Transient immunological control during acute hepatitis C virus infection: ex vivo analysis of helper T-cell responses.

Hepatitis C virus (HCV) readily sets up persistence after acute infection. Cellular immune responses are thought to play a major role in control of the virus. Failure of CD4+ T-cell responses in acute disease is associated with viral persistence but the dynamics of this are poorly understood. We aimed to assess such responses using a novel set of Class II tetrameric complexes (tetramers) to study helper T-cells ex vivo in acute disease. We analysed the HCV-specific CD4+ T-cell response in a patient with acute hepatitis c infection. We were able to track the virus-specific CD4+ T-cells directly ex vivo with HLA DR4 tetramers. Proliferative responses were absent initially, recovered as viral load dropped and were lost again during relapse. Longitudinal tetramer analyses showed expanded populations of antiviral CD4+ T-cells throughout acute infection despite lack of proliferation. A pattern of transient CD4+ T-cell proliferative responses as HCV is partially controlled is observed. Failure to control virus is associated with emergence of 'dysfunctional' CD4+ T-cell populations. Failure to control HCV in acute disease may relate to the capacity to sustain efficient immune responses as virus attempts to 'bounce back' after partial control.

The structural interactions between T cell receptors and MHC-peptide complexes place physical limits on self-nonself discrimination.

The activation and expansion of T cells in an antimicrobial immune response is based on the ability of T cell receptors (TCR) to discriminate between MHC-bound peptides derived from different microbial agents as well as self-proteins. However, the specificity of T cells is constrained by the limited number of peptide side chains that are available for TCR binding. By considering the structural requirements for peptide binding to MHC molecules and TCR recognition of MHC-peptide complexes, we demonstrated that human T cell clones could recognize a number of peptides from different organisms that were remarkably distinct in their primary sequence. These peptides were particularly diverse at those sequence positions buried in pockets of the MHC binding site, whereas a higher degree of similarity was present at a limited number of peptide residues that created the interface with the TCR. These T cell clones had been isolated from multiple sclerosis patients with human myelin basic protein, demonstrating that activation of such autoreactive T cells by microbial peptides with sufficient structural similarity may contribute to the disease process. Similar findings have now been made for a variety of human and murine T cell clones, indicating that specificity and cross-reactivity are inherent properties of TCR recognition. The observations that particular TCR are highly sensitive to changes at particular peptide positions but insensitive to many other changes in peptide sequence are not contradictory, but rather the result of structural interactions in which a relatively flat TCR surface contacts a limited number of side chains from a peptide that is deeply embedded in the MHC binding site.

Detailed analysis of the effects of Glu/Lys beta69 human leukocyte antigen-DP polymorphism on peptide-binding specificity.

The polymorphism at position beta69 of the human leukocyte antigen (HLA)-DP molecule has been associated with susceptibility to several immune disorders and alloreactivity. Using molecular modeling, we have predicted a detailed structure of the HLA-DP2 molecule (carrying Glubeta69) complexed with class II associated invariant chain derived peptide (CLIP) and compared it with the form carrying Lys at beta69 (HLA-DP2K69). Major changes between the two models were observed in the shape and charge distribution of pocket 4 and of the nearby pocket 6. Consequently, we analyzed in detail the peptide-binding specificities of both HLA-DP molecules expressed as recombinant proteins. We first determined that the minimum peptide-binding core of CLIP for both HLA-DP2 and DP2K69 is represented by nine aminoacids corresponding to the sequence 91-99 of invariant chain (Ii). We then assessed the peptide-binding specificities of the two pockets and determined the role of position beta69, using competition tests with the Ii-derived peptide CLIP and its mutated forms carrying all the aminoacidic substitutions in P4 and P6. Pocket 4 of HLA-DP2 showed high affinity for positively charged, aromatic, and polar residues, whereas aliphatic residues were disfavored. Pocket 4 of the DP2K69 variant showed a reduced aminoacid selectivity with aromatic residues most preferred. Pocket 6 of HLA-DP2 showed high affinity for aromatic residues, which was increased in DP2K69 and extended to arginine. Finally, we used the experimental data to determine the best molecular-modeling approach for assessing aminoacid selectivity of the two pockets. The results with best predictive value were obtained when single aminoacids were evaluated inside each single pocket, thus, reducing the influence of the overall peptide/ major histocompatibility complex interaction. In conclusion, the HLA-DPbeta69 polymorphism plays a fundamental role in the peptide-binding selectivity of HLA-DP. Furthermore, as this polymorphism is the main change in the pocket 4 area of HLA-DP, it could represent a supertype among HLA-DP molecules significantly contributing to the selection of epitopes presented in the context of this HLA isotype.

Equivalent induction of telomerase-specific cytotoxic T lymphocytes from tumor-bearing patients and healthy individuals.

Although high frequencies of T lymphocytes specific for certain tumor-associated antigens have been detected in some cancer patients, increasing evidence suggests that these T cells may be functionally defective in vivo and fail to induce meaningful clinical responses. One strategy to overcome this limitation is to target novel antigens that are ignored during the natural antitumor immune response but are nevertheless capable of triggering effector T-cell responses against tumors after optimal presentation by antigen-presenting cells. Here, we show that the telomerase catalytic subunit (hTERT)-a nearly universal tumor antigen identified by epitope deduction rather than from patient immune responses-is immunologically ignored by patients despite progressive tumor burden. Nevertheless, HLA-A2-restricted CTLs against hTERT are equivalently induced ex vivo from patients and healthy individuals and efficiently kill human tumor cell lines and primary tumors. Thus, telomerase-specific T cells from cancer patients are spared functional inactivation because of immunological ignorance. These findings support clinical efforts to target the hTERT as a tumor antigen with broad therapeutic potential.

Insights into autoimmunity gained from structural analysis of MHC-peptide complexes.

The structural and functional properties of HLA-DQ and -DR molecules that confer susceptibility to several common autoimmune diseases, such as type 1 diabetes, rheumatoid arthritis and multiple sclerosis, have been defined. The relevant polymorphisms directly affect interaction with peptides, which provides strong support for the hypothesis that these diseases are peptide-antigen driven. Several studies indicate that structural modifications of peptides can affect MHC class II binding and/or TCR recognition and should be considered in the analysis of T cell responses in autoimmune diseases.

Conserved CDR3 regions in T-cell receptor (TCR) CD8(+) T cells that recognize the Tax11-19/HLA-A*0201 complex in a subject infected with human T-cell leukemia virus type 1: relationship of T-cell fine specificity and major histocompatibility complex/peptide/TCR crystal structure.

We investigated the T-cell receptor (TCR) repertoire of CD8(+) T cells that recognize the Tax11-19 immunodominant epitope of Tax protein expressed by human T-cell leukemia virus (HTLV-1) that is implicated in the disease HTLV-1-associated myelopathy (HAM/TSP). A panel of Tax11-19-reactive CD8(+) T-cell clones was generated by single-cell cloning of Tax11-19/HLA-A*0201 tetramer-positive peripheral blood lymphocytes from an HTLV-1-infected individual. The analyses of TCR usage revealed that the combination of diverse TCR alpha and beta chains could be used for the recognition of Tax11-19 but the major population of T-cell clones (15 of 24 clones) expressed the TCR V beta 13S1 and V alpha 17 chain. We found striking similarities in CDR3 regions of TCR alpha and beta chains between our major group of CD8(+) T-cell clones and those originating from different subjects as previously reported, including TCRs with resolved crystal structures. A 3-amino-acid sequence (PG-G) in the CDR3 region of the V beta chain was conserved among all the Tax11-19-reactive T-cell clones expressing V beta 13S1 and V alpha 17 chains. Conserved amino acids in the CDR3 region do not directly contact the Tax11-19 peptide, as corroborated by the crystal structure of B7-TCR, a TCR that is almost identical to VB13S1 clones isolated in this study. Analysis of fine peptide specificity using altered peptide ligands (APL) of Tax11-19 revealed a similar recognition pattern among this panel of T-cell clones. These data suggest that the PG-G amino acids in the CDR3 beta loop provide a structural framework necessary for the maintenance of the tertiary TCR structure.

Analysis of the relationship between viral infection and autoimmune disease.

The clinical association between viral infection and onset or exacerbation of autoimmune disorders remains poorly understood. Here, we examine the relative roles of molecular mimicry and nonspecific inflammatory stimuli in progression from infection to autoimmune disease. Murine herpes virus 1 (HSV-1 KOS) infection triggers T cell-dependent autoimmune reactions to corneal tissue. We generated an HSV-1 KOS point mutant containing a single amino acid exchange within the putative mimicry epitope as well as mice expressing a TCR transgene specific for the self-peptide mimic to allow dissection of two pathogenic mechanisms in disease induction. These experiments indicate that viral mimicry is essential for disease induction after low-level viral infection of animals containing limited numbers of autoreactive T cells, while innate immune mechanisms become sufficient to provoke disease in animals containing relatively high numbers of autoreactive T cells.

Structure of a human insulin peptide-HLA-DQ8 complex and susceptibility to type 1 diabetes.

The class II major histocompatibility complex (MHC) glycoproteins HLA-DQ8 and HLA-DQ2 in humans and I-A(g7) in nonobese diabetic (NOD) mice are the major risk factors for increased susceptibility to type 1 diabetes. Using X-ray crystallography, we have determined the three-dimensional structure of DQ8 complexed with an immunodominant peptide from insulin. The similarity of the DQ8, DQ2 and I-A(g7) peptide-binding pockets suggests that diabetes is caused by the same antigen-presentation event(s) in humans and NOD mice. Correlating type 1 diabetes epidemiology and MHC sequences with the DQ8 structure suggests that other structural features of the P9 pocket in addition to position 57 contribute to susceptibility to type 1 diabetes.

Structural basis of molecular mimicry.

Infectious agents are thought to play an important role in the development of autoimmune diseases. Sequence similarity between infectious agents and self-proteins (molecular mimicry) has been proposed as a mechanism for the induction of autoimmunity [1]. However, it has been difficult to identify microbial peptides that activate autoreactive T cells using conventional sequence alignments. This chapter reviews progress made in the identification of such microbial peptides based on the analysis of structural features that are important for TCR recognition of MHC-bound peptides [2].

Anergy induction by dimeric TCR ligands.

T cells that recognize particular self Ags are thought to be important in the pathogenesis of autoimmune diseases. In multiple sclerosis, susceptibility is associated with HLA-DR2, which can present myelin-derived peptides to CD4(+) T cells. To generate molecules that target such T cells based on the specificity of their TCR, we expressed a soluble dimeric DR2-IgG fusion protein with a bound peptide from myelin basic protein (MBP). Soluble, dimeric DR2/MBP peptide complexes activated MBP-specific T cells in the absence of signals from costimulatory or adhesion molecules. This initial signaling through the TCR rendered the T cells unresponsive (anergic) to subsequent activation by peptide-pulsed APCs. Fluorescent labeling demonstrated that anergic T cells were initially viable, but became susceptible to late apoptosis due to insufficient production of cytokines. Dimerization of the TCR with bivalent MHC class II/peptide complexes therefore allows the induction of anergy in human CD4(+) T cells with a defined MHC/peptide specificity.

Binding of conserved islet peptides by human and murine MHC class II molecules associated with susceptibility to type I diabetes.

The major histocompatibility complex (MHC) is the most important susceptibility locus for type I diabetes in humans and NOD mice. NOD mice express a single MHC class II molecule (I-Ag7) which carries a unique beta chain sequence. In humans, DQ alleles that encode DQ8 and DQ2 confer the highest risk for the disease. Soluble DQ8 and I-Ag7 were used to directly compare the binding specificity of these MHC molecules. Peptides from three islet antigens--insulin, GAD 65 and HSP 60--bound to both CQ8 and I-Ag7. These peptides included epitopes that are immunodominant in NOD mice, namely insulin (9-23), GAD (206-220) and HSP 60 (441-460). All of these peptide sequences are highly conserved between the human and murine antigens. The binding specificity of DQ8 and I-Ag7 was similar, but not identical, since two peptides eluted from splenocytes of NOD mice did not bind to DQ8. DQ8 formed long-lived complexes with the majority of these peptides, indicating that DQ8 is not a poor peptide binder. These results demonstrate functional similarities between human and murine MHC class II molecules that confer susceptibility to type I diabetes.

Visualization of myelin basic protein (MBP) T cell epitopes in multiple sclerosis lesions using a monoclonal antibody specific for the human histocompatibility leukocyte antigen (HLA)-DR2-MBP 85-99 complex.

Susceptibility to multiple sclerosis (MS) is associated with the human histocompatibility leukocyte antigen (HLA)-DR2 haplotype, suggesting that major histocompatibility complex class II-restricted presentation of central nervous system-derived antigens is important in the disease process. Antibodies specific for defined HLA-DR2-peptide complexes may therefore be valuable tools for studying antigen presentation in MS. We have used phage display technology to select HLA-DR2-peptide-specific antibodies from HLA-DR2-transgenic mice immunized with HLA-DR2 molecules complexed with an immunodominant myelin basic protein (MBP) peptide (residues 85-99). Detailed characterization of one clone (MK16) demonstrated that both DR2 and the MBP peptide were required for recognition. Furthermore, MK16 labeled intra- and extracellular HLA-DR2-MBP peptide complexes when antigen-presenting cells (APCs) were pulsed with recombinant MBP. In addition, MK16 inhibited interleukin 2 secretion by two transfectants that expressed human MBP-specific T cell receptors. Analysis of the structural requirement for MK16 binding demonstrated that the two major HLA-DR2 anchor residues of MBP 85-99 and the COOH-terminal part of the peptide, in particular residues Val-96, Pro-98, and Arg-99, were important for binding. Based on these results, the antibody was used to determine if the HLA-DR2-MBP peptide complex is presented in MS lesions. The antibody stained APCs in MS lesions, in particular microglia/macrophages but also in some cases hypertrophic astrocytes. Staining of APCs was only observed in MS cases with the HLA-DR2 haplotype but not in cases that carried other haplotypes. These results demonstrate that HLA-DR2 molecules in MS lesions present a myelin-derived self-peptide and suggest that microglia/macrophages rather than astrocytes are the predominant APCs in these lesions.

Kinetics of T-cell receptor binding by bivalent HLA-DR. Peptide complexes that activate antigen-specific human T-cells.

Monovalent major histocompatibility complex-peptide complexes dissociate within seconds from the T-cell receptor (TCR), indicating that dimerization/multimerization may be important during early stages of T-cell activation. Soluble bivalent HLA-DR2.myelin basic protein (MBP) peptide complexes were expressed by replacing the F(ab) arms of an IgG2a antibody with HLA-DR2.MBP peptide complexes. The binding of bivalent HLA-DR2.peptide complexes to recombinant TCR was examined by surface plasmon resonance. The bivalent nature greatly enhanced TCR binding and slowed dissociation from the TCR, with a t((1)/(2)) of 2.1 to 4.6 min. Soluble bivalent HLA-DR2.MBP peptide complexes activated antigen-specific T-cells in the absence of antigen presenting cells. In contrast, soluble antibodies to the TCR.CD3 complex were ineffective, indicating that they failed to induce an active TCR dimer. TCR/CD3 antibodies induced T-cell proliferation when bound by antigen presenting cells that expressed Fc receptors. In the presence of dendritic cells, bivalent HLA-DR2. MBP peptide complexes induced T-cell activation at >100-fold lower concentrations than TCR/CD3 antibodies and were also superior to peptide or antigen. These results demonstrate that bivalent HLA-DR. peptide complexes represent effective ligands for activation of the TCR. The data support a role for TCR dimerization in early TCR signaling and kinetic proofreading.

Selective activation and expansion of high-affinity CD4+ T cells in resistant mice upon infection with Leishmania major.

Using multimers of MHC class II molecules linked to a peptide derived from the Leishmania LACK antigen, we have compared the fate of parasite-specific CD4+ T cells in resistant and susceptible mice transgenic for the beta chain of a LACK-specific TCR. Activated T cells were readily detected in the draining lymph nodes of infected animals. Although the kinetics of activation and expansion were similar in both strains, T cells from susceptible and resistant mice expressed low- and high-affinity TCR, respectively. As T cells from resistant mice produced more IFN-gamma and less IL-4 than those from susceptible animals, our results suggest that differences in TCR usage between MHC-matched animals may influence the development of the antiparasite immune response.

A humanized model for multiple sclerosis using HLA-DR2 and a human T-cell receptor.

Multiple sclerosis (MS) is a complex chronic neurologic disease with a suspected autoimmune pathogenesis. Although there is evidence that the development of MS is determined by both environmental influences and genes, these factors are largely undefined, except for major histocompatibility (MHC) genes. Linkage analyses and association studies have shown that susceptibility to MS is associated with genes in the human histocompatibility leukocyte antigens (HLA) class II region, but the contribution of these genes to MS disease development less compared with their contribution to disorders such as insulin-dependent diabetes mellitus. Due to the strong linkage disequilibrium in the MHC class II region, it has not been possible to determine which gene(s) is responsible for the genetic predisposition. In transgenic mice, we have expressed three human components involved in T-cell recognition of an MS-relevant autoantigen presented by the HLA-DR2 molecule: DRA*0101/DRB1*1501 (HLA-DR2), an MHC class II candidate MS susceptibility genes found in individuals of European descent; a T-cell receptor (TCR) from an MS-patient-derived T-cell clone specific for the HLA-DR2 bound immunodominant myelin basic protein (MBP) 4102 peptide; and the human CD4 coreceptor. The amino acid sequence of the MBP 84-102 peptide is the same in both human and mouse MBP. Following administration of the MBP peptide, together with adjuvant and pertussis toxin, transgenic mice developed focal CNS inflammation and demyelination that led to clinical manifestations and disease courses resembling those seen in MS. Spontaneous disease was observed in 4% of mice. When DR2 and TCR double-transgenic mice were backcrossed twice to Rag2 (for recombination-activating gene 2)-deficient mice, the incidence of spontaneous disease increased, demonstrating that T cells specific for the HLA-DR2 bound MBP peptide are sufficient and necessary for development of disease. Our study provides evidence that HLA-DR2 can mediate both induced and spontaneous disease resembling MS by presenting an MBP self-peptide to T cells.

pH-dependent peptide binding properties of the type I diabetes-associated I-Ag7 molecule: rapid release of CLIP at an endosomal pH.

MHC class II molecules and invariant chain assemble at a neutral pH in the endoplasmic reticulum and are transported to a low pH compartment where the invariant chain is trimmed to the class II-associated invariant chain peptide (CLIP). For many major histocompatibility complex class II molecules, DM is required for rapid removal of CLIP, which allows binding of antigenic peptides. Since I-Ag7 confers susceptibility to type I diabetes in NOD mice, the biochemical requirements for peptide loading were examined using soluble I-Ag7 expressed in insect cells. I-Ag7 formed long-lived complexes with naturally processed peptides from transferrin and albumin, whereas several peptides that represent T cell epitopes of islet autoantigens were poor binders. I-Ag7-peptide complexes were not sodium dodecyl sulfate (SDS) resistant, indicating that SDS sensitivity may be an intrinsic property of I-Ag7. Complexes of I-Ag7 and CLIP formed at a neutral pH, but rapidly dissociated at pH 5. This rapid dissociation was due to a poor fit of M98 of CLIP in the P9 pocket of I-Ag7, since substitution of M98 by a negatively charged residue greatly enhanced the stability of the complex. These biochemical properties of I-Ag7 result in the rapid generation of empty molecules at an endosomal pH and have a global effect on peptide binding by I-Ag7.

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.

Binding motifs of copolymer 1 to multiple sclerosis- and rheumatoid arthritis-associated HLA-DR molecules.

Copolymer 1 (Cop 1, poly (Y, E, A, K)) is a random synthetic amino acid copolymer effective in the treatment of relapsing forms of multiple sclerosis (MS). Cop 1 binds promiscuously, with high affinity and in a peptide-specific manner to purified MS-associated HLA-DR2 (DRB1*1501) and rheumatoid arthritis-associated HLA-DR1 (DRB1*0101) or HLA-DR4 (DRB1*0401) molecules. In the present work at least 95% of added Cop 1 could be bound to recombinant "empty" HLA-DR1 and -DR4, and 80% could be bound to HLA-DR2 proteins. Amino acid composition, HPLC profiles, and sequencing patterns of Cop 1 eluted by acid extraction from HLA-DR molecules were similar to those of the unseparated Cop 1. Protruding N-terminal ends of Cop 1 bound to HLA-DR1, -DR2, or -DR4 molecules were then treated with aminopeptidase I, followed by elution, HPLC, and pool sequencing. In contrast to untreated or unbound Cop 1, this material exhibited distinct motifs at some positions with increases in levels of E at the first and second cycles, of K at the second and third cycles, and of Y (presumably at P1 of the bound peptide) at the third to fifth cycles, regardless of the HLA-DR molecule employed. No preference was seen at the following cycles that were mainly A. These first pooled HLA-DR binding epitopes provide clues to the components of Cop 1 that are biologically active in suppressing MS and possibly rheumatoid arthritis.