Establishment of stable CD8 + suppressor T cell clones and the analysis of their suppressive function

Abstract

Stable CD8 + suppressor T cell (Ts) clones were established by a relatively simple method. Keyhole limpet hemocyanin (KLH)-primed spleen cells from C3H mice were depleted of B cells and CD4 + T cells by panning and cytotoxic treatment, and the resulting CD8 + T cells were periodically stimulated with antigen and irradiated syngeneic spleen cells followed by manifestation interleukin-2 (IL-2) containing medium. T cell clones with a definite suppressor function were established by limiting dilution. They were defined as classical effector type Ts of CD8 + phenotype as they had constant and definite suppressor functions in antigen-induced T cell proliferation and specific antibody response against T cell-dependent antigens without detectable cytotoxic activity against both antigen presenting cells (APC) and helper T cells (Th). They showed no helper activity for B cells and produced no detectable helper type lymphokines such as IL-2 and IL-4. CD8 + Ts clones were able to inhibit the antigen-induced IL-2 production of normal and cloned T cells. Their suppressive activity was antigen-nonspecific and major histocompatibility complex-unrestricted. CD8 + Ts clones were also able to suppress the proliferative response of Th clones induced by immobilized anti-T cell receptor (TcR) and anti-CD3 mAbs but not the response induced by concavalin A (ConA) and IL-2. All the CD8 + T cell clones established independently utilized the TcR Vβ8 gene. Syngeneic antigen presenting cells could induce proliferation of these CD8 + clones, which was blocked by anti-CD8 and anti-I-A k monoclonal antibody (mAb) but not by anti-class I mAbs. The stimulation of CD8 + T3 clones with immobilized anti-CD3 resulted in the release of a suppressor factor(s) that potently inhibited the antigen-induced proliferation of CD4 + Th clones and the in vitro secondary antibody formation.