Improved Protocol for Establishing CD4⁺ Hybridomas Specific for Human Class II MHC/Peptide Complex

Autoreactive CD4⁺ T cells are shaped by MHC class II-dependent selection, and HLA-DQ8 is a major susceptibility allele for type 1 diabetes and celiac disease. To define how HLA-DQ8 influences the autoreactive CD4⁺ T-cell repertoire, we generated T-cell hybridomas from HLA-DQ8 humanized mice using a BW5147 Nur77-GFP (BW-GFP) platform that enables sensitive quantification of antigen-induced T-cell receptor (TCR) signaling. The frequency of autoreactive conventional CD4⁺ hybridomas observed in HLA-DQ8 mice was higher than previously reported in C57BL/6 mice in our earlier study, suggesting that HLA-DQ8 shapes an autoreactive repertoire. However, because antigen presentation in this system is restricted by human HLA-DQ8 while hybridomas express murine CD4, we considered that CD4-MHC interspecies mismatch might affect signal strength and influence the apparent magnitude of autoreactivity. To address this limitation, we engineered a BW-GFP fusion partner expressing an optimized version of human CD4 (hCD4), restoring optimal CD4-HLA-DQ8 interactions. Hybridomas generated with this modified platform from both regulatory (Treg) and conventional (non-Treg) CD4⁺ T cells exhibited enhanced responses to HLA-DQ8/peptide complexes compared with hybridomas that do not express hCD4. This approach improves the reactivity and physiological accuracy of screening mouse-derived CD4 hybridomas specific to self and foreign antigens presented by human class II MHC complexes. Key features • Improved BW5147 Nur77-GFP thymoma with optimized human CD4 enhances CD4-HLA class II compatibility in HLA-DQ8 humanized hybridoma systems. • BW modification preserves fusion efficiency with Treg and conventional CD4 T cells, comparable to the parental BW line (n = 10 times for validation). • Optimized for studying HLA-DQ8-restricted autoimmunity and adaptable to other HLA-transgenic mouse models.