ANALYSIS OF HUMAN T-CELL RESPONSES TO GROUP-A STREPTOCOCCI USING FRACTIONATED STREPTOCOCCUS-PYOGENES PROTEINS

Cell extract and spent culture supernatant proteins from Streptococcus pyogenes Manfredo strain (type M5) were each separated to give 22 nar row range molecular weight fractions by blot-elution from SDS-polyacry lamide gels. fluted samples and unfractionated proteins were screened for T cell stimulatory activity using human peripheral blood mononucle ar cells (PBMC) from healthy adults in proliferation assays. Responses were measured in 4- and 7d cultures. Responses to a wide range of cel l extract proteins were revealed by fractionation, the degree of respo nse to each fraction varying between donors. Unfractionated culture su pernatant proteins elicited proliferative responses by PBMC from all i ndividuals examined. Responses to culture supernatant fractions contai ning 25-33 kDa proteins could be attributed ro known superantigens. Fu rthermore, samples from culture supernatants containing higher molecul ar weight fractions (> 45 kDa) elicited responses in 50% of donors in 7d cultures, suggesting that these fractions contained common recall a ntigens. The efficacy of using electroeluted samples to identify T lym phocyte stimulatory proteins was confirmed by demonstrating that a kno wn superantigen of S. pyogenes Manfredo strain, streptococcal pyrogeni c exotoxin C (SPEC), could be fractionated successfully using this met hod and its activity recovered. Our results show that human T cell res ponses to group A streptococci involve a remarkably wide range of both cell-associated and released streptococcal proteins.