TCR Repertoire Diversity Assessed with Immunosequencing Is Associated with Patient Mortality Following Cord Blood Transplant

Abstract In order to study the clinical impact of T-cell receptor (TCR) diversity in the setting of umbilical cord blood transplantation (UCBT), we retrospectively analyzed samples from 76 patients in 2 independent study cohorts at separate institutions. At Fred Hutchinson Cancer Research Center (FHCRC), we followed 34 patients with hematological malignancies who underwent myeloablation and primarily double UCBT (2 single cord). This cohort was composed of 11 pediatric and 23 adult patients (median age, 26.5yrs), primarily with acute leukemia (n=26), 50% of whom had evidence of MRD at UCBT. They received fludarabine, cytoxan or treosulfan, and total body irradiation (TBI) with cyclosporine and mycophenylate mofetil as GvHD prophylaxis. At Dana-Farber Cancer Institute (DFCI), we followed 42 adult patients (median age, 52.3 yrs), 19 with acute leukemia, 18 of whom had evidence of disease at UCBT. All patients received reduced-intensity conditioning with fludarabine, melphalan, and anti-thymocyte globulin (ATG) followed by double UCBT with tacrolimus and sirolimus as GvHD prophylaxis. DFCI participants were selected to have predominantly cord T cell chimerism. We analyzed peripheral blood pre-transplant, and at 1, 2, 3, 6 and 12 months after UCBT, based on sample availability. We performed high-throughput sequencing of rearranged (TCR) loci to track presence and frequency of individual T cell clones in each patient across time-points, as well as to calculate estimated diversity of the TCR repertoire as a whole. We correlated our measure of TCR repertoire diversity with clinical outcome, using one-year survival as our primary endpoint. Diversity of the T-cell repertoire can provide a measure of immune competence. We hypothesized that restoration of TCR repertoire diversity would be associated with overall survival after UCBT. For the combined group of 63 patients with samples available 3 months after UCBT, 19 subsequently died within 1 year of transplant. These patients had significantly lower TCR repertoire diversity at 3 months than patients who survived at least one year (data are given as percentiles within each cohort due to differing amounts of blood available for analysis; median 28th percentile in patients who died vs. median 56th percentile for patients who survived 1 year, p-value = 0.003 by permutation test). When analyzed as 2 independent cohorts, the 29 patients at FHCRC with samples at 3 months after UCBT demonstrated significantly lower TCR repertoire diversity in those who died within 1 year of UCBT versus those who survived beyond 1 year (median TCR diversity 12,000 vs. 27,000, p-value = 0.015), as did the 34 patients with samples available 3 months after transplant at DFCI (median TCR diversity 1,200 vs. 6,400, p-value = 0.027). Between 100 days and 1 year after dUCBT 6 patients died of non-relapse causes (NRM) and 4 with relapse at FHCRC and 6 patients died of NRM and 3 with relapse at DFCI. Correlation of TCR diversity with clinical outcomes such as NRM, relapse, GvHD and correlation with clinical outcomes beyond 1 year are ongoing. In this study, we demonstrate that measurement of TCR repertoire diversity generated using high-throughput sequencing genes at 3 months after UCBT is significantly correlated with subsequent mortality within the first year. This correlation was demonstrated separately in two unrelated cohorts of UCBT recipients at different transplant centers of differing ages and conditioning regimens and in combined analysis of all recipients. Low T cell diversity in the peripheral blood as soon as 3 months may therefore be an early indicator of inadequate immune reconstitution and could potentially be used to tailor monitoring and therapy after UCBT in several clinical contexts. Disclosures Emerson: Adaptive Biotechnologies: Employment, Equity Ownership. Sherwood:Adaptive Biotechnologies: Employment, Equity Ownership. Carlson:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties.

Download Full-text

T Cell Repertoire Diversity After Umbilical Cord Transplant Predicts Mortality From Infection

Blood ◽

10.1182/blood.v120.21.4202.4202 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4202-4202 ◽

Cited By ~ 1

Author(s):

Colleen Delaney ◽

Ryan O Emerson ◽

Filippo Milano ◽

Anna Sherwood ◽

Adrienne Papermaster ◽

...

Keyword(s):

T Cell ◽

High Throughput ◽

High Throughput Sequencing ◽

T Cell Repertoire ◽

Cell Repertoire ◽

T Cell Clones ◽

Cell Clones ◽

Tcr Repertoire ◽

Repertoire Diversity ◽

Equity Ownership

Abstract Abstract 4202 Background In a transplant study run at Fred Hutchinson Cancer Research Center, 34 patients with high risk hematological malignancies underwent a myeloablative procedure and were subsequently transplanted with double umbilical cord blood units. Peripheral blood samples were collected from each patient before myeloablation, and at 28, 56, 100, 180, and 360 days post-transplant. Methods At each time point, we used the immunoSEQ platform to perform high-throughput sequencing of rearranged T Cell Receptor (TCR) loci. Using immunoSEQ data, we were able to track the presence and frequency of individual TCR clones in each patient across time-points, as well as measuring the diversity of the TCR repertoire as a whole. We correlated our measure of TCR repertoire diversity with clinical outcomes in this cohort. Results The study produced two primary results. First, using the ability to track clones, the reconstituting TCR repertoire is shown to oscillate wildly with nearly complete turnover of the T cell repertoire occurring at least monthly after CB transplant. The largest T cell clones present in each blood draw drop below detection within weeks, contrasting with control data in which the top clones in healthy patients are not only observed in multiple subsequent time-points, but remain at high frequency. The second result is a test of the hypothesis that diversity of the T cell repertoire is a measure of immunocompetence, as a clinical application of high-throughput sequencing. Of the 34 patients, six died between Day 100 and Day 360 of non-relapse causes. At both Day 56 and Day 100, the diversity of the T cell repertoires of those six patients were far lower than the T cell repertoire diversity values of the remaining patients (P-value = 0.015). Conclusions We have demonstrated that the reconstitution of clinical immunity in cord blood transplantation patients is characterized by a highly unstable T cell compartment with very rapid turnover of T cell clones. Despite the transience of individual T cell clones, however, by two months after transplant T cell repertoire diversity as measured by high-throughput TCR sequencing accurately predicts risk of non-relapse mortality. Disclosures: Emerson: Adaptive Biotechnologies: Employment, Equity Ownership. Sherwood:Adaptive Biotechnologies: Employment, Equity Ownership. Carlson:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties.

Download Full-text

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

Blood ◽

10.1182/blood-2019-128913 ◽

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.

Download Full-text

Characterization of the TCR β Chain CDR3 Repertoire in Subarachnoid Hemorrhage Patients with Delayed Cerebral Ischemia

International Journal of Molecular Sciences ◽

10.3390/ijms21093149 ◽

2020 ◽

Vol 21 (9) ◽

pp. 3149 ◽

Cited By ~ 1

Author(s):

Bong Jun Kim ◽

Dong Hyuk Youn ◽

Youngmi Kim ◽

Jin Pyeong Jeon

Keyword(s):

Subarachnoid Hemorrhage ◽

T Cell ◽

Cerebral Ischemia ◽

High Throughput Sequencing ◽

Delayed Cerebral Ischemia ◽

Adaptive Immune Response ◽

Cell Receptor ◽

P Value ◽

Repertoire Diversity ◽

Β Chain

Little is known of the adaptive immune response to subarachnoid hemorrhage (SAH). This study was the first to investigate whether T cell receptor (TCR) immune repertoire may provide a better understanding of T cell immunology in delayed cerebral ischemia (DCI). We serially collected peripheral blood in five SAH patients with DCI. High-throughput sequencing was used to analyze the TCR β chain (TCRB) complimentary determining regions (CDR) 3 repertoire. We evaluated the compositions and variations of the repertoire between admission and the DCI period, for severe DCI and non-severe DCI patients. Clonality did not differ significantly between admission and DCI. Severe DCI patients had significantly lower clonality than non-severe DCI patients (p value = 0.019). A read frequency of 0.005% ≤ – < 0.05% dominated the clonal expansion in non-severe DCI patients. Regarding repertoire diversity, severe DCI had a higher diversity score on admission than non-severe DCI. The CDR3 lengths were similar between admission and DCI. Among 728 annotated V-J gene pairs, we found that the relative frequencies of two V-J pairs were different at the occurrence of DCI than at admission, with T cells increasing by over 15%. TCRB CDR3 repertoires may serve as biomarkers to identify severe DCI patients.

Download Full-text

Driving T Cell Development in the Thymus.

Blood ◽

10.1182/blood.v104.11.3860.3860 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3860-3860

Author(s):

Cristina M. Joao ◽

Brenda M. Ogle ◽

Marilia Cascalho ◽

Jeffrey L. Platt

Keyword(s):

T Cell ◽

B Cells ◽

B Cell ◽

T Cell Development ◽

Cell Development ◽

Thymic Epithelial Cells ◽

P Value ◽

Tcr Repertoire ◽

Repertoire Diversity ◽

Deficient Mice

Abstract Background: Classic reports on lymphocyte development hold that B and T cells develop independently. This concept derives in part from the observation that patients with pure B cell immunodeficiency and hypogammaglobulinemia have a normal thymus and T cell numbers. Our recent findings however challenge this concept. We found that T cell development depends not only on the interaction of T cell precursors with thymic epithelial cells but also on other cells. Here we report that those other cells are B cells. Aims: The purpose of this study was to determine whether B cells drive T cell development and TCR diversification in the thymus. Methods: We compared the number of sub-populations of thymocytes and TCR repertoire diversity in B-cell deficient and B-cell proficient mice and in B cell deficient mice following immunoglobulin (Ig) injections. Total leucocytes numbers were determined with a Coulter counter and numbers of thymocytes sub-populations were calculated by flow cytometry analysis. TCR repertoire diversity was measured by a novel method based on hybridization of TCR Vβ specific cRNA on a gene chip platform. Results: In B-cell deficient mice the number of thymocytes was four times reduced and TCR Vβ chain diversity was up to one million times lower compared with wild type mice. Numbers and diversity were restored by treatment of the mice with gamma globulin (see table). Conclusions: T cell development and diversification is driven by B cells. Mice Number of total thymocytes (mean ± standard deviation) p Value β V TCR diversity of thymocytes (median; min.-max.) p Value C57BL/6 (wild mice) 1.3 x 108 ± 5.1 x 107N=7 4.7 x 106; 1.0 x 105 − 1.1 x 108N=5 JH−/− (B cell immunodeficient mice) 3.1 x 107 ± 1.7 x107N=7 0.002 5.9 x 102; 3.6 x 102 − 1.1 x 103N=5 0.0002 JH−/− treated with Ig 3.9 x 107 ± 1.4 x106N=2 0.20 1.1 x 105; 2.7 x 100 − 7.7 x 105N=4 0.08

Download Full-text

Analytical Validity of Deep Sequencing to Characterize Tissue-Infiltrating Lymphocytes

Blood ◽

10.1182/blood.v120.21.3286.3286 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3286-3286

Author(s):

David Scott Johnson ◽

Andrea Löhr ◽

Walter Mathias Howell ◽

Andro Hsu ◽

Robert S. Negrin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Peripheral Blood ◽

Genomic Dna ◽

Employment Equity ◽

Cell Clones ◽

Tcr Repertoire ◽

Repertoire Sequencing ◽

Equity Ownership ◽

Infiltrating Lymphocytes

Abstract Abstract 3286 Background. Lymphocytes infiltrate most human tissues, comprising a critical component of the adaptive immune response. Infiltrating lymphocyte populations have previously been associated with outcomes for a variety of clinical issues, including cancer, alloimmunity, and autoimmunity. Quantitative detection of lymphocyte infiltration has potential as a clinical biomarker of disease activity. Furthermore, locally infiltrating disease-associated clones can be monitored longitudinally in peripheral blood, enabling less invasive follow-up. Deep sequencing of lymphocyte variable regions is an ideal candidate for clinical characterization of tissue-infiltrating lymphocytes. Tissue biopsies produce low genomic DNA yields generally and also contain only a small percentage of cells that have undergone variable region recombination. In this study, we first performed two analytical validity experiments to determine the sensitivity of T cell receptor (TCR) repertoire sequencing, and then made measurements of infiltrating lymphocytes in non-small cell lung carcinomas (NSCLCs), gastrointestinal (GI) biopsies, and cerebrospinal fluid (CSF). Methods. The first analytical validity experiment was designed to determine whether we could accurately characterize rare T cells in a background of cells with unrearranged TCRs. We therefore spiked genomic DNA from three unique T cell clones into background unrearranged human genomic DNA derived from immortal lung cancer carcinoma cells. The dilution series was designed such that the high end of the range surveyed the equivalent of 40,000 T cells, and the low end of the range surveyed the equivalent of 1,200 T cells. We then performed commercial TCR repertoire sequencing (GigaMune Rep-Seq™). The second analytical validity experiment was designed to determine reproducibility and accuracy across a dilution series. Here, we spiked two TCR clones into blood genomic DNA from a normal human individual. These DNA mixes were created at clonal cell to normal blood genomic DNA ratios of 1:1000, 1:100, 4:100, and 1:10. Then, we subjected these DNA mixtures to commercial TCR repertoire sequencing in triplicate. Finally, as proof-of-principle, we used deep TCR sequencing to characterize tissue-infiltrating lymphocytes and peripheral blood from patients with NSCLC, multiple sclerosis (MS), and graft-versus-host disease (GVHD). Results. In the two analytical validity experiments, we detected all expected clones, at quantities as low as 50 copies per reaction. In the first experiment, as a metric of detection noise, we calculated a normalized count of primer-dimer reads (“background”) across the dilution series. The background for the lowest spike-in mixture was 96% lower than a no-template negative control. In the second analytical validity experiment, the average variation coefficient across replicate measurements was 10%; regression analysis of observed versus expected TCR counts had an r2of 0.93. In our proof-of-principle experiments, we were able to detect oligoclonal TCR in CSF biopsies from two MS subjects. Each CSF contained a single clone at >39% of the total TCR repertoire. Matched peripheral blood from one of these subjects contained the CSF-infiltrating clone at 8% of the total TCR repertoire. We were also able to reproducibly detect TCR clones in GI biopsies from two GVHD subjects. For each of the GI biopsies, top GI-infiltrating clones represented >10% of the total peripheral blood TCR repertoire +30 days after GVHD diagnosis. Finally, we were able to detect tumor-infiltrating TCR clones in NSCLCs and matched peripheral blood from two subjects. In one of the NSCLC subjects, three of the top five tumor-infiltrating clones were among the four most common TCR clones in matched peripheral blood. Conclusions. TCR repertoire sequencing can be used to quantitatively, sensitively, and accurately detect tissue-infiltrating T cell clones. We have shown that tissue-infiltrating T cell clones proliferate significantly in the peripheral blood, across disease conditions as divergent as MS, GVHD, and NSCLC. In the future, we will conduct analytical and clinical validity studies that will eventually be used as evidence to justify use of deep TCR sequencing as a clinical diagnostic in autoimmunity, alloimmunity, and oncology. Disclosures: Johnson: GigaGen Inc.: Employment, Equity Ownership. Löhr:GigaGen Inc.: Employment, Equity Ownership. Howell:GigaGen Inc.: Employment, Equity Ownership. Hsu:GigaGen Inc.: Employment, Equity Ownership. Meyer:GigaGen Inc.: Consultancy, Equity Ownership.

Download Full-text

Blood T-cell receptor diversity decreases during the course of HIV infection, but the potential for a diverse repertoire persists

Blood ◽

10.1182/blood-2011-11-395384 ◽

2012 ◽

Vol 119 (15) ◽

pp. 3469-3477 ◽

Cited By ~ 27

Author(s):

Paul D. Baum ◽

Jennifer J. Young ◽

Diane Schmidt ◽

Qianjun Zhang ◽

Rebecca Hoh ◽

...

Keyword(s):

T Cell ◽

Hiv Infection ◽

T Cell Receptor ◽

Cell Receptor ◽

Cd4 T Cell ◽

Tcr Repertoire ◽

Repertoire Diversity ◽

Tcr Diversity ◽

Cell Subpopulations ◽

T Cell Subpopulations

HIV infection results in a decrease in circulating CD4+ T-cell and naive T-cell numbers. If such losses were associated with an erosion of T-cell receptor (TCR) repertoire diversity in the peripheral T-cell pool, this might exacerbate the state of persistent immunodeficiency. Existing methods for the analysis of the TCR repertoire have demonstrated skewed distributions of TCR genes in HIV-infected subjects but cannot directly measure TCR diversity. Here we used AmpliCot, a quantitative assay based on DNA hybridization kinetics, to measure TCR diversity in a cross-sectional comparison of 19 HIV-infected persons to 18 HIV-uninfected controls. HIV-infected persons had a 10-fold decrease in total TCR repertoire diversity in 1.5 mL of blood compared with uninfected controls, with decreased diversity correlating most closely with a lower CD4+ T-cell percentage. Nonetheless, the TCR repertoire diversity of sort-purified T-cell subpopulations in HIV-infected and HIV-uninfected subjects was comparable. These observations suggest that the TCR repertoire diversity changes in whole blood during HIV disease progression are primarily the result of changes in the number and proportion of T-cell subpopulations and that most HIV-infected persons may retain a sufficiently diverse TCR repertoire to permit immune reconstitution with antiretroviral therapy alone, without thymopoiesis.

Download Full-text

T cell immunity does not age in a long-lived rodent species

10.1101/259374 ◽

2018 ◽

Cited By ~ 1

Author(s):

M. Izraelson ◽

T.O. Nakonechnaya ◽

A.N. Davydov ◽

M.A. Dronina ◽

D.A. Miskevich ◽

...

Keyword(s):

T Cell ◽

Healthy Aging ◽

Cell Receptor ◽

T Cell Immunity ◽

Cell Immunity ◽

Large Numbers ◽

Tcr Repertoire ◽

Repertoire Diversity ◽

Accurate Comparison ◽

Tcr Diversity

Numerous studies have demonstrated that the percentage of naïve T cells and diversity of T cell receptor (TCR) repertoire decrease with age, with some findings likewise suggesting that increased repertoire diversity may be associated with longer lifespan and healthy aging. In this work, we have analyzed peripheral TCR diversity from humans, mice, and blind mole-rats (Spalax spp.)—long-lived, hypoxia- and cancer-tolerant rodents. We employed a quantitative approach to TCR repertoire profiling based on 5’RACE with unique molecular identifiers (UMI) to achieve accurate comparison of repertoire diversity, which also required development of specific wet lab protocol and TCR gene reference for Spalax. Our direct comparison reveals a striking phenomenon. Whereas TCR diversity of mice and humans decreases with age, resulting primarily from the shrinkage of the naive T cell pool, Spalax TCR diversity remains stable even for the animals that reach extreme old age (15-17 years). This indicates that T cell immunity does not meaningfully age in long-lived rodents, at least in terms of the classical understanding of immunosenescence, which is associated with the accumulation of large numbers of memory clones. We suggest that the extraordinary longevity of Spalax may be attributable at least in part to the distinctive organization of their T cell immunity. Our findings should therefore encourage a close re-examination of the contribution of immunosenescence to life span in mammals.

Download Full-text

Clinical Determinants of T-Cell Receptor Diversity after Allogeneic Hematopoietic Stem Cell Transplantation for Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2019-129308 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1997-1997

Author(s):

David Ritchie ◽

Eric Wong ◽

Rachel Koldej ◽

James Anton Kuzich ◽

Piers Blombery ◽

...

Keyword(s):

T Cell ◽

Peripheral Blood ◽

Research Funding ◽

Unrelated Donor ◽

Post Transplant ◽

Significant Difference ◽

Tcr Repertoire ◽

Repertoire Diversity ◽

Unrelated Donors ◽

Tcr Diversity

T-cell reconstitution after allogeneic haematopoietic stem cell transplantation (alloSCT) is critical for protection against infection and to mediate the graft versus leukemia (GVL) effect against hematological malignancies including acute myeloid leukemia (AML). T-cell reconstitution post-alloSCT is significantly impacted by exogenous factors including T-cell depleting strategies, immunosuppressive medications and the prohibitive effects of graft versus host disease (GVHD) and infection. The early T-cell repertoire post-alloSCT is oligoclonal and clinical events such as infection and GVHD may adversely impact recovery of a diverse TCR repertoire. The objectives of this study were to investigate clinical determinants of TCR diversity at day 100 after alloSCT and the impact of TCR diversity on risk of early AML relapse after alloSCT. Methods Twenty-nine patients who underwent HLA-matched sibling or unrelated donor alloSCT were included in this cohort comprising 16 patients with AML relapse at day 100 to 180 post-alloSCT and 13 control patients who did not relapse post-alloSCT. All patients received unmanipulated peripheral blood or bone marrow stem cells. Anti-thymocyte globulin was administered to all patients receiving unrelated donor stem cells as per institutional practice. Surveillance for cytomegalovirus (CMV) viremia in peripheral blood (plasma) was monitored twice weekly using polymerase chain reaction (PCR) and pre-emptive therapy with intravenous ganciclovir or oral valganciclovir was commenced in patients with plasma viral load of 400 copies/mL or greater. Peripheral blood samples were obtained at day 100 (early time-point). Eleven patients had follow-up samples 1-2 years post-transplant (late time-point). T-cells were isolated using immunomagnetic separation. Following DNA extraction, TCRβ loci deep amplicon sequencing was performed using LymphoTrack TRB. Sequence assembly, annotation and error correction was performed by MiXCR. TCR diversity was quantified using inverse Simpson's diversity index (1/D). Results TCRβ sequencing of the entire cohort of 29 patients was performed with a mean of 454516 sequence reads per patient. Median time from transplant for the early post-alloSCT timepoint was 99 days. Median TCR repertoire diversity (1/D) early post-transplant was 104.3 (IQR 46.5-398.4). TCR diversity was significantly greater in patients who received T-cell replete transplants from matched sibling donors compared with T-cell depleted transplants from unrelated donors (siblings 130.1 [IQR 54-1017] vs unrelated donors 64 [IQR 28.9-96.9]; P=0.04). Early TCR diversity was significantly reduced in recipients who were CMV seropositive prior to transplant compared with seronegative patients (77.5 [IQR 42.4-127.5] vs 718.8 [IQR 75.5-1884]; P=0.01). Twenty patients (69%) developed CMV viremia, defined as any detectable CMV virus in peripheral blood, prior to day 100 post-alloSCT. Early TCR diversity was significantly reduced in patients with CMV viremia within the first 100 days post-alloSCT (83.5 [IQR 42.4-131.5] vs 964.4 [IQR 71.7-2399]; P=0.02). There was no significant difference in TCR diversity at day 100 in patients who had prior acute GVHD compared to those who did not. There was no significant difference in early TCR diversity at the time of AML relapse compared with patients who remained in remission (78.4 [IQR 38-799.2] vs 132.8 [IQR 59.5-753.6]; P=0.22), suggesting that a restricted TCR repertoire early post-transplant is not a mechanism of AML relapse. Eleven patients had serial samples analysed at early (day 100) and late (between 1-2 years post-transplant) timepoints. All patients remained free of leukemia relapse between these two timepoints. Patients with early CMV viremia (prior to day 100) continued to have a significantly reduced TCR diversity late post-transplant compared with patients who did not have early CMV reactivation (33.2 [IQR 27.3-61.4] vs 3868 [IQR 1421-4565]; P=0.006), indicating that early CMV viremia had a persistent effect on post-transplant T-cell recovery extending to 1-2 years post-transplant. Conclusion T-cell depletion and CMV viremia are key determinants of early TCR repertoire diversity post-alloSCT. CMV viremia has persistent and deleterious effects on TCR repertoire late post-transplant. TCR diversity does not impact early AML relapse post-alloSCT. Disclosures Ritchie: Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy; BMS: Research Funding; Takeda: Research Funding; Beigene: Research Funding; Imago: Research Funding; Novartis: Honoraria; Sanofi: Honoraria. Koldej:NanoString Technologies: Other: Travel grant. Blombery:Novartis: Consultancy; Janssen: Honoraria; Invivoscribe: Honoraria.

Download Full-text

Immunosenescence and Autoimmunity: Exploiting the T-Cell Receptor Repertoire to Investigate the Impact of Aging on Multiple Sclerosis

Frontiers in Immunology ◽

10.3389/fimmu.2021.799380 ◽

2021 ◽

Vol 12 ◽

Author(s):

Roberta Amoriello ◽

Alice Mariottini ◽

Clara Ballerini

Keyword(s):

Multiple Sclerosis ◽

T Cell ◽

T Cell Receptor ◽

High Throughput Sequencing ◽

Cell Receptor ◽

Receptor Repertoire ◽

Age Related ◽

Tcr Repertoire ◽

Repertoire Diversity ◽

The Impact

T-cell receptor (TCR) repertoire diversity is a determining factor for the immune system capability in fighting infections and preventing autoimmunity. During life, the TCR repertoire diversity progressively declines as a physiological aging progress. The investigation of TCR repertoire dynamics over life represents a powerful tool unraveling the impact of immunosenescence in health and disease. Multiple Sclerosis (MS) is a demyelinating, inflammatory, T-cell mediated autoimmune disease of the Central Nervous System in which age is crucial: it is the most widespread neurological disease among young adults and, furthermore, patients age may impact on MS progression and treatments outcome. Crossing knowledge on the TCR repertoire dynamics over MS patients’ life is fundamental to investigate disease mechanisms, and the advent of high- throughput sequencing (HTS) has significantly increased our knowledge on the topic. Here we report an overview of current literature about the impact of immunosenescence and age-related TCR dynamics variation in autoimmunity, including MS.

Download Full-text

Detection Of Recurrent/Persistent Disease By T-Cell Receptor Repertoire Profiling In Patients With Mature T-Cell Neoplasm

Blood ◽

10.1182/blood.v122.21.2614.2614 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2614-2614

Author(s):

Anna Sherwood ◽

Harlan Robins ◽

Jonathan R. Fromm ◽

Harvey A. Greisman ◽

Daniel E. Sabath ◽

...

Keyword(s):

T Cell ◽

High Throughput ◽

High Throughput Sequencing ◽

Recurrent Disease ◽

Cell Lineage ◽

T Cell Repertoire ◽

Cell Repertoire ◽

Employment Equity ◽

Equity Ownership ◽

Clinical Measures

Abstract Identification of recurrent or persistent disease in T-cell neoplasms is important for individualized patient care. While patients with T-cell lineage lymphomas and leukemias are a small subset of all lymphoma and leukemia patients, the incidence of refractory disease in these patients can be higher than patients with B-cell lineage neoplasms. We recently developed a method to sequence the diversity of the TCR CDR3 rearrangements (Blood. 2009; 114(19): 4099-107) that exploits the capacity of high-throughput sequencing (HTS) to document the diverse repertoire of TCRB CDR3 chains simultaneously. These assays can describe both the breadth of T-cell repertoire and quantify individual clones. For example, we have shown in that T-cell clones as rare as 1:100,000 total T cells can be accurately quantified (Journal of Immunological Methods 2012;375:14-9). This technology thus provides a potential opportunity to track the presence and frequency of clones in the context of an evolving, adaptive immune system, during the course of ongoing therapy. While our recent study demonstrated the first application of high-throughput sequencing to the evaluation of minimal residual disease in precursor acute T lymphoblastic leukemias (Sci. Transl. Med. 4(134):134ra63. 2012), we focus here on mature T-cell neoplasms to demonstrate the broad potential of this technology to contribute to the post-therapeutic monitoring of T cell neoplasia. We amplified the TCRB repertoire of 35 index samples to identify high-frequency TCRB rearrangements. Clones were classified as neoplastic if occurring at a proportion greater than 7 standard deviations above the mean frequency of the most abundant rearranged TCRB in control samples of either blood, bone marrow, or lymphoid tissues. Samples that lacked a detectable TCRB clone were excluded. For each patient, at least one subsequent follow-up sample was available. For these 59 samples, we sequenced the TCRB repertoire to screen for the corresponding index clone. We find that for most samples, high-throughput sequencing concurs with currently available, routine clinical measures of disease, such as clinical flow cytometry or PCR-based evaluation of TCRG rearrangement. High-throughput sequencing of TCRB was concordant in 46 samples with identification of the index clones and in 7 additional samples without the identification of the index clones. However, 5 of 59 samples were only positive for recurrent disease based on HTS only, and 1 of the 59 samples was only positive for recurrent disease based on current diagnostic technology but not HTS. We find that for most samples, high-throughput sequencing concurs with currently available, routine clinical measures of disease, such as clinical flow cytometry or PCR-based evaluation of TCRG rearrangement and T-cell repertoire analysis may be useful for clinical laboratory evaluation of patients with T-cell neoplasms.Table 1High-throughput sequencingPositiveNegativeRoutine clinical testingPositive461Negative57 Disclosures: Sherwood: Adaptive Biotechnologies: Employment, Equity Ownership. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Emerson:Adaptive Biotechnologies: Employment, Equity Ownership. Rieder:Adaptive Biotechnologies: Employment, Equity Ownership. Wood:Becton Dickinson and Company, NJ, USA: Research Funding.

Download Full-text