HLA class II allele-specific allorecognition of CD4 T-cell subsets. MLR cultures were tested for allo-HLA reactivity in IFN-γ ELISpot assays on d12 (i.e. 5 days after first allo-HLA restimulation on d7). (A) Reactivity to original K562-HLA mismatch stimulator cells, as well as to parental K562 cells and K562 transfectant cells carrying an irrelevant HLA allele. To demonstrate HLA-restriction of detected reactivity, monoclonal antibodies blocking T-cell receptor-HLA interactions (and IgG isotype controls) were used. Representative results with naïve-enriched CD4 T-cell subsets of donor 372 for allo-HLA-DQB1*06:02 are shown (B) Numbers of IFN-γ spot-forming cells from d12 cultures of MLR started with sorted subsets (CD45RA, CD45RO, CD62L, CCR7) or total CD4 T cells from six healthy donors. HLA class II mismatch alleles used for allostimulation were HLA-DRB1*07:01 (upper panel) and HLA-DQB1*06:02 (lower panel). (C) Box plots and P values of data presented in (B); for explanation see legend to Figure 2C. If PBMC numbers were limited, MLR stimulations were restricted to fewer allo-HLA alleles.