Quantification of alloreactive CD4⁺ T cells responding to permissive or nonpermissive DPB1 TCE3 or TCE4 disparities. Classical 1-way MLRs were set up between R-S pairs of unrelated volunteers selected for the same patient and matched between each other for 10/10 of the HLA-A, -B, -C, -DRB, and -DQB1 alleles, but mismatched for -DPB1. R cells consisted of peripheral blood mononuclear cells (PBMCs), while S cells in most cases were PBMCs depleted of CD3⁺ T cells, at a ratio of 1:1. After 2 rounds of stimulation in the presence of 150 IU/mL IL-2, CD4⁺ T cells were rechallenged overnight with B lymphoblastoid cells (BLCLs) from R, S, or from third-party donors sharing only 1 mismatched DPB1 allele with S. Responding T cells were quantified by FACS analysis for surface expression of the activation marker CD137. In several cases, CD4⁺ T cells expressing CD137 upon challenge with DPB1 typed third-party BLCLs were FACS-sorted and their specificity for the relevant DP alloantigen was confirmed (not shown). (A) Exemplative analysis of alloreactive CD4⁺ T cells responding to a permissive or a nonpermissive DPB1 mismatch on the same S cell. R and S cells carried DPB1*02:02, 04:01 and DPB1*04:02, 09:01, respectively, and thus S cells presented 1 TCE3/4 permissive (DPB1*04:02) and 1 TCE3/4 nonpermissive (DPB1*09:01) mismatch. (B) Mean percentage of CD4⁺ T cells responding to permissive or nonpermissive DPB1 mismatches according to TCE3 (left panel) or TCE4 (right panel), in a series of 24 MLRs. At the moment of testing, cultures contained a mean of 53.75% ± 24.94% CD4⁺ T cells. The mean percentage of T cells expressing CD137 in response to autologous R-BLCLs was 2.06% ± 1.62%. The percentage of T cells responding specifically to allogeneic read-out BLCLs was calculated as the total percentage of CD137⁺ T cells after allogeneic stimulation minus the percentage of CD137⁺ T cells after autologous stimulation. Fully allogeneic R-S pairs (n = 8) were used as positive controls and yielded a mean of 17.53% ± 8.28% specifically responding CD4⁺ T cells, with a mean of 44.75% ± 25.17% CD4⁺ T cells. Pairwise comparison of the results obtained in the different groups was performed by the Kruskal-Wallis test followed by the Dunn multiple comparison posttest. (Left panel) In the TCE3 permissive group (n = 15), the mismatched DPB1 allele expressed by S was encoded by DPB1*02:01 (n = 4), 02:02 (n = 1), 04:01 (n = 5), 04:02 (n = 3), 11:01 (n = 1), 13:01 (n = 1). In the TCE3 nonpermissive group (n = 9), the mismatched DPB1 allele expressed by S was encoded by DPB1*03:01 (n = 3), 09:01 (n = 2), 10:01 (n = 3) or 17:01 (n = 1). The frequency of CD4⁺ T cells specifically responding to TCE3 permissive mismatches was significantly lower compared with TCE3 nonpermissive mismatches (*P < .05) and compared with fully mismatched third-party alloantigens (***P < .001). (Right panel) In the TCE4 permissive group (n = 10), the mismatched DPB1 allele expressed by S was encoded by DPB1*04:01 (n = 5), 04:02 (n = 3), 11:01 (n = 1), 13:01 (n = 1). In the TCE4 nonpermissive group (n = 15), the mismatched DPB1 allele expressed by S was encoded by DPB1*02:01 (n = 4), 02:02 (n = 1), 03:01 (n = 3), 09:01 (n = 2), 10:01 (n = 3) or 17:01 (n = 1). The frequency of CD4⁺ T cells specifically responding to TCE4 permissive mismatches was significantly lower compared with TCE4 nonpermissive mismatches (*P < .05) and compared with fully mismatched third-party alloantigens (***P < .001).