Peptide recognition by two HLA-A2/Tax11-19-specific T cell clones in relationship to their MHC/peptide/TCR crystal structures.

The crystal structures of two human TCRs specific for a HTLV-I Tax peptide bound to HLA-A2 were recently determined, for the first time allowing a functional comparison of TCRs for which the MHC/peptide/TCR structures are known. Extensive amino acid substitutions show that the native Tax residues are optimal at each peptide position. A prominent feature of the TCR contact surface is a deep pocket that accommodates a tyrosine at position 5 of the peptide. For one of these TCRs, this pocket is highly specific for aromatic residues. In the other TCR structure, this pocket is larger, allowing many different residues to be accommodated. The CTL clones also show major differences in the specificity for several other peptide residues, including side chains that are not directly contacted by the TCR. Despite the specificity of these clones, peptides that are distinct at five or six positions from Tax11-19 induce CTL activity, indicating that substantial changes of the peptide surface are tolerated. Human peptides with limited sequence homology to Tax11-19 represent partial TCR agonists for these CTL clones. The distinct functional properties of these CTL clones highlight structural features that determine TCR specificity and cross-reactivity for MHC-bound peptides.

Two human T cell receptors bind in a similar diagonal mode to the HLA-A2/Tax peptide complex using different TCR amino acids.

The three-dimensional structure of a human alphabeta T cell receptor (TCR), B7, bound to the HLA-A2 molecule/HTLV-1 Tax peptide complex was determined by x-ray crystallography. Although different from the A6 TCR, previously studied, in 16 of the 17 residues that contact HLA-A2/Tax, the B7 TCR binds in a similar diagonal manner, only slightly tipped and rotated, relative to the A6 TCR. The structure explains data from functional assays on the specificity differences between the B7 and A6 TCRs for agonist, partial agonist, and null peptides. The existence of a structurally similar diagonal binding mode for TCRs favors mechanisms based on the formation of geometrically defined supramolecular assemblies for initiating signaling.

Analysis of the expression of peptide-major histocompatibility complexes using high affinity soluble divalent T cell receptors.

Understanding the regulation of cell surface expression of specific peptide-major histocompatibility complex (MHC) complexes is hindered by the lack of direct quantitative analyses of specific peptide-MHC complexes. We have developed a direct quantitative biochemical approach by engineering soluble divalent T cell receptor analogues (TCR-Ig) that have high affinity for their cognate peptide-MHC ligands. The generality of this approach was demonstrated by specific staining of peptide-pulsed cells with two different TCR-Ig complexes: one specific for the murine alloantigen 2C, and one specific for a viral peptide from human T lymphocyte virus-1 presented by human histocompatibility leukocyte antigens-A2. Further, using 2C TCR- Ig, a more detailed analysis of the interaction with cognate peptide-MHC complexes revealed several interesting findings. Soluble divalent 2C TCR-Ig detected significant changes in the level of specific antigenic-peptide MHC cell surface expression in cells treated with gamma-interferon (gamma-IFN). Interestingly, the effects of gamma-IFN on expression of specific peptide-MHC complexes recognized by 2C TCR-Ig were distinct from its effects on total H-2 Ld expression; thus, lower doses of gamma-IFN were required to increase expression of cell surface class I MHC complexes than were required for upregulation of expression of specific peptide-MHC complexes. Analysis of the binding of 2C TCR-Ig for specific peptide-MHC ligands unexpectedly revealed that the affinity of the 2C TCR-Ig for the naturally occurring alloreactive, putatively, negatively selecting, complex, dEV-8-H-2 Kbm3, is very low, weaker than 71 microM. The affinity of the 2C TCR for the other naturally occurring, negatively selecting, alloreactive complex, p2Ca-H-2 Ld, is approximately 1000-fold higher. Thus, negatively selecting peptide-MHC complexes do not necessarily have intrinsically high affinity for cognate TCR. These results, uniquely revealed by this analysis, indicate the importance of using high affinity biologically relevant cognates, such as soluble divalent TCR, in furthering our understanding of immune responses.

Longitudinal study of myelin basic protein-specific T-cell receptors during the course of multiple sclerosis.

This study analyzed the stability of the myelin basic protein (MBP)-specific T-cell receptor (TCR) repertoire during the course of multiple sclerosis (MS) in three patients who were monitored for three years by gadolinium-enhanced magnetic resonance imaging. Bulk-culture T-cell lines (TCLs) were generated from 3-4 time points for each patient, including times of active and quiescent disease. TCR analysis of these TCLs indicated that both the V alpha and V beta usage was similar over time for each patient. Sequencing of TCRs demonstrated conserved complementarity-determining region 3 (CDR3) sequences within TCLs that expressed the same V alpha segment over time, although the J alpha usage was different for each TCR. This indicates that the population of MBP-reactive T-cells is changing during the course of MS, but that host and/or environmental factors may be selecting T-cells with particular MHC/peptide binding domains.

Human T cell leukemia virus type I (HTLV-I)-specific CD8+ CTL clones from patients with HTLV-I-associated neurologic disease secrete proinflammatory cytokines, chemokines, and matrix metalloproteinase.

Human T cell leukemia virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive neurologic disease characterized by marked degeneration of the spinal cord and the presence of infiltrating CD8+ T cells and macrophages. HAM/TSP patients have very high frequencies of HTLV-I-specific CD8+ CTL in peripheral blood and in cerebrospinal fluid. In this study, we show that HAM/TSP patients also have elevated levels of peripheral blood CD8+ T cells that produce intracellular IFN-gamma. To address the potential role of soluble mediators secreted by CD8+ T cells in the pathogenesis of HAM/TSP, we have analyzed the capacity of a panel of nine HTLV-I-specific CD8+ CTL clones derived from three HAM/TSP patients to secrete cytokines, chemokines, and matrix metalloproteinases. The results demonstrate that the majority of these CTL clones secrete IFN-gamma, TNF-alpha, macrophage-inflammatory protein-1alpha and -1beta, IL-16, and matrix metalloproteinase-9. These findings indicate that HTLV-I-specific CD8+ CTL are an important source of proinflammatory soluble mediators that may contribute significantly to the pathogenesis of HAM/TSP.

Assembly, specific binding, and crystallization of a human TCR-alphabeta with an antigenic Tax peptide from human T lymphotropic virus type 1 and the class I MHC molecule HLA-A2.

T lymphocytes use TCR-alphabeta to bind and to recognize complexes of antigenic peptides bound to MHC proteins located at the surface of APCs. We have assembled and crystallized this intercellular complex of TCR/peptide/MHC from soluble human TCR-alphabeta and soluble peptide/HLA-A2 complexes. The soluble TCR-alphabeta binds specifically to its in vivo ligand, the complex of HLA-A2, and a peptide from the Tax protein of human T lymphotropic virus type 1. The soluble TCR also binds in vitro to an altered peptide ligand, which appears to be a partial agonist in T cell assays as determined by its ability to elicit different cytolytic and lymphokine secretion responses. Heterodimerization and the antigenic specificity of the TCR do not require its interchain disulfide bond, transmembrane segments, or glycosylations. Crystals of the TCR/peptide/HLA-A2 complex diffract x-rays, providing the means to study in atomic detail the mechanism of Ag-specific cell-cell recognition between T cells and target cells.

Structure of the complex between human T-cell receptor, viral peptide and HLA-A2.

Recognition by a T-cell antigen receptor (TCR) of peptide complexed with a major histocompatibility complex (MHC) molecule occurs through variable loops in the TCR structure which bury almost all the available peptide and a much larger area of the MHC molecule. The TCR fits diagonally across the MHC peptide-binding site in a surface feature common to all class I and class II MHC molecules, providing evidence that the nature of binding is general. A broadly applicable binding mode has implications for the mechanism of repertoire selection and the magnitude of alloreactions.

Differential activation of human autoreactive T cell clones by altered peptide ligands derived from myelin basic protein peptide (87-99).

We have examined the functional consequences induced by interaction of DR2a-restricted myelin basic protein (MBP) (87-99)-specific T cell clones (TCC) with altered peptide ligands (APL) derived from MBP peptide (87-99). The immunodominant MBP peptide (87-99) has been implicated as a candidate antigen in multiple sclerosis (MS) by several lines of evidence. In the present study, we have defined the T cell receptor (TCR) contact residues for DR2a-restricted, (87-99)-specific T helper type 1 T cells to design APL suitable to modify the functions of such T cells potentially relevant for the pathogenesis of MS. We show that neutral (L-alanine substitutions) or conservative exchanges of the primary and secondary TCR contact residues lead to various alterations of T cell function, ranging from differences in interleukin-2 receptor up-regulation to anergy induction and TCR antagonism. The potential usefulness of APL as an immunomodulating therapy for DR2+ MS patients is discussed.

Analysis of the T-cell receptor repertoire of human T-cell leukemia virus type 1 (HTLV-1) Tax-specific CD8+ cytotoxic T lymphocytes from patients with HTLV-1-associated disease: evidence for oligoclonal expansion.

Human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive neurological disease characterized by marked degeneration of the spinal cord and the presence of antibodies against HTLV-1. Patients with HAM/TSP, but not asymptomatic carriers, show very high precursor frequencies of HTLV-1-specific CD8+ T cells in peripheral blood and cerebrospinal fluid, suggestive of a role of these T cells in the pathogenesis of the disease. In HLA-A2+ HAM/TSP patients, HTLV-1-specific T cells were demonstrated to be directed predominantly against one HTLV-1 epitope, namely, Tax11-19. In the present study, we analyzed HLA-A2-restricted HTLV-1 Tax11-19-specific cytotoxic T cells from three patients with HAM/TSP. An analysis of the T-cell receptor (TCR) repertoire of these cells revealed an absence of restricted variable (V) region usage. Different combinations of TCR V alpha and V beta genes were utilized between, but also within, the individual patients for the recognition of Tax11-19. Sequence analysis of the TCR showed evidence for an oligoclonal expansion of few founder T cells in each patient. Apparent structural motifs were identified for the CDR3 regions of the TCR beta chains. One T-cell clone could be detected within the same patient over a period of 3 years. We suggest that these in vivo clonally expanded T cells might play a role in the pathogenesis of HAM/TSP and provide information on HTLV-1-specific TCR which may elucidate the nature of the T cells that infiltrate the central nervous system in HAM/TSP patients.

Treatment of experimental encephalomyelitis with a peptide analogue of myelin basic protein.

Following induction of experimental encephalomyelitis with a T-cell clone, L10C1, that is specific for the myelin basic protein epitope p87-99, the inflammatory infiltrate in the central nervous system contains a diverse collection of T cells with heterogeneous receptors. We show here that when clone L10C1 is tolerized in vivo with an analogue of p87-99, established paralysis is reversed, inflammatory infiltrates regress, and the heterogeneous T-cell infiltrate disappears from the brain, with only the T-cell clones that incited disease remaining in the original lesions. We found that antibody raised against interleukin-4 reversed the tolerance induced by the altered peptide ligand. Treatment with this altered peptide ligand selectively silences pathogenic T cells and actively signals for the efflux of other T cells recruited to the site of disease as a result of the production of interleukin-4 and the reduction of tumour-necrosis factor-alpha in the lesion.

Limited T cell receptor usage by HTLV-I tax-specific, HLA class I restricted cytotoxic T lymphocytes from patients with HTLV-I associated neurological disease.

T cell receptor (TCR) V alpha and V beta chain usage of HTLV-I tax-specific, HLA class I restricted CD8+ cytotoxic T cells (CTL) was determined from lymphocytes obtained from peripheral blood of patients with HTLV-I associated neurological disease. To characterize TCR repertoire, CD8+ lymphocytes from peripheral blood were cloned in limiting dilution, and the resulting wells were screened for HTLV-I-specific precursor CTL activity. RNA was isolated from HLA-A2 restricted HTLV-I tax peptide-specific (tax 11-19; LLFGYPVYV) CD8+ CTL lines and cDNA was analyzed by PCR amplification using V alpha and V beta chain family-specific oligonucleotide primers. The results indicate that CD8+ cytotoxic T cell lines from HLA-A2 HAM/TSP patients express a limited repertoire of T cell receptor chains which may correlate with duration and severity of disease. The restricted use of TCR genes expressed by antigen-specific CTL may play a critical role in the pathogenesis of HAM/TSP and may be of value in developing immunotherapeutic strategies that focus on eliminating these cells or inhibiting their activity.

The role of T cells in multiple sclerosis: implications for therapies targeting the T cell receptor.

The cause of multiple sclerosis (MS) is unknown, but an immunopathological process with both endogenous and exogenous factors contributing to disease seems likely. Considerable recent attention, triggered predominantly by findings in the animal model, experimental allergic encephalomyelitis (EAE), which resembles MS, has focused on the role of T cells in MS. Findings in the animal model have raised the possibility that demyelination could be produced by CD4+ T cells specific for myelin proteins and expressing a limited set of T cell receptor (TCR) molecules. Thus, specific therapies targeting T cells or more specifically the TCR could represent an effective treatment of MS as has been demonstrated in EAE. However, current studies of patients with MS indicate that the immunological mechanisms in MS are considerably more complicated than in EAE. The evidence for a pivotal role for T cells in MS and the characteristics of these T cells particularly with respect to TCR usage and potential for therapies directed at the TCR will be examined in this review.

Heterogeneity of T-cell receptor alpha-chain complementarity-determining region 3 in myelin basic protein-specific T cells increases with severity of multiple sclerosis.

The pathogenesis of multiple sclerosis (MS) is thought to involve a T-cell-mediated autoimmune process. Experimental allergic encephalomyelitis (EAE), an animal model resembling MS, can be induced by immunization with myelin antigens such as myelin basic protein. The T-cell antigen receptor (TCR) usage in EAE is highly restricted in some strains of animals and experimental treatments targeting the TCR have been successful in EAE. Examination of the TCR beta-chain variable-region (V beta) usage of MBP-specific T-cell lines in MS patients has produced conflicting results. Our previous studies of TCR alpha-chain variable-region usage in monozygotic twins demonstrated a general skewing of the TCR repertoire in individuals with MS. This skewing became apparent only after stimulation with antigens; in peripheral blood lymphocyte preparations from individuals with MS V alpha 8-bearing T cells were preferentially selected by stimulation with myelin basic protein. In the present study we examined complementarity-determining region 3 of those V alpha 8-positive TCRs. Marked sequence heterogeneity was found in all individuals with severe MS. In contrast, restricted areas of complementarity-determining region 3 were found in healthy control individuals and in individuals with a mild form of MS. Sequences from tetanus toxoid-specific V alpha 8-positive T cells generated from the same individuals were relatively homogeneous within individuals regardless of disease activity and were distinct from the sequences of complementarity-determining region 3 in myelin basic protein-stimulated lines. These findings suggest that disease severity may be associated with increased heterogeneity of myelin antigen-specific T cells and could reflect an impaired ability of the immune system to down-regulate these anti-self responses.

Skewed T-cell receptor repertoire in genetically identical twins correlates with multiple sclerosis.

Although the cause of multiple sclerosis (MS) is unknown, it is thought to involve a T cell-mediated autoimmune mechanism. Susceptibility to the disease is influenced by genetic factors such as genes of the HLA and T-cell receptor (TCR) complex. Other evidence for a genetic influence includes the low incidence in certain ethnic groups, the increased risk if there are affected family members and the increased concordance rate for disease in monozygotic twin pairs (26%), compared to dizygotic twins. Epidemiological studies indicate that there may be an additional role for environmental factors. Although the target antigen(s) are not yet identified, several myelin or myelin-associated proteins have been suspected, among them myelin basic protein. A lack of genetically comparable controls has impaired the analysis of the T-cell response in MS patients and caused disagreement on TCR usage in the disease. Here we analyse the role of TCR genes in MS by comparing TCR usage in discordant versus concordant monozygotic twins in response to self and foreign antigens. We find that after stimulation with myelin basic protein or tetanus toxoid, control twin sets as well as concordant twin sets select similar V alpha chains. Only the discordant twin sets select different TCRs after stimulation with antigens. Thus exogenous factors or the disease shape the TCR repertoire in MS patients, as seen by comparison with unaffected genetically identical individuals. This skewing of the TCR repertoire could contribute to the pathogenesis of MS and other T-cell-mediated diseases.

Presentation of endogenous peptides to MHC class I-restricted cytotoxic T lymphocytes in transport deletion mutant T2 cells.

The ability of minigene-encoded viral peptide epitopes to be presented by class I molecules in the absence of MHC-encoded transporters has been evaluated in mutant T2 cells. These cells have a large deletion in the class II MHC region that includes the known transporter protein for antigenic peptides and proteasome genes and they are defective in presenting viral epitopes to CTL. T2 cells that express minigenes encoding the influenza virus matrix peptide 58-66 (GILGFVFTL) and two HTLV 1 Tax peptides 11-19 (LLFGYPVYV) and 12-19 were lysed by HLA-A2-restricted peptide-specific CTL. Minigene expression of a HLA-A2-restricted HIV reverse transcriptase peptide 476-484 (ILKEPVHGV) with three charged residues sensitized T2 cells poorly for lysis by HIV-specific CTL unless the peptide was preceded by an endoplasmic reticulum translocation signal sequence. Expression of an influenza virus nucleoprotein peptide 383-391 (SRYWAIRTR) with three charged arginine residues did sensitize HLA-B27+ T2 cells for lysis by peptide-specific CTL. These and other results with endogenously expressed peptide analogs in which hydrophobic and charged amino acids were interchanged demonstrate that antigenic peptides can be translocated from the cytoplasm into the class I Ag presentation pathway independent of MHC-encoded transporters; and that peptide hydrophobicity appears not to be a major determinant in selecting peptides for this alternate pathway.

Sequence motifs important for peptide binding to the human MHC class I molecule, HLA-A2.

Previous studies have indicated that most HLA-A2-binding peptides are 9 amino acid (aa) residues long, with a Leu at position 2 (P2), and a Val or Leu at P9. We compared the binding properties of different peptides by measuring the rate of dissociation of beta 2-microglobulin from peptide-specific HLA-A2 complexes. The simplest peptide that we identified that could form HLA-A2 complexes had the sequence (in single letter aa code) GLFGGGGGV, indicating that three nonglycine aa are sufficient for binding to HLA-A2. To determine whether most nonapeptides that contained Leu at P2 and Val or Leu at P9 could bind to HLA-A2, we tested the binding of nonapeptides selected from published HIV and melanoma protein sequences, and found that six of seven tested formed stable HLA-A2 complexes. We identified an optimal antigenic undecapeptide from the cytomegalovirus gB protein that could form stable HLA-A2 complexes that contained apparent anchor residues at P2 and P11 (sequence FIAGN-SAYEYV), indicating that the spacing between anchor residues can be somewhat variable. Finally, we tested the importance of every aa in the influenza A matrix peptide 58-66 (sequence GILGFVFTL) for binding to HLA-A2, by using Ala-substituted and Lys-substituted peptides. We found that multiple positions were important for stable binding, including P2, P3, P5-P7, and P9. We conclude that the P2 and P9 anchor residues are of prime importance for peptide binding to HLA-A2. However, other peptide side chains (especially at P3) contribute to the stability of the interaction. In certain cases, the optimal length for peptide binding can be longer than 9 residues.

Presentation of three different viral peptides is determined by common structural features of the human lymphocyte antigen-A2.1 molecule.

To assess whether similar or dissimilar molecular features of class I human lymphocyte antigen (HLA) molecules determine the presentation of structurally diverse peptides, we have examined the influence of different pockets within the HLA-A2.1 molecule on the presentation of three different viral peptides. The influenza virus M1 58-66, HTLV-I Tax peptide 12-19, and HCMV gB 619-628 are minimal peptides that induce HLA-A2.1-restricted non-cross-reactive CTL responses. The influence of distinct structural features of HLA-A2.1 on peptide presentation was analyzed using a panel of 14 HLA-A2 mutants each with single amino acid substitutions in one of six pockets that are located in the peptide binding site. Ten of the 14 mutants showed concordant effects on the presentation of all three peptides to their peptide-specific CTL lines. Four of the mutants had a negative effect on the presentation of only one or two of these viral peptides. These findings indicate that common structural features in HLA-A2 determine the binding and conformation of different peptides, and help to provide a plausible explanation for how diverse peptides bind to HLA-A2.

Presentation of three different viral peptides, HTLV-1 Tax, HCMV gB, and influenza virus M1, is determined by common structural features of the HLA-A2.1 molecule.

To determine whether similar or dissimilar molecular features of class I molecules are involved in the presentation of structurally distinct peptides, we have investigated the influence of different pockets of the HLA-A2.1 molecule on the presentation of three different viral peptides. HTLV-I Tax peptide 12-19, HCMV gB 619-628, and influenza M1 58-66 are minimal peptides that induce HLA-A2.1-restricted noncross-reactive CTL. A detailed analysis of the structural features of HLA-A2.1 that are involved in peptide presentation was undertaken using a panel of 11 HLA-A2 mutants with single amino acid substitutions within pockets present in the peptide binding site. Nine of the 11 mutants affected presentation of each of the three peptides, whereas the other two mutants had negative effects on presentation of only two of these viral peptides. These results indicate that common structural features in HLA-A2 determine the binding of different peptides, and help to provide a plausible explanation for how structurally diverse peptides bind to HLA-A2.

Diversity in fine specificity and T cell receptor usage of the human CD4+ cytotoxic T cell response specific for the immunodominant myelin basic protein peptide 87-106.

Multiple sclerosis (MS), a human demyelinating disease, is thought to be caused by an autoimmunologic process, and myelin basic protein (MBP) is considered a likely autoantigen. Studies of T cell lines (TCL) responding to different parts of the MBP molecule have indicated that amino acids 87 through 106 contain an immunodominant epitope of MBP. We have demonstrated previously that amino acids 89 through 99 represent the core of this 87-106 peptide epitope. Importantly, this epitope is not only encephalitogenic in SJL/J mice and Lewis rats but also has been shown to be recognized by human cytotoxic TCL in the context of four HLA-DR molecules that are associated with MS in different geographic areas. If the immune response to MBP peptide 87-106 was homogeneous with respect to epitope specificity and TCR usage, specific immunotherapies targeting the interaction of peptide, MHC, and TCR might be possible. In this study, the fine specificity of 29 CD4+ cytotoxic, long term, and limiting dilution TCL that had been generated against whole MBP and were derived from four MS patients and two healthy relatives was dissected using truncated and alanine-substituted peptides for the 87-106 peptide. In addition, the TCR alpha and beta chain usage of 15 CD4+ TCL was determined. Using truncated peptides, the presence of several nested immunogenic epitopes within amino acids 87 to 106 was demonstrated. TCL with identical restriction elements and similar responses to truncated peptides could be differentiated further using alanine-substituted peptides. Finally, heterogeneity of TCR usage was shown not only for those lines that differed in their peptide specificity but also for some that showed identical responses and were restricted by the same HLA-DR antigen. In conclusion, the CD4+ cytotoxic T cell response to the immunodominant MBP peptide 87-106 demonstrates a high degree of heterogeneity at the level of fine specificity and TCR usage. These findings indicate that specific immunotherapies aimed at TCR in MS will probably be more complicated than previously anticipated.