The repertoire of CD4+ CD28- T cells in rheumatoid arthritis.

BACKGROUND: While oligoclonality of circulating CD4- CD8 and of CD8+ T cells is not uncommon, clonal dominance within the CD4 compartment is not frequently found in healthy individuals. In contrast, the majority of patients with rheumatoid arthritis (RA) have clonally expanded CD4+ T cell populations. Previous studies have demonstrated that these clonogenic CD4+ T cells do not express the CD28 molecule. To examine the correlation between CD28 expression and clonal proliferation, we have analyzed the T cell receptor (TCR) diversity of CD4+ CD28- T cells in normal individuals and in RA patients. MATERIAL AND METHODS: The size of the peripheral blood CD4+ CD28- compartment was determined in 30 healthy individuals and 30 RA patients by two-color FACS analysis. In 10 RA patients and five controls with more than 2.5% CD4+ CD28- T cells, TCR BV gene segment usage was analyzed with 19 BV-specific antibodies. Oligoclonality was assessed in sorted CD4+ CD28+ and CD28- T cells using TCR BV-BC-specific polymerase chain reaction and size fractionation. Clonal dominance was confirmed by direct sequencing. RESULTS: The CD4+ CD28- T cell compartment was expanded to more than 2.5% in 70% of the RA patients and 30% of the normal individuals. Compared with the CD4+ CD28+ T cells, the TCR BV gene segment usage among CD4+ CD28- cells was grossly skewed with the dominance of single BV elements. Molecular TCR analysis provided evidence for oligoclonality in 17 of 21 expanded BV elements. In two unrelated RA patients who shared both HLA-DRB1 alleles, the TCR beta-chain sequences of dominant clonotypes were highly conserved. CONCLUSIONS: Oligoclonality is a characteristic feature of CD4+ CD28- T cells which are expanded in some healthy individuals and in the majority of RA patients. The lack of CD28 expression is a common denominator of CD4+, CD8+, and CD4- CD8- T cells prone to develop clonal dominance. The limited TCR diversity of clonal CD4+ CD28- populations in RA patients suggests that these T cells recognize a limited spectrum of antigens. The fact that the majority of individuals with marked expansions and oligoclonality of CD4+ CD28- T cells are RA patients suggests a role for these unusual lymphocytes in the pathogenetic events leading to RA.     

Dynamic monitoring the TCR CDR3 spectratypes in patients with metastatic CRC treated with a combination of bevacizumab, irinotecan, fluorouracil, and leucovorin

In the present study, either modified IFL regimen (modified irinotecan, fluorouracil and leucovorin, mIFL) alone or in combination with bevacizumab was used to treat patients with metastatic colorectal cancer (CRC). Treatment efficacy was assessed using coupled tomography imaging diagnosis. The toxicity accompany with treatment was evaluated, as well as T cell receptor (TCR) repertoire before and several cycles after therapy was dynamically monitored by analyzing the complementarity-determining region 3 (CDR3) length distribution within CD4⁺ and CD8⁺ T cell subsets. The degrees of normalization of the T cell repertoire in CRC patients treated with the two methods were compared. The results showed that mIFL combined with bevacizumab was more effective in treating patients with metastatic CRC, and was accompanied by an increase in side effects such as proteinuria and hematuria. An even more restricted CDR3 profile in patients with metastatic CRC compared with healthy control has been detected. A prominent usage of TCR β chain variable (BV) gene BV12 and BV16 families within the CD4⁺ T cell subset and BV19 and BV21 families within the CD8⁺ T cell subset have been found before treatment. Moreover, CD8⁺ T cells showed more restricted patterns than CD4⁺ T cells, especially in patients before treatment. For patients with stable disease (SD) or partial remission (PR) after treatment, a less restricted CDR3 profile in post-treatment compared with pre-treatment has been found, but the opposite result was observed for patients with progressive disease (PD). The less restricted CDR3 pattern suggested a trend toward normalization of the TCR repertoire. The normalization of TCR repertoire significantly increased in patients treated with mIFL in combination with bevacizumab, but slightly in patients treated with mIFL alone. The results demonstrate a positive correlation between post-therapy TCR repertoire normalization and remission of metastatic CRC.     

Tetramer-guided analysis of TCR beta-chain usage reveals a large repertoire of melan-A-specific CD8+ T cells in melanoma patients

The assessment of the TCR repertoire expressed by tumor-specific CD8+ T lymphocytes has been hampered to date by the difficulty of targeting the analysis to lymphocytes directed against a single epitope. In the present study we have used fluorescent A2/Melan-A tetramers in conjunction with anti-CD8 and anti-TCR beta-chain variable (BV) mAbs to analyze by flow cytometry the BV segment usage by Melan-A-specific CD8+ T cells in tumor-infiltrated lymph nodes (TILN) and tumor-infiltrating lymphocytes (TIL) from A2 melanoma patients. Analysis of TILN populations revealed small proportions of A2/Melan-A tetramer+ cells expressing many different BV together with over-representation of A2/Melan-A tetramer+ cells expressing certain BVs. The BV usage by A2/Melan-A tetramer+ lymphocytes in TIL was more restricted than that in TILN. Moreover, the predominant BV segments were quite distinct in populations derived from different patients. A2/Melan-A tetramer+ cells expressing the dominant BVs found in TILN could also be found in the corresponding peptide-stimulated autologous PBMC, although A2/Melan-A tetramer+ lymphocytes expressing additional BVs were also identified. Together, these results suggest that a large and diverse repertoire of Melan-A-specific T cells using different BV TCR segments is available in A2 melanoma patients.     

Limited T Cell Receptor Repertoire Diversity in Tuberculosis Patients Correlates with Clinical Severity

Background

The importance of CD4⁺ and CD8⁺ T cells in protection against tuberculosis (TB) is well known, however, the association between changes to the T cell repertoire and disease presentation has never been analyzed. Characterization of T-cells in TB patients in previous study only analyzed the TCR β chain and omitted analysis of the Vα family even though α chain also contribute to antigen recognition. Furthermore, limited information is available regarding the heterogeneity compartment and overall function of the T cells in TB patients as well as the common TCR structural features of Mtb antigen specific T cells among the vast numbers of TB patients.

Methodology/Principal Findings

CDR3 spectratypes of CD4⁺ and CD8⁺ T cells were analyzed from 86 patients with TB exhibiting differing degrees of disease severity, and CDR3 spectratype complexity scoring system was used to characterize TCR repertoire diversity. TB patients with history of other chronic disease and other bacterial or viral infections were excluded for the study to decrease the likely contribution of TCRs specific to non-TB antigens as far as possible. Each patient was age-matched with a healthy donor group to control for age variability. Results showed that healthy controls had a normally diversified TCR repertoire while TB patients represented with restricted TCR repertoire. Patients with mild disease had the highest diversity of TCR repertoire while severely infected patients had the lowest, which suggest TCR repertoire diversity inversely correlates with disease severity. In addition, TB patients showed preferred usage of certain TCR types and have a bias in the usage of variable (V) and joining (J) gene segments and N nucleotide insertions.

Conclusions/Significance

Results from this study promote a better knowledge about the public characteristics of T cells among TB patients and provides new insight into the TCR repertoire associated with clinic presentation in TB patients.     

Persistence of EBV Antigen-Specific CD8 T Cell Clonotypes during Homeostatic Immune Reconstitution in Cancer Patients

Persistent viruses are kept in check by specific lymphocytes. The clonal T cell receptor (TCR) repertoire against Epstein-Barr virus (EBV), once established following primary infection, exhibits a robust stability over time. However, the determinants contributing to this long-term persistence are still poorly characterized. Taking advantage of an in vivo clinical setting where lymphocyte homeostasis was transiently perturbed, we studied EBV antigen-specific CD8 T cells before and after non-myeloablative lympho-depleting chemotherapy of melanoma patients. Despite more advanced T cell differentiation, patients T cells showed clonal composition comparable to healthy individuals, sharing a preference for TRBV20 and TRBV29 gene segment usage and several co-dominant public TCR clonotypes. Moreover, our data revealed the presence of relatively few dominant EBV antigen-specific T cell clonotypes, which mostly persisted following transient lympho-depletion (TLD) and lymphocyte recovery, likely related to absence of EBV reactivation and de novo T cell priming in these patients. Interestingly, persisting clonotypes frequently co-expressed memory/homing-associated genes (CD27, IL7R, EOMES, CD62L/SELL and CCR5) supporting the notion that they are particularly important for long-lasting CD8 T cell responses. Nevertheless, the clonal composition of EBV-specific CD8 T cells was preserved over time with the presence of the same dominant clonotypes after non-myeloablative chemotherapy. The observed clonotype persistence demonstrates high robustness of CD8 T cell homeostasis and reconstitution.     

Conventional and Regulatory CD4+ T Cells That Share Identical TCRs Are Derived from Common Clones

Results from studies comparing the diversity and specificity of the TCR repertoires expressed by conventional (Tconv) and regulatory (Treg) CD4⁺ T cell have varied depending on the experimental system employed. We developed a new model in which T cells express a single fixed TCRα chain, randomly rearranged endogenous TCRβ chains, and a Foxp3-GFP reporter. We purified CD4⁺Foxp3^- and CD4⁺Foxp3⁺ cells, then performed biased controlled multiplex PCR and high throughput sequencing of endogenous TCRβ chains. We identified >7,000 different TCRβ sequences in the periphery of 5 individual mice. On average, ~12% of TCR sequences were expressed by both conventional and regulatory populations within individual mice. The CD4⁺ T cells that expressed shared TCR sequences were present at higher frequencies compared to T cells expressing non-shared TCRs. Furthermore, nearly all (>90%) of the TCR sequences that were shared within mice were identical at the DNA sequence level, indicating that conventional and regulatory T cells that express shared TCRs are derived from common clones. Analysis of TCR repertoire overlap in the thymus reveals that a large proportion of Tconv and Treg sharing observed in the periphery is due to clonal expansion in the thymus. Together these data show that there are a limited number of TCR sequences shared between Tconv and Tregs. Also, Tconv and Tregs sharing identical TCRs are found at relatively high frequencies and are derived from common progenitors, of which a large portion are generated in the thymus.     

Oligoclonality in the human CD8+ T cell repertoire in normal subjects and monozygotic twins: implications for studies of infectious and autoimmune diseases.

BACKGROUND: We have previously demonstrated CD8+ T cell clonal dominance using a PCR assay for the CDR3 length of T cell receptors belonging to a limited number of TCRBV segments/families. In this study, we have modified this approach in order to analyze more comprehensively the frequency of oligoclonality in the CD8+ T cell subset in 25 known TCRBV segments/families. In order to assess the relative roles of genes and environment in the shaping of a clonally restricted CD8+ T cell repertoire, we have analyzed clonal dominance in the CD8+ T cell population of monozygotic twins, related siblings, and adoptees. MATERIALS AND METHODS: Oligoclonality was assessed in the CD8+ T cell subsets using a multiplex PCR approach to assay for CDR3 length variation across 25 different TCRBV segments/families. Specific criteria for oligoclonality were established, and confirmed by direct sequence analysis of the PCR products. This assay was used to investigate the CD8+ T cell repertoire of 56 normal subjects, as well as six sets of monozygotic (MZ) twins. RESULTS: Seventy-two percent of normal subjects (n = 56) had evidence of oligoclonality in the CD8+ T cell subset, using well-defined criteria. Although MZ twins frequently displayed CD8+ T cell clonal dominance, the overall pattern of oligoclonality was very diverse within each twin pair. However, we occasionally observed dominant CD8+ T cell clones that were highly similar in sequence in both members of some twin pairs. Not a single example of such similarity was observed in normal controls or siblings. CONCLUSIONS: Oligoclonality of circulating CD8+ T cells is a characteristic feature of the human immune system; both host genetic factors and environment shape the pattern of oligoclonality in this T cell subset. The high frequency of this phenomenon in normal subjects provides a background with which to evaluate CD8+ T cell oligoclonality in the setting of infection or autoimmune disease. Further phenotypic and functional characterization of these clonally expanded T cells should provide insight into normal immune homeostasis.     

Contribution of the T cell receptor BJ gene to recognition of the P91A tumor antigen in DBA/2 mice

 To understand specific immune responses against a tumor, it is important to characterize T cells that recognize the tumor antigen. The mouse P91A antigen is one of the well-defined tumor antigens that is expressed on the P911 cell line, and T cells responding to the antigen in DBA/2 mice were reported to be restricted to BV8S2/S3 families in their T cell receptor (TCR) BV gene usage. We have further characterized the P91A-responding T cells in DBA/2 mice, focusing on TCR BJ gene usage and using the polymerase chain reaction/enzyme-linked immunosorbent assay and DNA sequencing studies of their third complementarity-determining (CDR3) regions. As a result, T cells with cytotoxic activity to the P91A antigen, induced from murine spleen cells both in vivo and in vitro, showed predominant use of the BJ2S1 gene segment in both BV8S2 and BV8S3 T cells compared to unmanipulated murine spleen cells. Sequencing studies of the CDR3 regions in the BV8S3 T cells revealed clonal expansion of T cells with the BV8S3-BJ2S1 combination in two of three DBA/2 mice tested. In the remaining mouse, clonal expansion was not detected despite predominant use of the BJ2S1 segment by these T cells. These data suggest that P91A-recognizing T cells would predominantly use the BV8S2/S3-BJ2S1 combination. Analysis of T cells with these TCR BV-BJ gene combinations may contribute to the evaluation, monitoring and development of a T-cell-mediated immunotherapeutic strategy. Received: 3 July 1997 / Accepted: 17 November 1997     

Cancer of the gastrointestinal tract results in a restricted T-cell repertoire dependent upon tumor differentiation

We investigated the influence of tumor tissue differentiation on the diversity of TCR repertoire. CDR3 spectratypes of CD4⁺ and CD8⁺ T cell subsets were analyzed from 27 patients with gastrointestinal tract tumors exhibiting varying degrees of differentiation. A CDR3 spectratype complexity scoring system was used to quantify the diversity of TCR repertoire. Each patient was matched with an age-matched healthy group to control for age variability. Results show that the complexity scores (TCR repertoire diversity) have a significant correlation with the degree of tumor differentiation, which provides useful information for understanding immune response in cancer patients.     

Skewed T-cell receptor repertoire: more than a marker of malignancy, a tool to dissect the immunopathology of inflammatory diseases

The highly diverse heterodimeric surface T cell receptor (TCR) gives the T lymphocyte its specificity for MHC-bound peptides needed to initiate antigen-recognition. In normal peripheral blood, spleen and lymph nodes, the TCR repertoire of the T lymphocytes is usually polyclonal. However, in malignancies such as leukemias, as well as in lymphoproliferative diseases of mature T cells, the TCR is a reflection of the clonality of the malignant cells and is therefore monoclonal. Several clinical conditions (mainly solid tumors and autoimmune diseases) have been described where the TCR repertoire is restricted. The ability to demonstrate clonal TCR usage provides a useful tool to dissect the immunopathology of inflammatory diseases. In this review we discuss these findings and propose to sub-divide diseases with restricted TCR repertoire into a group of conditions in which there is a known TCR ligand, as opposed to diseases in which the restricted TCR repertoire is the result of impaired T-cell development. This classification sheds light on the pathogenesis of several inflammatory diseases.     

Structural basis of biased T cell receptor recognition of an immunodominant HLA-A2 epitope of the SARS-CoV-2 spike protein

CD8⁺ T cells play an important role in vaccination and immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Although numerous SARS-CoV-2 CD8⁺ T cell epitopes have been identified, the molecular basis underpinning T cell receptor (TCR) recognition of SARS-CoV-2-specific T cells remains unknown. The T cell response directed toward SARS-CoV-2 spike protein–derived S^269–277 peptide presented by the human leukocyte antigen (HLA)-A∗02:01 allomorph (hereafter the HLA-A2^S269–277 epitope) is, to date, the most immunodominant SARS-CoV-2 epitope found in individuals bearing this allele. As HLA-A2^S269–277-specific CD8⁺ T cells utilize biased TRAV12 gene usage within the TCR α-chain, we sought to understand the molecular basis underpinning this TRAV12 dominance. We expressed four TRAV12⁺ TCRs which bound the HLA-A2^S269–277 complex with low micromolar affinity and determined the crystal structure of the HLA-A2^S269–277 binary complex, and subsequently a ternary structure of the TRAV12⁺ TCR complexed to HLA-A2^S269–277. We found that the TCR made extensive contacts along the entire length of the S^269–277 peptide, suggesting that the TRAV12⁺ TCRs would be sensitive to sequence variation within this epitope. To examine this, we investigated cross-reactivity toward analogous peptides from existing SARS-CoV-2 variants and closely related coronaviruses. We show via surface plasmon resonance and tetramer studies that the TRAV12⁺ T cell repertoire cross-reacts poorly with these analogous epitopes. Overall, we defined the structural basis underpinning biased TCR recognition of CD8⁺ T cells directed at an immunodominant epitope and provide a framework for understanding TCR cross-reactivity toward viral variants within the S^269–277 peptide.     

Double-Negative BV8S3 T Cells Expanded in MRL lpr/lpr Lymph Nodes Conserve TCRBJ Gene Usage of Single-Positive BV8S3 T Cells

Enlarged lymph nodes of mice with lpr mutation consist predominantly of CD4^?CD8^? (double-negative: DN) T cells. Among them, TCRBV8S3 (Vβ 8.3) T cells are overrepresented as compared to those in single-positive (SP) T cells. To address the question of whether the expansion of oligoclonal T cells is responsible for the increase in TCRBV8S3 cells, we examined the TCRBJ gene repertoires of BV8S3 DN and SP T cells from multiple MRL lpr/lpr mice. The BJ repertoires of BV3 (Vβ3), BV8S1 (Vβ8.1) and BV8S2 (Vβ8.2) were studied for comparison with those of BV8S3 T cells. The employed method, which was based on a PCR-ELISA technique, was newly developed and allowed us to make a precise quantitation of TCRBJ gene usage of the multiple lymphocyte samples. The results showed that there were no biases of the BJ gene usage by BV8S3 DN T cells as well as other BV T cells. Furthermore, the BJ gene usage of CD4 and CD8 BV8S3 T cells was conserved by the DN T cells. It is suggested that the BV8S3 DN T cells were not expanded by specific antigens. The expansion may result from aberrant regulation specific to the BV8S3-expressing T cells.     

Human CD8+ T Cells from TB Pleurisy Respond to Four Immunodominant Epitopes in Mtb CFP10 Restricted by HLA-B Alleles

CD8⁺ T cells are essential for host defense to Mycobacterium tuberculosis (Mtb) infection and identification of CD8⁺ T cell epitopes from Mtb is of importance for the development of effective peptide-based diagnostics and vaccines. We previously demonstrated that the secreted 10-KDa culture filtrate protein (CFP10) from Mtb is a potent CD8⁺ T cell antigen but the repertoire and dominance pattern of human CD8 epitopes for CFP10 remained poorly characterized. In the present study, we undertook to define immunodominant CD8 epitopes involved in CFP10 using a panel of CFP10-derived 13–15 amino acid (aa) peptides overlapping by 11 aa. Four peptides in CFP10 were observed to induce significant CD8⁺ T cell responses and we further determined the size of the epitopes involved in each individual peptide tested. Four 9 aa CD8 epitopes were finally identified and deleting a single amino acid from the N or C terminus of either peptide markedly reduced IFN-γ production, suggesting that they are minimum of CD8 epitopes. In the individuals tested, each epitope represented a single immunodominant response in CD8⁺ T cells. The epitope-specific CD8⁺ T cells displayed effector or effector memory phenotypes and could upregulate the expression of CD107a/b upon antigen stimulation. In addition, we found that epitope-specific CD8⁺ T cells shared biased usage of T cell receptor (TCR) variable region of β chain (Vβ) 12, 9, 7.2 or Vβ4 chains. As judged from HLA-typing results and using bioinformatics technology for prediction of MHC binding affinity, we found that the epitope-specific CD8⁺ T cells are all restricted by HLA-B alleles. Our findings suggest that the four epitopes in CFP10 recognized by CD8⁺ T cells might be of importance for the development of Mtb peptide-based vaccines and for improved diagnosis of TB in humans.     

Characterization of the inflammatory cell populations in normal colon and colonic carcinomas

Little is known about the nature of the mucosa-associated immune system within the normal colon, or about the immune response to colon carcinoma. In this study inflammatory cells (ICs) in 14 normal colons and 14 carcinomas were characterized. Overall inflammation, lymphocytes, plasma cells, neutrophils, and eosinophils were graded in routine H & E sections. Frozen sections were stained by an immunoperoxidase technique using antibodies to the T cell associated antigens CD2, CD7, CD4, CD8, and T cell receptors αβ and γδ. B cells were identified with CD20, macrophages with CD68, and Class II antigen with anti-HLA DR. Each cell type was semiquantitatively graded in 10 high power fields (HPFs) in the lumenal half (LH) or basal half (BH) of the normal mucosae, and in epithelium or stroma of the carcinomas. In normal colons, ICs were more frequent in LH than in BH. Plasma cells, lymphocytes and monocytes predominated. Subtyping of lymphocytes showed that CD4⁺ TCR αβ⁺ T lymphocytes were most numerous in the lamina propria. Lymphocytes within the epithelium were CD8⁺ T cells. Around carcinomas the overall grade of ICs was 1+ in the majority of cases. Plasma cells, CD4⁺ and CD8⁺ cells with the TCR αβ receptor, and macrophages were most frequent. Lymphoid aggregates of both T and B cells were frequent. Conclusions: 1. Normal colon contains a diffuse lumenally oriented population of TCR αβ⁺ CD4+ T cells, plasma cells, macrophages and class II antigen-expressing cells in the lamina propria. Intraepithelial lymphocytes are of the T suppressor phenotype. CD4+ T cells, macrophages and HLG-DR⁺ cells predominate in the response to colon carcinomas.     

Chronic soluble antigen sensitization primes a unique memory/effector T cell repertoire associated with th2 phenotype acquisition in vivo.

Although much progress has been made in characterization of the signaling pathways that control Th cell commitment, little is known about the early events that govern differentiation of IL-4-producing T lymphocytes in vivo. We have previously shown that chronic administration of low dose, soluble hen egg white lysozyme (HEL) induced the selective development of Ag-specific Th2 in genetically predisposed BALB/c mice. Here, we show that these memory/effector Th2 cells express a unique TCR Vbeta repertoire, different from the TCR Vbeta profile of primary effector cells from HEL-adjuvant-primed mice. This Th2-associated repertoire contains a highly frequent public clonotype characterized by preferred TCR AV and BV gene segment usage along with conserved sequences in the third hypervariable regions of both TCR chains. This Th2 clonotype, which is not recruited in primary effector T cells from HEL-adjuvant-immunized mice, recognized an IA(d)-restricted HEL determinant, preferentially processed by dendritic cells, but not by B cells. Thus, IL-4-producing CD4 T cells that expand following chronic Ag sensitization emerge from a distinct pool of precursors, supporting the hypothesis that ligand-TCR interactions play a crucial role in the regulation of Ag-specific Th2 cell development in vivo.     

Analysis of the TCR beta variable gene repertoire in chimpanzees: identification of functional homologs to human pseudogenes

Chimpanzees are used for a variety of disease models such as hepatitis C virus (HCV) infection, where Ag-specific T cells are thought to be critical for resolution of infection. The variable segments of the TCR alphabeta genes are polymorphic and contain putative binding sites for MHC class I and II molecules. In this study, we performed a comprehensive analysis of genes that comprise the TCR beta variable gene (TCRBV) repertoire of the common chimpanzee Pan troglodytes. We identified 42 P. troglodytes TCRBV sequences representative of 25 known human TCRBV families. BV5, BV6, and BV7 are multigene TCRBV families in humans and homologs of most family members were found in the chimpanzee TCRBV repertoire. Some of the chimpanzee TCRBV sequences were identical with their human counterparts at the amino acid level. Notably four successfully rearranged TCRBV sequences in the chimpanzees corresponded to human pseudogenes. One of these TCR sequences was used by a cell line directed against a viral CTL epitope in an HCV-infected animal indicating the functionality of this V region in the context of immune defense against pathogens. These data indicate that some TCRBV genes maintained in the chimpanzee have been lost in humans within a brief evolutionary time frame despite remarkable conservation of the chimpanzee and human TCRBV repertoires. Our results predict that the diversity of TCR clonotypes responding to pathogens like HCV will be very similar in both species and will facilitate a molecular dissection of the immune response in chimpanzee models of human diseases.     

Characterization of T Cell Receptors of Th1 Cells Infiltrating Inflamed Skin of a Novel Murine Model of Palladium-Induced Metal Allergy

Metal allergy is categorized as a delayed-type hypersensitivity reaction, and is characterized by the recruitment of lymphocytes into sites of allergic inflammation. Because of the unavailability of suitable animal models for metal allergy, the role of T cells in the pathogenesis of metal allergy has not been explored. Thus, we developed a novel mouse model for metal allergy associated with infiltration of T cells by multiple injections of palladium (Pd) plus lipopolysaccharide into the footpad. Using this model, we characterized footpad-infiltrating T cells in terms of phenotypic markers, T cell receptor (TCR) repertoires and cytokine expression. CD3+ CD4+ T cells accumulated in the allergic footpads 7 days after Pd challenge. The expression levels of CD25, interleukin-2, interferon-γ and tumor necrosis factor, but not interleukin-4 and interleukin-5, increased in the footpads after challenge, suggesting CD4+ T helper 1 (Th1) cells locally expanded in response to Pd. Infiltrated T cells in the footpads frequently expressed AV18-1 and BV8-2 T cell receptor (TCR) chains compared with T cells in the lymph nodes and exhibited oligoclonality. T-cell clones identified from Pd-allergic mouse footpads shared identical CDR3 sequences containing AV18-1 and BV8-2. These results suggest that TCR AV18-1 and BV8-2 play dominant and critical parts in the antigen specificity of Pd-specific Th1 cells.     

Characterization of CD8+ T lymphocytes that persist after peripheral tolerance to a self antigen expressed in the pancreas

As a result of expression of the influenza hemagglutinin (HA) in the pancreatic islets, the repertoire of HA-specific CD8+ T lymphocytes in InsHA transgenic mice (D2 mice expressing the HA transgene under control of the rat insulin promoter) is comprised of cells that are less responsive to cognate Ag than are HA-specific CD8+ T lymphocytes from conventional mice. Previous studies of tolerance induction involving TCR transgenic T lymphocytes suggested that a variety of different mechanisms can reduce avidity for Ag, including altered cell surface expression of molecules involved in Ag recognition and a deficiency in signaling through the TCR complex. To determine which, if any, of these mechanisms pertain to CD8+ T lymphocytes within a conventional repertoire, HA-specific CD8+ T lymphocytes from B10.D2 mice and B10.D2 InsHA transgenic mice were compared with respect to expression of cell surface molecules, TCR gene utilization, binding of tetrameric KdHA complexes, lytic mechanisms, and diabetogenic potential. No evidence was found for reduced expression of TCR or CD8 by InsHA-derived CTL, nor was there evidence for a defect in triggering lytic activity. However, avidity differences between CD8+ clones correlated with their ability to bind KdHA tetramers. These results argue that most of the KdHA-specific T lymphocytes in InsHA mice are not intrinsically different from KdHA-specific T lymphocytes isolated from conventional animals. They simply express TCRs that are less avid in their binding to KdHA.