Broad Vβ repertoire in CD4⁺ T cells with regulatory activity generated by ex vivo CD40:CD40L blockade.

CD4⁺ T cells with regulatory activity generated by ex vivo CD40:CD40L blockade have a broad Vβ repertoire. BALB/c CD4⁺ T cells were tolerized to B6 as described in “Materials and methods.” At the end of culture, total cellular RNA was isolated and cDNA was prepared from control cultured or tolerized cells, as well as from freshly purified naive CD4⁺ T cells. As a measure of repertoire size, spectratyping for 17 distinct Vβ families was performed, and CDR3-size usage was quantified by scanning densitometry. Three Vβ family members (Vβ4, Vβ6, and Vβ8) are illustrated. As compared with freshly purified CD4⁺ T cells, control cultured cells had restricted Vβ4 and Vβ5 but not Vβ8 usage, consistent with preferential Vβ use in response to alloantigen stimulation. In contrast, Vβ4 and Vβ5 usage was broader in tolerized cultures, indicating that tolerization and the generation of regulatory cells occurred without substantial repertoire restriction.