Genetic evidence for difference between intracellular and extracellular peptides in influenza A matrix peptide-specific CTL recognition.

During the course of extensive mutagenesis of HLA-A2.1, we examined influenza A matrix peptide (FMP)-specific CTL recognition of HMy2.C1R (C1R) cells expressing mutant HLA-A2.1 molecules, sensitized with synthetic peptide, FMP 58-66, (exogenous peptide), or infected with influenza A virus (endogenous peptide). Most mutants showed equivalent presentation of exogenous and endogenous peptides to FMP-specific CTL. However, five of the mutants differed in this property. Two of the five mutants, F9L and T134K, present exogenous peptide to FMP-specific CTL, but fail to present endogenous peptide to CTL. Western blot analysis using anti-matrix protein Ab indicates that the matrix protein is expressed in these mutants after infection with virus. Interestingly, transfection of these two mutants with a minigene encoding FMP 58-66 results in efficient lysis by FMP-specific CTL. Peptide-binding assays demonstrate that the two mutations dramatically decrease the binding of FMP. However, these mutants bind FMP as well as wild type in the presence of exogenously added human beta 2-m, suggesting that the lower affinity for beta 2-m leads to the inability to present endogenous peptide. The remaining three mutants, Y27N, Q32K, and S132C, fail to present exogenous peptide, but present endogenous peptide to FMP-specific CTL. Pulse-chase analyses followed by endoglycosidase-H treatment show that the rate of maturation and processing of the five mutant HLA-A2 molecules in C1R cells is identical to that of wild type. Overall, this study suggests that the assembly and subsequent recognition of endogenous peptide differs from that of exogenous peptide

Analysis of interactions in a tapasin/class I complex provides a mechanism for peptide selection

We examined interactions in a soluble tapasin (TPN)/HLA-B(*)0801 complex to gain mechanistic insights into the functions of TPN. Results show that TPN acts as a chaperone by increasing the ratio of active-to-inactive peptide-deficient HLA-B(*)0801 molecules in solution. TPN causes peptides to associate and dissociate faster owing to its effect on widening the binding groove of HLA-B(*)0801 molecules. Our data indicate that a TPN-assisted mechanism of peptide selection relies on disruption of conserved hydrogen bonds at the C-terminal end of the groove. Peptide sequence-dependent interactions along the entire length of the groove also play a role in this mechanism. We suggest that TPN influences presentation of antigenic peptides according to a mechanistically complicated process in which bound candidate peptides that are unable to conformationally disengage TPN from class I molecules are excluded from the repertoire. Overall, these studies unify our understanding of the functions of TPN