Kinetics of expression and subset distribution of the TNF superfamily members CD40 ligand and Fas ligand on T lymphocytes in cattle

Abstract

CD40 and Fas are members of the tumor necrosis factor receptor (TNFR) superfamily. CD40 and Fas play key roles in T cell-B cell interactions. Cross linkage of these molecules induces cell activation and cell death, respectively. The interaction of CD40 with its ligand (CD40L), which is expressed on activated T cells, plays a pivotal role in the generation of the T-dependent (TD) immune response, and FasL-bearing T cells, which have been shown to be predominantly of either the TH0 or TH1 type, have the potential to induce the apoptotic death of Fas expressing B cells. We investigated bovine CD40L mRNA expression in established T cell clones by RT-PCR and Southern blotting. T cells analyzed included CD4+ TH0 and TH1 cell subpopulations, CD8+, and gamma/delta T cells stimulated with either specific antigen or Con A. All CD4+ clones but not all CD8+ or gamma/delta T cell receptor (TCR)-bearing clones expressed mRNA for CD40L. To determine the activation requirements for CD40L expression in cattle, we examined the kinetics and induction requirements for CD40L transcription in peripheral blood T cells using a phorbol ester and/or ionomycin, immobilized mouse anti-bovine CD3, or Con A. Our results demonstrate that CD40L mRNA appears relatively early after activation (1 h) and peaks at 2-4 h poststimulation. A rise in intracellular calcium concentration mediated by ionomycin treatment alone was sufficient to induce CD40L mRNA expression at relatively high levels. Ionomycin treatment in combination with other agonists (anti-CD3, PMA) did not enhance CD40L mRNA expression above levels obtained with ionomycin alone. The bovine Fas ligand gene was partially cloned and mRNA expression determined by RT-PCR in a panel of T cell clones. Our results demonstrate that TH0 and TH1 bovine T cell clones expressed Fas ligand transcripts although only one gamma/delta T cell clone did. This expression was upregulated within 3 h after mitogen stimulation and reduced by 24 h.