Regulation of T cell activation and death by the affinity of TCR for peptide/MHC complexes

Abstract

The aim of this study is to investigate the role of the affinity of peptide:MHC/TCR interaction in the regulation of T cell activation, death and repertoire selection. Three aspects pertinent for our understanding of this issue have been analyzed.

First, we analyzed the effect of partially agonistic peptides on the activation and survival of CTL clones specific for a highly immunogenic HLA A11-restricted peptide epitope derived from the EBV nuclear Ag 4(EBNA 4), IVTDFSVIK (designated IVT). Several analogues with substitutions of TCR contact residues were able to trigger cytotoxic activity without induction of IL-2 mRNA and protein or T cell proliferation. Triggering with these partial agonists in the absence of exogenous IL-2 resulted in down-regulation of the cytotoxic potential of the specific CTLs. One analogue selectively triggered apoptosis as efficiently as the original epitope, subdividing the partial agonists into apoptosis-inducing and non-inducing ligands. Analysis of early T cell activation events did not reveal significant differences between the two types of analogue peptides. These results demonstrate that some partial agonists can dissociate the induction of CTL death from CTL activation.

Then, we characterized the apoptotic programs induced by the immunogenic peptide and its partially agonistic analogues in the IVT-specific CTL clones. Our major finding is that CTL triggering with partially agonistic peptide ligands can initiate death receptor dependent and independent apoptotic programs in the effector cells. In contrast to classical AICD, death receptors are not essential for the elimination of CTLs activated with partially agonistic peptides. In addition, death receptor independent apoptosis requires caspases other than caspase 3 and 8. Induction of anti-apoptotic BCl-2 and BCl-XL expression is associated with resistance to this form of apoptosis. Also, IL-2 enhances classical and inhibits death receptor independent AICD. We concluded that TCR triggering not accompanied by IL-2 production may result in elimination of T-lymphocytes in death receptor independent manner. Our data demonstrated that engagement of TCR by MHC-peptide complexes can trigger diverse apoptotic programs of AICD and that the choice between these programs is determined by the agonistic potency of MHC- peptide ligand.

Second, the molecular basis of different outcomes of CTLs stimulation with. immunogenic and partially agoistic peptide ligands was analyzed. The role of MHC:peptide/TCR affinity in the regulation of T cell activation was characterized using tetramer technology. Our results demonstrated that the All complexes assembled with the partial agonist dissociated from the surface of IVT-specific CTLs with a faster kinetics as compared with complexes containing the immunogenic peptide. We also showed that the efficiency of CTL recognition correlates with the stability of interaction between the specific TCR and MHC:peptide complex. Tetramer binding and secretion of INFgamma were shown to be compatible with T-cell activation by partially agonistic peptides. In conclusion, our results indicate that the affinity of TCR/MHC:peptide interaction determines the strength of TCR signalling, extent of CTL activation as well as the apoptosis pathway that operates in CTLs in the course of AICD.

The third aim of our study was to investigate the influence of the affinity of TCR/MHC interaction on the selection and maintenance of TCR repertoire of peptide specific CTLs. Using tetramer technology, we investigated the restriction of TCR usage among CTL responses against a subdominat EBNA 4 derived peptide referred to as AWF. In agreement with the earlier findings, ex vivo analysis of AVF-specific CTLs using AVF-containing HLA All tetramers revealed the same degree of conservation of the AVF-specific response both in healthy virus carriers and in the course of primary EBV infection. Tetramer binding and dissociation experiments performed with AVF-specific: CTLs or CTLs expressing a very diverse set of TCRs and specific to another immunodominant A 11-restricted EBV-derived peptide epitope did not support a model of affinity driven selection of restricted TCR repertoires. Characterization of individuals that fail to mount responses to the immunodominant A11-restricted CTL epitope but efficiently respond to the AVF-peptide argued against interclonal competition as the reason for the observed TCR conservation. Collectively, our data confirm the existence of naturally induced peptide-specific CTL responses with highly restricted TCR usage.

Our data do not support a major role for affinity of MHC:TCR interaction in the selection of structurally conserved TCR-repertoires and suggest that such conservations may be due to structural constrains in MHC:peptide/TCR interactions or endogenous pre-selection of certain clonotypes.