Costimulation of T cell activation involves both the B7:CD28 as well as the CD40 ligand (CD40L):CD40 pathway. To determine the importance of these pathways to in vitro and in vivo T cell activation, a direct comparison was made of the responses of TCR transgenic T cells lacking either CD28 or CD40L. In vitro, CD28−/− T cells showed a greater reduction in proliferative responses to Ag than did CD40L−/− T cells. The absence of CD28 resulted in defective Th2 responses, whereas CD40L−/− T cells were defective in Th1 development. In vivo, CD28−/− T cells failed to expand upon immunization, whereas CD40L−/− T cells could not sustain a response. These results suggest that CD28 is critical for initiating T cell responses, whereas CD40L is required for sustained Th1 responses. The different functional roles of these costimulatory pathways may explain why blocking B7:CD28 and CD40L:CD40 interactions has an additive effect in inhibiting T cell responses.

The frequency of clonally expanded and persistent T cells recognizing the immunodominant autoantigenic peptide of myelin basic protein (MBP)p85-99 was directly measured ex vivo in subjects with typical relapsing remitting multiple sclerosis (MS). T cells expressing mRNA transcripts encoding T cell receptor (TCR)-α and -β chains found in T cell clones previously isolated from these subjects recognizing the MBPp85-99 epitope were examined. In contrast to frequencies of 1 in 105–106 as measured by limiting dilution analysis, estimates of the T cell frequencies expressing MBPp85-99–associated TCR chain transcripts were as high as 1 in 300. These high frequencies were confirmed by performing PCR on single T cells isolated by flow cytometry. MBPp85-99 TCR transcripts were present in IL-2 receptor α–positive T cells which were induced to undergo Fas-mediated cell death upon antigen stimulation. These data demonstrate that at least a subpopulation of patients with MS can have a very high frequency of activated autoreactive T cells.

Tolerance in vivo is maintained by multiple mechanisms that function to prevent autoimmunity. An encounter of CD4+ T cells with a circulating self-Ag leads to partial thymic deletion, the development of CD25+ regulatory T cells (Tregs), and functional anergy in the surviving CD25− population. We have compared anergic and regulatory T cells of the same Ag specificity generated in vivo by the systemic self-Ag. Anergic cells are unresponsive to the self-Ag that induces tolerance, but upon transfer into a new host and immunization, anergic cells can induce a pathologic autoimmune reaction against tissue expressing the same Ag. Tregs, in contrast, are incapable of mediating harmful reactions. To define the basis of this functional difference, we have compared gene expression profiles of anergic and regulatory T cells. These analyses show that Tregs express a distinct molecular signature, but anergic cells largely lack such a profile. Anergic cells express transcripts that are associated with effector differentiation, e.g., the effector cytokines IL-4 and IFN-γ. Anergic cells do not produce these cytokines in response to self-Ag, because the cells exhibit a proximal signaling block in response to TCR engagement. Thus, anergy reflects an aborted activation pathway that can readily be reversed, resulting in pathologic effector cell responses, whereas Treg development follows a distinct developmental pathway that extinguishes effector functions.