Lower proportions of CD4+CD25high and CD4+FoxP3, but not CD4+CD25+CD127low FoxP3+T cell levels in children with autoimmune thyroid diseases

The essence of autoimmune thyroid disease (AITD) is loss of tolerance of own tissues caused by malfunction of T lymphocytes, which affects the production of antibodies reacting with particular cell structures and tissues. Foxp3⁺ regulatory T cells (Tregs) take part in the regulation of immune response and play a leading role in developing immune tolerance through active suppression. The aim of the study was to estimate the expression of CD4+CD25^high, CD4+CD25+CD127^lowFoxP3⁺ and CD4+ FoxP3 T cells in patients with Graves' disease (GD) (n = 24, median age 15.5 years), in patients with Hashimoto's thyroiditis (HT) (n = 30, median age 15 years) in comparison with sex- and age-matched healthy control subjects (n = 30, median age 15 years). Polychromatic flow cytometry using a FACSCalibur (BD Biosciences) cytometer was applied to delineate T regulatory cell populations. In untreated patients with Graves' disease and HT we observed a significant decrease in CD4+FoxP3 (p < 0.001, p < 0.01) and CD4+CD25^high (p < 0.016, p < 0.048) T lymphocytes as compared to the healthy control subjects. After 6–12 months of L-thyroxine therapy in HT cases these phenotypes of Tregs were normalized, yet no such changes were observed during GD therapy. The analysis of CD4+CD25+CD127^lowFoxP3⁺T cells in the peripheral blood revealed comparable percentages of these cells in patients with thyroid autoimmune diseases to the controls. We conclude that the reduction number of Tregs with CD4+CD25^high and CD4+FoxP3 phenotype suggests their role in initiation and development of autoimmune process in thyroid disorders.     

HIV-1感染者CD4+T细胞受体基因多样性特点及其与病毒载量的相关性

目的 分析HIV-1感染者CD4+T细胞受体(TCR)基因的多样性特征及其与病毒载量的相关性.方法 应用抗CD4单克隆抗体从25份HIV-1感染者和10份HIV-1阴性对照样本外周血单个核细胞(PBMC)中分离CD4+T细胞,提取细胞总RNA,然后通过逆转录及巢式多聚酶链反应(nestedPCR)对TCR 22个Vβ基因家族的互补决定区3(CDR3)进行扩增,利用ABI3700测序仪对扩增的PCR产物进行扫描,定最分析HIV-1感染者TCRCDR3区多样性变化特征及其与病毒载量的相关性.结果 HIV-1感染者CD4+T细胞TCR CDR3区平均D(distance)值显著高于正常对照组(P＜0 05),TCR Vβ基因各家族CDR3长度谱型成寡克隆分布,TCR CDR3区的紊乱与病毒载量呈正相关(r=0 494,P＜0 05);HIV-1感染引起TCR多样性的改变不仅表现在不同Vβ基因家族上,而且也表现在CDR3长度上,其中感染者Vβ8、Vβ22、Vβ23基因家族的变化与正常人差异有统计学意义.结论 HIV-1感染能引起CD4+T细胞TCR基因多样性的减少及高斯(Gaussian)分布的破坏,TCR CDR3区的紊乱与病毒载量呈正相关.

Abstract：

Objective To assess the impact of the virus on the complementary determining region 3 (CDR3) length diversity of T cell receptor(TCR) Vβ repertoires of CD4+ T lymphocytes and to explore its association with viral load in individuals with HIV-1 infection. Methods The TCR repertoire was examined using spectratyping of CDR3 length diversity within CD4+ T cells in HIV infected and healthy adults. Separation of CD4+ T cells from peripheral blood mononuclear cells ( PBMCs) was carried out by using immunomagnetic beads coated with anti-CD4 antibody. Total RNAs from the purified CD4 + T lymphocytes were isolated and used to perform nested-PCR amplifications in CDR3 of 22 TCR gene families. CDR3 diversity and its association with viral load in individuals with HIV-1 infection were analyzed. Results An average diversity for all CDR3 profiles in CD4+ T cells from 25 HIV-infected individuals was significantly different as compared to 10 age-matched healthy donors (P＜0.05) with the HIV-infected individuals losing diversity in the CDR3 profiles. There was positive correlation between changes in TCR CDR3 diversity and viral load (r = 0. 494, P ＜ 0. 05). The changes in CDR3 length diversity of Vβ families in HIV-infected individuals, particular in Vβ8, Vβ22, Vβ23 were statistically different from the healthy controls. Conclusion HIV-1 infection might induce the loss of TCR Vp repertoire diversity and disrupt the CDR3 Gaussian distributions within CD4 + T cells. There should be positive correlation between changes in TCR CDR3 diversity and the viral load in HIV-1 infected patients.     

CD8+ CD122+ regulatory T cells contain clonally expanded cells with identical CDR3 sequences of the T‐cell receptor β‐chain

We identified CD8⁺ CD122⁺ regulatory T cells (CD8⁺ CD122⁺ Treg cells) and reported their importance in maintaining immune homeostasis. The absence of CD8⁺ CD122⁺ Treg cells has been shown to lead to severe systemic autoimmunity in several mouse models, including inflammatory bowel diseases and experimental autoimmune encephalomyelitis. The T‐cell receptors (TCRs) expressed on CD8⁺ CD122⁺ Treg cells recognize the target cells to be regulated. To aid in the identification of the target antigen(s) recognized by TCRs of CD8⁺ CD122⁺ Treg cells, we compared the TCR diversity of CD8⁺ CD122⁺ T cells with that of conventional, naive T cells in mice. We analysed the use of TCR‐Vβ in the interleukin 10‐producing population of CD8⁺ CD122⁺ T cells marked by high levels of CD49d expression, and found the significantly increased use of Vβ13 in these cells. Immunoscope analysis of the complementarity‐determining region 3 (CDR3) of the TCR β‐chain revealed remarkable skewing in a pair of Vβ regions, suggesting the existence of clonally expanded cells in CD8⁺ CD122⁺ T cells. Clonal expansion in Vβ13⁺ cells was confirmed by determining the DNA sequences of the CDR3s. The characteristic TCR found in this study is an important building block for further studies to identify the target antigen recognized by CD8⁺ CD122⁺ Treg cells.     

Evidence for the persistence of monoclonal expansions of CD8+ T cells following primary simian immunodeficiency virus infection

A longitudinal study of the CD8⁺ TCR variable (Vβ) chain repertoire was performed in rhesus macaques experimentally infected with simian immunodeficiency virus (SIV) using both TCR Vβ chain-specific monoclonal antibodies and TCR β chain CDR3 length analysis. Expansions of subpopulations of CD8⁺ T cells were detected during the acute phase of SIV infection. In all monkeys studied, monoclonal expansions persisted for at least 18 months and increasingly dominated the repertoire of CD8⁺ T cells expressing the relevant Vβ chain. This study shows that persistent CD8⁺ T cell expansions develop in response to a virus infection. This is important not only for our understanding of the T cell response to viruses but also for understanding the factors that determine the normal CD8⁺ TCR repertoire.     

Oligoclonal T cell expansions in patients with Behçet's disease

Behçet's disease (BD) is a multisystem disorder with oral and genital ulcers, mucocutaneous, ocular, joint, vascular and central nervous system involvement. In this study, the peripheral T cell repertoire was analysed in patients with BD with MoAbs against T cell receptor (TCR) Vβ gene products in CD4⁺ and CD8⁺ T cell compartments, and these were compared with rheumatoid arthritis (RA) patients and healthy controls (HC). In the CD4⁺ T cell compartment, oligoclonal TCR Vβ expression was observed in 56% of BD (10/18), 71% of RA (5/7) patients and 21% (3/14) of HC. In the CD8⁺ T cell group 50% of BD (9/18), 57% of RA patients and 28% of HC (4/14) had an oligoclonal TCR repertoire. An increase of TCR Vβ5.1 subset was observed in five BD patients among CD8⁺ T cells. Other elevations of TCR Vβ subsets were heterogeneously distributed with one to three different Vβ subsets. Our results suggest an antigen-driven oligoclonal increase of T cells in BD. There was no overall increase in any Vβ group to suggest a superantigen effect. Analysis of the responsible antigens causing the increase in T cell subsets may give insights into the aetiopathogenesis of BD and immunomodulation of these T cells may lead to new treatments.     

T cell‐derived leptin contributes to increased frequency of T helper type 17 cells in female patients with Hashimoto's thyroiditis

Leptin modulates T cell function and plays an important role in autoimmune diseases. Our study aimed to explore the role of leptin and T helper type 17 (Th17) cells in Hashimoto's thyroiditis patients. Twenty‐seven patients with Hashimoto's thyroiditis (HT) and 20 healthy controls were enrolled into the current study. A modest increase of plasma leptin in HT patients and the CD4⁺ T cell‐derived leptin from HT patients was stronger than that from healthy controls. In HT patients, there are no statistically significant correlations between plasma leptin concentrations and the percentage of Th17 cells or the level of retinoic acid‐related orphan receptor γt (RORγt), but strong positive correlations were observed between CD4⁺ T cell‐derived leptin and the percentage of Th17 cells or the level of RORγt mRNA, and additionally significantly up‐regulated leptin, interleukin (IL)17 and RORγt mRNA levels in the thyroid tissue. Furthermore, neutralization of leptin decreases the frequency of Th17 cells in vitro. Current study has revealed an increased leptin involvment in Hashimoto's thyroiditis associated with an increased number of Th17 cells.     

Lack of selective Vβ deletion in peripheral CD4+ T cells of human immunodeficiency virus-infected infants

To investigate the possibility of superantigen-mediated deletions of T cells expressing particular T cell receptor Vβ (TcR Vβ) gene segments during human immunodeficiency virus (HIV) infection, TcR Vp usage in CD4⁺ and CD8⁺ subsets was analyzed in a cohort of infants maternally infected by HIV and in a group of healthy neonates. We used a semi-quantitative anchored polymerase chain reaction technique together with cytofluorographic analysis with anti-Vβ monoclonal antibodies. The representation of the 24 vβ families in CD4⁺ and CD8⁺ T cells from normal neonates was very similar to that in adults. Preferential expression of Vβ2 in the CD4⁺ subset was observed in both the neonates and in healthy adults. The representation of the 24 Vβ families in peripheral CD4⁺ T cells from the HIV-infected infants showed no selective vβ deletion, even when the CD4⁺ subset was globally depleted. Moreover, the main characteristics of the control group (predominance of certain Vβ families and Vβ2 skewing towards the CD4⁺ subset) were also present in all the HIV-infected infants.     

Altered balance between self‐reactive T helper (Th)17 cells and Th10 cells and between full‐length forkhead box protein 3 (FoxP3) and FoxP3 splice variants in Hashimoto's thyroiditis

T helper type 17 (Th17) cells play a pathogenic role in autoimmune disease, while interleukin (IL)‐10‐producing Th10 cells serve a protective role. The balance between the two subsets is regulated by the local cytokine milieu and by the relative expression of intact forkhead box protein 3 (FoxP3) compared to FoxP3Δ2, missing exon 2. Th17 and Th10 cell differentiation has usually been studied using polyclonal stimuli, and little is known about the ability of physiologically relevant self‐antigens to induce Th17 or Th10 cell differentiation in autoimmune thyroid disease. We subjected mononuclear cells from healthy donors and patients with Hashimoto's thyroiditis (HT) or Graves' disease (GD) to polyclonal stimulation, or stimulation with human thyroglobulin (TG), human thyroid peroxidase (TPO), or Esherichia coli lipopolysaccharide (LPS). TPO and LPS induced increased differentiation of naive CD4⁺CD45RA⁺CD45R0^– T cells from HT patients into Th17 cells. Th10 cell proportions were decreased in HT after polyclonal stimulation, but were comparable to those of healthy donors after antigen‐specific stimulation. Taken together, our data show that an increased Th17 : Th10 ratio was found in HT patients after stimulation with thyroid‐specific self‐antigens. We also observed an elevated baseline production of IL‐6 and transforming growth factor (TGF)‐β1 and of mRNA encoding FoxP3Δ2 rather than intact FoxP3. This may contribute to the skewing towards Th17 cell responses in HT.     

Evidence for selective in vivo expansion of synovial tissue-infiltrating CD4+ CD45RO+ T lymphocytes on the basis of CDR3 diversity

In this study we have analyzed the TCR V and V_ß regionsat the DNA level in the CD4⁺CD45RO⁺ memory T cell populationof synovial tissue infiltrating T lymphocytes of three rheumatoidarthritis (RA) patients and one patient with chronic arthritis.Cell lines of CD4⁺CD45RO⁺, CD4⁺CD45RO^–, CD8⁺CD45RO⁺ andCD8⁺CD45RO^– T lymphocyte populations were generated followingFACS cell sorting of freshly isolated synovial tissue mononuclearcell infiltrates (STMC) and of freshly isolated peripheral bloodmononuclear cells (PBMC) of these patients. The phenotyplc andmolecular analyses have revealed the following. (I) The TCRrepertoires of tissue infiltrating T lymphocytes in the varioussubsets were extensive on the basis of TCR V gene family usage.(II) Furthermore, each patient displayed individual specificTCR V gene expression patterns in the various STMC and PBMCderived T cell subsets. However, the majority of these arthritispatients manifested increased expression of multiple TCR V genefamilies in the synovial tissue derived CD4⁺CD45RO^– Tcell population when compared with the peripheral blood derivedCD4⁺CD45RO⁺ subset. Of these gene families, we found enhancedexpression of the TCR V7 and V_ß11 gene segments insynovial tissue to be shared by all four patients analyzed.OH) Nucleotlde sequence analysis of the CDR3 regions of a numberof TCR V regions in the CD4⁺CD45RO⁺ T cell subsets has revealedthat the CDR3 regions comprised within synovial tissue derivedTCR V regions differed from those found in peripheral bloodderived TCR V regions. These differences in CDR3 diversity mightbe the consequence of a specific interaction with particularMHC-peptlde complexes expressed at the site of inflammation.(Iv) The CDR3 region analysis also showed individual specificamlno acid motifs within the N-D-N regions of all analyzed TCRV_ß genes derived from PBMC as well as STMC.     

Preselection TCR repertoire predicts CD4+ and CD8+ T-cell differentiation state

T cells must display diversity regarding both the cell state and T-cell receptor (TCR) repertoire to provide effective immunity against pathogens; however, the generation and evolution of cellular T-cell heterogeneity in the adaptive immune system remains unclear. In the present study, a combination of multiplex PCR and immune repertoire sequencing (IR-seq) was used for a standardized analysis of the TCR β-chain repertoire of CD4⁺ naive, CD4⁺ memory, CD8⁺ naive and CD8⁺ memory T cells. We showed that the T-cell subsets could be distinguished from each another with regard to the TCR β-chain (TCR-β) diversity, CDR3 length distribution and TRBV usage, which could be observed both in the preselection and in the post-selection repertoire. Moreover, the Dβ-Jβ and Vβ-Dβ combination patterns at the initial recombination step, template-independent insertion of nucleotides and inter-subset overlap were consistent between the pre- and post-selection repertoires, with a remarkably positive correlation. Taken together, these results support differentiation of the CD4⁺ and CD8⁺ T-cell subsets prior to thymic selection, and these differences survived both positive and negative selection. In conclusion, these findings provide deeper insight into the generation and evolution of TCR repertoire generation.     

T cell repertoire in patients with multiple myeloma and monoclonal gammopathy of undetermined significance: Clonal CD8+ T cell expansions are found preferentially in patients with a low tumor burden

The T cell receptor (TCR) variable (V) gene repertoire was analyzed in patients with monoclonal gammopathy of undetermined significance (MGUS) (n = 17), multiple myeloma (MM) stage I (n = 16), MM stages II/III (n = 31) and agematched controls (n = 27) by immunofluorescence and flow cytometry using a panel of mouse monoclonal antibodies (mAb) (n = 10) against TCR Vα and Vβ gene products. T cell expansion was defined as a value ? thrice the normal median value for each respective TCR V mAb. Fifty-three percent of all patients displayed CD8⁺ expansion(s) as compared to 30% of age-matched controls (p = 0.001). Within the CD4 subset, 18% of the patients displayed T cell expansion(s) in comparison to 11% of the controls (not significant). Interestingly, the CD8⁺ expansion(s) were more frequently noted in patients with a low tumor burden (MGUS/MMI) (73%) as compared to those with advanced disease (MM II/III) (32% and control donors (30%) (p < 0.01). Likewise, multiple CD8⁺ expansions (two or more) were more common in MGUS/MM I patients than in MM II/III and controls (p = 0.01). The T cell expansions were stable over time in patients with a stable disease. A high degree of clonality of the expansions was detected by TCR CDR3 fragment length analysis, determination of Jβ gene usage and nucleotide sequencing. The frequent finding of oligoclonal CD8⁺ T cell expansions in patients with a low tumor mass, but not in patients with advanced disease justifies further work in order to identify the relevance of expanded CD8⁺ T cells. In one patient with T cell reactivity against the autologous myeloma idiotype, two expansions within the CD8 population (Vβ3 and Vβ5.2 respectively) displayed no reactivity against the idiotype. Instead, idiotype recognition was confined to a CD8 non-expanded Vβ22⁺ T cell population, with a highly restricted TCR usage (CDR3 fragment length analysis).     

Amplification of TCRβ gene rearrangements from micromanipulated single cells: T cells rosetting around Hodgkin and Reed-Sternberg cells in Hodgkin's disease are polyclonal

Despite accounting for only a minor fraction of all cells in Hodgkin's lymphoma tissue, the Hodgkin and Reed-Sternberg (HRS) cells represent the malignant tumor cell clone in Hodgkin's disease (HD). By far the most abundant cell type in the tumor tissue are CD4⁺ T cells. Some of them intimately associate with HRS cells forming rosettes around them. This study addresses the question whether the rosetting phenomenon reflects a specific interaction between T and HRS cells by asking whether the rosettes are composed of T cells expressing a restricted TCR repertoire. Single rosetting T cells were micromanipulated from frozen sections of tumor tissue in two cases of nodular sclerosing HD and one case of lymphocyte predominant HD. TCR Vβ gene rearrangements were amplified from these single cells by PCR. Of 83 potentially functional Vβ gene rearrangements obtained altogether from the three cases, 81 were found to be clonally unrelated. Furthermore, they did not show signs of selection of the receptor chains for recognition of common epitopes: The usage of Vβ and Jβ gene segments as well as the distribution of complementarity-determining region (CDR) 3 lengths was similar to what was seen in a collection of 60 Vβ gene rearrangements from blood of healthy donors and no recurrent CDR3 amino acid motifs were found. These data suggest that the HRS cells attract CD4⁺ T cells nonspecifically.     

INCREASE IN PERIPHERAL NATURAL KILLER CELL ACTIVITY IN PATIENTS WITH AUTOIMMUNE THYROID DISEASE

Changes in the activity and number of natural killer (NK) cells in peripheral blood in patients with autoimmune thyroid disease were examined. NK activity was measured in a 4-hr ⁵¹Cr-release assay and the number of NK cells was analyzed with FITC-conjugated monoclonal antibodies by use of an automated flow cytometer. NK activity in patients with untreated Graves' disease (n=25, 39.7+13.5%, P<0.05) and Hashimoto's thyroiditis (n=18, 41.0±14.2%, P<0.05) was high compared to the activity in non-pregnant controls (n=61, 32.6±15.0%). NK activity in patients with postpartum Graves' thyrotoxicosis (n=11, 48.6±18.9%) was markedly increased compared to the activity in non-pregnant controls (P<0.01) and in postpartum controls (n=29, 33.8±15.2%, P<0.05), although the mean ages of each group did not differ significantly. Moreover, NK activities in the thyrotoxic state were significantly higher than those in the euthyroid state in the same patients with postpartum Graves' thyrotoxicosis or with postpartum destructive thyrotoxicosis. The number of CD16 positive cells increased in patients with postpartum Graves' thyrotoxicosis. However the number of CD 16 and CD57 positive cells were normal in all other groups of patients. These results indicate that an increase of NK activity is associated with exacerbation of autoimmune thyroid disease both in Hashimoto's thyroiditis and in Graves' disease and suggest that NK cells might have an important role for the control of disease activity in autoimmune thyroid disease.     

Decreases in alpha beta T cell receptor negative T cells and CD8 cells, and an increase in CD4+ CD8+ cells in active Hashimoto''s disease and subacute thyroiditis.

We examined peripheral lymphocyte subsets in patients with autoimmune thyroid disease, or subacute thyroiditis, in the active stage when possible. During destructive thyrotoxicosis arising from alpha beta T cell receptor (TCR) negative T (WT31-CD3+) cells and CD8 (CD4-CD8+) cells decreased and those of CD4+CD8+ cells increased slightly, resulting in proportional increases in CD4 (CD4+CD8-) cells, non-T, non-B (CD5-CD19-) cells, and the CD4/CD8 cell ratio. Changes were similar in active subacute thyroiditis. During stimulative thyrotoxicosis in active Graves' disease, the numbers of such T lymphocyte subsets were not changed, but only the number of CD5+ B (CD5+CD19+) cells increased markedly, resulting in proportional decreases in total T (CD3+) cells, alpha beta+ TCR T (WT31+CD3+) cells, CD8 cells, and non-T, non-B cells. A serial study of some of the patients showed opposite changes in alpha beta TCR- T cells, the CD4/CD8 cell ratio, and CD5+ B cells between the active stages of Graves' and Hashimoto's diseases. alpha beta TCR- T cells were mostly gamma delta TCR+ T (IIF2+ CD3+) cells in these patients. These data suggest that alpha beta TCR-T (gamma delta TCR+ T), CD8, and CD4+ CD8+ cells are important in thyroid destruction in Hashimoto's disease and subacute thyroiditis, and that CD5+ B cells are important in thyroid stimulation in Graves' disease.     

Skewing of TCR V genes to CD4+ and CD8+ subsets of T lymphocytes is not determined by amino acid composition of CDR3

TCR V genes show differing expression patterns, termed skewing, in CD4+ and CD8+ subsets of T lymphocytes. To determine which elements of the TCR V regions contribute to these observed TCR V gene skewing patterns, we have performed an in-depth analysis, taking advantage of RT-PCR and DNA sequencing, which was focused on the multi-member TCRBV6 gene family. These studies allowed us to evaluate the contributions of the various elements, that constitute the TCR beta chain variable region, to the observed TCR V gene skewing patterns. The results of these analyses revealed that within the TCRBV6 family individual members exhibited differing skewing patterns, i.e. TCRB6S7 was significantly skewed towards the CD4+ T cell subset, whereas TCRBV6S5 was significantly skewed towards the CD8+ subset. Scrutiny of the usage of TCRBV6 family members in combination with TCRBJ gene usage and amino acid composition of CDR3 did not reveal obvious structural characteristics which would explain the differing skewing patterns between TCRBV6S7 and TCRBV6S5. Further examination of these TCR V regions showed that the CDR1 and 2 regions within these TCRBV elements were composed of different amino acids. These observations suggests that these components contribute to the observed TCR V gene skewing patterns.      

Preferential Positive Selection of T Lymphocytes which Express Two Different TCRα Chains, an Endogenous and a Transgenic

A hallmark of positive selection in T-cell receptor (TCR)-transgenic mice is a strong skewing towards the CD4⁺ or the CD8⁺ subset, depending on the class II or I restriction of the TCR, respectively. However, previous experiments in TCR transgenic mice specific for an Ig light chain (λ2³¹⁵)/I-E^d class II molecule did not fit into this scheme because the authors observed an anomalous skewing towards CD8. In this paper, the authors show that endogenous TCRα chains are expressed on > 90% of CD4⁺ and CDS⁺ cells in this particular transgenic strain, even on a selecting H-2^d haplotype. Endogenous TCRQ chains are first detected when double-positive thymocytes down-regulate either CD4 or CD8. Endogenous Vα seems to influence generation of T-cell subsets because CD4⁺ and CD8⁺ cells express different frequencies of endogenous Vα2 and Vα8. In the absence of endogenous TCRα chains in recombination-deficient TCR-transgenic severe combined immunodeficiency (SCID) mice, a strong skewing towards CD4⁺ T cells is seen, but such mice are severely T-cell deficient. As an explanation for these results, the authors suggest that the transgenic TCR has a too low affinity for efficient positive selection, therefore, TCRa gene rearrangements proceed. Endogenous TCRa paired with transgenic TCRβ could bind to class I or class II molecules, enhance positive selection and thereby production of CD4⁺ or CD8⁺ cells. Most of the ‘mismatched’ CD8⁺ cells are λ2³¹⁵-specific and I-E^d class II restricted, and may function as idiotype-specific suppressors of B cells. These results may help explain the origin of dual TCRα T cells. Furthermore, the authors suggest that T cells ‘mismatched’ for co-receptor/TCR MHC-specificity may be enriched among dual TCRa T cells.     

Contributions of CD4+, CD8+, and CD4+CD8+ T cells to skewing within the peripheral T cell receptor beta chain repertoire of healthy macaques.

Diversity in the peripheral T cell receptor repertoire of rhesus (Macaca mulatta) and pig-tailed macaques (Macaca nemestrina) has been studied by examining the profile of CDR3 lengths in TCR beta chains. Expressed CDR3 length distribution profiles for individual TCRBV families were obtained from total peripheral blood mononuclear cells (PBMC) and T cell subsets isolated from PBMC. These studies reveal that the T cell receptor repertoire of PBMC from healthy macaques often exhibits skewing in TCRBV family CDR3 profiles. The skewing of TCRBV family CDR3 profiles was evident as discrete expanded length(s) and was detected in up to 50% of the PBMC profiles. Analyses of separated T cell populations demonstrated that the CD8+ T cell subset was responsible for the majority of observed skewing in CDR3 length profiles. However, CD4+ T cells were also shown to contribute to the skewed peripheral PBMC repertoire in these animals. While certain TCRBV families frequently displayed skewed profiles, there was no concordance in the particular CDR3 lengths expanded among the different animals. Furthermore, an additional feature of the peripheral blood of the animals studied was the presence of an unusual population of extrathymic CD4 and CD8+ (double-positive) T cells (up to 9.6% in the PBMC of rhesus macaques). The double-positive T cells could be differentiated from CD4 single-positive and CD8 single-positive T cells by their increased surface expression of LFA-1 and decreased CD62L expression. The percentage of the double-positive T cells was higher in rhesus than pig-tailed macaques and contributed substantially to the peripheral T cell repertoire.     

Alterations in T-cell receptor Vbeta repertoire of CD4 and CD8 T lymphocytes in human immunodeficiency virus-infected children

Perturbations in the T-cell receptor (TCR) Vbeta repertoire were assessed in the CD4 and CD8 T lymphocytes of human immunodeficiency virus (HIV)-infected children who were receiving therapy during the chronic phase of infection by flow cytometry (FC) and PCR analysis. By FC, representation of 21 TCR Vbeta subfamilies was assessed for an increased or decreased percentage in CD4 and CD8 T cells, and by PCR, 22 TCR Vbeta subfamilies of CD4 and CD8 T cells were analyzed by CDR3 spectratyping for perturbations and reduction in the number of peaks, loss of Gaussian distribution, or clonal dominance. The majority of the TCR Vbeta subfamilies were examined by both methods and assessed for deviation from the norm by comparison with cord blood samples. The CD8-T-lymphocyte population exhibited more perturbations than the CD4 subset, and clonal dominance was present exclusively in CD8 T cells. Of the 55 total CD8-TCR Vbeta families classified with clonal dominance by CDR3 spectratyping, only 18 of these exhibited increased expression by FC. Patients with high numbers of CD8-TCR Vbeta families with decreased percentages had reduced percentages of total CD4 T cells. Increases in the number of CD4-TCR Vbeta families with increased percentages showed a positive correlation with skewing. Overall, changes from normal were often discordant between the two methods. This study suggests that the assessment of HIV-induced alterations in TCR Vbeta families at cellular and molecular levels yields different information and that our understanding of the immune response to HIV is still evolving.     

Abnormal T cell receptor V gene usage in myasthenia gravis: prevalence and characterization of expanded T cell populations

The usage of T cell receptor (TCR) Vα/Vβ chains on cells from 38 patients with myasthenia gravis (MG) was determined by flow cytometry. There was a decreased number of cells expressing Vβ2 in CD8⁺ and Vβ3 in CD4⁺ cells in patients compared with healthy individuals. Abnormal expansions of T cells using particular TCR Vα/Vβ gene products were found in 18/38 patients. A significantly higher usage of Vβ13 was observed but there was no restriction with regard to other TCR Vα/Vβ. Expanded cells belonging to both CD4⁺ and CD8⁺ were present in MG patients while restricted to the CD8⁺ population in healthy individuals. To elucidate the role of the expanded populations, we studied characteristics of the expanded and non-expanded T cells from MG patients who had persistent T cell expansions over more than 2 years. The cells were analysed with regard to phenotype, cytokine secretion, cytokine mRNA expression and reactivity with the autoantigen, the acetylcholine receptor. The characteristics of the expanded populations in MG clearly differed from those found in healthy individuals. More cells in the CD4⁺ expanded populations expressed HLA-DR and there was also a tendency for higher expression of CD25, CD28 and CD57. The number of cells spontaneously secreting cytokines was higher in the expanded populations. A dominant Th1-type cytokine secretion and mRNA expression was noted. Autoantigen-reactive CD4⁺ T cells were largely restricted to the expanded populations.