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Ligation of CD46 on human T cells prevented recruitment of the microtubule organizing center, CD3, and perforin to the interface with the antigen-presenting cell and caused a reduction in IFN- production

Ligation of CD46 on human T cells prevented recruitment of the microtubule organizing center, CD3, and perforin to the interface with the antigen-presenting cell and caused a reduction in IFN- production. and caused a reduction in IFN- production. In human NK cells, similar changes in polarity induced by CD46 ligation inhibited the recruitment of the microtubule organizing center and perforin to the interface with target cells and correlated with reduced killing. These data indicate that external signals can alter lymphocyte polarization toward antigen-presenting cells or target cells, inhibiting lymphocyte function. can impact on IS formation and signaling. Results Lymphocytes Polarize Toward the Site of CD46 Ligation. To determine whether ligation of CD46 on lymphocytes can mediate polarity changes, we incubated CTLs with anti-CD46-coated beads. We observed microtubule organizing center (MTOC) polarization to the AZD3264 cellCbead interface in 92% of cells conjugated to anti-CD46-coated beads (Fig. 1and and and = 25). (Scale bar: 10 m.) (= 50). (= 8 fields of 100 target cells per field). Ligation of CD46 by Bystander Cells Expressing Measles Hemagglutinin Alters T Cell Polarity and Signaling. During measles infection, bystander cells transmit the virus through cellCcell contact, with only small amounts of free virus in circulation (23). Furthermore, immunosuppression is mediated by cell surface contact with virus rather than infection of immune cells (14, 24), suggesting that the effects of measles on immune cell function can be mimicked by using a cell line expressing measles hemagglutinin on its surface (LH cells) to ligate CD46 on T cells. We therefore assessed the effects of LH cells on T cell polarization. In conjugates formed between LH and T cells, CD46 was tightly capped at the point of contact, with the MTOC polarized to the site of contact in 92% of cells (Fig. 5for 10 min) and processed as above. For Ab ligation, cells were incubated with 4C10 g/ml CD46 (1840), CD28, or TfR antibody for 30 min at 37C. Confocal images were acquired and processed by using a BX61 microscope (Olympus, Melville, NY) with Olympus Fluorview FV1000 laser-scanning confocal and software (Olympus, Japan). Live-cell imaging was carried out as described (16). Movies were compiled from individual images and play at four frames AZD3264 per sec. Scoring of polarization was performed blind on at least 50 conjugates (25 for allogeneic conjugates), and molecules were considered polarized toward the target cell if 50% of fluorescence was detected at the interface. The 51Cr-labeled cells (2 104) were incubated with peripheral blood mononuclear cells enriched for NK cells (50%) for 4 h at 37C and 51Cr (cpm) released into the supernatant was detected by using a Wallac Wizard 1470 automatic counter (PerkinElmer Life Sciences, Boston, MA). Human IFN- was measured by ELISA (Pierce, Rockford, IL). values were determined by two-tailed Rabbit polyclonal to ACBD4 Student’s test. Supplementary Material Supporting Information: Click here to view. Acknowledgments We thank C. Clarke, P. Humbert, AZD3264 K. Poetter, and M. Smyth for critical reading of the manuscript; Y. Hayakawa for helpful discussion; Denis Gerlier (Institut Fdratif Laennec, Lyon, France) for LH cells and MCI20.6 antibody; and Bruce Loveland (Austin Research Institute, Victoria, Australia) for CD46 antibody and recombinant CD46. This work was supported by National Health and Medical Research Council (NHMRC) Grants 251619, 350461, and 400043; Australian Research Council Grant DP0451224; a AZD3264 Wellcome Fellowship (to S.M.R.); an R. D. Wright Fellowship (to N.J.W.); and an NHMRC Senior Principal Research Fellowship (to J.A.T.). Abbreviations APCantigen-presenting cellCTLcytotoxic T lymphocyteDCdendritic cellISimmunological synapseLHL cells expressing measles hemagglutininMTOCmicrotubule organizing centerTCRT cell receptorTfRantitransferrin receptor. Footnotes The authors declare no conflict of interest. This article is a PNAS direct submission..