A Longitudinal Analysis of the T-Cell Receptor Vβ Repertoire in Aplastic Anemia Patients at Initial Presentation, Remission, and Relapse.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2402-2402
Author(s):  
Yunfeng Cheng ◽  
Yong Tang ◽  
Spencer Green ◽  
Keyvan Keyvanfar ◽  
Tullia Bruno ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cell ◽  
Aplastic Anemia ◽  
T Cell Receptor ◽  
Immunosuppressive Treatment ◽  
Cell Receptor ◽  
Initial Presentation ◽  
Cell Counts ◽  
Therapeutic Decisions

Abstract Aplastic anemia is a bone-marrow-failure syndrome characterized by low blood-cell counts and a fatty bone marrow. In most cases, no obvious etiological factor can be identified, but clinical responses to immunosuppressive treatment (IST) strongly suggest an immune pathophysiology. Our previous study of T-cell receptor (TCR) Vβ (variable region of β-chain) repertoire usage by flow cytometry suggested that aplastic anemia results from antigen-specific lymphocyte attack on hematopoietic progenitors (Risitano et al. Lancet2004; 364:355). In the current work, 7 patients were investigated for Vβ pattern expression before first immunosuppresive treatment, at the remission, and again on relapse. The TCR Vβ repertoire was analyzed for CD4+ and CD8+ subsets, separately, by flow cytometry, using a monoclonal antibody set of 22 different Vβ chains. Most patients had very different patterns of Vβ usage from healthy individuals, and all but one showed expansion of at least one Vβ family before immunosuppressive treatment (Vβ family expansions were defined as 2 standard deviations (SD) from the means in controls). The median number of expanded Vβ families was 4 per patient among CD8CD28dim effector cells. At remission, almost all the initially expanded Vβ subfamilies decreased to less than 2SD of controls. At relapse, most of the expanded Vβ subsets were increased again. However, 5/7 patients showed new expanded Vβ subsets at recurrence of cytopenias, suggesting antigenic spread of new epitopes recognized by immune systems. Although no common pattern of specific expanded Vβ subsets could be identified among different patients, some Vβ subfamilies appeared to be more frequently involved (Vβ 5.1 and Vβ 5.2 were expanded in 4 of 7 patients both at initial presentation and relapse ). These data suggest that monitoring Vβ subsets in aplastic anemia, and potentially in other immune-mediated human diseases of a similar pathophysiology could be used to guide individual therapeutic decisions and in the development of new treatments.

Ehrlichiosis Mimicking T-Cell Lymphoma/Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4343-4343
Author(s):  
Ashok Malani ◽  
Robert Weigand ◽  
Vicram Gupta ◽  
Lawrence Hertzberg ◽  
Gautam Rangineni
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cell ◽  
T Cell Receptor ◽  
Gene Rearrangement ◽  
Bone Marrow Cells ◽  
Cell Lymphoma ◽  
Cell Receptor ◽  
T Cell Lymphoma ◽  
Pcr Analysis

Abstract Immunophenotyping by flow cytometry has revolutinized the diagnosis of blood cell disorders such as leukemias and lymphomas and is now commonly used in diagnosis and prognosis of such patients. We describe a case of human ehrlichiosis mimicking T-cell lymphoma/leukemia based on flow cytometry of bone marrow cells and confirmed by T-cell receptor gene rearrangement (TCR) by polymerase chain reaction (PCR). Treatment with doxycycline reversed these findings. A 20-year-old, Amish female presented with fatigue, fever, chills, sweating, low back pain, and lower abdominal pain for 2 days. She admitted to multiple bites from ticks 2 weeks prior to presentation and also reported having numerous animals such as cats, dogs, cows, goats, horses at her farm where she lived. Clinical exam was significant for fever of 101.4 F, heart rate of 118/min, BP of 80/60 mm Hg and a distended urinary bladder which was treated by catheter drainage. Relevant laboratory tests are shown in table 1. Table 1 Hemoglobin 9.7 12–16 gm/dl WBC 0.8 4–10.8 k/mm3 Platelets 16 150–400 k/mm3 Segments 62% 50–75% Lymphocytes 15% 20–40% Sodium 140 125–135 mmol/L AST 126 0–37 IU/L ALT 71 0–65 IU/L Alk. Phos. 49 50–136 IU/L LDH 691 91–190 IU/L Chest radiograph, Ultrasound and Computed tomography scan of the abdomen were within normal limits. With a provisional diagnosis of septic shock and suspicion for Ehrlichiosis, therapy with intravenous(IV) fluids, vasopressors and doxycycline was initiated. Blood was cultured and a sample was forwarded to CDC for analysis of tick borne infections. In order to evaluate and exclude blood disorders like leukemia and lymphoma in a patient with fever and pancytopenia, a bone marrow aspiration and biopsy was performed. It showed cytologically abnormal-appearing, large sized lymphocyte population with irregular nuclear membranes. Flow cytometry of the bone marrow cells revealed 8–10% of phenotypically abnormal T-cells with abnormally weak intensity of membrane surface CD3, CD5, and CD7 expression and negativeCD4 and CD8 expression. These cells also expressed HLA-DR and CD38 at uncommonly bright intensity and there were no CD34 benign immature B-cells. Cytogenetics however was normal. Interestingly, PCR analysis was positive for clonal TCR gamma gene rearrangement. These results were reported as consistent with involvement of marrow by a peripheral T-cell lymphoma/leukemia T-Cell receptor PCR analysis T-Cell receptor PCR analysis Since the patient was steadily improving with IV Doxycycline, we decided to wait and repeated the bone marrow aspiration a week later. This time the bone marrow exam was found to be normal morphologically, on flow cytometry and TCR gamma gene rearrangement by PCR. Patient was discharged on oral doxycycline after a stay of 13 days in the hospital. The blood test for ehrlichiosis from CDC was reported 3 weeks later as positive for Ehrlichia chaffeensis by PCR. Patient is doing well 6 months after the illness. This case illustrates that Ehrlichiosis can transiently cause T cell abnormalities resulting in false positive analysis on flow cytometry and TCR gamma gene rearrangement, thereby leading to false positive diagnosis of Ehrlichiosis. Reconfirmation with repeat studies need to be done before considering active treatment for lymphoma/leukemia.

T-cell Receptor β Chain Variability in Bone Marrow and Peripheral Blood in Severe Acquired Aplastic Anemia

1997 ◽  
Vol 23 (1) ◽  
pp. 110-122 ◽  
Author(s):  
Chantal Y. Manz ◽  
Pierre-Yves Dietrich ◽  
Valérie Schnuriger ◽  
Catherine Nissen ◽  
Aleksandra Wodnar-Filipowicz
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Aplastic Anemia ◽  
T Cell Receptor ◽  
Peripheral Blood ◽  
Cell Receptor ◽  
Β Chain

scholarly journals Oligoclonality and subpopulation structure of bone marrow T-cells in patients with aplastic anaemia

2020 ◽  
Vol 65 (4) ◽  
pp. 417-430
Author(s):  
A. V. Abramova ◽  
I. V. Galtseva ◽  
E. A. Mikhailova ◽  
N. M. Kapranov ◽  
Yu. O. Davydova ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Cell Subpopulation ◽  
Cytotoxic T Cell ◽  
Double Negative ◽  
Cell Subpopulations

Introduction. The main pathogenetic mechanism of the development of aplastic anemia (AA) is a violation of the immune regulation of hematopoiesis.Aim: to study of the subpopulation composition of T-cells and the repertoire of the T-cell receptor in AA patients.Patients and Methods. The study included AA patients (n = 40) without prior immunosuppressive therapy in 2018–2020. The T-cell subpopulation structure and T-cell receptor Vβ-family (TCR-Vβ) oligoclonality were studied in samples of bone marrow using flow cytometry.Results. We report characteristic properties of T-cell subpopulations of bone marrow in all AA patients: elevated counts of cytotoxic T-cells, effector CD4+ and CD8+ cells, CD4+ memory cells, which may suggest a long-term antigenic stimulation with subsequent activation of these cell subpopulations resulting in hyperexpression of pro-inflammatory cytokines. Diminishing of naive CD4+ and CD8+ cells, regulatory and double negative T-cells may indicate a relaxing control of cytokine-producing T-cells. A relationship has been established between the AA severity and counts of effector, regulatory, double negative and PD-1 positive T-cells. A highest count of potentially cytokine-producing T-cells and lowest count of cells involved in T-cell activity regulation were observed in very severe AA patients. Studies of the TCR-Vβ repertoire revealed oligoclonal expansion in the cytotoxic T-cell subpopulation.Conclusion. Enrichment in selected Vβ families suggests autoreactive T-cell clonality and attests to the immune nature of AA. A dynamic TCR-Vβ repertoire assay may be recommended in the disease monitoring. Flow cytometry helps identify valuable biomarkers for T-cell clone monitoring in AA and a better assessment of the disease progression.

Unexpected Monoclonal T Cell Line Derived From the Bone Marrow of a Multiple Myeloma Patient.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4905-4905
Author(s):  
Debora Levy ◽  
Graciela Aparecida Brocardo ◽  
Carla Rosa Teixeira de Godoy ◽  
Camila da Cruz Gouveia Linardi ◽  
Jorge Maria Ruiz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cell ◽  
B Cell ◽  
Cell Line ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Scid Mice ◽  
Bm Niche

Abstract Abstract 4905 Background Multiple Myeloma (MM) is a mature B-cell lymphoproliferative disorder in which the microenvironment of the bone marrow (BM) is important in its pathophysiology. BM niche is composed of osteoblasts, endothelial cells, stromal cells, adipocytes, extracellular matrix proteins and lymphocytes. The interaction between MM cells and BM triggers multiple proliferative and antiapoptotic signaling pathways. Moreover, an adequate microenvironment could increase survival and chemoresistance of MM cells to current therapies. However, our knowledge in this field is still poor and many details are unknown. Therefore, there is a strong need to further study the MM BM niche and understand how it influences MM cell growth, survival and development of resistance to chemotherapy. Methods BM (with 31.6% of plasma cells) was obtained from a 67-yr old woman (EC DS IIIA, ISS II) before treatment. Whole BM was cultivated in RPMI with10% FBS. After 36 days several colonies were observed and the cells had a fibroblast-like appearance. They were transferred again to another flask and after 30 days, small rounded floating cells were seen in the media or attached to the adherent fibroblast-like cells. The floating cells were transferred to another flask. They were named MOX cells. Immunophenotyping was done by flow cytometry before and after 9 days of culture and in the MOX cells. PCR was performed to detect clonality in gamma T cell receptor (TCR). In other experiment, seven Nude/SCID mice were injected IV or subcutaneously (SC) with MOX cells (4×106/100mL PBS) to test for tumorigenicity. After 15 and 30 days blood was drawn and examined for the presence of these cells. Animals were sacrificed at the 30th day when immunophenotyping was performed in blood and bone marrow. Results Cells prior to culture were CD45-, CD19-, CD38+++ and CD56+++ compatible with plasma cell phenotype. MOX cells had a doubling time of 20 h, DNA index of 1,19 (hyperdiploid) with 22.5% of cells in S, 13.88% in G2-M and 63.62% in G0-G1 phase. Unexpectedly, flow cytometry results of MOX cells showed a phenotype of mature T cell (CD45+++/CD38+CD3+/CD4+/CD8-/CD7+/CD5+/CD2-/TCRab+). The analysis of V(D)J rearrangement in T cell receptor demonstrated that the MOX cells were monoclonal biallelic to TCR gamma gene. We demonstrated the malignant feature of the MOX cells in Nude/SCID mice. MOX cells were observed in peripheral blood from all animals 15 and 30 days after IV or SC injection of MOX cells. After 30 days MOX cells were found in the BM in a considerable amount (10%). Conclusions 1) Monoclonal malignant T cells were unexpectedly obtained from the BM of a patient with MM, a neoplasia of the B-cell linage. 2) This new cell line is potentially useful for future studies in this field. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Multiplex, Droplet Digital PCR Assay for the Detection of T-Cell Receptor Excision Circles and Kappa-Deleting Recombination Excision Circles

2019 ◽  
Vol 66 (1) ◽  
pp. 229-238 ◽  
Author(s):  
Tracie Profaizer ◽  
Patricia Slev
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
T Cell Receptor ◽  
Reference Gene ◽  
Cell Receptor ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Blood Samples ◽  
Healthy Donors ◽  
Excision Circles

Abstract BACKGROUND T-cell receptor excision circles (TREC) and κ-deleting recombination receptor excision circles (KREC) concentrations can be used to assess and diagnose immune deficiencies, monitor thymic and bone marrow immune reconstitution, or follow responses to drug therapy. We developed an assay to quantify TREC, KREC, and a reference gene in a single reaction using droplet digital PCR (ddPCR). METHODS PCR was optimized for 3 targets: TREC, KREC, and ribonuclease P/MRP subunit p30 (RPP30) as the reference gene. Multiplexing was accomplished by varying the target's fluorophore and concentration. Correlation with clinical results was evaluated using 47 samples from healthy donors, 59 samples with T-cell and B-cell markers within the reference interval from the flow cytometry laboratory, 20 cord blood samples, and 34 samples submitted for exome sequencing for severe combined immunodeficiency disease (SCID). RESULTS The limit of the blank was 4 positive droplets, limit of detection 9 positive droplets, and limit of quantification 25 positive droplets, or 2.0 copies/μL. TREC and KREC copies/μL were as expected in the healthy donors and cord blood samples and concordant with the healthy flow cytometry results. Of the samples from the SCID Panel, 56.5% had a TREC count <20 copies/μL and 17.7% had a KREC count <20 copies/μL, suggestive of low T- and B-cell numbers, respectively. CONCLUSIONS Our multiplex ddPCR assay is an analytically sensitive and specific method for the absolute quantification of TREC and KREC. To the best of our knowledge, this paper is the first to describe the simultaneous quantification of TREC, KREC, and a reference gene by use of ddPCR.

T-cell receptor pharmacodynamics associated with survival and response to tremelimumab (T) in combination with durvalumab (D) in patients (pts) with unresectable hepatocellular carcinoma (uHCC).

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 4087-4087
Author(s):  
Patricia McCoon ◽  
Young S Lee ◽  
Robin Kate Kelley ◽  
Violeta Beleva Guthrie ◽  
Song Wu ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Single Agent ◽  
Clonal Expansion ◽  
Cell Receptor ◽  
Combination Regimen ◽  
Cell Counts ◽  
Significant Difference ◽  
Dose Dependent ◽  
Cell Clonal Expansion

4087 Background: Study 22, a phase 2 clinical study (NCT02519348) evaluating T (anti-CTLA-4) and D (anti-PD-L1) as monotherapies and in combination indicated the best efficacy-safety profile with a novel combination regimen containing a single, priming dose of T (T300+D). Additionally, an expansion of proliferative CD8+ lymphocytes at Day 15 was observed with T300+D that was associated with improved response. Here, an exploratory molecular analysis of peripheral blood T cell receptors is presented. Methods: Immune-checkpoint inhibitor-naïve pts were randomized to 1 of 2 T+D combinations: T300+D (T 300 mg [1 dose] + D 1500 mg, then D every 4 weeks [Q4W]) or T75+D (T 75 mg Q4W + D 1500 mg Q4W [4 doses], then D Q4W); or single agent D (1500 mg Q4W) or T (750 mg Q4W [7 doses] then Q12W). DNA was isolated from PAXgene-preserved whole blood collected at baseline and on Day 29 during the first cycle of Q4W dosing, and then underwent CDR3 sequencing of T-cell receptor β using the immunoSEQ Assay (Adaptive Biotechnologies, Seattle, WA). Associations with objective response rate (ORR) and overall survival (OS) were evaluated. Results: The number of evaluable pts, samples, and overall ORR and OS are provided (Table). Immunosequencing analysis did not reveal significant differences in baseline T-cell clonality across arms. Increased T-cell clonal expansion at Day 29 appeared to be T dose dependent (Table), with no significant difference in the median expansion between the D and T75+D arms. Across all arms, responders had a larger median number of expanded T-cell clones on Day 29 than nonresponders (77.5 vs 40), and this greater expansion trended with longer OS (Table). Further evaluation by arm demonstrated an increase in T-cell clonal expansion in responders vs nonresponders in the T300+D arm. Pts with T-cell expansion above the median in the T300+D and T75+D arms also exhibited longer OS. Both newly expanded and total expanded clones on Day 29 vs Day 1 were associated with improved OS. Conclusions: The observed T dose-dependent increase in T-cell clonal expansion trended with improved ORR and longer OS, with the greatest overall benefit seen with T300+D vs T75+D, D and T. This is consistent with the previously reported observation that T300+D led to the highest median proliferating CD8+ T-cell counts and radiographic response. Further work is needed to differentiate the relative contributions of CD4 and CD8 clonal expansion to increased efficacy. T300+D and D are being evaluated in the phase 3 HIMALAYA study (NCT03298451) in uHCC vs sorafenib. Funding: AstraZeneca. Clinical trial information: NCT02519348. [Table: see text]

scholarly journals Evidence for extrathymic generation of intermediate T cell receptor cells in the liver revealed in thymectomized, irradiated mice subjected to bone marrow transplantation.

1995 ◽  
Vol 182 (3) ◽  
pp. 759-767 ◽  
Author(s):  
K Sato ◽  
K Ohtsuka ◽  
K Hasegawa ◽  
S Yamagiwa ◽  
H Watanabe ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
T Cell ◽  
T Cell Receptor ◽  
Marrow Transplantation ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
Phenotypic Characterization ◽  
Receptor Cells

In addition to the major intrathymic pathway of T cell differentiation, extrathymic pathways of such differentiation have been shown to exist in the liver and intestine. In particular, hepatic T cells of T cell receptors or CD3 of intermediate levels (i.e., intermediate T cell receptor cells) always contain self-reactive clones and sometimes appear at other sites, including the target tissues in autoimmune diseases and the tumor sites in malignancies. To prove their extrathymic origin and self reactivity, in this study we used thymectomized, irradiated (B6 x C3H/He) F1 mice subjected to transplantation of bone marrow cells of B6 mice. It was clearly demonstrated that all T cells generated under athymic conditions in the peripheral immune organs are intermediate CD3 cells. In the case of nonthymectomized irradiated mice, not only intermediate CD3 cells but also high CD3 cells were generated. Phenotypic characterization showed that newly generated intermediate CD3 cells were unique (e.g., interleukin 2 receptor alpha-/beta+ and CD44+ L-selectin-) and were, therefore, distinguishable from thymus-derived T cells. The precursor cells of intermediate CD3 cells in the bone marrow were Thy-1+ CD3-. The extrathymic generation of intermediate CD3 cells was confirmed in other combinations of bone marrow transplantation, C3H --> C3H and B10.Thy1.1 --> B6.Thy1.2. The generated intermediate CD3 cells in the liver contained high levels of self-reactive clones estimated by anti-V beta monoclonal antibodies in conjunction with the endogenous superantigen minor lymphocyte-stimulating system, especially the combination of B6 --> (B6 x C3H/He) (graft-versus-host-situation).(ABSTRACT TRUNCATED AT 250 WORDS)

Prognostic value of T-cell receptor γ rearrangement in peripheral blood or bone marrow of patients with peripheral T-cell lymphomas

2008 ◽  
Vol 49 (2) ◽  
pp. 237-246 ◽  
Author(s):  
Christian SchüTzinger ◽  
Harald Esterbauer ◽  
Gregor Hron ◽  
Cathrin Skrabs ◽  
Martin Uffmann ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
T Cell Receptor ◽  
Peripheral Blood ◽  
Prognostic Value ◽  
Cell Receptor ◽  
T Cell Lymphomas ◽  
Peripheral T Cell Lymphomas
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