DM-resistant and DM-sensitive antigens as recognized by the natural CD4⁺ T-cell repertoire. Purified CD4⁺ T cells were seeded in one 96-well plate, each well containing 2000 different T cells. CD4⁺ T cells were nonspecifically expanded and tested for recognition of HeLa–invariant chain (Ii) and HeLa-Ii/DM transduced with different HLA class II molecules. T-cell pools with no or low (< 200 pg/mL IFN-γ) recognition of both HeLa variants are depicted as “nonreactive.” The remaining pools were separated into DM-sensitive and DM-resistant phenotype, based on the difference in recognition between HeLa-Ii and HeLa-Ii/DM. Pools with > 50% decrease in recognition of HeLa-Ii/DM versus HeLa-Ii were assigned DM-sensitive, whereas all other pools were counted as DM-resistant. CD4⁺ T-cell libraries were generated from 3 different donors, and CD4⁺ T-cell pools were tested against HeLa-Ii and HeLa-Ii/DM variants transduced with HLA-DQB1*0603/A*0103 (2 donors), DQB1*0501/A*0101 (1 donor), DRB1*0301 (1 donor), DRB1*1301 (2 donors), DRB3*0101 (1 donor), and DRB3*0202 (1 donor). Representative results are shown for 2 HLA-DQ alleles (A) and 4 HLA-DR molecules (B). The total number and percentages of DM-resistant and DM-sensitive pools for each HLA class II allele are indicated.