Our epidemiologic studies on invasive Group A Streptococci (GAS) infections identified specific HLA class II haplotypes/alleles conferring high-risk or protection from streptococcal toxic shock syndrome with a strong protection conferred by the DRB1* 15/DQB1* 06 haplotype. We used HLA-transgenic mice to provide an in vitro and in vivo validation for the direct role of HLA class II allelic variation in streptococcal toxic shock syndrome. When splenocytes from mice expressing the protective HLA-DQB1* 06 (DQ6) allele were stimulated with a mixture of streptococcal superantigens (SAgs), secreted by the prevalent M1T1 strain, both proliferative and cytokine responses were significantly lower than those of splenocytes from mice expressing the neutral DRB1* 0402/DQB1* 0302 (DR4/DQ8) alleles (p< 0.001). In crisscross experiments, the presentation of SAgs to pure T cells from either the DQ6 or the DR4/DQ8 mice resulted in significantly different levels of response depending on the HLA type expressed on the APCs. Presentation by HLA-DQ6 APCs elicited significantly lower responses than the presentation by HLA-DR4/DQ8 APCs. Our in vitro data were supported by in vivo findings, as the DQ6 mice showed significantly longer survival post-iv infection with live M1T1 GAS (p< 0.001) and lower inflammatory cytokine responses as compared with the DR4/DQ8 mice (p< 0.01). The data presented here provide evidence for a direct role of HLA class II molecules in modulating responses to GAS SAgs and underscore the dominant role of HLA class II allelic variation in potentiating the severity of GAS systemic infections.