Eur. J. Immunol. 1992. 22: 1935-1938 zyxwvutsrq Short paper zyxwvu In zyxwv vivo response to SEB 1935 Thomas Herrmann., Selene Baschieri., Rosemary K. Lees and H. Robson MacDonald Ludwig Institute for Cancer Research, Lausanne Branch, Epalinges and C.R.E. ENEA Casaccia., Rome zyxwvutsrqp In vivo responses of CD4+ and CDS+ cells to bacterial superantigens Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that binds to major histocompatibility complex (MHC) class zyx I1 molecules and specifically activates T cells bearing VpS T cell receptor domains. We have compared several aspects of the response of CD4+ and CD8+ Tcell subsets to SEB in vivo. VpS+ cells in both subsets proliferated to a similar extent upon SEB injection. Furthermore, mRNA for interferon-y was induced in both subsets with similar kinetics and SEB dose-response. Finally CD8+ (but not CD4+) Tcells from SEB-injected mice exhibited SEB-specific lysis of h4HC class II-bearing target cells. Collectively, these data indicate that the CD4 zyxw : MHC class I1 interaction confers no detectable selective advantage to CD4+ cells in the in vivo response to SEB. The observed effector functions of both subsets may contribute to SEB-induced immunopathology. 1 Introduction Superantigens are operationally defined as substances that stimulate Tcells via the variable domain of the Tcell receptor fi chain (TcR Vp). Two main categories of super- antigens have been defined : bacterial superantigens, such as the exotoxins of Staphylococcus aureus [l], and retrovi- ral superantigens, such as the Mls genes encoded within the 3' long terminal repeat of mouse mammary tumor virus [2]. Since presentation of superantigens in vitro is dependent upon MHC class I1 molecules, it is frequently assumed that CD4+ cells (which recognize conventional peptide antigens in association with MHC class 11) respond better to super- antigens than CD8+ cells [3]. In this regard we and others have previously shown that CDS+ cells can respond to both bacterial [4, 51 and Mls [6-81 superantigens in vitro; however, it could be argued that these responses are unphysiological (due to the high cytokine titers achieved in culture). We, therefore, undertook a direct comparison of in vivo responses of CD4+ and CD8+ T cells to the bacterial superantigen staphylococcal enterotoxin B (SEB).We show here that CD8+ T cells proliferate and exhibit specific effector functions (IFN-y mRNA accumulation and cyto- toxicity) following in vivo administration of SEB. 2 Materials and methods 2.1 Injections and cell cycle analysis Adult (8-12 weeks) BALB/c mice were injected i.p. with SEB (Toxin Technology, Madison, WI) at the doses indi- [I 104651 Institute of Virology and Immunology, University of Wurzburg, VersbacherstraBe 7. W-8700 Wurzburg, FRG Correspondence: H. Robson MacDonald, Ludwig Institute for Cancer Research, Ch. des Boveresses 155, CH-1066 Epalinges, Switzerland Abbreviation: SEB: Staphylococcal enterotoxin B cated. Lymph node cells from control or SEB-injected mice were depleted of CD4+ or CD8+ cells by treatment with IgM mAb RL172.4 (anti-CD4) or 3.168.1 (anti-CDS), respectively, in the presence of rabbit complement. After removal of dead cells on a Ficoll gradient, the remaining viable cells were stained with FITC-conjugated mAb directed against Vp6 (44-22-1) or VpS (F23.1) and fixed in 70% ethanol overnight. Fixed cells were then stained with the DNA-binding dye propidium iodide (50 pg/ml) in the presence of RNase A (100U/ml) and analyzed on a FACScan (Becton Dickinson, Mountain View, CA) equip- ped with a doublet discrimination module. DNA content histograms gated on V$+ or Vp6+ cells in both CD8- depleted (CD4+) and CD4-depleted (CD8+) subsets were obtained using Lysis I1 software. Data are expressed as the percentage of cells in the S + G2/M phase of the cell cycle. 2.2 Cell sorting Spleen cells from BALBk mice injected 48 h previously with 10 pg SEB were double stained with FITC-conjugated anti-CD8 mAb (H35-17.2) and PE-conjugated anti-CD4 mAb (GK-1.5). CD4+ and CD8+ populations were then sorted on a FACStar Plus (Becton Dickinson) and tested directly for SEB-dependent cytolytic activity. Purity of sorted cells was > 99%. 2.3 Cytolytic assay Spleen cells from control or SEB-injected mice were usually enriched for CD8+ cells by treatment with mAb RL172.4 (anti-CD4) and 14-4-4s (anti-I-E) plus complement. SEB- dependent cytolysis was measured on the MHC class II- expressing human B cell lymphoma target Raji as described IS]. Briefly, various numbers of splenic effector cells from SEB-injected or control mice were mixed with 2000 51Cr- labeled Raji cells for 4 h at 37 "C in the precence or absence of SEB (1 pg/ml). Percent specific lysis was calculated as described [5]. zyxw 0 VCH Verlagsgescllschaft mbH, D-6940 Weinheim, 1992 0014-2980/Y2/0707-1935$3.50 + .25/0