A Tale of Two T Cells
Authors: Charles A Janeway
Journal: Immunity (1998)

In this issue of Immunity, Don Wiley and colleagues (4Ding Y-H Smith K.J Garboczi D.N Utz U Biddison W.E Wiley D.C Immunity. 1998; 8 (this issue): 403-411Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar) describe a remarkable finding: the structure of a T cell receptor (TCR) in which 16 of 17 peptide/major histocompatibility complex (MHC) contact residues differ from those of the original TCR analyzed by this team (10Garboczi D.N Ghosh P Utz U Fan Q.R Biddison W.E Wiley D.C Nature. 1996; 384: 134-141Crossref PubMed Scopus (1203) Google Scholar), yet the same peptide/MHC complex in the same orientation is bound by both TCRs. These two TCRs were derived from two different patients, although the significance of this factor is not clear. Because TCR specificity is believed to be determined by the amino acids that make contact with the antigenic complex of peptide and MHC molecule, how is it possible that both TCRs can recognize the same peptide/MHC complex? There are two types of answer to this question. The first is the subject of the Wiley laboratory's thorough discussion of their results. The second answer, as I believe and will argue in this minireview, is that the strong similarity in antigen recognition between these two HLA-A2/Tax peptide recognition sites that differ in nearly all of their peptide/MHC contact residues is due to positive selection on a single self-peptide/self-MHC complex. Self-peptide/self-MHC complexes are expressed on thymic cortical epithelial cells (22Laufer T.M DeKoning J Markowitz J.S Lo D Glimcher L.H Nature. 1996; 383: 81-85Crossref PubMed Scopus (334) Google Scholar), and such complexes drive positive selection within the thymus. They are also expressed in the periphery, apparently by dendritic cells (2Broker T J. Exp. Med. 1997; 186: 1223-1232Crossref PubMed Scopus (339) Google Scholar), and there they drive the long-term survival of peripheral T cells. Although TCRs such as these that differ extensively in contact residues may represent an extreme case, there is evidence from other systems that such “synonymous” TCRs exist in several different settings, possibly accounting for several phenomena such as immunodominance (3Busch D.H Pamer E.G J. Immunol., in press. 1998; Google Scholar) and the role of the MHC in autoimmunity. The two TCRs in question both recognize the same Tax peptide bound to HLA-A2, an MHC class I molecule, and therefore must come from CD8 T cells. When positive selection of CD8 T cells was analyzed in vitro, it was found that CD8 T cells of non-TCR transgenic mice are selected most strongly by the mixture of self-peptides bound to self-MHC class I molecules (1Ashton-Rickardt P.G Van Kaer L Schumacher T.N.M Ploegh H.L Tonegawa S Cell. 1993; 73: 1041-1049Abstract Full Text PDF PubMed Scopus (250) Google Scholar, 15Hogquist K.A Gavin M.A Bevan M.J J. Exp. Med. 1993; 177: 1469-1473Crossref PubMed Scopus (210) Google Scholar). These investigators destroyed the ability of MHC class I molecules to present peptides for positive selection, either by interfering with transport of the peptides across the endoplasmic reticulum, using TAP (transporter associated with antigen processing) knockout mice, or by interfering with their presentation in the context of naturally conformed MHC class I molecules, using β2m (β2-microglobulin) knockout mice. In both cases, restoration of one or a few peptides had widely divergent results in fetal thymic organ culture (FTOC): some peptides did not affect positive selection at all, whereas others restored a considerable degree of positive selection, although all of them increased MHC class I expression. In the TAP knockout mice, a mixture of two peptides that bound to different MHC class I molecules could restore positive selection of CD8 T cells to 30%–50% of normal levels. However, when peptides eluted from thymic stroma were used to reconstitute peptide/MHC complex expression in the TAP knockout system, full restoration of positive selection was observed in FTOC. In the case of the β2m knockout mice, mixtures of synthetic peptides of increasing complexity restored positive selection of the numbers of CD8 T cells to near normal. Thus, it was established early that the selection of CD8 T cells occurred on peptide/MHC complexes, and that the most effective peptides for positive selection of CD8 T cells were those eluted from the MHC molecules expressed in the normal thymus. These studies were extended to include specific T cells by using mice transgenic for a TCR of known specificity. In this approach, a single peptide could be used to influence selection of the TCR in FTOC. In the most compelling case, it was determined that positive selection could be driven by peptides that interact with the receptor but fail to activate mature T cells, but not by agonist peptides that activate them, since these would be expected to induce CD8 T cell deletion (20Jameson S.C Hogquist K.A Bevan M.J Nature. 1994; 369: 750-752Crossref PubMed Scopus (204) Google Scholar). Subsequently, the same group used a “dulling” assay combined with sophisticated molecular characterization to determine the nature of one naturally occurring self-peptide that is a candidate for a peptide that may positively select transgenic thymocytes (16Hogquist K.A Tomlinson A.J Kieper W.C McGargill M.A Hart M.C Naylor S Jameson S.C Immunity. 1997; 6: 389-399Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Recently, it was shown that specific recognition of thymic self-peptides can induce positive intrathymic selection of CD8 cytotoxic T cells (17Hu Q Bazemore Walker C.R Girao C Opferman J.T Sun J Shabanowitz J Hunt D.F Ashton-Rickardt P.G Immunity. 1997; 7: 221-231Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Thus, it is clear that in the case of CD8 T cells, positive intrathymic selection on self-peptide/self-MHC complexes can produce a mature TCR repertoire. Similar studies have been carried out in the MHC class II–mediated positive selection of CD4 T cells; complex mixtures of peptides drive a broad and highly variable TCR repertoire in vivo, while single peptide/MHC class II mice produced either by covalent linkage of a known MHC class II–binding peptide (18Ignatowicz L Kappler J Marrack P Cell. 1996; 84: 521-529Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar) or by deletion of the α chain of the peptide exchange factor H-2M (9Fung-Leung W.P Surh C.D Lilhedahl M Pang J Leturcq D Peterson P.A Webb S.R Karlson L Science. 1996; 271: 1278-1281Crossref PubMed Scopus (242) Google Scholar, 24Martin W.E Hicks G.G Mendiratta S.K Leva H.I Ruley E.H Van Kaer L Cell. 1996; 84: 543-550Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 27Miyazaki T Wolf P Tourne S Waltzinger C Dierich A Barois N Ploegh H Benoist C Mathis D Cell. 1996; 84: 531-541Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar) select a repertoire that is dominated by mature T cells that respond to self-MHC stimulators expressing a random set of self-peptides. These data originally were interpreted to mean that negative selection was the dominant force in shaping the mature TCR repertoire, since the majority (up to 70%) of such cells reacted to syngeneic stimulator cells with a random set of self-peptides, and such cells would normally have been deleted during their maturation in normal mice. This interpretation was tested in an elegant experiment published in Cell (34Zerhann J Held W Raulet D.H Cell. 1997; 188: 627-636Abstract Full Text Full Text PDF Scopus (274) Google Scholar), in which T cells were raised in the absence of all MHC molecules by using MHC class I– and class II–deficient mice; in such mice, no mature T cells develop. However, when fetal thymocytes were incubated with anti-TCR and anti-CD4 antibodies, the thymocytes became CD4 T cells that apparently had no constraints on their usage of TCR genes by either positive or negative selection. Such cells had a “normal” level of alloreactivity and also “autoreactivity” in that they reacted to any tested haplotype at the 1%–10% level, which has been known to be the frequency of alloreactive T cells since this number was first reported by two groups (8Ford W.L Atkins R.C Nature (New Biol.). 1971; 234: 178-180Crossref PubMed Scopus (94) Google Scholar, 32Wilson D.B Blythe J Nowell P.C J. Exp. Med. 1978; 129: 1157-1167Google Scholar). When T cells were raised in the presence of I-Ab, this had no influence on their responses to other alleles at I-A, but eliminated the response to I-Ab. This demonstrated that MHC recognition was an intrinsic property of TCR genes rather than the product of either positive or negative selection. However, the system used could not answer questions about the role of positive or negative selection in normal settings. An alternative interpretation to the results in the single-peptide mice was that the normal TCR repertoire, which is positively selected on self-peptide/self-MHC class II complexes (22Laufer T.M DeKoning J Markowitz J.S Lo D Glimcher L.H Nature. 1996; 383: 81-85Crossref PubMed Scopus (334) Google Scholar), was constrained by positive selection in the presence of a single or limited number of peptide/MHC class II proteins. That such constraints occur in the thymi of single-peptide mice was reported recently in a study in which the sequences of α chain joins were highly constrained in postselection thymic cells as well as in peripheral T cells of β chain transgenic mice deficient in the peptide exchange factor H-2M (30Sant'Angelo D.B Waterbury P.G Cohen B.E Martin W.D Van Kaer L Hayday A.C Janeway Jr., C.A Immunity. 1997; 7: 517-524Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Similar constraints were not observed in the absence of the β chain transgene or in the presence of H-2M. Nevertheless, these mice generated normal numbers of T cells with a normal distribution of T cell maturational phenotypes. This led to a very different interpretation of the results obtained with single-peptide mice; rather than selecting a broad repertoire of TCRs, the repertoire was highly constrained. The former conclusion was based on the finding of a diverse set of β chains in both the TCRs of single-peptide mice and those of normal mice. Nevertheless, even though they produced all of their TCRs with a single β chain sequence, the mice had an identical number of thymocytes and peripheral T cells and equivalent T cell responses to I-Ab–bearing stimulator cells when compared to the wild-type, H-2M–deficient mice. The process of β selection relies on the presence of an in-frame β chain join that can encode β chain protein. β chain protein can form a pre-TCR by pairing with the pre-Tα chain and CD3 (28Mombaerts P Clarke A.R Rudnicki M.A Iacomini J Itohara S Lafaille J.J Wand L Ichikawa Y Jaenisch R Hooper M.L et al.Nature. 1992; 360: 225-231Crossref PubMed Scopus (962) Google Scholar, 23Mallick C.A Dudley E.C Viney J.L Owen M.J Hayday A.C Cell. 1993; 73: 513-519Abstract Full Text PDF PubMed Scopus (183) Google Scholar, 5Dudley E.C Petrie H.T Shah M.L Owen M.J Hayday A.C Immunity. 1994; 1: 83-93Abstract Full Text PDF PubMed Scopus (257) Google Scholar, 7Fehling H.J Krukova A Saint-Ruf C von Boehmer H Science. 1995; 375: 795-798Google Scholar, 14Hoffman E.S Passoni L Crompton T Leu T.M.J Schatz D.G Koff A Owen M.J Hayday A.C Genes Dev. 1996; 10: 948-962Crossref PubMed Scopus (277) Google Scholar). T cells normally rearrange their β chains before proceeding with α chain gene rearrangement. The process called β selection, which should be distinguished from positive intrathymic selection since it does not require specific self-peptide/self-MHC complexes, requires only that the β chain be rearranged in the correct reading frame in order to generate β chain protein. This apparently explains earlier results in which a transgenic β chain that lacked a V region could promote the development of double-positive thymocytes (19Jacobs H Vanderputte D Tolkamp L de Vries E Borst J Burns A Eur. J. Immunol. 1994; 24: 934-939Crossref PubMed Scopus (143) Google Scholar). The pre-TCR is functional since it allows double-negative thymocytes that have productively rearranged their TCR β chains to respond to unknown stimuli that lead to expansion of the pool of thymocytes by driving several rounds of division. During this proliferative phase of thymocyte maturation, RAG-2 protein is degraded, and Rag1 and Rag2 mRNAs are repressed, preventing further β chain gene rearrangement, since the rearrangements are dependent on the expression of both RAG proteins. Also during this process, the CD4 and CD8 coreceptor genes are turned on. Once the cells cease dividing and revert to a small double-positive state, the α chains begin to rearrange. Then positive selection occurs on those few T cells whose αβ TCRs are specific for the correct combination of self-peptide bound to self-MHC class II molecules. When a transgenic TCR β chain is inserted into the germline of a mouse, all of the T cells can undergo β selection because they are already provided with a transgene-encoded rearranged β chain. Thus, by fixing the β chain by means of transgenesis, analysis of TCR α chain sequence yields information about the specificity of the TCR repertoire in mature, single-positive thymocytes as well as peripheral, mature CD4 T cells. During the characterization of T cell hybrids derived from single-chain TCR transgenic mice prepared from the rearranged β chain of a clone that was specific for the 81–104 peptide of cytochrome c, immunization with this peptide produced a collection of α chains that were identical at the amino acid level to the α chain in the parental clone but differed in nucleotide sequence (21Jorgensen J.L Esser U Fazekas de St. Groth B Reay P Davis M.M Nature. 1992; 355: 224-230Crossref PubMed Scopus (491) Google Scholar). Similar results were obtained when TCR β chain transgenic mice that were derived from the D10 TCR were immunized with peptide 134–146 of conalbumin to which D10 responds (29Sant'Angelo D.B Waterbury P.G Preston-Hurlburt P Yoon S.T Medzhitov R.M Hong S-c Janeway Jr., C.A Immunity. 1996; 4: 367-376Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). When peptides mutant toward the N-terminus were used for immunization of these mice, then the sequence of the α chain obtained from peptide-specific T cell hybrids differed from that of the parental clone at the amino acid level, but all of the hybrids produced the same amino acid sequence, although it was encoded in several different nucleotide sequences (21Jorgensen J.L Esser U Fazekas de St. Groth B Reay P Davis M.M Nature. 1992; 355: 224-230Crossref PubMed Scopus (491) Google Scholar, 29Sant'Angelo D.B Waterbury P.G Preston-Hurlburt P Yoon S.T Medzhitov R.M Hong S-c Janeway Jr., C.A Immunity. 1996; 4: 367-376Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). However, when α chain transgenic mice were immunized with peptides that had single amino acid changes near the C-terminus of the peptide, it was found that the accompanying β chains used different Vβ gene segments (21Jorgensen J.L Esser U Fazekas de St. Groth B Reay P Davis M.M Nature. 1992; 355: 224-230Crossref PubMed Scopus (491) Google Scholar) or used the the same Vβ gene segment with various V(D)J junctions (29Sant'Angelo D.B Waterbury P.G Preston-Hurlburt P Yoon S.T Medzhitov R.M Hong S-c Janeway Jr., C.A Immunity. 1996; 4: 367-376Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). These results are consistent with crystal structures of TCR/MHC class I/peptide complexes, which show that the C-terminal half of the peptide is contacted mainly by the CDR3 of the TCR β chain, with minimal contacts with CDR1 and none with CDR2 (10Garboczi D.N Ghosh P Utz U Fan Q.R Biddison W.E Wiley D.C Nature. 1996; 384: 134-141Crossref PubMed Scopus (1203) Google Scholar), although no such structures exist for TCR/MHC class II/peptide complexes. These results are direct predictions of the β selection hypothesis in the two single-chain transgenic mice, since a transgenic β chain can drive β selection as efficiently as in a wild-type mouse. In fact, there are indications that the transgenic β chain may be more efficient than normal β chains, because all of the expressed transgene-encoded rearranged β chains are in-frame, whereas a fraction of cells with naturally rearranged β chains will have β chains that are not in the correct reading frame, and such cells will die as a result of their failure to produce a pre-TCR. By contrast, in an α chain transgenic mouse, the process of β selection will not be efficient, since the α chain needs not to rearrange but only to pair with the in-frame β chain and recognize a self-peptide/self-MHC ligand. Another example of apparently synonymous receptors comes from immunization of normal mice with moth cytochrome c, in which TCRs characterized by the use of the same Vβ3 and Vα11 used in all such responses were obtained in measurable quantities; however, there was some variability in the amino acid residues in the respective CDR3s. Although this was used as a way to track T cells after priming with moth cytochrome c peptide, it was yet another example of synonymous TCRs recognizing the same peptide using similar but not identical CDR3s (26McHeyzer-Williams M.G Davis M.M Science. 1995; 268: 106-110Crossref PubMed Scopus (403) Google Scholar). As there are examples of TCRs that recognize the same peptide while they differ in sequence and V gene segment usage, so there are examples of single TCRs that can recognize a variety of different peptides. This work on peptide recognition began as an exploration of the flexibility of the TCR. This excercise produced several interesting findings. The first is that most peptides could be modified at certain positions and retain T cell receptor recognition, while modifications at certain other positions prevented recognition altogether. In general, peptides that could still bind to the presenting MHC molecule retained the ability to stimulate a particular T cell clone, while modification of TCR contacting residues, especially the central amino acid at positions 4 or 5 of the peptide, were very sensitive to changes in sequence, and most residues substituted at this position were inactive in stimulating the cognate T cell clone. The second finding of interest is the frequent observation of alterations in peptide sequence that could not activate a cloned T cell line fully but that could induce a partial activation program. This made it obvious that such altered ligands were recognized by the TCR, but only a partial signal was delivered. Such peptides are generally referred to as partial agonists, partial antagonists, or altered peptide ligands. The third finding of interest is that some of these altered peptide ligands were actually fully antagonistic of T cell activation by the wild-type peptide ligand presented on competent antigen-presenting cells. Such peptides are referred to as “antagonist” peptides, to distinguish them from so-called “agonist” peptides, which induce full activation of the cognate T cells. After several laboratories had investigated single amino acid changes in peptides, it was then asked how many residues were really essential to bind a given TCR (6Evavold B.D Sloan-Lancaster J Wilson K.J Rothbard J.B Allen P.M Immunity. 1995; 2: 655-663Abstract Full Text PDF PubMed Scopus (202) Google Scholar)? It was found that peptides with as few as one conserved amino acid residue could be recognized by a given TCR, when this residue was positioned properly in the peptide, and the peptide was constructed so that it bound to the same MHC molecule. The only residue that literally could not be changed was the central amino acid residue of the peptide, which makes complex interactions with the CDR3 regions of both chains of the TCR (11Garcia K.C Degano M Stanfield R.L Brunmark A Jackson M.R Peterson P.A Teyton L Wilson I.A Science. 1996; 274: 209-219Crossref PubMed Scopus (1060) Google Scholar, 12Garcia K.C Degano M Pease L.R Huang M Peterson P.A Teyton L Wilson I.A Science. 1998; 279: 1166-1172Crossref PubMed Scopus (592) Google Scholar, 29Sant'Angelo D.B Waterbury P.G Preston-Hurlburt P Yoon S.T Medzhitov R.M Hong S-c Janeway Jr., C.A Immunity. 1996; 4: 367-376Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Thus, there is remarkable flexibility in peptide structure that can lead to T cell recognition. Another study involved cloned T cell lines from patients with multiple sclerosis (33Wucherpfennig K.W Strominger J.L Cell. 1995; 80: 695-705Abstract Full Text PDF PubMed Scopus (1283) Google Scholar). Using these T cell lines, the authors searched a database of viral proteins from unrelated infectious agents for peptides that were candidates to stimulate these clones. From this search they found seven different peptides that could stimulate the T cell clones derived from various patients. The sequences of these peptides bore only a passing resemblance to the peptide to which the T cell clones were stimulated in vitro during their generation. Their interpretation was that the infectious organism had activated an immune response that by chance cross-reacted with a self-antigen. This speculation, which has been around for many years, is referred to as “molecular mimicry.” Whether molecular mimicry plays a pathogenetic role in any autoimmune disease is an open question at the present time. I return last to the case that focused our attention in the first place on the mystery of how two TCRs that are so different can bind the same peptide/MHC class I ligand in a similar orientation (10Garboczi D.N Ghosh P Utz U Fan Q.R Biddison W.E Wiley D.C Nature. 1996; 384: 134-141Crossref PubMed Scopus (1203) Google Scholar, 4Ding Y-H Smith K.J Garboczi D.N Utz U Biddison W.E Wiley D.C Immunity. 1998; 8 (this issue): 403-411Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). In my opinion, positive intrathymic selection on the same peptide can, in normal mice, lead to what appears to be a highly diverse repertoire of TCRs, due to the initial rearrangement of the TCR β chain. This leads to T cells with similar recognition properties but diverse TCRs. This is seen also in single-peptide mice in which a diversity of TCRs recognize similar peptides. Presumably, the sequences of these TCRs will be highly distinct from one another, although how distinct they are is unclear at the present time. Suffice it to say that if two such different TCRs can recognize the same exact peptide/MHC complex, then attempts to modify T cell responses using strategies targeted at a particular TCR are unlikely to succeed. However, strategies that use antigen to manipulate the immune response look more favorable than they did initially, since the range of peptides that can be recognized appears to be limited, and within these limitations, perhaps the most important is the repertoire of self-peptides expressed within the thymus. These examples of “synonymous” TCRs dramatically affirm that it is the repertoire of self-peptides that selects the TCR that determine TCR specificity for a given antigenic peptide. This property, which has recently been described as degeneracy or cross-reactivity in the TCR repertoire (25Mason D Immunol. Today, in press. 1998; Google Scholar), is a characteristic feature of TCRs. Immune responses, then, are derived from TCRs that are selected on a limited set of self-peptides but have different cross-reactivities with different foreign peptide/self-MHC complexes. The recent finding that mice expressing a limited set of self-peptides due to deletion of the H-2Ma gene cannot positively select several different TCR transgenes (13Grubin C.E Kovats S DeRoos P Rudensky A.Y Immunity. 1997; 7: 197-208Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 31Surh C.D Lee D.-S Fung-Leung W.-p Karlsson L Sprent J Immunity. 1997; 7: 209-219Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) confirms that it is self-peptide bound to self-MHC molecules that is required to generate a normal TCR repertoire. In the case of the TCRs described by Wiley and colleagues (10Garboczi D.N Ghosh P Utz U Fan Q.R Biddison W.E Wiley D.C Nature. 1996; 384: 134-141Crossref PubMed Scopus (1203) Google Scholar, 4Ding Y-H Smith K.J Garboczi D.N Utz U Biddison W.E Wiley D.C Immunity. 1998; 8 (this issue): 403-411Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar), this is carried to an extreme, in which only 1 of 17 TCR contacts is conserved, and the conserved amino acid makes a relatively unimportant contact at that. It will be interesting to examine other cases of TCRs that are unrelated but nevertheless recognize the same peptide/MHC ligand, as well as the 2C TCR that recognizes several highly distinct ligands with the same TCR. The molecular views of this interaction continue to fascinate and surprise us, and the surprises are likely to continue until we have assembled enough such structures to see general rules emerging (if they exist at all!).