The subset of M1-paired MHC class II conformers is critical for CD4+ T cell activation

Major histocompatibility complex class II molecules present exogenous antigen-derived peptide to CD4+ T cells leading to T cell activation as well as signaling into the antigen-presenting cell. Class II is an αβ heterodimer that forms 2 distinct conformers via differential pairing of transmembrane domain GxxxG motifs (i.e. M1- and M2-paired class II). Previous studies with mouse I-Ak and human HLA-DR class II used conformer-specific monoclonal antibodies (mAbs) to define unique immunobiological properties of each conformer. Here, we report that AMS-32.1, a widely used anti-I-Ad mAb, is-specific for the 20% of M1-paired I-Ad class II expressed by the cell, and 34-5-3S anti-I-Ad mAb is non-conformer specific. While AMS-32.1 engagement of M1-paired I-Ad elicits B cell calcium signaling, 34-5-3S coengagement of M1 and M2-paired I-Ad blocks this response, indicating M2-paired class II inhibits M1-paired class II signaling. In vitro, AMS-32.1 blocks >90% of T cell activation by ovalbumin peptide-I-Ad complexes, even under conditions where peptide is loaded onto both conformers. In vivo, AMS-32.1 blocks accumulation of ovalbumin-specific CD4+ T cells in the spleen and lymph nodes. In a murine model of hepatitis B virus replication, in which major histocompatibility complex class II-restricted T cell-dependent humoral responses are key to antigen clearance, AMS-32.1 blocks hepatitis B surface antigen seroconversion, indicating that M1-paired class II is central to the in vivo development of CD4+ T cell-dependent antibody responses. Overall, this study reveals that AMS-32.1 is specific for M1-paired I-Ad class II and that M1-paired class II is critical for CD4+ T cell activation both in vitro and in vivo.