As long ago as the early 1970s, more than a decade before the study by Townsend et al. (1) at Oxford that is showcased in this issue, Doherty and Zinkernagel (2) discovered that CTLs recognize H-2 Ags that have been altered in some way by viral infection. What may seem curious is that it took so long to determine that this alteration was rendered by the binding of small peptide fragments to MHC class I (MHC-I)2 molecules.

Because of the requirements of the endogenous Ag processing pathway, the cytotoxic T cell response to viral infections has played a major role in elucidating the features that govern CTL recognition. Early studies were dominated by the study of a few viruses, including lymphocytic choriomeningitis virus (LCMV), Sendai, and influenza. Influenza virus played a key role in these studies because there is a convenient mouse model for infection. In addition, there are a variety of viral strains for mapping immune recognition, including a number that are no longer human pathogens that are more convenient for laboratory use. The discovery that MHC restriction was required for CTL responses to viral infections (2) was thus followed by intensive studies on the CTL response to influenza infection. Early studies focused on the envelope protein of influenza, hemagglutinin, which was expressed on the surface of virally infected cells and which induced a vigorous cytotoxic T cell response (3). Consequently, for many years, it was assumed that CTLs recognized an intact viral protein on the surface of an infected cell that was physically associated with the MHC-I Ag (3). However, as early as 1977, CTLs that were cross-reactive for all type A viruses, but not type B viruses, had been identified by scientists at the Wistar Institute (4) and by the Askonas laboratory (5) at the National Institute for Medical Research, where Alain Townsend started his research career. This suggested that another Ag common to all A subtypes, which have serologically distinct hemagglutinin molecules, also might induce CTL responses in mice. Furthermore, several major advances in the early to mid 1980s appeared to be quite inconsistent with the notion that CTLs recognize cell-bound viral proteins. These led Townsend and colleagues to seek an alternative ligand.

One advance was the cloning of the TCR by Davis and colleagues (6, 7) and independently by Tak Mak’s laboratory in 1984. As more sequences became available, it became obvious that it was impossible to determine from these primary structures whether the T cell was MHC-I or MHC-II restricted (8). They all bore striking homology to Ig variable and constant domains. In addition, around this time, it was determined that MHC-II-restricted helper T cells appeared to recognize small peptides. In contrast to cytotoxic T cell recognition, studies on helper T cells usually used small model Ags such as lysozyme, cytochrome c, insulin, and OVA. At that time, immunization of mice with these small soluble proteins always resulted in MHC-II-restricted responses, whereas it was necessary to use virus infection to raise CTLs. It was known in the late 1970s/early 1980s that Th epitopes to these proteins were contained within smaller fragments of the molecule (9, 10). In 1983, Shimonkevitz et al. (11) showed that tryptic digests of OVA, but not intact protein, presented by syngeneic spleen cells that were incapable of taking up the peptide into the cell, were recognized by cloned T cell hybridomas. This confirmed that degradation of Ag into peptides was both a necessary and sufficient condition for MHC-II-restricted recognition. By the mid 1980s, many MHC-II T cell epitopes had been at least partially mapped as peptide fragments ranging in length from 7 to 20 residues (12). But how could MHC-I-restricted TCRs interact with a two-protein complex and MHC-II-restricted TCRs interact with MHC-II plus a small peptide when they appeared, at least from their sequences, to be so structurally similar?

A third compelling piece of counterevidence to the theory that CTLs recognized membrane-bound protein came from the finding of the Oxford group, and of Yewdell et al. (13), that influenza-infected mice responded not only to membrane-bound glycoproteins but also to nucleoprotein (NP), which is not exported to the cell membrane and cannot access the Golgi. This explained the specificity of cross-reactive CTL responses in mice because NP is conserved among all type A viruses. So how could a viral protein that is not membrane bound and does not have a signal sequence be transported to the surface of the cell for CTL recognition? One possibility is that the viral sequence contains other signals for membrane transport. In an article published a year before this one, Townsend et al. (14) eliminated this possibility, located a 59-residue region of the NP protein that appeared to be recognized by CTLs, and came to the important conclusion that nonmembrane viral proteins might be degraded in the cytosol into peptides that were then exported from the cell. As they state in the introduction of the “Pillars of Immunology” paper, “Such degraded viral proteins may then become available for recognition by CTL in association with class I MHC molecules in a way similar to that in which helper T cells recognize denatured or degraded proteins with class II molecules.” To test this hypothesis, they set out to show that CTLs could recognize target cells incubated with short synthetic peptides from the NP sequence. They showed that human CTL clones specific for the 1934 NP sequence recognized one region of the molecule, 335–349. They extensively analyzed the mouse recognition of the molecule using mouse Db-restricted CTL clone F5, which is specific for the 1968 flu NP and three clones that were specific for the 1934 virus isolate. Using a series of synthetic peptides from the region 325–386 that they had previously mapped (14), they further narrowed the epitope to 366–379 for clone F5 and 365–380 for the CTLs specific for the 1934 NP. The NP proteins differ at two consecutive amino acid positions in this region of the molecule. Later work by Rammensee and colleagues (15), who sequenced naturally processed peptides eluted from purified MHC-I molecules, showed that the ideal epitope sequence is 366–374 or ASNENMDAM in the 1934 sequence.

In 1987, the Wiley laboratory solved the crystal structure of the human MHC-I molecule, HLA-A2 (16). They found that the MHC-I molecule had a unique fold with a groove formed between two α-helices, which could provide a binding site for processed foreign Ags. Electron-dense material, unresolvable in the crystal structure, was found in this site in the crystals of purified HLA-A2. This was postulated to be peptides that coisolated with the MHC-I molecule. This inspired Rammensee and colleagues (17) to biochemically characterize this material. By sequencing the mixture of material that they acid-eluted from isolated MHC-I alleles they found that the peptides were of a very limited size, 8–10 residues. In addition, each MHC-I allele appeared to bind peptides with specific motifs that displayed conserved residues at unique positions, which could provide the specificity for binding to individual MHC alleles (17). Interestingly, and in contrast, sequencing of a mixture of peptides eluted from MHC-II molecules did not reveal such motifs. Instead, individual peptides isolated from a mixture showed that the MHC-II/peptide complex included peptides that were of varying lengths (18).

The important conclusion of this “Pillars of Immunology” paper was that TCR interaction with MHC-I or MHC-II Ags at the surface of the cell was probably very similar, and that the element that “altered self” was probably a peptide derived from the whole protein Ag. How the peptide changed the H-2 molecule so that it could now be recognized by CTL was revealed by a flurry of publications from the Wiley and Wilson laboratories in the early to mid 1990s, which showed how these peptides fitted into grooves in the MHC molecules (reviewed, at that time, in Ref.19). These structures also explained why enzymatic digests of peptides are more easily loaded onto MHC-II molecules than MHC-I. The MHC-II peptide binding groove is more open-ended and thus more forgiving in the length of the peptide sequence that it can accommodate. In contrast, the peptides that bind to MHC-I molecules are of 8–12 residues and are more firmly anchored and buried in the structure. Peptides of such a precise length are unlikely to arise in enzymatic digests of proteins. What still remained a mystery was how the infected cell degraded peptides and how they managed to combine with the MHC-I molecule. Townsend and colleagues knew that the pathway by which peptides associated with MHC-I differed from the exogenous pathway of Ag processing. First of all, NP protein could not be substituted for peptides nor was the pathway sensitive to chloroquine. This “Pillars of Immunology” article thus predicted the ubiquitin-proteosome pathway because they reasoned that “If a proteolytic system does exist that is involved in presentation of antigen to class I restricted CTL, it should be present in all the cell types capable of being recognized by CTL, act on newly synthesized viral proteins, and be resistant to the effects of chloroquine and other agents thought to inhibit lysosomal degradation of proteins.”

2

Abbreviations used in this paper: MHC-I, MHC class I; LCMV, lymphocytic choriomeningitis virus; NP, nucleoprotein.

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