A recently developed limiting dilution (LD) method was used to analyze the frequency and specificity of IL2-producing cells within alloantigen-stimulated human CD4+ and CD8+ T cell subsets. Cell sorter-separated CD4+ and CD8+ responder cells were cocultured under LD conditions with HLA class I and/or class II different Epstein Barr virus (EBV)-transformed lymphoblastoid cells line (LCL) stimulator cells in the absence of additional factors. After 3 days, IL2 in cell-free culture supernatants was measured by a colorimetric assay on IL2-dependent murine CTLL cells. Under these conditions, one out of 200-500 CD4+ and one out of 300 to 1000 C8+ T cells produced IL2 when stimulated by HLA class I and class II disparate LCL. By using selected responder and stimulator cells differing only in HLA class I (A, B, C) or class II (DR) antigens, it was found that CD4+ T cells produced IL2 in response to HLA class II antigens, while CD8+ T cells produced IL2 in response to HLA class I antigens. Surprisingly, high frequencies of IL2-secreting CD4+ T cells were noted in certain HLA-DR-identical responder-stimulator combinations. To investigate whether HLA class II antigens other than DR (i.e., DQ or DP) activate CD4+ cells to IL2 secretion, we analyzed a set of HLA-A,B,C and -DR,DQ-identical responder-stimulator cells which differed only in DP antigens. In several of these instances, we measured high frequencies (f = 1/1000 to 1/2000) of HLA-DP-reactive CD4+ IL2 producers, while the frequencies in LD cultures stimulated with autologous LCL were low (f = 1/10,000 to 1/30,000). The specificity of alloantigen-activated IL2-secreting T cells was assayed by restimulation with the original or HLA-mismatched third-party LCLs. CD4+ responder cells could be efficiently and specifically restimulated to IL2 production after a resting period of 3 to 4 days, while CD8+ cells were refractory to restimulation under these conditions. Together these data demonstrate that: 1) CD4+ and CD8+ cells are stimulated to IL2 production by HLA class II and class I antigens, respectively; 2) alloantigen-activated CD4+ IL2 producers are highly specific for stimulating HLA antigens as shown by a split culture and restimulation approach; and 3) significant numbers of CD4+ IL2-producing T cells can be activated by selected HLA-DR-identical, DP-different stimulator cells.
