Eur. J. Immunol. zyxwvutsrq 1988.18: 559-564 Immune responses to a 14-kDa streptococcal antigen zy 559 zy Raymond Fellowes, Farida Fortune, Lesley A. Bergmeier and Thomas Lehner Department of Immunology, United Medical and Dental Schools of Guy’s and St. Thomas’s Hospitals, London Bridge, London The effect of immunization with a 14-kDa streptococcal antigen on primate T cell and B cell responses A streptococcal antigen (SA) of 185 kDa was isolated from zyxw Streptococcus mutuns and this antigen induced in vitro helper, suppressor and contrasuppressor activities with primate peripheral blood lymphocytes. The 185-kDa SA was then treated by sodium dodecyl sulfate and yielded a 4-kDa SA which was capable of eliciting only helper activity. We have now cleaved the 185-kDa SA with cyanogen bromide, in an attempt to identify suppressor and contrasuppressor determinants. A 14-kDa SA was sepa- rated from the cyanogen bromide digest and its ability to elicit T cell and B cell functional activities was tested in rhesus monkeys. Whereas the 185-kDa SA (and 4- kDa SA) elicited high serum anti-SA antibodies and the CD4 cells showed an increase in DNA synthesis, this was not demonstrable with the 14-kDa SA. However, the 14- kDa SA, unlike the 185-kDa SA, activated a significant proportion of CD4 and CD8 cells to bind the Viciu villosu lectin (VV) and this is a characteristic feature of con- trasuppressor cells. We then studied the effect of sequential immunization of mon- keys with the 14-kDa SA, followed by the 185-kDa SA. The results of this showed suppression of the CD4 proliferative response, in the presence of a normal antibody production. We suggest that the split tolerance between the T cell proliferative and B cell differentiating functions might be interpreted on the basis of suppressor CD8 cells inhibiting the CD4 proliferative phase and the W-adherent CD8 cells contrasuppres- sing B cell antibody formation. 1 Introduction In a comprehensive investigation of the cell surface protein antigens of Streptococcusmutans we have identified 4 antigens [l]. The native 185-kDa streptococcal antigen (SA) ID1 appears to be of major significance, as it is immunogenic [2] and may function as a virulence factor [3] and adhesin [4]. Immunization with this SA elicits T cell helper and suppressor functions [5], and prevents the development of dental caries [61. We have then identified and isolated a 4-kDa SA on polyacryl- amide gel electrophoresis of sodium dodecyl sulfate (SDS)- urea-treated native 185-kDa SA [7]. This low molecular mass SA showed antigenic cross-reactivity with the native 185-kDa SA peptide. However, whereas the 185-kDa SA elicits both helper and suppressor activities, the 4-kDa SA peptide elicits only helper activity [8]. These results were comparable with those reported for P-galactosidase, in that a large 90 amino acid (AA) peptide (3-92) induces both helper and suppressor T cell activities, whereas a 9 AA peptide (44-52) activates only helper activity [9]. An 11 AA peptide (27-37) which induces suppressor function was also identified. In an attempt to identify a SA determinant which elicits sup- pression we subjected the 185-kDa SA to cyanogen bromide [I 64681 Correspondence: Thomas Lehner, Department of Immunology, United Medical and Dental Schools, Guy’s Hospital, London SE19RT, GB Abbreviations: AET-SRBC: 2-Amino-ethylisothiouronium bromide- treated sheep red blood cells E’; E-: AET-SRBC rosetting; non- rosetting mononuclear cells HBSS Hanks’ buffered salt solution MEC: Monocyte-enriched cells PBS: Phosphate-buffered saline PRS: Pooled rhesus monkey serum SA Streptococcal antigen W Vicia vilZosa lectin cleavage and found a 14-kDa SA which shares the SA MI determinant with the native 185-kDa SA but was only weakly immunogenic [lo]. The objectives of this investigation were to investigate the immunogenicity of the 14-kDa SA in nonhu- man primates. The proliferative responses of T cells reconsti- tuted with monocytes, from rhesus monkeys which were immunized with the 185-kDa, 4-kDa or 14-kDa SA, were com- pared. Whereas the 185-kDa and 4-kDa SA elicited significant T cell proliferative responses and serum IgG and IgA anti- bodies, the 14-kDa SA induced a low T cell proliferative response and a low serum antibody titer to the 185-kDa SA. However, immunization first with the 14-kDa SA, followed by the 185-kDa SA, depressed the T (CD4) cell proliferative response but retained the capacity to elicit a high serum anti- body titer to the 185-kDa SA. Furthermore, immunization with the 1CkDa SA induced a significant increase in Viciu villosu lectin (W)-binding T cells [ l l , 121, but a low lympho- proliferative response and low levels of anti-SA antibodies. We suggest a contrasuppressor hypothesis in which the 14-kDa SA induces CD8 cells to suppress the CD4 cell proliferative response, but contrasuppression by CD8-W-binding cells of the CD4 helper function in antibody formation. 2 Materials and methods 2.1 Animals Fifteen rhesus monkeys, each weighing between 3-4 kg, were divided into 5 groups with 3 animals in each group (Table 1). Each animal in the first group was immunized with 10 pg of 185-kDa SA, in the second group with 10 pg of 4-kDa SA, and in groups 3 and 4 with 10 pg of 14-kDa SA. Three animals were first immunized with 10 pg of the 14-kDa SA, followed by 10 pg of the 185-kDa SA. The antigens were mixed with aluminium hydroxide (Alu Gel S, Wniscience Ltd., London, GB) and injected S.C. into one arm and one leg (0.5 mi each) on the 3 occasions indicated (Table 1). zyx 0 VCH VerlagsgesellschaftmbH, D-6940 Weinheim, 1988 zyxwvut 00 zyxw 14-2980/88/0404-0559$02.50/0