1+ T cells, and T cells. ULBPs are expressed by a variety of leukemias, carcinomas, melanomas, and tumor cell lines. ULBP expression correlates with improved survival in cancer patients, however, the nature of the immune response that ULBPs elicit is not well understood. We report that ectopic expression of ULBP1 or ULBP2 on murine EL4 or RMA tumor cells elicits potent antitumor responses in syngeneic C57BL/6 and SCID mice. Although binding of ULBP3 to murine NKG2D could not be demonstrated in vitro, ULBP3 can also stimulate antitumor responses, suggesting that ULBP3 binds to murine NKG2D or possibly another receptor in vivo. ULBP expression was found to recruit NK cells, NK1.1+ T cells, and T cells to the tumor. IL-15 was found to strongly enhance the immune response directed against ULBP-expressing tumors. Tumors can evade NKG2D immunity by down-regulating expression of NKG2D. Our data suggest that IL-15 may be useful for overcoming this tumor-evasion strategy. Together, these results demonstrate that ULBP expression can elicit a potent immune response and suggest that ULBPs, alone or in combination with IL-15, can be exploited for antitumor therapy.

T cells cultured for 3 h with antigen-presenting cells (APCs) stimulated syngeneic IL-2-activated NK cells as measured via a standard chromium-release assay. Discrete caps containing both TCR and MHC-I had formed on the surface of these activated T cells. When conjugates were formed between NK cells and these activated T cells, >80% of the contact sites were in the MHC-I d i m region outside the TCR-MHC-I cap. Stimulation with phorbol myristate acetate plus lonomycin, which bypasses the need for cell surface events during activation, did not induce either cap formation or NK cell activation. Further, the addition of the protein transport inhibitor Brefeldin A did not block activation of NK cells. MHC-I is the major inhibitory ligand recognized by NK cells. One possible mechanism for the activation of NK cells by TCR-MHC-I-capped T cells is that aggregation of MHC-I into one region leaves the remaining T cell surface denuded of ligands for NK-inhibitory receptors. As a test of this hypothesis, we aggregated MHC-I on T cells with plate-bound anti-MHC-I mAb. This treatment conferred upon the T cells the capacity to activate NK cells, suggesting that MHC-I clustering could contribute to the observed phenomenon.