Recentwork has demonstrated that non-stimulatory endogenous peptides can enhanceT-cell recognition, a finding that is unexplained by the classical model ofCD8, TCR, and MHC interaction. To investigate this phenomenon, we havedeveloped a tetracycline-inducible CHO cell system where cognate single-chainMHC-peptide is expressed at low levels and non-stimulatory single-chainMHC-peptide is constitutively expressed. Mutations in the cognate ornon-stimulatory MHC can therefore be tested to identify the interactionsimportant in this enhancement. We have tested co-receptor binding and TCRbinding separately. Our results show that mutation of the CD8 binding site onthe non-stimulatory MHC neutralizes T-cell activation enhancement. Furthermore,H-2Kb non-stimulatory MHC provide a stronger activation enhancementthan H-2Db non-stimulatory MHC, which directly correlates activationenhancement with higher in vitroaffinity of CD8 for H-2Kb relative to H-2Db. We testedthe potential importance of a TCR interaction with the non-stimulatoryMHC-peptide by mutations in the a-helix of H-2Kbthat affect recognition of antigen by this TCR. These did not affect aid forantigen recognition unless the CD8-binding site of the cognate MHC wasinactivated. Thus, co-receptor binding is the prime determinant of the abilityof non-cognate MHC-peptide to enhance antigen recognition, but an effect of TCRinteraction can be identified when CD8 interaction is removed from theequation. We tested the importance of TCR versus CD8 binding in another system,by replacing the a1a2 domains of H-2Kbwith those of H-2TL, which cannot present peptides to TCR. This TL-Kbchimera was able to enhance antigen recognition provided the CD8-binding siteon the Kb-derived a3 domain was not mutated.Ourresults show that the non-cognate CD8-MHC interaction is important in enhancingantigen recognition, and that interaction between the non-stimulatory MHC andTCR is dispensable as long as the CD8 binding site on the cognate MHC moleculeis intact. Future work will address specific models for how the CD8 and TCRinteractions cooperate in T cell activation, any sub-threshold signaling thatoccurs due to the non-cognate CD8-MHC interaction, the dynamics of Lckrecruitment and activation with CD8 in the non-cognate setting, and theimportance of non-cognate CD8-MHC interactions in immune responses, for exampleto herpesviruses that have mechanisms to reduce MHC expression. We will alsouse FRET between fluorescent-labeled cognate and non-cognate MHC-peptidemolecules to investigate how closely opposed they are during the enhancedantigen recognition.

Refined model for CD8,
TCR, and MHC interactions at the immunological synapse

240th National Meeting (2010)

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Refined model for CD8, TCR, and MHC interactions at the immunological synapse