Superantigenicity of helper T-cell mitogen (SPM-2) isolated from culture supernatants of Streptococcus pyogenes

A superantigen (Streptococcus pyogenes mitogen-2; SPM-2) that stimulates human helper T cells bearing unique types of variable domains of T-cell receptor β-chain (TCR Vβ) was isolated from the culture supernatant of S. pyogenes strain T12. The active molecule isolated by diethylaminoethyl (DEAE)-cellulose chromatography and isoelectric focusing was a protein with a molecular weight (MW) of 29 000 and isoelectric point (pI) of 6.0. This new superantigen was found to activate preferentially Vβ4⁺, 7⁺, and 8⁺ T cells, whereas recombinant streptococcal pyrogenic exotoxin A and C activated Vβ12⁺ and Vβ2⁺ T cells, respectively, as determined by flow cytometry and reverse transcriptase-polymerase chain reaction (RT PCR) methods. This proliferative response was significantly inhibited by anti-HLA-DR monoclonal antibody, and required paraformaldehyde-fixed antigen-presenting cells (APC), indicating that this action is dependent on major histocompatibility complex (MHC) class II molecules without processing. Analysis of the aminoterminal amino acid sequence of the molecule failed to find any identical or significantly homologous proteins. We have previously reported that cytoplasmic membrane-associated protein (CAP), a streptococcal superantigen isolated from the cell membranes of S. pyogenes T12 strain, stimulated mainly Vβ8⁺ T cells. Both SPM-2 and CAP preferentially stimulated helper T cells, and rabbit antiserum against SPM-2 completely neutralized the T-cell-stimulating activities of CAP, suggesting that SPM-2 and CAP belong to a family of streptococcal mitogenic proteins. The SPM-2 activity with stimulation of vβ8⁺ T cells was detected extensively in the culture fluids of group A streptococci, but not in those of other streptococcal species, including groups B and D streptococci, and most of the activities detected were completely inhibited by anti-SPM-2 serum. These results indicate that SPM-2 may be a newly discovered superantigen molecule, which can be commonly synthesized by group A streptococci.