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The molecular basis underlying the specificity of alloreactive T cells for peptide-major histocompatibility complex ligands has been elusive. Here we describe a screen of 60 I-E(k)-alloreactive T cells and 83 naturally processed peptides that identified 9 reactive T cells. Three of the T cells responded to multiple, distinct peptides that shared no sequence homology. These T cells recognized each peptide-major histocompatibility complex ligand specifically and used a distinct constellation of I-E(k) contact residues for each interaction. Our studies show that alloreactive T cells have a 'germline-encoded' capacity to recognize multiple, distinct ligands and thus show 'polyspecificity', not degeneracy. Our findings help to explain the high frequency of alloreactive T cells and provide insight into the nature of T cell specificity.

TCRs exhibit a high degree of Ag specificity, even though their affinity for the peptide/MHC ligand is in the micromolar range. To explore how Ag specificity is achieved, we studied murine T cells expressing high-affinity TCRs engineered by in vitro evolution for binding to hemoglobin peptide/class II complex (Hb/I-Ek). These TCRs were shown previously to maintain Ag specificity, despite having up to 800-fold higher affinity. We compared the response of the high-affinity TCRs and the low-affinity 3.L2 TCR toward a comprehensive set of peptides containing single substitutions at each TCR contact residue. This specificity analysis revealed that the increase in affinity resulted in a dramatic increase in the number of stimulatory peptides. The apparent discrepancy between observed degeneracy in the recognition of single amino acid-substituted Hb peptides and overall Ag specificity of the high-affinity TCRs was examined by generating chimeric peptides between the stimulatory Hb and nonstimulatory moth cytochrome c peptides. These experiments showed that MHC anchor residues significantly affected TCR recognition of peptide. The high-affinity TCRs allowed us to estimate the affinity, in the millimolar range, of immunologically relevant interactions of the TCR with peptide/MHC ligands that were previously unmeasurable because of their weak nature. Thus, through the study of high-affinity TCRs, we demonstrated that a TCR is more tolerant of single TCR contact residue substitutions than other peptide changes, revealing that recognition of Ag by T cells can exhibit both specificity and degeneracy.

To identify the structural basis of Ag fine specificity, TCR sequences from a panel of Hb(64-76)/I-Ek-specific T cells were compared and found to be restricted in variable (V) gene usage, predominantly using BV1 or BV15 and AV4 or AV10 genes. TCRA and TCRB junctional sequences were extremely diverse. No conservation of length or position was found, which distinguishes this response from others, but correlates with the range of fine specificities that these T cells display. A remarkable subtlety in the recognition of Hb(64-76) was revealed from the study of the response to the D73 variant of Hb(64-76), which contains a conservative change in an MHC anchor residue not affecting the binding affinity to I-Ek. To one group of T cells this determinant was non-cross-reacting with Hb(64-76), whereas another recognized both Ags. Interesting, they all used a different constellation of TCRBV genes than that found in Hb(64-76) recognition. To limit the variability in the anti-Hb(64-76) TCR repertoire, transgenic mice expressing a fixed TCRB rearrangement from a Hb(64-76)-specific T cell were used. In Hb(64-76)-specific TCR from these mice, the endogenous alpha-chains pairing with the transgenic beta-chain were highly restricted in their AV gene usage. A comparison of two pairs of closely related T cells of these endogenous TCR variants, one differing by a single, conservative substitution in the complementarity-determining region 3 and the other containing a positional switch of two amino acids, revealed dramatically different fine specificities. Overall, these findings highlight the exquisite sensitivity of the TCR- peptide/MHC interaction to subtle alterations in any of the components.