T-cell Receptor Vβ Repertoire Research Articles

Increased proinflammatory cytokine gene expression in the colonic mucosa of coeliac disease patients in the early period after gluten challenge.

Activation of T cells in the intestinal mucosa in response to gluten exposure is thought to play a key role in the pathogenesis of coeliac disease. Moreover, the response of the rectal mucosa to gluten challenge has been considered a useful predictor of gluten sensitivity in coeliac disease. In the present study, we assessed early changes in the expression of proinflammatory cytokine genes and the T cell receptor (TCR) Vbeta repertoire in the rectal mucosa of coeliac disease patients following experimental gluten challenge. Cytokine gene expression was assessed in rectal mucosal biopsies from coeliac disease subjects and controls before and after rectal gluten challenge using quantitative reverse transcription polymerase chain reaction analysis, and the TCR Vbeta repertoire was characterized using a multiprobe RNase protection assay. Marked up-regulation of expression of the C-X-C chemokine IL-8, the proinflammatory cytokine IL-1beta, and the C-C chemokine monocyte chemotactic protein-1 occurred within 24 h of rectal gluten challenge in coeliac disease subjects, but not in controls. Furthermore, these changes occurred in the absence of parallel changes in the expressed repertoire of TCR Vbeta genes in the rectal mucosa. Thus, an increased expression of proinflammatory cytokine genes precedes the expansion of antigen-specific T cell populations in the early period following experimental exposure of the rectal mucosa of coeliac disease patients to gluten. These findings provide new insights into pathways that may be involved in the activation or reactivation of coeliac disease.

TCR Vbeta repertoire analysis in CD56+ CD16(dim/-) T-cell large granular lymphocyte leukaemia: association with CD4 single and CD4/CD8 double positive phenotypes.

We report 10 patients with T-cell large granular lymphocyte (LGL) leukaemia: four patients had CD16+ CD56- LGL lymphocytes (typical for LGL leukaemia), and six patients had CD56+ CD16(dim/-) LGL lymphocytes (atypical). Among the CD56+ CD(dim/-) patients, LGL lymphocytes were CD4+ CD8- in one patient, CD4/CD8 double positive (DP) in three, and CD4- CD8+ in two. The CD4+ CD8dim DP cells expressed a CD8alphaalpha homodimer. T-cell receptor (TCR) Vbeta complementarity-determining region 3 (CDR3) size distribution analysis and direct sequencing identified at least 1 in-frame clonal TCR Vbeta transcript in each patient; three patients had two or three different clonal sequences. To determine whether these transcripts were translated into cell surface TCR, we performed flow cytometric analysis using Vbeta monoclonal antibodies (mAbs). A single Vbeta protein was identified in patients, even those with multiple in-frame transcripts. Previous and present results suggest that CD56+ CD16(dim/-) LGL leukaemia is more common than previously thought, and is associated with unusual phenotypes. When assessed using only molecular techniques, the monoclonal status of this disease may be misinterpreted as oligoclonal; thus, flow cytometric analysis using Vbeta mAb is quite useful. Because mAbs do not cover the entire Vbeta repertoire, assessing clonality using a combination of molecular methods and mAbs is preferable.

Thymus transplantation in complete DiGeorge syndrome: immunologic and safety evaluations in 12 patients

AbstractComplete DiGeorge syndrome is a fatal condition in which infants have no detectable thymus function. The optimal treatment for the immune deficiency of complete DiGeorge syndrome has not been determined. Safety and efficacy of thymus transplantation were evaluated in 12 infants with complete DiGeorge syndrome who had less than 20-fold proliferative responses to phytohemagglutinin. All but one had fewer than 50 T cells/mm3. Allogeneic postnatal cultured thymus tissue was transplanted. T-cell development was followed by flow cytometry, lymphocyte proliferation assays, and T-cell receptor Vβ (TCRBV) repertoire evaluation. Of the 12 patients, 7 are at home 15 months to 8.5 years after transplantation. All 7 survivors developed T-cell proliferative responses to mitogens of more than 100 000 counts per minute (cpm). By one year after transplantation, 6 of 7 patients developed antigen-specific proliferative responses. The TCRBV repertoire showed initial oligoclonality that progressed to polyclonality within a year. B-cell function developed in all 3 patients tested after 2 years. Deaths were associated with underlying congenital problems. Risk factors for death included tracheostomy, long-term mechanical ventilation, and cytomegalovirus infection. Adverse events in the first 3 months after transplantation included eosinophilia, rash, lymphadenopathy, development of CD4-CD8- peripheral T cells, elevated serum immunoglobulin E (IgE), and possible pulmonary inflammation. Adverse events related to the immune system occurring more than 3 months after transplantation included thrombocytopenia in one patient and hypothyroidism and alopecia in one other patient. Thymic transplantation is efficacious, well tolerated, and should be considered as treatment for infants with complete DiGeorge syndrome.

T-cell receptor repertoire in healthy Sardinian subjects

The T-cell receptor (TCR) Vβ gene usage of CD4 + and CD8 + T-cell subpopulations was evaluated by flow cytometric analysis and by CDR3 spectratyping in healthy subjects belonging to Sardinian population, which is ethnically homogeneous and genetically distant from all other Italian and Caucasoid groups. As described in healthy Caucasian subjects, we found a nonrandom Vβ gene usage and in some Vβ families a significant skewed reactivity toward CD4 + T cells. Moreover, different subjects showed expansions in some Vβ subfamilies, mainly in the CD8 + T cells. By CDR3 spectratyping analysis we found a significantly higher degree of skewness in the TCR Vβ repertoire of CD8 + than in that of CD4 + T cells. The similarity found in the TCR Vβ gene usage between the population examined and other Caucasoid groups suggest that the shape of the TCR repertoire is more influenced by rearrangement processes than ethnic background. However, genetic polymorphisms may condition the expression levels of some Vβs, determining the variability of the TCR repertoire between different populations. Finally, the profound perturbations evidenced in the CD8 + T cell subpopulation could be related to a different response to the antigenic stimulation between CD8 + and CD4 + T lymphocytes.

Immune Reconstitution and Tolerance after Allogeneic Hematopoietic Stem Cell Transplantation

We have evaluated recovery of CD56 positive and other cell types following allogeneic stem cell transplantation and have found that the recovery of CD56 positive cells was faster than other lymphoid cells after allogeneic stem cell transplantation, while the recovery of CD4 positive cells was markedly delayed. Chimerism analysis showed that mixed chimerism was often observed in younger (<30 years old) patients. Mixed chimerism in older (≥30 years old) patients was associated with rejection and relapse, while this was not found in younger patients. Among the chimerism of various cell populations, donor-derived CD56-positive cells are important in early engraftment when determined in allogeneic nonmyeloablative stem cell transplantation (allo-NST), regardless of the proportion of donor-derived CD3-positive cells. Complementarity-determining region three (CDR3) size spectratyping in T-cell receptor (TCR) chain subfamilies (Vβ) showed that high level of diversity in TCR Vβ repertoire is important for a late rejection and skewed TCR Vβ repertoire is correlated with the occurrence of graft-versus-host disease (GVHD) especially chronic GVHD. Expression of inhibitory natural killer (NK) cell receptors such as CD158b and CD94/NKG2A on peripheral CD3-negative and -positive cells were increased in parallel with GVHD. Interestingly, these cells appeared to control GVHD, while preserving graft-versus-leukemia (GVL) effect. Analysis of cytokine gene expression in peripheral blood mononuclear cells showed that type 1 helper T cells (Th1)-derived cytokines increased in severe GVHD, while Th2-derived cytokines such as IL-4, IL-10 and IL-13 increased in mild GVHD. These results indicate that Th2 cells suppress GVHD, although Th1 cells augment GVHD. Taken together, evaluation of immune reconstitution and tolerance in patients receiving allogeneic stem cell transplantation from the various viewpoints is essential and useful to obtain better clinical outcome.

Early increase of CD4+ CD45RA+ and CD4+ CD95- cells with conserved repertoire induced by anti-retroviral therapy in HIV-infected patients.

Administration of anti-retroviral drugs induces a decrease of viral load associated with increase of CD4+ cell count in most HIV-infected patients. To investigate the early changes in CD4+ cell phenotype induced by anti-retroviral therapy, six patients with CD4+ cell count > 100/mm3 and never treated with anti-HIV therapy were enrolled and blood samples collected several times within 14 days from the initiation of therapy with Zidovudine plus Didanosine. CD4+ cell count and HIV viraemia were investigated at each time point, as well as the expression of CD45RA, CD45RO and CD95/Fas molecules on CD4+ cells, and the T cell receptor (TCR) Vbeta repertoire of CD4+ cells. All patients showed a rapid and dramatic decrease in viral load with a corresponding increase of CD4+ cell count. The main remodelling of CD4+ cell subpopulations took place in the first 14 days of therapy, and consisted of: (i) increased CD4+CD45RA+/CD4+CD45RO+ ratio; (ii) decrease of CD95/Fas expression. The rise in absolute number of CD4+CD45RA+ cells was paralleled by an increase of CD4+CD95/Fas- cells and accounted for most of the early increment of CD4+ cell count. The TCR Vbeta repertoire of CD4+ cells was conserved after anti-HIV therapy, with the exception of two patients with expanded CD4+Vbeta12+ cells, which also tested CD45RA+ and CD95/Fas-. These experiments show that newcomer CD4+ lymphocytes are CD45RA+CD95/Fas- cells, suggesting that blocking HIV replication causes an early and antigen-independent proliferation of possibly 'naive' cells unprimed for CD95/Fas-mediated apoptosis. These cells expressed a conserved and widespread TCR repertoire, suggesting that their capability for antigenic recognition is intact.

T cell receptor repertoire and function in patients with DiGeorge syndrome and velocardiofacial syndrome.

DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS) are associated with chromosome 22q11.2 deletion. Limited information is available on the T cell receptor (TCR) Vbeta repertoire. We therefore investigated TCR Vbeta families in lymphocytes isolated from blood and thymic samples of seven patients with DGS and seven patients with VCFS, all with 22q11.2 deletion. We also studied activities related to TCR signalling including in vitro proliferation, anti-CD3-induced protein tyrosine phosphorylation, and susceptibility to apoptosis. Reduced CD3+ T cells were observed in most patients. Spontaneous improvement of T cell numbers was detected in patients, 3 years after the first study. Analysis of CD4+ and CD8+ TCR Vbeta repertoire in peripheral and thymic cells showed a normal distribution of populations even if occasional deletions were observed. Lymphoproliferative responses to mitogens were comparable to controls as well as anti-CD3-induced protein tyrosine phosphorylation. Increased anti-CD3-mediated apoptosis was observed in thymic cells. Our data support the idea that in patients surviving the correction of cardiac anomalies, the immune defect appears milder than originally thought, suggesting development of a normal repertoire of mature T cells.

Modification of T-cell receptor Vβ repertoire in response to allergen stimulation in peanut allergy

Background: Peanut is one of the most common foods causing allergic reactions and is the most common cause of fatal and near-fatal food-related anaphylaxis. Little is known of the immunologic mechanisms that underlie peanut allergy. Objectives: In this study we examined clonality of the T-cell response (TCR) to peanut in MHC class II identical, peanut allergy–discordant sibling pairs. Methods: Four sibling pairs were investigated. The TCR repertoire was analyzed before and after in vitro stimulation of PBMCs with crude peanut or PHA, as control for general/nonspecific reactivity. Eighteen TCR-Vβ families were examined by flow cytometry. Where significant differences in incidence of particular TCR-Vβ families were observed, PCR familyspecific cDNA amplification and gene scanning were performed. Results: After stimulation with peanut, no selective expansion of any TCR-Vβ subpopulation was observed with flow cytometry, in either the peanut-allergic or nonallergic siblings, with the exception of 1 peanut-allergic subject who demonstrated a significant increase of TCR-Vβ11+ cells (0.3%-5.9% of the total CD3+ cells). However, gene scanning revealed predominant single-size PCR products for TCRBV11 in all peanut-allergic subjects after peanut stimulation. TCRBV11 polyclo-nality was observed in allergic and nonallergic subjects before peanut stimulation and in nonallergic subjects after peanut stimulation. In comparison, all subjects, before and after stimulation with peanut, showed polyclonality for TCRBV2.Conclusions: Our results argue for clonal or oligoclonal TCRs to crude peanut and indicate that changes in the TCRBV11 subpopulation are restricted to peanut-allergic subjects after stimulation with crude peanut allergen. (J Allergy Clin Immunol 2001;107:1089-94.)

Changes in the pattern of TCR V beta repertoire expression after bone marrow transplant is linked to the HLA haplotype in humans.

Neutrophil, monocyte, natural killer- and B-cell number and function are rapidly restored after bone marrow transplant (BMT), whereas T-cell reconstitution is often quite delayed. Our hypothesis was that V beta T-cell receptor (TCR) repertoire diversity among recipients of allogeneic BMT is influenced by the expression of major and minor HLA antigens in the host. The study population comprised unmanipulated and CD34(+)-selected allogeneic bone marrow grafts, autologous peripheral blood stem cell transplants and recipients of volunteer unrelated donor (VUD) bone marrow transplants. Using flow cytometry, the relative frequencies of 18 V beta TCR families were determined and ranked for each time point studied. Comparisons and correlations were made between paired blood samples obtained within a single patient over time, and between donors and their recipients. The pattern of the V beta TCR repertoire from allogeneic recipients and their HLA-matched donors was very similar, with a correlation coefficient (CC) of 0.59. This similarity was not as marked in VUD pairs (CC = 0.32). By 3 months after transplant, the pattern of the V beta TCR repertoire in recipients of HLA-matched sibling transplants was more similar to the pattern seen in pretransplant recipients than to the donor pattern (CC = 0.40 vs. 0.31). Our data suggest that both major and minor HLA antigens influence V beta TCR repertoire diversity and reconstitution after BMT.

Distinct T‐cell clonal expansion in the vicinity of tumor cells in plasmacytoma

BACKGROUND Although various clinical observations suggested that myeloma cell growth might be modulated by the immune system, evidence supporting the in situ immunogenicity of myeloma cells remains scarce. The authors reasoned that if there is any specific T-cell/tumor cell interaction in myeloma, it is most likely reflected in the T-cell population in the vicinity of the tumor cells. METHODS The authors used a molecular method to compare the T-cell populations in the vicinity of tumor cells with those in the peripheral blood in patients with plasmacytomas and multiple myeloma. RESULTS Six patients were studied. When compared with the peripheral blood from the corresponding patients, T cells in the vicinity of tumor cells in five of the six patients showed significant contraction of the T-cell receptor (TCR) Vβ repertoire. In addition to this, the T cells isolated from the sites of the tumor cells from four of these five patients also demonstrated significant increase in the number of TCR Vβ gene families with restricted number of CDR3 size peaks and loss of the normal CDR3 size gaussian distribution pattern. These findings were observed in patients who experienced recurrence after allogeneic stem cell transplantation and also in those who had autologous stem cell transplant. They also were found in previously untreated myeloma patients. In all six patients, distinct TCR Vβ recurring transcripts indicative of a T-cell clonal expansion were found in the vicinity of the tumor cells and either absent or detected at only a low frequency in the peripheral blood. CONCLUSIONS Our results provide evidence for an in situ local T-cell clonal expansion in the vicinity of tumor cells and support the presence of specific T-cell/tumor cell interaction in myeloma. Cancer 2001;91:900–8. © 2001 American Cancer Society.

Distinct T-cell clonal expansion in the vicinity of tumor cells in plasmacytoma

Although various clinical observations suggested that myeloma cell growth might be modulated by the immune system, evidence supporting the in situ immunogenicity of myeloma cells remains scarce. The authors reasoned that if there is any specific T-cell/tumor cell interaction in myeloma, it is most likely reflected in the T-cell population in the vicinity of the tumor cells. The authors used a molecular method to compare the T-cell populations in the vicinity of tumor cells with those in the peripheral blood in patients with plasmacytomas and multiple myeloma. Six patients were studied. When compared with the peripheral blood from the corresponding patients, T cells in the vicinity of tumor cells in five of the six patients showed significant contraction of the T-cell receptor (TCR) Vbeta repertoire. In addition to this, the T cells isolated from the sites of the tumor cells from four of these five patients also demonstrated significant increase in the number of TCR Vbeta gene families with restricted number of CDR3 size peaks and loss of the normal CDR3 size gaussian distribution pattern. These findings were observed in patients who experienced recurrence after allogeneic stem cell transplantation and also in those who had autologous stem cell transplant. They also were found in previously untreated myeloma patients. In all six patients, distinct TCR Vbeta recurring transcripts indicative of a T-cell clonal expansion were found in the vicinity of the tumor cells and either absent or detected at only a low frequency in the peripheral blood. Our results provide evidence for an in situ local T-cell clonal expansion in the vicinity of tumor cells and support the presence of specific T-cell/tumor cell interaction in myeloma.

Neonatal dexamethasone treatment induces long-lasting changes in T-cell receptor Vβ repertoire in rats

Glucocorticoids are frequently administered for the prevention of chronic lung disease in infants with respiratory distress syndrome. However, neonatal treatment may have consequences for immune functioning in the long-term. Here we demonstrate that neonatal glucocorticoid treatment has long-lasting effects on mRNA expression of several Vβ genes within the CD4 and CD8 T cell subset in rats. Changes in the peripheral T cell Vβ repertoire may be a consequence of altered intrathymic selection events in which corticosterone plays an important role. Indeed, here we show that neonatal glucocorticoid treatment affects corticosterone production by thymic epithelial cells during neonatal life. In conclusion, changes in T cell Vβ repertoire after neonatal glucocorticoid treatment may contribute to altered immune reactivity in later life.

Evidence for a disease-promoting effect of Staphylococcus aureus–derived exotoxins in atopic dermatitis

Background: The skin of patients with atopic dermatitis (AD) exhibits a striking susceptibility to colonization with Staphylococcus aureus. Some strains of S aureus secrete exotoxins with T-cell superantigen activity (toxigenic strains), and abnormal T-cell functions are known to play a critical role in AD. Objective: Our purpose was to examine the impact of superantigen production by skin-colonizing S aureus on disease severity. Methods: In a cross-sectional study of 74 children with AD, the presence and density of toxigenic and nontoxigenic strains of S aureus was correlated with disease severity. In a subgroup of patients the T-cell receptor Vβ repertoire of peripheral blood and lesional T cells was investigated and correlated with individual superantigen activity of skin-colonizing S aureus.Results: Fifty-three percent of children with AD were colonized with toxigenic strains of S aureus producing staphylococcal enterotoxin C, staphylococcal enterotoxin A, toxic shock syndrome toxin-1, staphylococcal enterotoxin B, and staphylococcal enterotoxin D in decreasing frequency. Children colonized with toxigenic S aureus strains had higher disease severity compared with the nontoxigenic and S aureus –negative groups. Patients colonized with toxigenic S aureus exhibited shifts in the intradermal T-cell receptor Vβ repertoire that correspond to the respective superantigen-responsive T-cell subsets. Conclusion: The data demonstrate that S aureus –released exotoxins can modulate disease severity and dermal T-cell infiltration. (J Allergy Clin Immunol 2000;105:814-9.)

Application of staphylococcal enterotoxin B on normal and atopic skin induces up-regulation of T cells by a superantigen-mediated mechanism

Background: The skin of patients with inflammatory skin diseases such as atopic dermatitis is frequently colonized with Staphylococcus aureus. Colonization with S aureus has been reported to exacerbate atopic dermatitis. Recent studies have demonstrated that S aureus isolated from the skin of patients with atopic dermatitis releases bacterial toxins that act as superantigens. We have previously applied the staphylococcal superantigen staphylococcal enterotoxin B (SEB) on intact human skin and found that the application led to induction of dermatitis. Objective: The purpose of the study was to determine whether superantigen-induced dermatitis is primarily due to a T cell–superantigen–mediated reaction or represents nonspecific cytokine-driven inflammation. Methods: We applied SEB, vehicle, and sodium lauryl sulfate on normal skin in healthy (n = 6) and atopic subjects (n = 6) and biopsy specimens were taken from all treated areas. The biopsy specimens from all subjects and peripheral blood from the atopic subjects were analyzed for the T-cell receptor (TCR) Vβ repertoire with mAbs against TCR Vβ 2, 3, 8.1, 12, 14, and 17. Results: From all subjects, both healthy and patients with atopic dermatitis, skin biopsy specimens from SEB-treated areas demonstrated selective accumulation of T cells expressing SEB-reactive TCR Vβ 12 and 17 (P < .05). This selective up-regulation was not found in the sodium lauryl sulfate–treated areas. Conclusion: Our data strongly support that superantigen-induced T-cell activation is involved in the dermatitis seen after experimental application of SEB on intact skin. (J Allergy Clin Immunol 2000;105:820-6.)

T-cell clonality of peripheral blood from patients with chronic hepatitis C before and after treatment with interferon α

Immune-mediated liver cell damage has been likely to occur in patients with chronic hepatitis C virus (HCV) infection. To clarify the T-cell clonality in patients with chronic hepatitis C, we analyzed T-cell receptor (TCR) Vβ chain messages expressed in peripheral blood mononuclear cells (PBMC) before and after treatment with interferon (IFN)-α. PBMC from 15 patients with chronic hepatitis C and from six healthy subjects were examined. The 15 patients were allocated to three groups based on their response to IFN-α treatment as follows: responders (group A, n=5), transient responders (group B, n=5), and non-responders (group C, n=5). Twenty-two TCR Vβ repertoires Vβ 1–Vβ 20 expressed in PBMC were analyzed by reverse-transcription polymerase chain reaction (RT-PCR) and single strand conformation polymorphism (SSCP). In responders, the mean total number of T-cell clones in 22 TCR Vβ repertoires was significantly decreased from 97.4±40.1 to 62.6±32.1 ( P<0.01) after treatment. In transient responders, although one patient showed a decrease, four patients showed an increase, with the mean total number of T-cell clones increasing from 66.4±28.0 to 79.8±28.8 after treatment. In non-responders, the mean total number of T-cell clones was almost unchanged between before and after treatment. No clear tendency towards the use of any specific Vβ was observed in any group of patients. These results suggest that T-cell clones accumulated in the peripheral blood of patients with chronic hepatitis C and that the number of clones may change in response to IFN-α treatment.

Generation of Functional Thymocytes in the Human Adult

Reconstituting the immune response will be critical for the survival of HIV-infected individuals once viral load is brought under control. While the adult thymus was previously thought to be relatively inactive, new data suggest it may play a role in T cell reconstitution. We examined thymopoiesis in adults up to 56 years of age and found active T cell receptor (TCR) rearrangement, generating a diverse TCR Vβ repertoire. The resulting thymocytes are functional and are capable of responding to costimulatory signals. These data demonstrate that the adult thymus remains active late in life and contributes functional T cells to the peripheral lymphoid pool.

‘Naı̈ve’ and ‘memory’ CD4 + T-cells and T-cell receptor (TCR) Vβ repertoire dynamics are independent of the levels of viremia following HIV seroconversion

The progression of ‘naı̈ve’ and ‘memory’ T-cells and the T-cell receptor Vβ (TCR Vβ) repertoire dynamics within the peripheral CD4 + T-cell compartment were studied in individuals following HIV seroconversion. Profound TCR Vβ repertoire perturbations were observed within the CD4 + T-cell pool in treatment-naive patients regardless of their levels of viremia during the first 6–8 months after seroconversion. The ratio of ‘naı̈ve’ to ‘memory’ CD4 + T-cells as well as the TCR Vβ repertoire dynamics did not appear to correlate with absolute numbers of CD4 + T-cells.

Rat peripheral T-cell receptor Vβ repertoire in F344-to-B10 (rat-to-mouse) mixed xenogeneic chimeras

In previous studies, we demonstrated that T cell receptor (TCR) Vβ8.2 and Vβ10, both of which are frequently used by encephalitogenic T cells, spectratypes expand oligoclonally in spinal cord lesions of Lewis rats with experimental autoimmune encephalomyelitis (EAE) and that the DSSYEQYF and WDGSGNVLYF sequences are predominantly found in the complementarity-determining region 3 (CDR3) of spectratype-derived TCR clones. However, it is unknown whether these CDR3 sequences are used only by Vβ8.2- and Vβ10-positive T cells or by encephalitogenic T cells bearing Vβs other than these Vβs. The present study was undertaken to address this issue using a new approach, i.e. CDR3 spectratyping and subsequent DNA hybridization with several probes corresponding to various parts of the CDR3 region. Consequently, we found that probes specific for the Vβ8.2 spectratype-derived Dβ and Jβ2.7 hybridized only with the Vβ8.2 spectratype in acute EAE and with the Vβ8.2 and Vβ12 spectratypes in chronic relapsing EAE. Similarly, a probe specific for the Vβ10 spectratype-derived Dβ hybridized only with the Vβ10 spectratype in both acute and chronic relapsing EAE. In contrast, a probe specific for Jβ1.3 hybridized with several Vβ spectratypes including Vβ8.2 and Vβ10 only during the early stage of the disease. These findings suggest that T cells bearing a few Vβs with a limited number of the CDR3 sequences are activated in acute and chronic relapsing EAE induced in Lewis rats. Characterization of the CDR3 region of pathogenic TCR by this approach may be of value for the screening of autoimmune disease-associated TCR and for the development of TCR-based specific immunotherapy.

Selective expansion of large lymphoblasts expressing Vβ6.1-3 or 7 in synovial fluid from patients with rheumatoid arthritis

The affected joints of patients with rheumatoid arthritis (RA) contain a large number of T cells, of which those expressing specific T cell receptor (TCR) Vβ chains are thought to play an important role in the pathogenesis of RA. The TCR Vβ repertoire of large lymphoblasts and small non-blasts from the synovial fluid (SF) and peripheral blood (PB) of RA patients, as well as their responsiveness to staphylococcal enterotoxin A (SEA), was investigated. TCR Vβ repertoire was examined with a semiquantitative reverse transcription-polymerase chain reaction assay, as was expression of genes encoding interleukin (IL)-2 and IL-4. Production of IL-2 and IL-4 was measured with a bioassay and an enzyme immunoassay system, respectively. Expression of TCR Vβ6.1-3, 7 and 15 was more frequent among large lymphoblasts than among small non-blasts in paired preparations from the 14 SF of 14 RA patients. TCR Vβ7 was expressed more frequently by large lymphoblasts than by small non-blasts in the PB of eight RA patients. The usage of TCR Vβ6.1-3 and 7 by large lymphoblasts in SF from RA patients significantly reduced in response to SEA stimulation. Both IL-2 production and gene expression as well as IL-4 production and gene expression in large lymphoblasts from the SF of RA patients increased to a lesser extent in response to SEA stimulation compared with the effects observed in paired small non-blasts. In summary, large lymphoblasts expressing TCR Vβ6.1-3, 7 and 15 were selectively expanded in SF of RA patients, and those bearing TCR Vβ6.1-3 or 7 were selectively unresponsive to SEA stimulation. These results indicate that SF lymphoblasts bearing Vβ6.1-3 or 7 were anergic with regard to stimulation with superantigen, suggesting the implication of a superantigen in previous activation of T cells in SF of RA patients.

T-cell receptor Vbeta gene expression in experimental lupus nephritis.

A limited T-cell receptor (TCR) Vbeta repertoire employed by autoreactive T cells may be related to the development and course of autoimmune diseases. Vbeta repertoire skewing has been observed not only in man, but also in animal models of several human autoimmune diseases, such as MRL-lpr mice, which spontaneously develop a systemic lupus erythematosus (SLE)-like disease. Murine chronic graft-versus-host disease (GVHD) is an inducible model for SLE, involving direct interaction between donor T cells and recipient B cells. It is not known whether Vbeta-specific T-cell subsets are pathogenically involved in this model. Retroviral superantigens such as Mls-1 are known to have a profound impact on the TCR Vbeta repertoire in mice. Restriction of the peripheral TCR repertoire may result from intrathymic expression of Mls-1, which causes deletion of T cells expressing Vbeta6, -7, -8.1, or -9. Mls-1 incompatibility between donor and recipient can be used to determine the involvement of these TCR Vbeta families in GVHD. In the present study we induced GVHD in several strain combinations to investigate TCR Vbeta gene expression during GVHD, and the effect of Mls-1 incompatibility on the TCR Vbeta repertoire. TCR Vbeta gene expression was determined using an RNase protection assay. Our results indicate that T cells expressing the Vbeta2 or Vbeta16 chain play an important pathogenetic role, while T cells bearing the Vbeta1 or Vbeta6 chain may be related to self-limitation of the lupus-like disease in this model.