Analysis Of T-cell Receptor Vβ Repertoire Research Articles

Clinical Usefulness of T-Cell Receptor Vβ Repertoire Analysis for Differentiating Multisystem Inflammatory Syndrome in Japanese Children From Toxic Shock Syndrome and Kawasaki Disease.

The specific expansion of T-cell receptor β chain variable region (TCR-Vβ21.3 + ) CD4 + and CD8 + T cells was observed in Japanese patients with multisystem inflammatory syndrome in children. In contrast, these findings were not observed in patients with toxic shock syndrome and Kawasaki disease. T-cell receptor β chain variable region repertoire analysis to detect specific expansion of Vβ21.3 + T cells might be useful for differentiating multisystem inflammatory syndrome in children from toxic shock syndrome and Kawasaki disease.

Rapid evaluation of T cell clonality in the diagnostic work-up of mature T cell neoplasms: TRBC1-based flow cytometric assay experience

The identification of mature T cell neoplasms by flow cytometry is often challenging, due to overlapping features with reactive T cells and limitations of currently available T cell clonality assays. The description of an antibody specific for one of two mutually exclusive T cell receptor (TCR) β-chain constant regions (TRBC1) provides an opportunity to facilitate the detection of clonal TCRαβ+ T cells based on TRBC-restriction. Here we prospectively analyzed 14 healthy controls and 63 patients with the flow cytometry protocol currently used for suspected T cell neoplasm implemented with immunostaining targeting TRBC1. Specimens were firstly classified in 3 groups based on clinical records data, laboratory findings and immunophenotypic features. T cell clonality was assessed by TCR Vβ repertoire analysis and the new rapid TRBC1 assay. Results showed that TRBC1 unimodal expression was unequivocally associated with samples presenting with immunophenotypic aberrancies. Moreover, we demonstrated that the use of TRBC1 is useful in solving uncertain cases and confirmed the high sensitivity of the method in identifying small T cell clones of uncertain significance (T-CUS). Finally, we found a high degree of concordance (97%) comparing the currently available clonality assessment methods with the proposed new method. In conclusion, our results provided real-life evidence of the utility of TRBC1 introduction in the flow cytometric clonality evaluation for the routine diagnostic work-up of T cell neoplasms.

Recurrent STAT3 Mutations in the Lymphocytes of Pure Red Cell Aplasia without T-Cell Large Granular Lymphocytic Leukemia

▪Background: Activating somatic mutations of STAT3 are one of the most frequently recognized genetic alterations in T-cell lymphoma including T-cell large granular lymphocytic leukemia (T-LGLL). T-LGLL is associated with certain proportions of patients (pts) with acquired aplastic anemia (AA) and/or myelodysplastic syndromes (MDS). Pure red cell aplasia (PRCA), especially acquired PRCA, is a syndrome defined by an anemia with marked reduction or absence of erythroid production associated with immunological mechanisms. PRCA is classified into primary and secondary types including T-LGLL. Previous reports have described that T-LGLL with STAT3 mutations was closely associated with PRCA. However, it is yet to be clear whether STAT3-mutated T cells would be involved in the pathogenesis of other cytopenias, such as AA, MDS, primary PRCA or secondary PRCA without (w/o) T-LGLL. In this study, we analyzed the profiles of STAT3 gene mutations in bone marrow failure syndrome (BMFS)s including AA/MDS and PRCA.Methods: Peripheral blood samples were collected from 94 patients with BMFSs, including AA, MDS, AA/PNH, and PRCA with or w/o T-LGLL. When possible, T-cell receptor (TCR) Vβ repertoire of the patients was determined by flow cytometry. DNA was extracted from mononuclear cells (MNC), and STAT3 Y640F and D661Y, two hot spot mutations, were detected with sensitive allele specific-PCR (AsPCR) methods. Then we performed deep amplicon sequencing analysis of STAT3 in PRCA pts. CD8+ T cells were sorted if possible, and also subjected to the analyses.Results: The recruited pts included 38 AA, 20 MDS, 7 AA/PNH, 11 PRCA with T-LGLL, and 18 PRCA w/o T-LGLL. Among PRCA pts w/o apparent T-LGLL, 10 had primary PRCA and 8 had secondary PRCA including 2 with thymoma. The clinical characteristics of PRCA are shown in Table 1. TCR Vβ repertoire analysis were performed in 8 PRCA w/o T-LGLL and 5 PRCA with T-LGLL. All pts with PRCA associated with T-LGLL showed restricted Vβ repertoire. On the other hand, only 1pt showed monoclonal Vβ pattern in PRCA w/o TLGLL. AsPCR did not detect Y604F or D661Y mutations in any of 65 pts with AA, MDS, or AA/PNH. In contrast, 2 pts with PRCA and 4 pts with PRCA-T-LGLL were positive for either of the mutations, suggesting STAT3 mutations may be unique to PRCA pts. Further analysis with deep amplicon analysis in the 29 PRCA pts revealed STAT3 mutations in 11 pts (61%) of PRCA w/o T-LGLL, which included 6 pts with primary PRCA and 5 pts with secondary PRCA associated with autoimmune diseases (n=2), 2 thymoma (n=2) and thymic cancer (n=1). The median variant allele frequency of STAT3 mutations was 2.3% (0.2-48.9). N538Y and I653V were most frequent mutations in PRCA w/o T-LGLL. Eight pts (73%) with T-LGLL-associated PRCA were also positive for STAT3 mutation. Thirty-nine % and 70% of STAT3 mutation were found in SH2 domain in PRCA w/o T-LGLL and PRCA with T-LGL, respectively. There was no difference in clinical features between pts with and w/o STAT3 mutations.Discussion: The high frequency of STAT3 mutations in PRCA pts regardless of etiology and the absence of the mutation in pts with AA/MDS suggest that STAT3 mutations may serve as a molecular marker of PRCA. The discordant results with regard to AA/MDS pts from a previous report which detected STAT3 mutations in 4.9% of AA/MDS patients by AsPCR methods (Jerez et al. BLOOD 2013) may be due to the difference in ethnic backgrounds of the pts studied. Given very low frequencies of STAT3 mutations in AA and MDS that were revealed by several previous studies, JAK/STAT signaling systems in the axis of STAT3 mutations may have unique roles in the pathogenesis of PRCA.Conclusion: STAT3 mutations are frequently detected in pts with PRCA even in pts w/o T-LGLL and the JAK/STAT pathway may therefore be therapeutic targets of PRCA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

T Cell Phenotype and T Cell Receptor Repertoire in Patients with Major Depressive Disorder.

While a link between inflammation and the development of neuropsychiatric disorders, including major depressive disorder (MDD) is supported by a growing body of evidence, little is known about the contribution of aberrant adaptive immunity in this context. Here, we conducted in-depth characterization of T cell phenotype and T cell receptor (TCR) repertoire in MDD. For this cross-sectional case–control study, we recruited antidepressant-free patients with MDD without any somatic or psychiatric comorbidities (n = 20), who were individually matched for sex, age, body mass index, and smoking status to a non-depressed control subject (n = 20). T cell phenotype and repertoire were interrogated using a combination of flow cytometry, gene expression analysis, and next generation sequencing. T cells from MDD patients showed significantly lower surface expression of the chemokine receptors CXCR3 and CCR6, which are known to be central to T cell differentiation and trafficking. In addition, we observed a shift within the CD4+ T cell compartment characterized by a higher frequency of CD4+CD25highCD127low/− cells and higher FOXP3 mRNA expression in purified CD4+ T cells obtained from patients with MDD. Finally, flow cytometry-based TCR Vβ repertoire analysis indicated a less diverse CD4+ T cell repertoire in MDD, which was corroborated by next generation sequencing of the TCR β chain CDR3 region. Overall, these results suggest that T cell phenotype and TCR utilization are skewed on several levels in patients with MDD. Our study identifies putative cellular and molecular signatures of dysregulated adaptive immunity and reinforces the notion that T cells are a pathophysiologically relevant cell population in this disorder.

CDR3 analysis of TCR Vβ repertoire of CD8+ T cells from chickens infected with Eimeria maxima

CD8+ T cells play a major role in the immune protection of host against the reinfection of Eimeria maxima, the most immunogenic species of eimerian parasites in chickens. To explore the dominant complementarity-determining regions 3 (CDR3) of CD8+ T cell populations induced by the infection of this parasite, sequence analysis was performed in this study for CDR3 of CD8+ T cells from E. maxima infected chickens. After 5days post the third or forth infection, intraepithelial lymphocytes were isolated from the jejunum of bird. CD3+CD8+ T cells were sorted and subjected to total RNA isolation and cDNA preparation. PCR amplification and cloning of the loci between Vβ1 and Cβ was conducted for the subsequent sequencing of CDR3 of T cell receptor (TCR). After the forth infection, 2 birds exhibited two same frequent TCR CDR3 sequences, i.e., AKQDWGTGGYSNMI and AGRVLNIQY; while the third bird showed two different frequent TCR CDR3 sequences, AKQGARGHTPLN and AKQDIEVRGPNTPLN. No frequent CDR3 sequence was detected from uninfected birds, though AGRVLNIQY was also found in two uninfected birds. Our result preliminarily demonstrates that frequent CDR3 sequences may exist in E. maxima immunized chickens, encouraging the mining of the immunodominant CD8+ T cells against E. maxima infection.

Flow Cytometric Immunophenotypic Assessment of T-Cell Clonality by Vβ Repertoire Analysis in Fine-Needle Aspirates and Cerebrospinal Fluid

Flow cytometric T-cell receptor V(β) repertoire analysis (TCR-V(β)-R) is a sensitive method to detect T-cell clonality; however, its implementation in low-cellularity specimens has not been established. We developed a strategy to use TCR-V(β)-R in cerebrospinal fluid (CSF) and fine-needle aspirate (FNA) specimens. Initially, full TCR-V(β)-R was evaluated in diagnostic/screening specimens from 8 patients with T-cell neoplasia to determine tumor-specific TCR-V(β) protein expression. Subsequently, an abbreviated, patient-specific TCR-V(β)-R evaluation was performed in 17 paucicellular specimens from the patients (8 CSF, 9 FNA) for staging and monitoring of minimal residual disease (MRD). A single cocktail containing 3 anti-V(β) antibodies (1 tumor-specific and 2 negative controls) in combination with other antibodies chosen to help gate on atypical T cells is highly sensitive and specific for detecting low-level neoplastic T-cell involvement in paucicellular specimens. This TCR-V(β)-R strategy is valuable in staging and evaluating MRD in patients with T-cell non-Hodgkin lymphoma.

Analysis of T Cell Cloanlity of Ph+ Acute Lymphoblastic Leukemia with Chronic Gvhd in Continuous Remission after Allogeneic Hematopoietic Stem Cell Transplantation

Chronic graft-versus-host disease (cGVHD) is one of the major complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, recent study suggested that the presence of cGVHD might reduce the risk of relapse in Ph+ acute lymphoblastic leukemia (ALL) after allo-HSCT. GVHD is mediated by proliferation of special T-cell clones, we analyzed the T cell receptor (TCR) Vβ repertoire and cloanlity in Ph+ ALL patients undergone cGVHD after allo-HSCT in order to find the special T-cell clones associated with continuous remission.Allogeneic transplantations were performed in the first complete remission (CR) (6 ALLP190+ patients). The numbers of BCR-ABL copies in peripheral blood samples were assessed with the real-time quantitative RT-PCR (RQ-PCR) test at diagnosis, and every month after HSCT. The quantity of BCR-ABL transcript was normalized for normal ABL expression and the result was expressed as the ratio of BCR-ABL copies to ABL copies. With monitoring BCR-ABL copies of 6 BCR-ABLP190+ ALL patients after HSCT (the mean ratio of BCR-ABLP190/ABL at diagnose was 0.41 ± 0.39%), we found that 4 patients with extensive cutancous cGVHD had no evidence of leukemia recurrence, and the BCRABL transcripts of patients with cGVHD were remaining 0 copy, while 2 patients without cGVHD relapsed (the mean ratio of BCR-ABLP190/ABL was 7.56 ± 2.49%) and even dead within six months of HSCT.The expression and cloanlity analysis of TCR Vβ repertoire was detected in peripheral blood samples from patients with cGVHD by RT-PCR and genescan technique. TCR Vβ repertoires with polyclonal pattern were identified in normal controls and donor groups. However, the skew expression pattern of TCR Vβ repertoire could be detected in patients with cGVHD even more than 1 year after allo-HSCT. Among the 24 Vβ subfamilies, there were only 11~17 Vβ subfamilies expressed in cGVHD patients. Oligoclonal or monoclonal expanded T cells were identified in TCR Vβ 1, 2, 3, 7, 8, 12, 14, 16~20, 22 and 23 subfamilies in 4 ALL patients (BCR-ABLP190 remission) with cGVHD. As a contrast, oligoclonal or monoclonal expanded T cells were identified in TCR Vβ 2, 7, 8, 14, 17, 20, and 22 subfamilies in 2 chronic myeloid leukemia patients (BCR-ABLP210 remission) with cGVHD.In conclusion, the predominant usage and clonal expansion of TCR Vβ subfamily T cells could be found in Ph+ ALL patients with cGVHD and these groups of specific T-cell clones (Vβ 1, 3, 12, 16, 18, 19 and 23) were potentially associated with cGVHD in ALL and might be responsible for maintaining CR in Ph+ ALL patients after allo-HSCT. Research Funding.

T-Cell Receptor Vβ Repertoire Analysis in Patients with Chronic Idiopathic Neutropenia: Evidence for Presence of Predominant T-Cell Clones with Possible Pathogenetic Significance.

Chronic idiopathic neutropenia (CIN) is an acquired disorder of granulopoiesis characterized by hypoplastic and left-shifted granulocytic series in the bone marrow (BM). It has been shown that CIN patients display increased number of activated T-lymphocytes with myelosuppressive properties in the BM and peripheral blood (PB) that induce Fas-mediated apoptotic death of the BM myeloid progenitor cells by producing interferon-γ and Fas-ligand. Epidemiological data have also shown female predominance and HLA-DRB1*1302 genetic background in CIN patients. The aim of the present study was to characterize T-cell receptor (TCR) Vβ repertoire of CD3+ cells in patients with CIN and explore the presence of dominant T-cell expansions with possible pathogenetic significance. Eighty patients with CIN were studied after informed consent. All patients had PB neutrophil counts below 1800/μL (mean 1411±380 neutrophils/μL, range 100-1799 neutrophils/μL), displayed negative anti-neutrophil antibody activity and were satisfying the previously reported diagnostic criteria for the disease. PB and BM samples from the patients were subjected to flow-cytometric analysis for the quantification of the TCR Vβ repertoire of the CD3+ cells (IOTest Beta Mark kit, Beckman-Coulter). Vβ family expansions were defined as above of 2SD (standard deviation) from the mean values of 85 healthy controls. DNA samples from immunomagnetically sorted PB and BM CD3+ cells were also subjected to multiplex polymerase chain reaction (PCR) analysis using the BIOMED2 protocol that covers all Vβ TCR gene rearrangements. We found that 54 patients, i.e. a proportion of 67.5% displayed one or more prominent clones in PB CD3+ cells. The Vβ16, Vβ12 and, in a lower proportion, the Vβ7.2 were the most frequently expanded subsets in PB, identified in 53.75%, 15% and 6.25% of CIN patients, respectively. Furthermore, CIN patients as a group displayed statistically increased proportion of Vβ16, Vβ12 and Vβ7.2 subsets within the CD3+ PB cells (1.96±1.30, 2.49±2.56 and 2.97±1.72 respectively) compared to controls (0.90±0.29, 1.66±0.54 and 1.47±1.03, respectively) (P<0.001, P<0.01 and P<0.001, respectively). In 20 patients, BM cells were also studied. Sixteen of 20 patients (80%) showed one or more prominent clones in CD3+ BM cells. In accordance with the PB data, the Vβ16, Vβ12 and Vβ7.2 were also the most frequently expanded subsets in the BM, identified in 65%, 10% and 10% of CIN patients, respectively. Further analysis showed that although more than one T-cell subset might be expanded in each patient, an identical expansion pattern was noted individually in BM and PB. Interestingly, a proportion of patients displayed prominent clones only in BM and not in PB but not vice versa, suggesting selective expansion or persistence of these subsets within the BM. None of the patients displayed monoclonal TCR Vβ expansion on multiplex PCR and heteroduplex analysis of BM and PB CD3+ lymphocyte subsets. In conclusion, in vivo dominant T-cell responses are identified in the majority of patients with CIN. These data substantiate further our hypothesis for the T-cell mediated immune nature of CIN and provide novel insights in the pathophysiology of the disease with possible therapeutic implications.

The Feature of Distribution and Clonality of TCR Vα and Vβ Repertoire in Cord Blood.

Analysis of T cell receptor (TCR) Vα and Vβ repertoire is one of the sensitive methods to understand the distribution and clonality of T cells from different samples, which is used to identify clonal expansion T cells that response to tumor associated antigens. Cord blood has been used successfully as a source of hematopoietic stem cells for transplantation. Recently, stem cells transplantation was demonstrated more important as immunotherapy against malignance cells. And cord blood T cells are resourceful for production the specific CTL to use in leukemia immunotherapy. But the feature of distribution and clonality of TCR Vα and Vβ subfamily T cells in cord blood is not yet clearly defined. In the present study, the CDR3 of 29 TCR Vα and 24 Vβ subfamily genes were analyzed in T cells from 10 cases of cord blood, which obtained at delivery from full-term healthy pregnancies, using RT-PCR and genescan technique. Peripheral blood of nine cases healthy adult volunteers served as controls. The results showed that 15–19 of 29 Vα and 9–15 of Vβ subfamily T cells could be identified in the most cases, whereas only 2–4 of Vα subfamilies expressed was found in 3 cases. The most frequently expressed Vα subfamilies were Vα 3 and Vα 10 (100%), Vα 4, 5, 6, 8, 12, 15, 17 and Vα 21 (70%), Vα1, 13, 25, and Vα26 (60%), with a lower expression rate found in Vα16, 24 and Vα 28 (10%). Vα 9 and Vα 29 were not detected in both CB and healthy adults. In analysis of TCR Vβ repertoire, Vβ3, 5, 8 and Vβ9 genes could be identified in all samples, Vβ7 and Vβ13 could be found in 90% of samples, whereas Vβ4, 6, 11, 12, 18, 23 and Vβ24 were absent in all samples, which could be detected in normal peripheral blood samples. Genescan analysis showed that all PCR products of TCR Vα and Vβ subfamilies from cord blood displayed a Gaussian distribution of CDR3 lengths (multi-peaks), which are corresponding to polyclonal rearrangement pattern, except for tow cases, which displayed oligoclonal peak in Vα 24 or Vα28 respectively. In contrast, some Vα subfamily products from 8 of 9 cases of healthy adult volunteers contained at least an oligoclonal peak in different Vα subfamilies respectively, however, multi-peaks were found in all PCR products of TCR Vβ from normal peripheral blood. In conclusion, more than 20% of TCR Vα or Vβ subfamily T cells were absent in cord blood. The majority of TCR Vα and Vβ subfamily T cells in cord blood displayed polyclonality. Occasional oligoclonal peaks are identified in some cases.

Specific Cytotoxicity and Clonal Expansion of TCR Vβ Subfamily T Cells Induced by PML-RARα Peptide.

The analysis of T cell receptor (TCR) Vβ repertoire is one of the sensitive methods to identify the clonal expansion T cells which response to tumor associated antigens. Understanding the clonality and restricted usage of TCR Vβ repertoire of expanded T-cells induced by PML-RARα peptide may be useful in helping design the new immunotherapeutic strategy specifically for acute promyelocytic leukemia (APL).The aim of the present study was to investigate the specific cytotoxicity and clonality of TCR Vβ repertoire in cord blood T cells induced by PML-RARα peptide (LSSCITQGKAIETQSSSSEE) in vitro. Cord blood mononuclear cells were amplified by IL-2, anti-CD3 and anti-CD28 antibody with different concentration (16.7μg /ml, 33.3μg /ml or 50μg /ml respectively) of synthetic PML-RARα peptide. The induced T cells were collected at different time points after culture (3, 6, 9, 10, 12 or 15 days). The expression and clonality of TCR Vβ subfamilies within induced T cells were analyzed by using RT-PCR and genescan technique. The cytotoxicity of induced T cells was detected by LDH release assay. The results showed that the best condition for T cells induction and amplication was at a concentration with 16.7μg /ml of PML-RARα peptide and at a culture duration with 10 to 15 day. TCR Vβ repertoire analysis showed that restricted expression and cloanl expansion of TCR Vβ subfamily cord blood T cells could be identified after induction by PML-RARα peptide. Clonal expanded T cells were found in Vβ13, Vβ14 and Vβ16 subfamlies respectively. The induced T cells were showed to have the specific cytotoxicity for NB4 cell line (effector cells: tagerted cells=20:1), the cytotoxicity rates were 49.65±6.7% (p<0.05) at day 10th and 73.13±8.42% (p<0.01) at day 15th after culture, which show statistical significance in compare to the control group (without PML-RARα induction). In conclusions, the PML-RARα peptide could induce the clonal expansion T cells from cord blood in vitro, which may have specific cytotoxicity for PML-RARα+ cells.

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.

Overlapping cases with psoriasis and Sjögren syndrome: a study of lymphocyte response to staphylococcal enterotoxin B

Sjögren syndrome (SjS) is an auto-immune disease and immunological mechanisms have recently been suggested in the pathogenesis of psoriasis; however, co-existence of these diseases is relatively rare. To determine whether or not there exist the nature of common underlying abnormalities, five patients with psoriasis and SjS were studied. Two patients had psoriasis vulgaris (PV), two had psoriasis arthropathica (PA), and the other had generalized pustular psoriasis (GPP). Lymphocyte response against staphylococcal enterotoxin B was examined by 3H-thymidine incorporation. In one patient with GPP, T- cell receptor (TCR) Vβ repertoire of infiltrating T-cells into the lesional skin was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Stimulation index (S.I.) level of patients with psoriasis and SjS did not show any significant increases compared with patients with psoriasis or SjS alone; however, one patient with PV and SjS, who showed several serological immune imbalances, demonstrated a significant increase of S.I. level. Analysis of TCR Vβ repertoire of the lesion of GPP showed strong expression of Vβ 14 and 16 with mild expression of V 13-2 and 19, which may suggest that TCR Vβ repertoire became oligoclonal in case of complication of these two disorders. In conclusion, our data did not suggest any common immunological responses to staphylococcal enterotoxin in induction of both psoriasis and SjS.