Treg tissue-homing subsets, PTDM, and transplant outcomes. (A) Multiparameter analysis of Treg tissue-homing subsets. Treg tissue-homing subset identification using 10-color multiparametric flow cytometry. Gates were initially set on CD3⁺ while excluding cells expressing CD14 or marking with the amine viability dye (data not shown). Viable CD4⁺ T cells were then selected by gating on CD4⁺CD8⁻ cells. To ensure adequate cell numbers for accurate gating, quadrants were created using 2-parameter comparisons assessing the expression of Foxp3, CD25, CD127, CD45R0, α4β7, and CLA by the total CD4⁺ cell population (data not shown). These gates were then sequentially applied to the CD4⁺ cell population to determine Treg subset frequency. (B-C) Overall survival and nonrelapse mortality of patients undergoing HSCT stratified by the presence of PTDM. (D-E) PTDM was associated with changes in Treg tissue-homing phenotype. The percentage of CLA⁺ Tregs and α₄β₇⁺ Tregs was determined by multiparametric flow cytometry at the time of neutrophil engraftment. Box plots define the values for median, range, and 25th and 75th percentiles. Two-tailed P values were calculated using the Mann-Whitney U test to assess for differences in the median percentage of CLA⁺ Tregs and α₄β₇⁺ among patients with or without PTDM.