Phenotypic and functional analysis of minor H alloantigen–specific T_REG. Results from 1 male donor (♂5) and 2 female donors (♀1 and ♀6) with minor H antigen disparate family members are shown. (A) Cell-surface expression of CD25 and CTLA-4 by CD8⁺ HA-1^A2 (top graph) or HY^A2 (center and bottom graphs) tetramer^dim staining T cells (indicated by circle) isolated by 2 consecutive rounds of FACS sorting from CD8-enriched PBMCs. (B) Defining the role of CTLA-4 in minor H alloantigen–driven bystander suppression of recall responses. Total PBMCs, obtained from the same blood sample as shown in panel A, were injected together with the recall antigens tetanus toxoid (TT) and diphtheria toxoid (D), allopeptide (HA-1 for ♂5; HY for ♀1 and ♀6) alone or a combination thereof into the footpads of CB17.SCID mice. Footpad swelling indicated as net swelling is depicted on the y-axis. Uncovering of recall antigen–induced footpad swelling was induced by coinjection of blocking CTLA-4 antibodies (αCTLA-4) or an isotype control antibody (iso). The percentages indicate the percentage of inhibition of the recall response (■) when minor H allopeptide is coinjected with or without blocking CTLA-4 antibody.