Superantigen-Dependent, Cell-Mediated Cytotoxicity Inhibited by MHC Class I Receptors on T Lymphocytes Joseph H. Phillips,* Jenny E. Gumperz, Peter Parham, Lewis L. Lanier Bacterial superantigens bind with high affinity to major histocompatibility complex (MHC) class 11 antigens on antigen-presenting cells and with T cell antigen receptor (TCR) P chains on T lymphocytes, which results in the T cell activation responsible for toxic shock syndrome and food poisoning. Many cytotoxic T lymphocyte (CTL) clones were shown to have receptors for human leukocyte antigen (HLA) class I molecules that inhibited superantigen-induced cytotoxicity against appropriate class I-bearing target cells. One type of inhibitory receptor, NKB1, was present on CD4+ and CD8+TCRaP+ CTL clones and blocked the killing of staphylococcal enterotoxin B (SEB)-coated targets bearing certain polymorphic HlA-B molecules. Expression of HLA-A, -B, and -C molecules on the SEB-coated targets also protected against cytolysis mediated by many NKB1 -negative T cell clones, suggesting the presence of additional inhibitory MHC class I receptors. These HLA class I receptors may limit tissue destruction and possibly autoimmunity caused by activated T lymphocytes. Superantigens are a group of unprocessed bacterial and viral proteins that activate T cells in vivo and in vitro (1) by directly binding to the lateral exposed surfaces of MHC class II molecules on antigen-pre- senting cells (APCs) and to the variable region of the TCR 13 chain (V,) on the responding T cells (2-7). Superantigens can thus interact with a large fraction of T cells, resulting in cellular activation, prolifera- tion, anergy, or deletion of specific T cell subsets (8-13). Bacterial superantigens in- clude staphylococcal enterotoxins A (SEA), B (SEB), C (SEC), D (SED), and E (SEE) and the toxic shock syndrome tox- in-1 (TSST-1), which are the causative agents for several human diseases such as food poisoning and toxic shock syndrome (1). Although structural analysis of super- antigens has defined how they bind to MHC class II and TCR molecules (2-5, 7), little is known about the immune regula- tion of superantigen-induced responses, par- ticularly the inhibitory mechanisms re- quired to limit superantigen-induced tissue pathology. Natural killer (NK) cells express recep- tors that bind polymorphic MHC class I molecules on potential target cells and pre- sumably transmit inhibitory signals that prevent NK cell-mediated cytotoxicity (14, 15). A subset of human NK cells express an MHC class I receptor (NKB1) that inhibits NK cell-mediated cytotoxicity against Ep- J. H. Phillips and L. L. Lanier, Department of Human Immunology, DNAX Research Institute of Molecular and Cellular Biology, Palo AJto, CA 94304, USA. J. E. Gumperz and P. Parham, Departments of Microbi- ology and Immunology and Cell Biology, Stanford Univer- sity, Stanford, CA 94305, USA. *To whom correspondence should be addressed. stein-Barr virus-transformed B lymphoblas- toid target cells that express HLA-B mole- cules of the Bw4 serotype (16, 17). Addi- tional inhibitory NK cell receptors may ex- ist that recognize polymorphic HLA-A and -C molecules (18-21). We have investigat- ed the expression of the NKB1 receptor on peripheral blood T cells and now present evidence which suggests that these inhibi- tory MHC class I receptors may regulate superantigen-induced T cell responses. NKB1 is expressed on a small percentage of freshly isolated peripheral blood T lym- phocytes (Fig. 1). Examination of lympho- cytes from 47 healthy, adult blood donors revealed NKB1 expression on 0.2 to 15% (mean, 1.6%) of CD3' T cells. Within the CD3+,NKB1+ T cell population, we de- tected T cells that expressed either CD4 or CD8 and the presence of TCRaj3+ and TCRVy8 T cells (Fig. 1). The NKB1 anti- gen present on the cell surface of T lym- phocytes is an -70-kD glycoprotein (22), indistinguishable from the NKB1 glycopro- tein immunoprecipitated from NK cells (16). To determine whether the NKB1 re- ceptor expressed on T lymphocytes regu- lates effector cell function, we established NKB1+,CD3+,TCRctl3+ T cell clones from the peripheral blood of healthy adults by single-cell sorting. These T cell clones sta- bly maintain expression of the NKB1 recep- tor, whereas T cell clones derived from NKB1-negative T cells do not acquire NKB1 during in vitro culture (22). The NKB1+ T cell clones (including both CD4+ and CD8+CD3+,TCRac3+ T cell clones) were assayed for the ability to kill the HLA class I-negative (class II-posi- tive) B lymphoblastoid cell line 721.221 (18) in the presence (but not in the absence) of SEB (Fig. 2). To determine whether ex- pression of HLA class I on the target cells affects their susceptibility to cytolysis, we tested these T cell clones for the ability to kill SEB-coated 721.221 target cells that were transfected with HLA-A, -B, or -C Fig. 1. Expression of NKB1 on peripheral blood T A lymphocytes. (A) Peripheral blood mononuclear 14 1 cells isolated from a healthy, adult donor were stained with fluorochrome-conjugated, isotype- 0 matched control mAbs (cig); fluoroscein isothio- ?102 102 cyanate (FITC)-conjugated mAb to NKB1 (DX9 hy- 10 bridoma) (16) and phycoerythrin (PE)-conjugated 10. 101 mAb to CD3 (mAb Leu 4); FITC-conjugated NKB1 10° 10 and PE-conjugated mAb to TCRy8 (mAb 11 F2); or 10° 101 1c2 103 104 10° 101 102 103 1(4 FITC-conjugated NKB1 and PE-conjugated mAb 04 - 104 to TCRapi (mAb WT31), as indicated, by the tech- niques described previously (16, 25). (B) Peripheral 1 1 blood mononuclear cells were stained with Cy- ihC21 Y1 2 Chrome-conjugated mAb to CD3 (Pharmingen, g s1O2 Q San Diego, CA), FITC-conjugated mAb to NKB1, 101 101 and PE-conjugated mAb to CD4 (mAb Leu 3a) or o a 1 CyChrome-conjugated mAb to CD3, FITC-conju- 10D101 102 103 1C 10 101 1o2 103 104 gated mAb to NKB1, and PE-conjugated mAb to NKB1 NKB1 CD8 (mAb Leu 2a). Samples were analyzed by flow B cytometry (25). An electronic gate was used to 10 10 identify CD3+ T lymphocytes, and the FITC and PE 103 fluorescence of T cells are shown. Data are dis- 103 played as contour plots of emitted fluorescence (4 cl 102 102 decade log scales). In the control sample, >98% of U 0 II the lymphocytes are in the lower left quadrant of the 101 contour plot. Results are shown for an individual 100 1 1.. 103 14 10 with a relatively high percentage (-10%) of NKB1 + 100 101 101 102 103 1 T cells. In a study of 47 healthy adult donors, 1.6% NKB1 NKB1 (mean, range <0.1 to 15%) of CD3+ T cells expressed the NKB1 receptor. The relative proportion of TCRaB+, TCRy8+,CD4+, and CD8+ T cells within the NKB1 + T cell population varied among donors, but all subsets were represented in most individuals. 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