Our results make two important and related points. First, the data show the exquisite functional specificity of CD8+ T cells, with different receptors from chronically HIV-infected patients, to variants of this immunodominant epitope. Second, we provide evidence in support of an induced fit mechanism of TCR binding, whereby large conformational changes in the peptide follow initial TCR engagement.
T cells cross-react with virus variants is a very important issue in infection with HIV and other variable viruses. Failure of T cells to recognize variants gives the chronically replicating virus multiple chances to escape from immune control. Here, we show that for a clinically relevant epitope, two thirds of random mutations are not recognized. Although much has been made of cross-reactive T cells in the arena of vaccine design, our data are in line with earlier studies on peptide variants of influenza matrix peptide presented by HLA-A2, HTLV-1 tax peptide presented by HLA-A2 and EBV EBNA-3 peptide presented by HLA-B8 (9
). Therefore, T cell responses to pathogenic viruses appear very sensitive to epitope change. However, Mason (61
) has argued that because the T cell receptor repertoire is limited by the total number of T cells in the body, yet responds to all pathogens, each receptor must be able to see many different peptides presented by the same MHC molecule. Indeed, there are well-known examples of T cell clones cross-reacting on different MHC–peptide complexes (3
), but the specific set of binding interactions made by the TCR to each of these peptide–MHCs must be different (56
) and we would expect each binding mode to be just as sensitive to variants of the relevant peptide. Thus, the paradox of the same TCR showing significant cross-reactivity, but also very fine specificity for recognition of a single epitope, can be resolved. For a minority of variant peptides, binding to the MHC is so impaired that the epitope fails to present. Most well-described escape mutations of HIV are of this type (13
) and such mutations reach fixation. However, we show here that most variant peptides still bind HLA-A2, but most are not recognized by T cells. Given that T cells can react to very low numbers of presented peptides on a cell (44
), this finding implies that the majority of randomly generated epitope mutations affect the interaction between TCR and bound peptide. Priming of new T cell clones could deal with the problem, but in ongoing HIV infection the capacity to make new primary T cell responses may be impaired (22
), possibly because of damage to dendritic cells or T helper cells. If we can generalize from our findings concerning a dominant HIV epitope, and from previous studies of this type, the implication is that a T cell response to HIV that fails to suppress virus replication sufficiently is doomed to select escape mutants. The demonstration that the HLA type of an infected person shapes the virus sequence is powerful support for this concept (63
One surprising finding in our structure–function analysis was that the apparently small variation in the epitope from SLFNTVATL to SLYNTVATL (a common mutation in vivo that is cross-recognized by many T cells) causes a major difference in structure. Yet the observed T cell cross-reactivity implies that one conformation, common to SLFNTVATL and SLYNTVATL, is recognized by the TCRs. This interpretation is strongly supported by the very similar patterns of T cell recognition for variants of the two peptides in which single amino acid changes are made to residues in the conformationally different regions. If peptides with either conformation pre–TCR-binding change to a single common conformation on TCR binding, the additional changes in peptide sequence would be sampled in essentially identical contexts. The alternative explanation, that both TCRs recognize two very different peptide structures and that many changes in the structurally distinct portions (P5-P6) of these peptides coincidently all have the identical effect on TCR recognition, is highly implausible. It follows that at least one of the peptides must undergo a substantial conformational change during TCR binding. Some previous observations support this hypothesis. Crystal structures of HLA-A2 HTLV-1 Tax epitope in complex with two different TCRs show a significant and identical change in the peptide conformation on comparison of the pMHC and TCR–pMHC structures (52
). In that case, the change in peptide conformation at P5-P6 rotated both side chains by ~90° on TCR binding. A subsequent series of crystal structures for TCR–pMHC complexes of altered peptide ligands also preserved the same TCR bound peptide conformation (65
). Clearly, the balance of interactions that determine the conformation of the central portion of the peptide in at least some pMHCs can be altered in response to TCR binding. For SLFNTVATL and SLYNTVATL kinetic analysis revealed, somewhat unexpectedly, that the kon
s (and the Δ‡
) of TCR binding the two peptide complexes were indistinguishable. Because significant conformation adjustment of the peptide would result in significant differences in the kon
, this implies that energetically significant conformational adjustments are not occurring before formation of the transition state complex. This means that the conformational changes probably occur as the transition state complex relaxes into the final complex. An unlikely alternative explanation for the identical kinetics is that both peptides change into a third common conformation in the transition state, and that, by coincidence, both conformational changes have identical energetic barriers.
All interacting molecules, including the TCR–pMHC complex form a high-energy transition state complex (66
) before relaxing into the final complex, and Wu et al. (60
) showed that the TCR forms contact with residues on the MHC α-helices in the transition state complex. They were unable to rule out formation of contacts with peptides in the transition state, but it was clear that TCR forms these in the final complex. Based on these findings, and structural studies of other TCR–pMHC systems, which provide clear evidence of conformational change upon binding, Wu et al. proposed that the TCR initially forms contacts with the MHC in the transition state, and that after this there is a conformational change of the TCR CDR3 loops and formation of contacts with peptide (60
). However, they provided no direct evidence for this induced fit mechanism and their data are also consistent with a preexisting equilibrium mechanism (57
), whereby conformational changes precede TCR binding to pMHC. Indeed, the very slow kon
of many TCR–pMHC interactions (59
), including the 2B4 TCR system studied by Wu et al., strongly suggests that conformation changes often precede formation of the transition state. Recently, Gakamsky et al. (67
) pointed out that published kinetic and structural data to date do not allow one to distinguish between the two main mechanisms of conformational change at the TCR–pMHC interface.
In contrast, the data presented here provide evidence that conformational changes at the TCR–pMHC binding interface can occur after formation of the transition state complex (). These findings support the proposal by Wu et al. that the TCR first docks on MHC molecules relatively independently of the peptide, followed by conformational adjustments as the TCR and peptide–MHC relax from the transition state into the final complex. The stability of the final complex and, therefore, TCR triggering will be very sensitive to epitope sequence variation.
Figure 7. Thermodynamic analysis of TCR binding to HLA-A2–SLFNTVATL and HLA-A2–SLYNTVATL. Reaction profile illustrating the energy changes as the G10 T cell receptor binds to HLA-A2 complexed to SLFNTVATL and SLYNTVATL. ΔGass measures the (more ...)
In conclusion, these studies give some insight into how initial TCR contact can be quite cross-reactive, but transient. If TCR and peptide–MHC can adjust conformation to fit each other, more stable binding occurs and the T cell is triggered. However, this phase of T cell activation is highly specific with minimal scope for cross-reactivity. Such fine specificity may protect the host from autoimmune reactions but a penalty is paid when a virus can persist and is poorly controlled, as replication and mutation will lead to frequent immune escape. For vaccine design, the susceptibility of T cells to virus variation is of immense importance (24
). Unless vaccines can stimulate very broad T cell responses specific for multiple epitopes, it is unlikely that vaccines that are based on one subtype will offer significant protection against another subtype of HIV where the proteins differ by 20–30% (i.e., more than one amino acid change per epitope). Even within subtypes, there is enough variation (~5%) to cause concern.