scholarly journals Clonal Expansion of Bone Marrow CD8+ T Cells in Acute Myeloid Leukemia Patients at New Diagnosis and Post Chemotherapy

2020 ◽  
Author(s):  
Zhongxin Feng ◽  
Qin Fang ◽  
Xingyi Kuang ◽  
Xin Liu ◽  
Ying Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Clonal Expansion ◽  
Cell Repertoire ◽  
Healthy Donors ◽  
Wide Range ◽  
Tcr Repertoire ◽  
Repertoire Diversity ◽  
New Diagnosis

Abstract Background: CD8+ T cells are crucial adaptive immune effectors and express receptors (T cell receptors, TCRs) that specifically recognize and eradicate tumor cells. The diversity of the TCR repertoire is generated by specialized genetic diversification mechanisms, leading to an extremely variable TCR repertoire capable of recognizing a wide range of antigens. However, the variations in CD8+ TCR diversity and their clinical implications in AML patients remain unknown.Methods: CD8+ T cells in 10 healthy donors and 31 AML patients at diagnosis and after chemotherapy were enriched using the Dynabeads CD8 Positive Isolation Kit. Flow cytometry were used for PD-1 expression level analysis of CD8+ T cells and CD8+PD-1+ and CD8+PD-1- T cell sorting. TCRβ deep sequencing was performed to analysis the CD8+ T cells clonal expansion and TCR repertoire diversity.Results: Diminished TCR repertoire diversity and expansional T cell clones were noted in the bone marrow of AML patients. In relapsed patients, T cells were found to be more clonally expanded post chemotherapy when compared to new diagnosis. Moreover, more significantly expanded TCRβ clonotypes were noted in CD8+ PD-1+ T cells than in CD8+ PD-1- T cells regardless of the time point of examination. Conclusions: Our systematic T-cell repertoire analysis may help better characterize CD8+ T cells pre- and post-chemotherapy in AML, which may provide insights into therapeutic strategies in hematological malignancies.

scholarly journals Oligoclonal Expansion and T-Cells Subpopulations in Patients with Aplastic Anemia at Different Stages of the Disease

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3864-3864
Author(s):  
Anastasia V. Abramova ◽  
Elena A. Mikhaylova ◽  
Zalina T. Fidarova ◽  
Yuliya O. Davydova ◽  
Nikolay M. Kapranov ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Aplastic Anemia ◽  
Memory T Cells ◽  
Clonal Expansion ◽  
Th Cells ◽  
Healthy Donors ◽  
Oligoclonal Expansion

Abstract Background. The main mechanism of the bone marrow (BM) failure in idiopathic aplastic anemia (AA) has an immunomediated character. Researching the T-cell clone's effect in the AA pathogenesis is very relevant at the present time. Oligoclonal expansion of T cells is frequent in AA and the identification of immunodominant T-cell clones can correlate with the disease activity and may possibly serve as response predictor to immunosuppressive therapy (IST). The aim. To identify T-cells subpopulations, expression of PD-1 and PD-L1 on T-cells and TCR-Vβ repertoires by flow cytometry in different groups of AA patients. Methods. Thirty AA patients (pts) with median age of 30.5 (19-71), m/f ratio 1:1,3 were divided in 3 groups: pts with newly diagnosed (ND) AA (n=13), pts with overall response to IST (OR) (n=10), non-response pts (NR) for 2 and more lines of IST (n=7). Flow cytometry was performed with BD FACS Canto II. We used commercial kit (IOTest® Beta Mark TCR Vb Repertoire) for evaluation of TCR-Vβ repertoire in the bone marrow (BM) of these patients. We performed analysis of BM samples from healthy donors as a control group (n=8). Due to low amount of donor samples the maximal value each of the 24 subclones (for CD4+ (T-helpers - Th) and CD8+ cells (T-cytotoxic cells - TCL)) was accepted as threshold. We concluded the presence of clonal expansion if TCR subclone exceeded this threshold. We identified different T-cell subpopulations in all 3 groups of AA and healthy donors by flow cytometry: double positive T-cells (CD3+CD4+CD8+), double negative T-cells (CD3+CD4- CD8-), Th (CD3+CD4+), TCL (CD3+CD8+), NK-T-cells (CD3+CD56+) out of CD3+ cells. Among Th and TCL cells was determined naive T-cells (CD28+CD95-), effector T-cells (CD28-CD95+), memory T-cells (CD28+CD95+), regulatory T-cells (CD4+CD127-CD25high) and subpopulations Th and TCL co-expressed PD-1 and PD-L1. Multiple comparisons were assessed by ANOVA or Kruskal Wallis test by GraphPad Prism software. Results. In our study all 30 AA patients had an immunodominant TCR-Vβ clones among Th and/or TCL cells. We identified the most common clonotypes in comparison with healthy donors - Vβ1, Vβ2, Vβ3 among the Th cells and Vβ3, Vβ9, Vβ13.1 among the TCL cells. In ND group Vβ1 was highly expanded in 5 (38.5%), Vβ3 - in 7 (53.8%) pts among Th, and Vβ3 - in 3 (23.1%) and Vβ9 - in 4 (30.8%) out of 13 pts among TCL. In OR group Vβ2 expansion was in 4 (40%) and Vβ3 - in 5 (50%) pts among Th; Vβ3 in 6 (60%) and Vβ9 in 6 (60%) out of 10 pts among TCL. In NR group the most frequent was Vβ13.1 clone in TCL - in 3 (42.9%) out of 7 pts. In NR group in overall clonal expansion was less frequent than in ND and OR groups. We also analyzed the previously mentioned subpopulations of T-cells in patients with AA in three groups (ND, OR, NR) compared to healthy donors (table 1). We obtained significant differences in the count of naive Th and TCL cells, memory T-cells in all three groups of AA patients compared to donors: proportion of naive Th and TCL cells was significantly higher and proportion of memory Th cells was lower in the donor group than in AA pts. The percent of TCL effectors was higher in ND AA pts compare to donors. We also found that cell count of activated Th (CD4+CD25+) was higher in the group of refractory pts. In OR pts proportion of PD-1-positive Th was higher than in donors. In NR pts Th and TCL co-expressed with PD-L1 were lower compare to donors (table 1). Conclusions. In our study we found immunodominant clonotypes in different AA pts and depletion of the pool of naive T cells. Dynamic observation of changes in the most common clonotypes in AA pts during treatment will provide suitable therapy tactics (allogenic bone marrow transplantation or IST). Disclosures No relevant conflicts of interest to declare.

scholarly journals Single Cell RNA Sequencing and V(D)J Profiling of T-Cell Large Granular Lymphocytosis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2404-2404
Author(s):  
Shouguo Gao ◽  
Zhijie Wu ◽  
Carrie Diamond ◽  
Bradley Arnold ◽  
Valentina Giudice ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Single Cell ◽  
Research Funding ◽  
Clonal Expansion ◽  
Rna Seq ◽  
Healthy Donors ◽  
Tcr Repertoire ◽  
Before And After ◽  
Repertoire Diversity

Abstract Introduction . T-cell large granular lymphocytosis (T-LGL) is a low grade lymphoproliferative disorder, often clinically manifest as bone marrow failure. Treatment with immunosuppressive therapies is effective, but the dominant clone may persist even in responding patients. The pathogenesis of T-LGL has not been fully elucidated. In this study, we performed single cell RNA sequencing (sc-RNA seq) and V(D)J profiling to discern clonotypes and gene expression patterns of T lymphocytes from T-LGL patients who were sampled before and after treatment. Methods. Blood was obtained from patients participating in a phase 2 protocol of alemtuzumab as second line therapy (NCT00345345; Dumitriu B et al, Lancet Haematol 2016). Leukapheresis was performed in 13 patients (M/F 7/6; median age 51 years, range 26-85) before and after 3-6 months alemtuzumab administration and in 7 age-matched healthy donors. Cryopreserved blood was enriched for T cells with the EasySep Human T cell Isolation Kit (Stem cell). sc-RNA seq was performed on the 10XGenomics Chromium Single Cell V(D)J + 5' Gene Expression platform, and sequencing obtained on the HiSeq3000 Platform. Barcode assignment, alignment, unique molecular index counting and T cell receptor sequence assembly were performed using Cell Ranger 2.1.1. Results. Four hundred fifty thousand cells from 13 patients and 107,000 cells from 7 healthy donors were profiled. We measured productive TCR chains (which fully span the V and J regions, with a recognizable start codon in the V region and lacking a stop codon in the V-J region, thus potentially generating a protein). We detected at least one productive TCR α-chain in 50%, one productive TCR β-chain in 69% and paired productive αβ-chains in 47% of all cells. There was loss of TCR repertoire diversity in patients which was quantified by Simpson's diversity index; most patients showed oligoclonal or, less frequently, monoclonal expansion of the TCR repertoire (Fig. A). Regardless of clinical response, alemtuzumab treatment did not correct the low TCR repertoire diversity. TCR repertoires can be classified as "public", when they express identical TCR sequences across multiple individuals, or "private", when each individual displays distinct TCR clonotypes. No TCRA or TCRB CDR3 homology among patients was observed: most TCR clonotypes appeared to be private. Our data suggests that T-LGL is etiologically heterogenous disease, consistent with T cell expansion in response to a variety antigens, in diverse HLA contexts, or randomly. Despite differences of TCR among patients and healthy donors, and the presence of large clones in patients, distribution of TCR diversity followed the power law distribution in healthy donors and patients (Fig. B, showing the negative linear relationship between logarithmic expression of clone frequency and clone size). The observed distribution is consistent with a somatic evolution model, in which cell fitness depends on cellular receptor response to specific antigens and stimulation of cells by cytokine and other signals from the environment; fitted clones have higher birth-death ratios and thus expand (Desponds J et al, PNAS 2016). CD4 and CD8 T cells can be virtually separated by imputation from their transcriptomes (Fig. C). Comparison of gene expression between patients and healthy donors showed dysregulation of genes involved in pathways related to the immune response and cell apoptosis, consistent with a pathophysiology of T cell clonal expansion. We used diffusion mapping, which localizes datapoints to their eigen components in low-dimesional space, to characterize sources contributing to the gene expression phenotype: the first component was mainly from T cell activation and the second was associated with TCR expression. In LGL the T cell transcriptome appeared to be shaped by both lineage development and TCR rearrangement. Conclusion. We describe at the single cell level T clonal expansion profiles in T-LGL, pre- and post-treatment. Single cell analysis allows accurate recovery of paired α and β chains in the same cell and demonstrates a continuum of cell lineage differentiation. We found a range of differences in transcriptome and TCR repertoires across patients. Transcriptome data, coupled with detailed TCR-based lineage information, provides a rich resource for understanding of the pathology of T-LGL and has implications for prognosis, treatment, and monitoring in the clinic. Figure. Figure. Disclosures Young: GlaxoSmithKline: Research Funding; CRADA with Novartis: Research Funding; National Institute of Health: Research Funding.

scholarly journals Focused Regulatory T Cell Repertoires Are Indicative of Successful GvHD Control in Experimental and Clinical Stem Cell Transplantation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2029-2029
Author(s):  
Ivan Odak ◽  
Solaiman Raha ◽  
Saleh Tavil ◽  
Christian R Schultze-Florey ◽  
Arnold Ganser ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Ex Vivo ◽  
Foxp3 Expression ◽  
Clonal Expansion ◽  
Tcr Repertoire ◽  
Suppression Assay

Abstract Acute Graft versus Host Disease (aGvHD) remains a major complication and leading cause of mortality after allogeneic stem cell or bone marrow transplantation (BMT). Current strategies for treatment are still based on unspecific immunosuppressive therapy. Over the last decade, there have been major advances in the field of adoptive immunotherapy using regulatory CD4+CD25+Foxp3+ T cells (Treg cells). Nonetheless, not much is known about the exact mechanisms of Treg-mediated suppression, and even less about the importance of T cell receptor (TCR) specificity and its diversity on the functionality of Tregs. We hypothesized that an optimal Treg TCR repertoire is necessary for successful prevention of aGvHD. To test this hypothesis, we sequenced the TCR repertoire of 8 patients who were diagnosed with aGvHD on day 30 post transplantation and compared it with the TCR repertoire of nine GvHD-free patients. Analysis of GvHD-free patients on day 30 (and 100 days-follow up) revealed a lower TCR diversity when compared to the patients suffering from GvHD. A more detailed analysis of the TCR repertoire showed that in patients without GvHD, fewer clonotypes were needed to comprise 50% of the whole repertoire as compared with samples from patients with GvHD (Figure 1A). Thus, expansion of protective clones indicates their potent immunosuppressive capabilities. Next, we employed a well-described murine model of allogeneic BMT (BL/6-->Balb/c) with co-injection of Tregs. Recipient Balb/c mice transplanted in this fashion were previously shown to be protected from aGvHD. However, the mechanisms involved in this Treg-mediated protection are not fully understood. Therefore, Tregs were FACS sorted from B6.Cg-Foxp3tm1Mal/J mice based on their Foxp3 expression. Recipient mice were transplanted with T-cell depleted bone marrow and a mixture of conventional T cells (Tconv) and Tregs in 1:1 ratio. Transferred Tregs were re-sorted on day 7 and day 14 from secondary lymphoid organs based on the congenic marker Thy 1.1 and Foxp3 expression. Using this model, we investigated the kinetics of the Treg TCR repertoire early after BMT in 5 independent experiments. We found a consistently similar narrowing of the repertoire and clonal expansion in mice protected from GvHD (Figure 1B). Diversity analysis using inverse Simpson Index also confirmed our findings. These data further support the notion that a clonal expansion of Tregs is necessary for an optimal immunosuppression of an allogeneic response, both in human and in mice. To test the functionality and phenotype of such expanded Tregs, they were re-sorted from BMT-recipient mice 14 days after transplantation. These Tregs were expanded using α-CD3 and α-CD28 antibodies and were functionality tested in an in vitro Treg suppression assay. Re-sorted Tregs after expansion showed expression of established Treg surface and intracellular markers such as Helios, CD25, GITR and CTLA-4. For the suppression assay, responder CD4 Tconv were stained with a proliferation tracking dye eFluor670 and stimulated in vitro with CD3 and CD28 beads in the presence of different ratios of re-sorted and expanded, or polyclonaly activated Tregs as the control. Allo-specific ex vivo Tregs exhibited a superior suppressive potential when compared with polyclonaly activated Tregs in vitro. Taken together, our current study highlights the importance of specific Treg driven allo-response in GvHD prevention. Further studies are needed, particularly in larger patient cohorts to confirm these findings. However, we propose that this approach might lead to identification and subsequent use of specific Treg clones with high immunosuppressive capacity for the prevention of aGvHD. Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees. Koenecke:abbvie: Consultancy; BMS: Consultancy; Roche: Consultancy; Amgen: Consultancy.

scholarly journals Characterization of human cytomegalovirus peptide–specific CD8+ T-cell repertoire diversity following in vitro restimulation by antigen-pulsed dendritic cells

Blood ◽  
2002 ◽  
Vol 99 (1) ◽  
pp. 213-223 ◽  
Author(s):  
Karl Peggs ◽  
Stephanie Verfuerth ◽  
Arnold Pizzey ◽  
Jenni Ainsworth ◽  
Paul Moss ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Viral Replication ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Repertoire Diversity ◽  
Clonal Composition

Under conditions of impaired T-cell immunity, human cytomegalovirus (HCMV) can reactivate from lifelong latency, resulting in potentially fatal disease. A crucial role for CD8+ T cells has been demonstrated in control of viral replication, and high levels of HCMV-specific cytotoxic T-lymphocytes are seen in immunocompetent HCMV-seropositive individuals despite very low viral loads. Elucidation of the minimum portion of the anti-HCMV T-cell repertoire that is required to suppress viral replication requires further study of clonal composition. The ability of dendritic cells to take up and process exogenous viral antigen by constitutive macropinocytosis was used to study HCMV-specific T-cell memory in the absence of viral replication. The specificity and clonal composition of the CD8+ T-cell responses were evaluated using HLA tetrameric complexes and T-cell receptor β chain (TCRBV) spectratypic analyses. There was a skewed reactivity toward the matrix protein pp65, with up to 40-fold expansion of CD8+ T cells directed toward a single peptide-MHC combination. Individual expansions detected on TCRBV spectratype analysis were HCMV-specific and composed of single or highly restricted numbers of clones. There was preferential TCRBV gene usage (BV6.1/6.2, BV8, and BV13 in HLA-A*0201+ individuals) but lack of conservation of CDR3 length and junctional motifs between donors. While there was a spectrum of TCR repertoire diversity directed toward individual MHC-peptide combinations between donors, a relatively small number of clones appeared to predominate the response in each case. These data provide further insight into the range of anti-HCMV responses and will aid the design and monitoring of adoptive immunotherapy protocols.

scholarly journals The MEK Inhibitor Trametinib Enhances Diverse T Cell Reconstitution with Suppressing Xenogeneic Graft-Versus-Host-Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1929-1929
Author(s):  
Hidekazu Itamura ◽  
Hiroyuki Muranushi ◽  
Takero Shindo ◽  
Kazutaka Kitaura ◽  
Seiji Okada ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Mek Inhibitor ◽  
Effector Memory ◽  
Graft Versus Host ◽  
T Cell Reconstitution ◽  
Tcr Repertoire ◽  
Repertoire Diversity ◽  
Equity Ownership

Introduction: Early immune reconstitution without severe graft-versus-host disease (GVHD) is required for the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We showed that MEK inhibitors suppress GVHD but retain antiviral immunity and graft-versus-tumor (GVT) effects (Shindo, Blood2013; Itamura, Shindo, JCI Insight2016). Furthermore, we have shown that they attenuate graft rejection but spare thymic function following rat lung transplantation (Takahagi, Shindo, Am J Respir Cell Mol Biol2019). Here we analyzed their effects on human polyclonal T cell reconstitution in xenogeneic transplant by evaluating T-cell receptor (TCR) repertoire diversity. Methods: As a xenogeneic GVHD model, human PBMCs were infused to NOD/Scid/JAK3null mice, immunodeficient mice lacking T/B/NK cells, after total body irradiation. Vehicle, tacrolimus, or the MEK inhibitor trametinib was administered from day 0 through 28 or day 15 through 28. Human TCR repertoire diversity was evaluated by an adapter ligation PCR method with next generation sequencing (Shindo, Oncoimmunol2018) in the liver, lung, and spleen. The assignment and frequencies of TCRαV/J clones were determined at the single-cell level. Their diversity and clonality were evaluated by Inv. Simpson's index 1/λ. Results: Trametinib prolonged their survival compared with vehicle (median survival: 88 vs 46 days, p<0.05). It enhanced engraftment of human leukocytes in peripheral blood (human CD45+cells: 11.0 vs 2.5%), but prevented their infiltration into the lung (human CD45+cells on day 60: 1.5 vs 6.5%). Treatment with vehicle resulted in skewed TCR repertoire with limited clones in the spleen, liver and lung. Interestingly, expansion of one specific clone (TRAV20/J10) was commonly observed, which might reflect the GVHD-inducing pathological clone (Fig. 1: 3D graphs show the frequencies of TCRαV/J clones). However, trametinib enabled diverse and polyclonal T cell engraftment without the TRAV20/J10 clone. While CD4+and CD8+T cells within injected human PBMCs mainly consisted of naïve (CD45RA+CD27+) and central memory (CD45RA-CD27+) T cells, infiltrating T cells in each organ showed effector memory (CD45RA-CD27-) T cell phenotype. Of note, CD8+T cells in the bone marrow, spleen, and lung of trametinib-treated recipients showed reduced effector memory T cells (CD45RA-CD27-) compared with vehicle-treated mice at day 28 (bone marrow 21.7 vs 74.7%, p<0.01; spleen 66.3 vs 88.7%, p<0.05; lung 33.0 vs 72.5%, p<0.05), which indicating that MEK inhibition suppresses functional differentiation of human T cells in vivo. Furthermore, trametinib treatment from day 14 to 28 still ameliorated clinical GVHD score, and maintained polyclonal T cell repertoire. Conclusions:GVHD can be characterized with skewed TCR repertoire diversity and expansion of pathological T cell clones in the target tissues. Trametinib suppresses GVHD but maintains polyclonal T cell reconstitution, even in established GVHD. These results explain the facts that MEK inhibitors separate GVHD from GVT effects/antimicrobial immunity. Furthermore, MEK inhibition enhances immune reconstitution after allo-HSCT, which would avoid post-transplant complications. Disclosures Shindo: Novartis: Research Funding. Kitaura:Repertoire Genesis Inc.: Employment. Okada:Bristol-Myers Squibb: Research Funding; Japan Agency for Medical Research and Development: Research Funding. Shin-I:BITS Co., Ltd: Equity Ownership. Suzuki:Repertoire Genesis Inc.: Equity Ownership. Takaori-Kondo:Celgene: Honoraria, Research Funding; Novartis: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Ono: Research Funding; Takeda: Research Funding; Kyowa Kirin: Research Funding; Chugai: Research Funding; Janssen: Honoraria; Pfizer: Honoraria. Kimura:Ohara Pharmaceutical Co.: Research Funding; Novartis: Honoraria, Research Funding.

scholarly journals Dynamics of TCR repertoire and T cell function in COVID-19 convalescent individuals

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Lingjie Luo ◽  
Wenhua Liang ◽  
Jianfeng Pang ◽  
Gang Xu ◽  
Yingying Chen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Critical Role ◽  
World Health ◽  
Cell Repertoire ◽  
Metabolic Functions ◽  
Tcr Repertoire ◽  
Discharged Patients ◽  
Health Organization

AbstractSARS-CoV-2 outbreak has been declared by World Health Organization as a worldwide pandemic. However, there are many unknowns about the antigen-specific T-cell-mediated immune responses to SARS-CoV-2 infection. Here, we present both single-cell TCR-seq and RNA-seq to analyze the dynamics of TCR repertoire and immune metabolic functions of blood T cells collected from recently discharged COVID-19 patients. We found that while the diversity of TCR repertoire was increased in discharged patients, it returned to basal level ~1 week after becoming virus-free. The dynamics of T cell repertoire correlated with a profound shift of gene signatures from antiviral response to metabolism adaptation. We also demonstrated that the top expanded T cell clones (~10% of total T cells) display the key anti-viral features in CD8+ T cells, confirming a critical role of antigen-specific T cells in fighting against SARS-CoV-2. Our work provides a basis for further analysis of adaptive immunity in COVID-19 patients, and also has implications in developing a T-cell-based vaccine for SARS-CoV-2.

scholarly journals TCR Repertoire Characterization for T Cells Expanded in Response to hRSV Infection in Mice Immunized with a Recombinant BCG Vaccine

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 233
Author(s):  
Emma Rey-Jurado ◽  
Karen Bohmwald ◽  
Hernán G. Correa ◽  
Alexis M. Kalergis
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
T Cell Response ◽  
Bcg Vaccine ◽  
Lung Damage ◽  
Cell Repertoire ◽  
Cell Immunity ◽  
Tcr Repertoire ◽  
Syncytial Virus

T cells play an essential role in the immune response against the human respiratory syncytial virus (hRSV). It has been described that both CD4+ and CD8+ T cells can contribute to the clearance of the virus during an infection. However, for some individuals, such an immune response can lead to an exacerbated and detrimental inflammatory response with high recruitment of neutrophils to the lungs. The receptor of most T cells is a heterodimer consisting of α and β chains (αβTCR) that upon antigen engagement induces the activation of these cells. The αβTCR molecule displays a broad sequence diversity that defines the T cell repertoire of an individual. In our laboratory, a recombinant Bacille Calmette–Guérin (BCG) vaccine expressing the nucleoprotein (N) of hRSV (rBCG-N-hRSV) was developed. Such a vaccine induces T cells with a Th1 polarized phenotype that promote the clearance of hRSV infection without causing inflammatory lung damage. Importantly, as part of this work, the T cell receptor (TCR) repertoire of T cells expanded after hRSV infection in naïve and rBCG-N-hRSV-immunized mice was characterized. A more diverse TCR repertoire was observed in the lungs from rBCG-N-hRSV-immunized as compared to unimmunized hRSV-infected mice, suggesting that vaccination with the recombinant rBCG-N-hRSV vaccine triggers the expansion of T cell populations that recognize more viral epitopes. Furthermore, differential expansion of certain TCRVβ chains was found for hRSV infection (TCRVβ+8.3 and TCRVβ+5.1,5.2) as compared to rBCG-N-hRSV vaccination (TCRVβ+11 and TCRVβ+12). Our findings contribute to better understanding the T cell response during hRSV infection, as well as the functioning of a vaccine that induces a protective T cell immunity against this virus.

scholarly journals Determinants governing T cell receptor α/β-chain pairing in repertoire formation of identical twins

2019 ◽  
Vol 117 (1) ◽  
pp. 532-540 ◽  
Author(s):  
Hidetaka Tanno ◽  
Timothy M. Gould ◽  
Jonathan R. McDaniel ◽  
Wenqiang Cao ◽  
Yuri Tanno ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
High Throughput ◽  
Monozygotic Twin ◽  
Amino Acid Sequences ◽  
Structural Constraints ◽  
Single Chain ◽  
Cell Repertoire ◽  
Tcr Repertoire ◽  
Β Chain

The T cell repertoire in each individual includes T cell receptors (TCRs) of enormous sequence diversity through the pairing of diverse TCR α- and β-chains, each generated by somatic recombination of paralogous gene segments. Whether the TCR repertoire contributes to susceptibility to infectious or autoimmune diseases in concert with disease-associated major histocompatibility complex (MHC) polymorphisms is unknown. Due to a lack in high-throughput technologies to sequence TCR α–β pairs, current studies on whether the TCR repertoire is shaped by host genetics have so far relied only on single-chain analysis. Using a high-throughput single T cell sequencing technology, we obtained the largest paired TCRαβ dataset so far, comprising 965,523 clonotypes from 15 healthy individuals including 6 monozygotic twin pairs. Public TCR α- and, to a lesser extent, TCR β-chain sequences were common in all individuals. In contrast, sharing of entirely identical TCRαβ amino acid sequences was very infrequent in unrelated individuals, but highly increased in twins, in particular in CD4 memory T cells. Based on nucleotide sequence identity, a subset of these shared clonotypes appeared to be the progeny of T cells that had been generated during fetal development and had persisted for more than 50 y. Additional shared TCRαβ in twins were encoded by different nucleotide sequences, implying that genetic determinants impose structural constraints on thymic selection that favor the selection of TCR α–β pairs with entire sequence identities.

Influence of Graft Versus-Host Disease Prophylaxis Regimen On T-Cell Repertoire Diversity Following Reduced-Intensity HLA-Matched Unrelated Donor Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3054-3054 ◽  
Author(s):  
Rachel B. Salit ◽  
Frances T. Hakim ◽  
Michael R. Bishop ◽  
Thea M. Friedman ◽  
Robert Korngold ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Reconstitution ◽  
Unrelated Donor ◽  
Matched Unrelated Donor ◽  
Cell Repertoire ◽  
Post Transplant ◽  
Gvhd Prophylaxis ◽  
Repertoire Diversity ◽  
Reduced Intensity

Abstract Abstract 3054 Background: A clearly superior graft-versus-host disease (GVHD) prophylaxis regimen has not been established for patients undergoing reduced intensity allogeneic hematopoetic stem cell transplantation (HSCT) from matched unrelated donors (URD). Encouraging results have been reported with both the combination of alemtuzumab and cyclosporine (AC) and the regimen of tacrolimus, methotrexate, and sirolimus (TMS) in the URD setting. These two regimens work by biologically distinct mechanisms and may have markedly different effects on immune reconstitution. T-cell receptor (TCR) spectratyping analysis, which provides information on antigen receptor diversity, is a valuable method for monitoring post-transplant immune reconstitution. As part of a randomized pilot study, we prospectively assessed the effects of AC vs. TMS on TCR Vb repertoire diversity in patients undergoing reduced intensity HLA-matched unrelated donor transplantation. Methods: Twenty patients (median age 53 yrs; range 24–70 yrs) with hematologic malignancies received reduced intensity conditioning (fludarabine 30 mg/m2/day and cyclophosphamide 1200 mg/m2/day IV Day -6 to -3) followed by a 10/10 HLA-matched unrelated donor T-cell replete mobilized peripheral blood allograft. Patients were randomized to receive either: AC (n=10): alemtuzumab 20 mg/day IV over 8 hours Days -8 to -4 and cyclosporine starting at Day -1 with a 10% per week taper starting at Day +100 or TMS (n=10): tacrolimus and sirolimus starting at Day -3 with a 33% taper at Day +63 and Day +119 and methotrexate 5 mg/m2 IV, Days +1, +3, +6, and +11. Blood samples were collected from the donor and patient at baseline and the patient at 1, 3, 6 and 12 months post-transplant for TCR spectratyping analysis. All comparisons are based on an exact Wilcoxon rank sum test; p values < 0.01 were significant because of multiple comparisons. Results: Patients on the AC arm had significantly fewer T-cells on Day +14 compared with the TMS arm (median CD3+ = 1 cells/μl vs 356 cells/μl; CD4+ = 0 cells/μl vs 243 cells/μl; CD8+ = 0 cells/μl vs. 59 cells/μl; each p<0.0001); there was less disparity at Day +28 (median CD3+ = 45 cells/μl vs. 398 cells/μl; CD4+ = 36 cells/μl vs. 218 cells/μl; CD8+= 5 cells/μl vs 152 cells/μl; each p 0.002). By Day +100, lymphocyte recovery was not appreciably different between the two arms (median CD3+ = 242 cells/μl vs. 445 cells/μl (p = 0.095): CD4+ = 106 cells/μl vs. 212 cells/μl (p=0.28); CD8+ = 72 cells/μl vs. 135 cells/μl (p = 0.03). NK-cell recovery was slightly less in the AC vs. TMS arm at Day +14 (median NK = 27 cells/μl vs. 70 cells/μl; p = 0.01) and at Day +28 (median NK = 29 cells/μl vs. 150 cells/μl; p=0.02). There was no difference by Day +100 (median NK = 124 cells/μl vs. 88 cells/μl; p=0.31). B-cell reconstitution was negligible in both arms through Day +100. Assessment of CD4+ TCR Vb repertoire diversity by spectratyping demonstrated significantly lower diversity in patients receiving AC at 1 (p = 0.0003), 3 (p = 0.0003) and 6 (p=0.003) months post transplant compared with patients receiving TMS. CD8+ TCR spectratyping similarly revealed significantly reduced diversity in the AC arm at 3 (p = 0.001) and at 6 months (p = 0.003), and a trend toward significance at 12 months (p = 0.07). On each of the 2 arms, 2 of 10 patients developed acute Grade II-IV GVHD. Of the 5 patients on the AC arm who were seropositive for CMV, all 5 reactivated CMV by PCR within the first 60 days and reactivated 2–5 times in the first year. In contrast, only 3 of 5 seropositive patients reactivated CMV on the TMS arm and only one reactivated in the first 60 days. Conclusions: Two factors may have contributed to the loss of repertoire diversity in the AC arm. First, the alemtuzumab regimen may have severely depleted the infused donor T-cells. Second, stimulation by reactivating virus may have induced expansion of CMV-specific memory and effector T-cells, resulting in a skewed and oligoclonal T-cell repertoire. Especially in CD8+ T-cells, CMV has been shown to produce significant oligoclonal expansion (including CD4+: CD8+ ratio inversion). The loss of T-cell numbers and repertoire may in turn have contributed to the prevalence of early CMV reactivation. Thus, despite the similarities in frequency of acute GVHD in this small sample, it appears that these two commonly used GVHD prophylaxis regimens have very different effects on post-transplant immune reconstitution in the first 6 months after allogeneic HSCT. Disclosures: No relevant conflicts of interest to declare.

T Cell Exhaustion and Downregulation of Cytotoxic NK Cells – an Immune Escape Mechanism in Adult Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3781-3781
Author(s):  
Eolia Brissot ◽  
Sawa Ito ◽  
Kit Lu ◽  
Carly Cantilena ◽  
B. Douglas Smith ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Research Funding ◽  
Immune Escape ◽  
Nk Cell ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Healthy Donors

Abstract Adult acute lymphoblastic leukemia (ALL) remains a therapeutic challenge with less than 40% long term survival. There is growing evidence that malignant diseases exert an “immune editing” effect which blocks antitumor immunity and permits tumor growth through immune evasion. Such tumor escape represents an obstacle for anticancer immunotherapy. In ALL such immune escape mechanisms are not well characterized. We therefore profiled cellular immunity in ALL, by characterizing the subsets of T cells, regulatory T cells (Treg), natural killers (NK) cells and γd T cells, using various functional markers including T cell exhaustion and NK cell activating or inhibitory molecules. Forty ALL patients were included in the study. The median age was 39 y (range, 18-75). Thirty-six presented with B-lineage ALL and 4 with T-lineage ALL. Mononuclear cells were isolated from blood (n=19) or bone marrow (n=21) at the onset of leukemia or at relapse. The median infiltration of blasts was 85% (range 24-96%). Healthy donor peripheral blood (n=12) and bone marrow (n=9), from age and gender matched population, were simultaneously analyzed as controls. Extra-and intra cellular staining were performed using using antibodies directed against CD3, CD4, CD8, CD45, CD45, CD45RA, CD45RO, CCR7, CD95, CD27, CD19, CD14, CD127, CD25, Foxp3, Helios, αβTCR, HLA-DR, CD117, CD20, CD10, CD22, CD34, LAG3, PD1, PDL1, CD56, NKG2A, NKG2C, NKG2D, KIR2DL1, KIR2DL3, CD57, CD33, CD11b, CD15, CD38 and CD24. Data were acquired on a BD LSRFORTESSA flow cytometer. The expression of programmed cell death 1 (PD-1, CD279) receptor on CD8+T cells was significantly increased in blood and bone marrow of ALL patients compared to healthy donors (p<0.0001 and p=0.004, respectively) (Fig. 1). Focusing on the different subsets, CD8+ effector memory T cells significantly over-expressed PD-1 in blood and bone marrow of ALL patients compared to healthy donors (p=0.008 and p=0.04, respectively). Moreover, there was a significant positive correlation between PD-1 expression on CD8+ effector memory T cells and blast infiltration (R2=0.23, 95%CI 0.026-0.76, p=0.04). Expression of the co-inhibitory receptor lymphocyte-activation gene 3 (LAG-3, CD223) was similar in ALL patients compared to healthy donors. A significantly higher frequency of T regulators (CD25+, CD127 low, Foxp3+) was found in bone marrow microenvironment in ALL patients (4.3% versus 1.6%, p=0.02). Concerning γd T cells, frequency was similar in blood and bone marrow of ALL patients compared with healthy donors. There was a significantly lower frequency of CD56dimNKG2A+KIR-CD57- (p=0.02) in the bone marrow of ALL patients indicating a maturation arrest. Interestingly, expression of the activating receptor NKG2D which plays an important role in triggering the NK cell–mediated tumor cell lysis was significantly reduced in NK cells of ALL patients while no difference in NK cell expression of NKG2C was found(Fig. 2). Adult patients with ALL show evidence of immune-editing of T cells and NK cells. This global immunosuppressive mechanism may contribute to the eventual escape of ALL from immune control. PD-1, overexpression, described in acute myeloid leukemia and chronic myeloid leukemia has been implicated in T-cell exhaustion and subsequent tumor immune evasion. Our data suggests similar immune escape mechanisms pertain in ALL. Effective antileukemia immunotherapy will require targeting one or more of these immunosuppressive pathways to achieve optimum results. Disclosures Fathi: Seattle Genetics, Inc.: Consultancy, Research Funding; Takeda pharmaceuticals International Co.: Research Funding; Exelixis: Research Funding; Ariad: Consultancy.

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