p2Ca

Peptide length variants p2Ca and QL9 present distinct conformations to L(d)-specific T cells

Recent advances have provided insights into how the TCR interacts with MHC/peptide complexes and a rationale to predict optimal epitopes for MHC binding and T cell recognition. For example, peptides of nine residues are predicted to be optimal for binding to H2-L(d), although 8 mer epitopes have also been identified. It has been predicted that 8 mer and 9 mer length variant peptides bound to L(d) present identical epitopes to T cells. However, in contrast to this prediction, we demonstrate here that the 8 mer peptide p2Ca and its 9 mer length variant QL9, extended by an N-terminal glutamine, assume distinct conformations when bound to L(d). We generated self-L(d)-restricted CTL clones specific for p2Ca that recognize L(d)/QL9 poorly if at all. This result is in sharp contrast to what has been observed with L(d)-alloreactive T cells that possess a much higher affinity for L(d)/QL9 than for L(d)/p2Ca. Alanine substitutions of the N-terminal residues of the QL9 peptide rescue detection by these self-L(d)/p2Ca-specific T cells, but decrease recognition by the L(d)-alloreactive 2C T cell clone. In addition, 2C T cell recognition of the p2Ca peptide is affected by different alanine substitutions compared with 2C T cell recognition of the QL9 peptide. These data clearly demonstrate that the p2Ca and QL9 peptides assume distinct conformations when bound to L(d) and, furthermore, demonstrate that there is flexibility in peptide binding within the MHC class I cleft.

Potentiation of T Cell Stimulatory Activity by Chemical Fixation of a Weak Peptide-MHC Complex

The stability of peptide-MHC complex (pMHC) is an important factor to shape the fate of peptide-specific T cell immune response, but how it influences on T cell activation process is poorly understood. To better understand that, we investigated various T cell activation events driven by L^d MHCI loaded with graded concentrations of P2Ca and QL9 peptides, respectively, with 2C TCR Tg T cells; the binding strength of P2Ca for L^d is measurably weaker than that of QL9, but either peptides in the context of L^d interact with 2C TCR with a similar strength. When their concentrations required for early T cell activation events, which occur within several minutes to an hour, were concerned, EC50s of QL9 were about 100 folds lower than those of P2Ca, which was expected from their association constants for L^d. When EC₅₀s for late activation events, which takes over several hours to occur, were concerned, the differences grew even larger (> 300 folds), suggesting that, due to weak binding, L^d/P2Ca dissociate from each other more easily to lose its antigenicity in a short time. Accordingly, fixation of L^d/P2Ca with paraformaldehyde resulted in a significant improvement in its immunogenicity. These results imply that binding strength of a peptide for a MHC is a critical factor to determine the duration of pMHC-mediated T cell activation and thus the attainment of productive T cell activation. It is also suggested that paraformaldehyde fixation should be an effective tool to ameliorate the immunogenicity of pMHC with a poor stability.

Naturally occurring low affinity peptide/MHC class I ligands can mediate negative selection and T cell activation

The affinity/avidity model for T cell development postulates that ligands with high affinity for the TCR are efficient in negative selection, whereas those with lower affinity/avidity favor positive selection. Using the 2C TCR transgenic model, we evaluated the efficacy of ligands with widely differing affinity for the TCR (3 x 10(3) to 2 x 10(6) M(-1)) in mediating thymocyte deletion. The relative affinities of the 2C TCR for the p2Ca/Ld, dEV-8/Kb, p2Ca-A3/Ld, and p2Ca/Kb ligands are approximately 1000:50:10:1, respectively. Here we show, using an in vitro assay, that the deletion of 2C CD4+ CD8+ thymocytes is mediated not only by p2Ca/Ld, but also by the lower affinity ligands dEV-8/Kb, p2Ca-A3/Ld, and p2Ca/Kb, albeit at relatively higher peptide concentrations. Deletion mediated by low affinity ligands required CD8, whereas high affinity ligand-mediated deletion was CD8 independent. The p2Ca/Kb and dEV-8/Kb ligands are naturally occurring in H-2b mice, and others have shown that p2Ca/Kb can induce the maturation of CD4- CD8+ 2C-TCR(high) thymocytes in fetal thymic organ culture. In this study we showed that in addition to deletion, the p2Ca/Kb and dEV-8/Kb ligands, in the presence of exogenous IL-2, induced mature 2C T cell proliferation, albeit at a lower level than that induced by the high affinity p2Ca/Ld ligand. Thus, the same low affinity ligands that can effect negative selection and, in the case of p2Ca/Kb, the maturation of CD8 single-positive thymocytes can also induce the activation of mature CD8 T cells.

A cytotoxic T lymphocyte clone can recognize the same naturally occurring self peptide in association with a self and nonself class I MHC protein

The alloreactive CD8+ cytotoxic T lymphocyte (CTL) clone 2C was previously shown to recognize complexes made up of the class I MHC (MHC-I) molecule Ld and an octapeptide (LSPFPFDL, termed p2Ca) isolated from tissues of H-2d mice. Because peptide p2Ca has also been found in BALB.B (H-2b) mice, the strain from which clone 2C originated, the question arises as to whether these T cells can recognize peptide p2Ca in association with a self MHC protein of the H-2b haplotype. Here we show that 2C CTL do indeed recognize peptide p2Ca in association with Kb on the surface of H-2b cells or on transfected cells expressing Kb, but that an approximately 1000-fold higher concentration of this peptide is required to sensitize Kb+ than Ld+ target cells for lysis by 2C cells. However, the peptide's binding to Kb was not much weaker than to Ld, with only an approximately 10-fold difference in the respective equilibrium constants. These results predict that the T cell receptor (TcR) of clone 2C has a much lower intrinsic affinity for p2Ca-Kb complexes than for p2Ca-Ld complexes, and they provide some quantitative limits on the requirements for triggering T cell-mediated autoimmune reactivity.

Sequence restrictions in T cell receptor beta-chains that have specificity for a self-peptide/Ld complex

Cytotoxic T lymphocytes that react with a complex of Ld and a ubiquitous self-peptide derived from the enzyme alpha-ketoglutarate dehydrogenase (p2Ca, LSPFPFDL) can be readily elicited by the addition of synthetic peptide to cultures of BALB/c spleen cells. As with other Ld-restricted CTL, the p2Ca-specific cells use predominantly the V beta 8.3 region. In addition, the p2Ca-specific cells use almost exclusively one of three J beta gene segments. Selection for these J beta regions appears to be related to the presence of a glutamic acid residue that is encoded at the 5' end of the J beta and is present within the CDR3. As p2Ca does not contain a complementary charged residue, this finding may suggest that the beta-chain CDR3 from p2Ca-specific CTL contacts one of the five basic residues located on the Ld helices. Together, the results support the possibility that CDR1 and/or CDR2 (within V beta 8.3) and the CDR3 may each contact the Ld molecule. In contrast to the V beta and J beta regions, the V beta D beta J beta junctions and V alpha J alpha repertoires were diverse. The diversity could explain why p2Ca-specific CTL have relatively high precursor frequencies allowing them to be generated rapidly in primary cultures.