Characterization of the immune response to a secondary encephalitogenic epitope of basic protein in Lewis rats. II. Biased T cell receptor V beta expression predominates in spinal cord infiltrating T cells.

The immune response of Lewis rat lymph node T cells to guinea pig myelin basic protein (GP-BP) in experimental allergic encephalomyelitis is directed primarily against a region of basic protein encompassed by residues 72-89. T cells that respond to this epitope are restricted by the RT1.B class II molecule of the MHC and use V beta 8.2 exclusively in their TCR. A second region of GP-BP, residues 87-99, also induces experimental allergic encephalomyelitis in Lewis rats but this response is restricted primarily by RT1.D. Elsewhere we describe the biologic characteristics of T cell clones responding to the synthetic peptide, s87-99, and to a related peptide, s85-99. We present a detailed analysis of TCR V beta gene expression among these clones, derived from the lymph node and spinal cord of immunized animals, and among spinal cord derived T cell clones reactive to GP-BP 72-89. We find that spinal cord-derived clones, reactive to s85-99 and to s87-99, use V beta 6 predominantly. In contrast, T cell clones derived from lymph nodes and reactive to the same peptides express multiple V beta genes including V beta 6. This difference in heterogeneity of V beta usage at the clonal level is also seen in T cell lines derived from spinal cord and immune lymph node. DNA sequence comparison of the CDR3 regions in V beta 6+ spinal cord clones revealed a conserved amino acid motif also found in the majority of V beta 6 sequences from the spinal cord anti-s85-99 line. Although V beta 6 was expressed in some lymph node-derived clones, only one contained a CDR3 region similar to that seen in spinal cord isolates. All spinal cord-derived T cell clones reactive to GP-BP 72-89 used V beta 8.2 and most (five of six) contained the AspSer residues in CDR3 previously shown to be associated with V beta 8.2 receptors expressed by the majority of lymph node T cells responding to GP-BP 72-89. These data indicate that TCR V beta usage in peripheral T cells responding to an autoantigen does not always predict the V beta usage among T cells at the site of an autoimmune attack. Possible explantations for the relative homogeneity in TCR V beta expression seen in T cell clones derived from the spinal cord are discussed.     

A common TCR V-D-J sequence in V beta 13.1 T cells recognizing an immunodominant peptide of myelin basic protein in multiple sclerosis.

T cell responses to the immunodominant peptide (residues 83-99) of myelin basic protein are potentially associated with multiple sclerosis (MS). This study was undertaken to examine whether a common sequence motif(s) exists within the TCR complementarity-determining region (CDR)-3 of T cells recognizing the MBP83-99 peptide. Twenty MBP83-99-reactive T cell clones derived from patients with MS were analyzed for CDR3 sequences, which revealed several shared motifs. Some V beta 13.1 T cell clones derived from different patients with MS were found to contain an identical CDR3 motif, V beta 13.1-LGRAGLTY. Oligonucleotides complementary to the shared CDR3 motifs were used as specific probes to detect identical target CDR3 sequences in a large panel of T cell lines reactive to MBP83-99 and unprimed PBMC. The results revealed that, in contrast to other CDR3 motifs examined, the LGRAGLTY motif was common to T cells recognizing the MBP83-99 peptide, as evident by its expression in the majority of MBP83-99-reactive T cell lines (36/44) and PBMC specimens (15/48) obtained from randomly selected MS patients. The motif was also detected in lower expression in some PBMC specimens from healthy individuals, suggesting the presence of low precursor frequency of T cells expressing this motif in healthy individuals. This study provides new evidence indicating that the identified LGRAGLTY motif is preferentially expressed in MBP83-99-reactive T cells. The findings have important implications in monitoring and targeting MBP83-99-reactive T cells in MS.     

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.     

Prolonged dominance of clonally restricted CD4(+) T cells in macaques infected with simian immunodeficiency viruses

The repertoire of functional CD4(+) T lymphocytes in human immunodeficiency virus type 1-infected individuals remains poorly understood. To explore this issue, we have examined the clonality of CD4(+) T cells in simian immunodeficiency virus (SIV)-infected macaques by assessing T-cell receptor complementarity-determining region 3 (CDR3) profiles and sequences. A dominance of CD4(+) T cells expressing particular CDR3 sequences was identified within certain Vbeta-expressing peripheral blood lymphocyte subpopulations in the infected monkeys. Studies were then done to explore whether these dominant CD4(+) T cells represented expanded antigen-specific cell subpopulations or residual cells remaining in the course of virus-induced CD4(+) T-cell depletion. Sequence analysis revealed that these selected CDR3-bearing CD4(+) T-cell clones emerged soon after infection and dominated the CD4(+) T-cell repertoire for up to 14 months. Moreover, inoculation of chronically infected macaques with autologous SIV-infected cell lines to transiently increase plasma viral loads in the monkeys resulted in the dominance of these selected CDR3-bearing CD4(+) T cells. Both the temporal association of the detection of these clonal cell populations with infection and the dominance of these cell populations following superinfection with SIV suggest that these cells may be SIV specific. Finally, the inoculation of staphylococcal enterotoxin B superantigen into SIV-infected macaques uncovered a polyclonal background underlying the few dominant CDR3-bearing CD4(+) T cells, demonstrating that expandable polyclonal CD4(+) T-cell subpopulations persist in these animals. These results support the notions that a chronic AIDS virus infection can induce clonal expansion, in addition to depletion of CD4(+) T cells, and that some of these clones may be SIV specific.     

Breast cancer is marked by specific,Public T-cell receptor CDR3 regions shared by mice and humans

The partial success of tumor immunotherapy induced by checkpoint blockade, which is not antigen-specific, suggests that the immune system of some patients contain antigen receptors able to specifically identify tumor cells. Here we focused on T-cell receptor (TCR) repertoires associated with spontaneous breast cancer. We studied the alpha and beta chain CDR3 domains of TCR repertoires of CD4 T cells using deep sequencing of cell populations in mice and applied the results to published TCR sequence data obtained from human patients. We screened peripheral blood T cells obtained monthly from individual mice spontaneously developing breast tumors by 5 months. We then looked at identical TCR sequences in published human studies; we used TCGA data from tumors and healthy tissues of 1,256 breast cancer resections and from 4 focused studies including sequences from tumors, lymph nodes, blood and healthy tissues, and from single cell dataset of 3 breast cancer subjects. We now report that mice spontaneously developing breast cancer manifest shared, Public CDR3 regions in both their alpha and beta and that a significant number of women with early breast cancer manifest identical CDR3 sequences. These findings suggest that the development of breast cancer is associated, across species, with biomarker, exclusive TCR repertoires.     

Clonal structure of rapid-onset MDV-driven CD4+ lymphomas and responding CD8+ T cells

Lymphoid oncogenesis is a life threatening complication associated with a number of persistent viral infections (e.g. EBV and HTLV-1 in humans). With many of these infections it is difficult to study their natural history and the dynamics of tumor formation. Marek's Disease Virus (MDV) is a prevalent α-herpesvirus of poultry, inducing CD4+ TCRαβ+ T cell tumors in susceptible hosts. The high penetrance and temporal predictability of tumor induction raises issues related to the clonal structure of these lymphomas. Similarly, the clonality of responding CD8 T cells that infiltrate the tumor sites is unknown. Using TCRβ repertoire analysis tools, we demonstrated that MDV driven CD4+ T cell tumors were dominated by one to three large clones within an oligoclonal framework of smaller clones of CD4+ T cells. Individual birds had multiple tumor sites, some the result of metastasis (i.e. shared dominant clones) and others derived from distinct clones of transformed cells. The smaller oligoclonal CD4+ cells may represent an anti-tumor response, although on one occasion a low frequency clone was transformed and expanded after culture. Metastatic tumor clones were detected in the blood early during infection and dominated the circulating T cell repertoire, leading to MDV associated immune suppression. We also demonstrated that the tumor-infiltrating CD8+ T cell response was dominated by large oligoclonal expansions containing both "public" and "private" CDR3 sequences. The frequency of CD8+ T cell CDR3 sequences suggests initial stimulation during the early phases of infection. Collectively, our results indicate that MDV driven tumors are dominated by a highly restricted number of CD4+ clones. Moreover, the responding CD8+ T cell infiltrate is oligoclonal indicating recognition of a limited number of MDV antigens. These studies improve our understanding of the biology of MDV, an important poultry pathogen and a natural infection model of virus-induced tumor formation.     

Preferential recognition of TCR hypervariable regions by human anti-idiotypic T cells induced by T cell vaccination

T cell responses to myelin basic protein (MBP) are potentially involved in the pathogenesis of multiple sclerosis (MS). Immunization with irradiated MBP-reactive T cells (T cell vaccination) induces anti-idiotypic T cell responses that suppress circulating MBP-reactive T cells. This T cell-T cell interaction is thought to involve the recognition of TCR expressed on target T cells. The study was undertaken to define the idiotypic determinants responsible for triggering CD8+ cytotoxic anti-idiotypic T cell responses by T cell vaccination in patients with MS. A panel of 9-mer synthetic TCR peptides corresponding to complementarity-determining region 2 (CDR2) and CDR3 of the immunizing MBP-reactive T cell clones were used to isolate anti-idiotypic T cell lines from immunized MS patients. The resulting TCR-specific T cell lines expressed exclusively the CD8 phenotype and recognized preferentially the CDR3 peptides. CDR3-specific T cell lines were found to lyze specifically autologous immunizing MBP-reactive T cell clones. The findings suggest that CDR3-specific T cells represented anti-idiotypic T cell population induced by T cell vaccination. In contrast, the CDR2 peptides were less immunogenic and contained cryptic determinants as the CDR2-specific T cell lines did not recognize autologous immunizing T cell clones from which the peptide sequence was derived. The study has important implications in our understanding of in vivo idiotypic regulation of autoimmune T cells and the regulatory mechanism underlying T cell vaccination.     

Large and dissimilar repertoire of Melan-A/MART-1-specific CTL in metastatic lesions and blood of a melanoma patient

It is widely accepted that the repertoire of Melan-A-specific T cells naturally selected in melanoma patients is diverse and mostly nonoverlapping among different individuals. To date, however, no studies have addressed the TCR profile in different tumor sites and the peripheral blood from the same patient. We compared the TCR usage of Melan-A-specific T cells from different compartments of a single melanoma patient to evaluate possible clonotype expansion or preferential homing over a 4-mo follow-up period. Using HLA-A2 peptide tetramers, CD8(+) T cells recognizing the modified Melan-A immunodominant ELAGIGILTV peptide were isolated from four metastatic lesions resected from a single melanoma patient, and their TCR repertoire was studied. A panel of T cell clones was generated by cell cloning of tetramer-positive cells. Analysis of the TCR beta-chain V segment and the complementarity-determining region 3 (CDR3) length and sequence revealed a large diversity in the TCR repertoire, with only some of the clones showing a partial conservation in the CDR3. A similar degree of diversity was found by analyzing a number of T cell clones obtained after sorting a Melan-A-specific population derived from PBLs of the same patient after in vitro culture with the immunodominant epitope. Moreover, clonotypes found at one site were not present in another, suggesting the lack of expansion and circulation of one or more clonotypes. Taken together, these results buttress the notion that the CTLs recognizing the immunodominant Ag of Melan-A comprise a high number of different clonotypic TCR, of which only some exhibit common features in the CDR3.     

The cell surface-expressed HSC70-like molecule preferentially reacts with the rat T-cell receptor Vdelta6 family

We previously showed that the cell surface-expressed Mr 70,000 heat shock cognate (hsc70, a constitutively expressed member of the hsp70 family) protein-like molecule (#067 molecule) interacts with rat CD3+, CD4-, CD8-, T-cell receptor (TCR)alphabeta-, natural killer recetor-P1- T cells. This 70hsc-like molecule was also suggested to present cellular peptide antigens to these T cells. In the present study, we identified the genetic structure of the TCR by establishing T-cell hybridomas between these T cells and mouse BW5147 cells. Our data indicated that these T cells preferentially used TCRs with the Vdelta6 family. Analysis of the nucleotide sequence of the CDR3 junctional portion showed that there are substantial diversities, with insertion of seven to nine amino acid residues. These data provide indirect evidences for our hypothesis that an hsc70-like molecule could be presented together with cellular peptide antigens to particular T cells with TCR gammadelta chains. Since the expression of this hsc70-like #067 antigen on the cell surface is usually induced along with cell transformation by activated oncogenes, T cells with the TCR Vdelta6 family are likely to contribute to host resistance to tumor cells.     

Complementarity-determining region 1 sequence requirements drive limited V alpha usage in response to influenza hemagglutinin 307-319 peptide

We have developed a T cell activation-based system that allows for the selection of TCRs with defined peptide/MHC specificities from libraries in which complementarity-determining region (CDR) sequences have been randomized by in vitro mutagenesis. Using this system, we have explored the sequence requirements for CDR1 and CDR2 of the TCR alpha-chain in a human T cell response characterized by restricted Valpha and Vbeta usage. Libraries of T cells expressing receptors built on the framework of a TCR specific for the influenza virus peptide hemagglutinin 307-319 presented by HLA-DR4, but with random sequences inserted at CDR1alpha or CDR2alpha, were selected for response to the same peptide/MHC ligand. A wide variety of CDR2alpha sequences were found to be permissive for recognition. Indeed, >25% of T cell clones chosen at random displayed a significant response. In contrast, a similar challenge of a randomized CDR1alpha library yielded only the parental sequence, and then only after multiple rounds of selection. T cell clones cross-reactive on closely related HLA alleles (subtypes of DR4) could be isolated from randomized libraries, but not clones restricted by more distantly related alleles such as HLA-DR1. These results indicate that, in the context of this T cell response, the structural requirements for recognition at CDR1alpha are significantly more restricted than at CDR2alpha. This system for mutation and selection of TCRs in vitro may be of use in engineering T cells with defined specificities for therapeutic applications.     

Class II-restricted T-cell clones to a synthetic peptide of influenza virus hemagglutinin differ in their fine specificities and in the ability to respond to virus.

Fifteen T-cell clones were derived from BALB/c or DBA/2 mice immunized with a synthetic peptide corresponding to the C-terminal 24 residues (residues 305 to 328) of the HA1 chain of H3 subtype influenza virus hemagglutinin. All of the clones proliferated when the peptide was presented in association with I-Ed. By using shorter homologs, it was shown that the T-cell response was focused predominantly on the region at the N-terminal end of the peptide encompassed by residues 306 to 319. Individual clones recognizing this region differed in their absolute requirements for residues at the extremities of the site and also in their patterns of efficiency of recognition of shorter homologs. One particular clone defined another site of T-cell recognition within residues 314 to 328. The response of the clones to peptide analogs identified certain residues within the sites that were critical for recognition, with the substitution Gln-311----Ser having a differential effect on clones responding to the N-terminal site. Only one of the clones responded well to influenza virus itself. This clone also required relatively low concentrations of the parent peptide for optimum stimulation and was suppressed by higher concentrations. The data demonstrate striking heterogeneity in the T-cell response even to a short synthetic peptide, with different T-cell clones recognizing slightly different but overlapping areas of the molecule.     

A single cell analysis of TCR AV24AJ18+ DN T cells.

The T-cell receptor (TCR) BV gene of human TCR AV24+ double-negative (DN) T cells, a novel subset of natural killer (NK) T cells, was investigated by single-cell sorting and single-cell polymerase chain reaction (PCR) methods. Seven of eleven TCR AV24+ DN T-cell clones utilized TCR BV8, three BV9, and one BV6. Six of seven TCR AV24/BV8+ DN T-cell clones had identical TCR beta and alpha chains, indicating that they were the same clone. All three TCR AV24/BV9+ DN T-cell clones also demonstrated the same amino acids in the CDR3 region. These findings strongly suggest that the usage of TCR beta and alpha chains on TCR AV24+ DN T cells is extremely restricted, supporting the notion that these cells recognize highly limited T-cell epitopes on antigens. All TCR AV24+ clones expressed the NKR-P1A mRNA, and so were true NK T cells. IL-2 and IL-4 mRNAs were detected in all clones, suggesting that the majority of these cells were Th0-type T cells. Six clones overexpressed Fas-ligand (Fas-L) mRNA and Fas antigen was detected on all clones at the mRNA level. In conclusion, TCR AV24+ DN T cells might recognize restricted T-cell epitopes on antigens and function as Th0-type T cells, inducer cells to Th1- or Th2-type T cells (regulatory T cells), and as Fas-L-positive cytolytic T cells.     

Oligoclonality of CD8+ T cells in breast cancer patients.

Substantial evidence has suggested that T cells play an important role in antitumor immunity. T cells with cytotoxic activity against tumors have been isolated from in vitro culture of tumor-infiltrated lymphocytes of cancer patients. In addition, clonal expansions of T cells have been identified in lesions of tumors by using a PCR-based CDR3 analysis of T cell receptors (TCR). Since the CDR3 region of the T cell receptor directly interacts with the antigen-MHC complex and is thus highly polymorphic, a dominant CDR3 length in a particular TCR V beta population will indicate the clonal expansion of a specific T cell clone. Utilizing this technique, we have analyzed the T cell repertoire in lymph nodes (LNs) and peripheral blood of 20 breast cancer patients. Our results show that in most cases, peripheral blood mononuclear cells (PB-MCs) and LN express dominant CD8+ T cell clones in different V beta gene families, and the number of dominant clones is higher in PBMC than in the LN. Furthermore, in 7 out of 16 patients' lymph nodes, there is a dominant V beta 18 T cell clonal expansion in the CD8+ T cell subset. The frequency of an oligoclonal expansion of V beta 18 CD8+ T cells in non-breast cancer lymph nodes is 1 out of 9, but no obvious motif in the CDR3 region of V beta 18 TCR can be identified. The prevalence of the clonal dominance found in breast cancer is discussed in the context of a possible tumor-related antigen stimulation.     

Conserved and heterogeneous lipid antigen specificities of CD1d-restricted NKT cell receptors

CD1d-restricted NKT cells use structurally conserved TCRs and recognize both self and foreign glycolipids, but the TCR features that determine these Ag specificities remain unclear. We investigated the TCR structures and lipid Ag recognition properties of five novel Valpha24-negative and 13 canonical Valpha24-positive/Vbeta11-positive human NKT cell clones generated using alpha-galactosylceramide (alpha-GalCer)-loaded CD1d tetramers. The Valpha24-negative clones expressed Vbeta11 paired with Valpha10, Valpha2, or Valpha3. Strikingly, their Valpha-chains had highly conserved rearrangements to Jalpha18, resulting in CDR3alpha loop sequences that are nearly identical to those of canonical TCRs. Valpha24-positive and Valpha24-negative clones responded similarly to alpha-GalCer and a closely related bacterial analog, suggesting that conservation of the CDR3alpha loop is sufficient for recognition of alpha-GalCer despite CDR1alpha and CDR2alpha sequence variation. Unlike Valpha24-positive clones, the Valpha24-negative clones responded poorly to a glucose-linked glycolipid (alpha-glucosylceramide), which correlated with their lack of a conserved CDR1alpha amino acid motif, suggesting that fine specificity for alpha-linked glycosphingolipids is influenced by Valpha-encoded TCR regions. Valpha24-negative clones showed no response to isoglobotrihexosylceramide, indicating that recognition of this mammalian lipid is not required for selection of Jalpha18-positive TCRs that can recognize alpha-GalCer. One alpha-GalCer-reactive, Valpha24-positive clone differed from the others in responding specifically to mammalian phospholipids, demonstrating that semi-invariant NKT TCRs have a capacity for private Ag specificities that are likely conferred by individual TCR beta-chain rearrangements. These results highlight the variation in Ag recognition among CD1d-restricted TCRs and suggest that TCR alpha-chain elements contribute to alpha-linked glycosphingolipid specificity, whereas TCR beta-chains can confer heterogeneous additional reactivities.     

Molecular analysis of alloreactive CTL post-hemopoietic stem cell transplantation

The development of laboratory tests for the diagnosis and monitoring of graft-vs-host disease (GVHD) is hampered by a lack of knowledge of minor histocompatibility Ags triggering alloresponses. We hypothesized that the unique molecular structure of the TCR could be used as a marker for the unidentified Ags and exploited for molecular monitoring of GVHD posttransplant. To identify alloreactive T cell clones, we performed in vitro allostimulation cultures for a cohort of patients undergoing hemopoietic stem cell transplantation and determined the sequence of the CDR3 of immunodominant alloreactive clones; 10 corresponding clonotypes restricted to activated T cells were identified. As an alternative method for the identification of alloreactive clones, molecular TCR analysis was applied to biopsies of GVHD-affected tissues. Culture- and biopsy-derived clonotypes were used to design sequence-specific quantitative PCR assays to monitor the levels of putative allospecific clonotypes in posttransplant blood samples and subsequent biopsies. Because of the rational design of the methods used to identify immunodominant clonotypes, we were able to follow the behavior of potentially GVHD-specific T cells during the transplant course. Based on our results, we conclude that molecular T cell diagnostics can be a powerful tool for monitoring immune responses posttransplantation.     

Identification of T-cell epitopes on E2 protein of rubella virus, as recognized by human T-cell lines and clones.

T-cell epitopes on the E2 protein of rubella virus were studied by using 15 overlapping synthetic peptides covering the E2 protein sequence. The most frequently recognized epitopes on E2 were E2-4 (residues 54 to 74), with 5 of 10 tested T-cell lines responding to it. Two CD4+ cytotoxic T-cell cloned isolated from one T-cell line responded strongly in proliferation assays with peptide E2-4 and were cytotoxic to target cells presenting the E2-4 determinant. Truncated peptides contained within the E2-4 peptide sequence were used to define the T-cell determinants. Results indicated that amino acid residues 54 to 65 were directly involved. Human cell lines with different HLA phenotypes were tested for the capacity to present the antigenic determinants. The results suggested that recognition of peptide E2-4 by T-cell clones was associated with HLA DR7.