Anti-HLA class II allele-specific immune reactivity in early post-dUCBT PB samples. Nineteen PBMC samples obtained from 11 patients at 1 to 6 months after dUCBT were propagated toward mismatched HLA class II alleles of the nonengrafting CBU and subsequently tested for alloreactivity toward mismatched and matched HLA class II alleles. The response was quantified by CD137 upregulation on CD4⁺ T cells and presented as fold increase (FI; %CD137⁺/CD4⁺) of reactivity toward HeLa cells transduced with (mis)matched HLA class II alleles relative to reactivity toward the nontransduced empty HeLa cell (control) (supplemental Figure 3A). In case >1 PBMC sample per patient was tested, the best result (ie, highest FI) was considered, generally obtained with the PBMC sample with the shortest follow-up after dUBCT. (A) Patient PBMCs were propagated toward mismatched alleles in a 14- to 21-day coculture and subsequently assayed toward the same mismatched alleles (as specified in the left column). The level of response is presented in a 4-color grading scale, as depicted and was as follows: DR alleles, FI = 11.6 (median; range, 2-84; n = 16); DQ alleles, FI = 8.9 (median; range, 4-87; n = 7); DP alleles, FI = 3.6 (median; range, 1.0-12; n = 10). The level of reactivity toward the HLA class II negative (empty) HeLa cell is shown at the bottom row of the figure, is presented as the mean % CD137⁺/CD4⁺ of 1 to 7 observations per patient (median %CD137⁺/CD4⁺, 2.2; range, 0.5-8.7; n = 11), and is depicted in a 4-color-grading scale. (B) Patient PBMCs were propagated toward 1 to 7 mismatched alleles per patient (as shown in A) and subsequently assayed toward a single matched allele of the engrafting CBU (as specified in the left column). The mean value of response of 1 to 7 observations per patient is presented. The level of response was as follows: DR alleles, FI = 1.6 (median; range, 1.2-3.6; n = 4); DQ alleles, FI = 2.3 (median; range, 1.3-4.3; n = 3); DP alleles, FI = 1.1 (median; range, 0.6-1.6; n = 5; see A).