Proc. Nati. Acad. Sci. USA Vol. 89, pp. 8035-8039, September 1992 Immunology

Superantigen staphylococcal enterotoxin B-induced T-helper cell activation is independent of CD4 molecules and phosphatidylinositol hydrolysis NAOKI OYAIZU*, NARENDRA CHIRMULE*, HIROSUKE YAGURA*, RAJENDRA PAHWAt, ROBERT A. GOODt, AND SAVITA PAHWA*§ *Department of Pediatrics, North Shore University Hospital-Cornell University Medical College, Manhasset, NY 11030; tSchneider Children's Hospital, Long Island Jewish Medical Center, New Hyde Park, NY 11042; and tDepartment of Pediatrics, All Children's Hospital, University of South Florida, St. Petersburg, FL 33701

Contributed by Robert A. Good, May 22, 1992

erative responses, interleukin 2 (IL-2) production, and IL-2 receptor (IL-2R) expression, stimulation with SEB does not trigger phosphatidylinositol (PI) hydrolysis or an increase of intracellular free calcium ion concentration ([Ca2+]i and (ii) the monoclonal antibody (mAb) Leu3a, which blocks conventional antigen-triggered Tb-cell activation, fails to inhibit SEB-elicited Th-cell activation. The results suggest that interaction ofTCR with conventional antigen or superantigen may be coupled to separate signal transduction pathways.

The role of the CD4 molecule in activation of ABSTRACT T-helper cells was examined by investigating the effect of an anti-CD4 monoclonal antibody (Leu3a) in conventional peptide antigen-specific cloned T-helper cells that are also reactive to staphylococcal enterotoxin B (SEB). These T-helper cell clones are CD4+/CD45RO+/T-cell antigen receptor «-chain variable region 12-positive and can respond to nominal peptide antigens and SEB by proliferation in the presence of class II major histocompatibility complex-expressing accessory cells. Although antigen and SEB were comparable in their ability to induce proliferative responses, interleukin 2 (IL-2) production, and IL-2 receptor a-chain expression, stimulatin with SEB failed to trigger phosphatidylinositol hydrolysis or a rise in the intracellular free calcium ion concentration. Leu3a treatment inhibited antigen-induced proliferative responses of T cells with concomitant suppression of IL-2 production and IL-2 receptor expression. In contrast, SEB-induced responses were unaffected by Leu3a. These findings indicate that the functional consequences of binding (ligation) of conventional antigen and of superantigen with the T-cell receptor are distinct in the context of both signal transduction pathways and participation of CD4 molecules.

MATERIALS AND METHODS Antibodies and Reagents. Reagents and sources were as follows: mAb to CD4 (Leu3a; IgGl) and to CD3 (Leu4; IgGl), Becton Dickinson; mAb OKT4 (IgG2b), Patricia Rao (Ortho Diagnostics); SEB, Toxin Technology (Madison, WI); phorbol 12-myristate 13-acetate, Sigma; the calcium ionophore ionomycin, Calbiochem. Cells. Antigen-specific human T-cell clones specific for tetanus toxoid and for purified protein derivative (PPD) were developed as described (7). The clones used, PPD3.2, PPD3.5, and Tt4.2, express the phenotype CD3+/CD4+/ CD45RO+ (UCHL1+). These clones also react with mAb to Cell Science, Cambridge, MA). They proliferate and V,12 (TIL-2 after stimulation with antigen in the presence of secrete irradiated autologous Epstein-Barr virus-transformed B cells as antigen-presenting cells (APC), designated EBV300 for clones PPD3.2 and PPD3.5 and EBV400 for Tt4.2. Addition of anti-HLA-DR mAbs abrogates these responses. Cloned Th cells were maintained with irradiated APC for a 5-day stimulation with antigen, followed by a 10-day rest without antigen. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood of healthy donors by Ficoll/ Hypaque density gradient centrifugation. T cells were separated from non-T cells by erythrocyte rosette formation and plastic Petri dish adherence. These nonadherent cells were >97% CD2+ and contained 80%o the binding of fluorescein-conjugated Leu3a to T cells but did not induce down-modulation of CD4 molecules (8). After washing, the cells were recultured with one of the following: medium alone, tetanus toxoid (2 ,ug/ml), PPD (2 ,ug/ml), Leu4 (1 ,ug/ml), phorbol 12-myristate 13-acetate (10 ng/ml) plus ionomycin (1 ,uM), or various concentrations of SEB and analyzed for proliferative responses, IL-2R expression, and IL-2 secretion. For proliferative responses 1 x 104 T cells were cultured with 4 x 104 APC in 0.2 ml of complete medium in round-bottomed 96-well plates with the indicated stimuli for 48 h, followed by a 16-h pulse of 1 pCi (1 Ci = 37 GBq) of [14C]thymidine. [14C]Thymidine incorporation was measured by liquid scintillation counting. IL-2R expression was determined with fluorescein isothiocyanate-labeled antiCD25 mAb (Becton Dickinson) by flow cytometry using an Epics C flow cytometer (Coulter). IL-2 was quantitated in a bioassay using the IL-2-dependent cell line CTLL-2. Units of IL-2 activity were calculated by comparing [14C]thymidine incorporation in CTLL-2 cells supported with supernatants with that supported by known concentrations of recombinant IL-2 (Genzyme). Paraformaldehyde Fixation of APC. In some experiments, APC were pulsed with antigens at 20 lZg/ml or SEB at 0.1 pg/ml at 37°C overnight and fixed with 1% (wt/vol) paraformaldehyde in Hanks' balanced salt solution followed by extensive washing. Inositol Phosphate Accumulation. Accumulation of inositol phosphates was measured as described (7, 9). Inositol monophosphate, inositol bisphosphate, and inositol trisphosphate were eluted as described (9). Total inositol phosphates were eluted with 0.1 M formic acid/1.2 M ammonium formate. Radioactivity was assessed by liquid scintillation counting. Measurement of Intracellular Calcium Accumulation. [Ca2W]i was measured by flow cytometry using the fluorescent indicator dye fluo-3 AM. Fluorescence of fluo-3-loaded cells was monitored at an excitation wavelength of 488 nm and an emission wavelength of 526 nm on an Epics C flow cytometer (Coulter). Maximal fluorescence was obtained by addition of 4 mM CaCl2 and minimum fluorescence was obtained by addition of 2 mM MnCl2. Intracellular calcium was calculated by the equation [Ca2+1] = Kd[(F - F/(Fma - F)], where Kd = 450 nM (7), F = observed fluorescence, Table 1. Leu3a inhibits antigen-induced, but not SEB-induced, proliferative responses of T-cell clones [3H]Thymidine incorporation, cpm x 10-3


Table 2. Leu3a selectively inhibits antigen-induced, but not SEB-induced, IL-2 secretion IL-2 secretion, units/ml Medium Cells Addition Leu3a OKT4 PPD3.2 Medium 3 ± 0.8 ND ND 84 ± 2.8 PPD (2 jg/ml) 1 ± 0.1 115 ± 7.1 SEB (10 ng/ml) 96 ± 5.6 104 ± 5.6 ND

Superantigen staphylococcal enterotoxin B-induced T-helper cell activation is independent of CD4 molecules and phosphatidylinositol hydrolysis.

The role of the CD4 molecule in activation of T-helper cells was examined by investigating the effect of an anti-CD4 monoclonal antibody (Leu3a) in co...
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