In 1991 Brian Evavold, at that time a postdoctoral fellow, and Paul Allen made a seminal report by introducing altered peptide ligands (APLs) into the field of Ag presentation and T cell activation (1). Since that time, numerous studies have attempted to explain the function of an APL. Other investigations have used APLs to identify different nuances of the immune response, even attempting to use them to alter peptide recognition by pathogenic T cells. I focus this commentary mostly on the series of studies on APLs by Paul Allen’s laboratory (2, 3).

Evavold and Allen examined a peptide from the hemoglobin β-chain bound to the I-Ek molecule, specifically to the 64–76 segment of the Hbbd allele. Such a peptide–MHC (pMHC) complex was recognized as a T cell epitope by mice bearing the Hbbs variant. A CD4 T helper-2 clone (2.102) produced IL-4 and proliferated in response to the Hb 64–76 peptide (Fig. 1). Their striking finding centered on the response to the peptide with one conservative substitution, that of aspartic acid for glutamic acid at position 73: IL-4 production was not impaired, but proliferation was affected. Thus, removing one methylene group of an amino acid anchoring side chain resulted in a profound functional change, leading the authors to conclude that “the T cell receptor has the capacity of differential signaling”. Follow-up papers extended the original findings to a wide range of T cell clones, including Th1 clones, and indicated that signaling by a T cell did not involve an all-or-none response but rather contained a spectrum of functional responses and events (4, 5).

FIGURE 1.

The Hb 64–76 peptide, which formed the basis of the Science paper. The 9-mer segment bound to I-Ek molecules is in bold. Arrows pointing up represent the TCR contact residues; arrows pointing down represent the MHC anchor residues. Residue 73 is circled.

FIGURE 1.

The Hb 64–76 peptide, which formed the basis of the Science paper. The 9-mer segment bound to I-Ek molecules is in bold. Arrows pointing up represent the TCR contact residues; arrows pointing down represent the MHC anchor residues. Residue 73 is circled.

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Important for the understanding of the first observation was a subsequent study examining the features of the Hb 64–76 interaction with I-Ek (6). At the time of the Science report, the structure of the class II MHC molecules had not been determined. Examining the binding and structural features of the APL made possible a precise identification of the changes brought about by the E73D change. Both wild-type and E73D-APL peptides bound equally well to the I-Ek molecule; functional analysis using APCs pulsed with each peptide disclosed similar off-rates. Glutamic acid 73 was an MHC anchor residue binding at the P6 pocket of I-Ek. However, each peptide adopted a different conformation, particularly centered on the region between the P5 and P8 residues. The absence of a methylene group in aspartic acid resulted in a change in the relative positions of the P6 and P8 residues of the peptide, both of which were solvent exposed. The P8 residue, a leucine, was an important TCR contact residue and showed a slight rotation, depending critically on the P6 anchor. This single change in an MHC anchor residue, therefore, induced a different topology at an important segment of the bound peptide.

In attempting to explain the T cell response to an APL, studies by Allen, with Kersh as lead author, followed (7). It became apparent that TCR affinities to the pMHC were changed when APLs were of low affinity, as is the case with most TCR–pMHC interactions. Further experimentation made the point that patterns of TCR ζ phosphorylation varied between APLs and the cognate peptide. The pattern of phosphorylation was thought to be unique to the APL and not a consequence of quantitative effects (8; see also Ref. 9).

The kinetic interaction of pMHCs, including APLs, with TCR has been difficult to study because of the low affinity of the interactions. However, a more recent study from Allen’s laboratory, together with David Kranz, is of particular importance. Persaud et al. (10) examined a number of binding parameters of a single-chain TCR (3L2) engineered to bind the Hb 64–76 peptide at higher affinity than the wild-type TCR. The study confirmed that a rather small change in the topology of the pMHC brought about by single-residue substitutions in the Hb 64–76 peptide resulted in marked change in binding thermodynamics; in this example, the strength of the interaction reflected the ensuing T cell response.

As would be expected, the APL findings came to the attention of those interested in various aspects of the T cell response to a pMHC complex. A number of questions were raised concerning their significance and whether the differential responses of the APL could be explained by conformational changes in the TCR engaging the APL pMHC or on various other aspects, such as strength of the binding of the TCR to the pMHC, differences in thresholds of activation, persistence of the TCR–pMHC interaction, or kinetic proofreading. It raised the important issue of the relationship between the functional response and the strength of the TCR interaction with either the wild-type or APL pMHC. These issues most notably included thymic differentiation in which the positive selecting ligand in the thymic APC, consider it an APL, may differ slightly from the agonist peptide. Yet the threshold of activation between the T cell recognizing the selecting ligand and the agonist peptide may also differ greatly. An analysis of these important discussions is beyond the scope of this commentary, but for examples see Refs. 1115.

Discussions also have focused on the role of APLs in infectious diseases and in the control of autoimmune responses. Several reports made the case that viral peptides could act as antagonistic APLs (16, 17). This is an important issue but one made complex by the diversity of relevant epitopes derived from the pathogen. In autoimmunity, APLs were tested for their ability to control disease, with some success experimentally (18, 19), but so far, none are in clinical trials (20). The broad repertory of TCR to autologous pMHC, as well as the diversity of pMHC present during the chronic autoimmune process, may preclude the therapeutic success of APLs. In summary, APLs continue to be a challenge. The Science paper opened an area of fruitful discussions that are still ongoing, centering on how subtle, or not so subtle, changes in a pMHC may affect the T cell response to it.

Disclosures The author has no financial conflicts of interest.

Abbreviations used in this paper:

APL

altered peptide ligand

pMHC

peptide–MHC.

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