High-throughput Sequencing Of T-cell Receptor Research Articles

Unleashed monocytic engagement in Sézary syndrome during the combination of anti-CCR4 antibody with type I interferon

AbstractSézary syndrome (SS) is an aggressive leukemic expansion of skin-derived malignant CD4+ T cells. Drug monotherapy often results in disease relapse because of the heterogenous nature of malignant CD4+ T cells, but how therapies can be optimally combined remains unclear because of limitations in understanding the disease pathogenesis. We identified immunologic transitions that interlink mycosis fungoides with SS using single-cell transcriptome analysis in parallel with high-throughput T-cell receptor sequencing. Nascent peripheral CD4+ T cells acquired a distinct profile of transcription factors and trafficking receptors that gave rise to antigenically mature Sézary cells. The emergence of malignant CD4+ T cells coincided with the accumulation of dysfunctional monocytes with impaired fragment crystallizable γ-dependent phagocytosis, decreased responsiveness to cytokine stimulation, and limited repertoire of intercellular interactions with Sézary cells. Type I interferon supplementation when combined with a monoclonal antibody targeting the chemokine receptor type 4 (CCR4), unleashed monocyte induced phagocytosis and eradication of Sézary cells in vitro. In turn, coadministration of interferon-α with the US Food and Drug Administration–approved anti-CCR4 antibody, mogamulizumab, in patients with SS induced marked depletion of peripheral malignant CD4+ T cells. Importantly, residual CD4+ T cells after Sézary cell ablation lacked any immunologic shifts. These findings collectively unveil an auxiliary role for augmenting monocytic activity during mogamulizumab therapy in the treatment of SS and underscore the importance of targeted combination therapy in this disease.

Composition and diversity analysis of the TCR CDR3 repertoire in patients with idiopathic orbital inflammation using high-throughput sequencing

BackgroundIdiopathic orbital inflammation (IOI) is a nonspecific orbital inflammatory disease with the third highest prevalence among orbital diseases, and its pathogenesis is associated with T-cell-mediated immune responses. This study aimed to investigate the differences in T-cell receptor (TCR) expression between IOI patients and healthy subjects by high-throughput sequencing and to characterize TCR expression in patients with IOI and with respect to glucocorticoid response.MethodsA total of 19 subjects were enrolled in this study and were divided into the idiopathic orbital inflammation group (IOI group, n = 13) and the healthy control group (HC group, n = 6), and within the IOI group were further divided into the glucocorticoid therapy sensitive group (IOI(EF) group, n = 6) and the glucocorticoid therapy ineffective group (IOI(IN) group, n = 7) based on the degree of effectiveness to glucocorticoid therapy. High-throughput TCR sequencing was performed on peripheral blood mononuclear cells of IOI patients and healthy control individuals using 5’ RACE technology combined with Unique Identifier (UID) digital tag correction technology. The TCR CDR3 region diversity, sharing patterns, and differential sequences between the IOI and HC groups, and between the IOI(EF) and IOI(IN) groups were analyzed.ResultsIt was found that the diversity of TCR CDR3 in the IOI group was significantly lower than that in the HC group, and the frequency of V gene use was significantly different between groups. The diversity of TCR CDR3 in patients in the IOI(EF) group was significantly lower than that in patients in the IOI(IN) group, and the frequency of V and J gene use was significantly different between the IOI(EF) group and the IOI(IN) group. Additionally, we found 133 nucleotide sequences shared in all IOI samples and screened two sequences with higher expression from them.ConclusionsOur results suggested that abnormal clonal expansion of specific T-cells exists in IOI patients and that TCR diversity may had an impact on the prognosis of glucocorticoid-treated IOI. This study may contribute to a better understanding of the immune status of IOI and provide new insights for T-cell -associated IOI pathogenesis, diagnosis and treatment prediction.

Mapping the spatial distribution of T cells in repertoire dimension

T cells mediate adaptive immunity in diverse anatomic compartments through recognition of specific antigens via unique T cell receptor (TCR) structures. However, little is known about the spatial distribution of an organism’s TCR repertoire. Here, using high-throughput TCR sequencing (TCRseq), we investigated the TCR repertoires of sixteen tissues in healthy C57B/L6 mice. We found that TCR repertoires generally classified into three categories (lymph nodes, non-lymph node tissues and small intestine) based on sequence similarity. Clonal distribution and diversity analyses showed that small intestine compartment had a more skewed repertoire as compared to lymph nodes and non-lymph node tissues. However, analysis of TRBV and TRBJ gene usage across tissue compartments, as well as comparison of CDR3 length distributions, showed no significant tissue-dependent differences. Interestingly, analysis of clonotype sharing between mice showed that although non-redundant public clonotypes were found more easily in lymph nodes, small intestinal CD4 + T cells harbored more abundant public clonotypes. These findings under healthy physiological conditions offer an important reference dataset, which may contribute to our ability to better manipulate T cell responses against infection and vaccination.

ICAT: diagnostic assessment tool of immunological history using high-throughput T-cell receptor sequencing

The pathogen exposure history of an individual is recorded in their T-cell repertoire and can be accessed through the study of T-cell receptors (TCRs) if the tools to identify them were available. For each T-cell, the TCR loci undergoes genetic rearrangement that creates a unique DNA sequence. In theory these unique sequences can be used as biomarkers for tracking T-cell responses and cataloging immunological history. We developed the immune Cell Analysis Tool (iCAT), an R software package that analyzes TCR sequencing data from exposed (positive) and unexposed (negative) samples to identify TCR sequences statistically associated with positive samples. The presence and absence of associated sequences in samples trains a classifier to diagnose pathogen-specific exposure. We demonstrate the high accuracy of iCAT by testing on three TCR sequencing datasets. First, iCAT successfully diagnosed smallpox vaccinated versus naïve samples in an independent cohort of mice with 95% accuracy. Second, iCAT displayed 100% accuracy classifying naïve and monkeypox vaccinated mice. Finally, we demonstrate the use of iCAT on human samples before and after exposure to SARS-CoV-2, the virus behind the COVID-19 global pandemic. We were able to correctly classify the exposed samples with perfect accuracy. These experimental results show that iCAT capitalizes on the power of TCR sequencing to simplify infection diagnostics. iCAT provides the option of a graphical, user-friendly interface on top of usual R interface allowing it to reach a wider audience.

Quantifying the Size and Diversity of the Human Alloresponse Via High-Throughput T Cell Receptor Sequencing

Alloreactive T lymphocytes are the primary mediators of the immune response in transplantation, both in the graft-versus-host and host-versus graft directions; however, the large size and diversity of the alloreactive T cell population has presented a great challenge for quantitative analysis of this important response. We have therefore developed and validated a method for defining the biologically-relevant alloreactive T cell repertoire for any HLA-mismatched human responder-stimulator pair by combining the in vitro mixed lymphocyte reaction with high-throughput T cell receptor (TCR) sequencing. Here we use this method to characterize the normal human alloresponse (n = 9) at an unprecedented level of resolution. We provide the first quantification of alloreactive T cell repertoire diversity comparing CD4 and CD8 populations. In addition to standard techniques for comparing population diversity such as entropy, clonality, and R20 (fraction of clones accounting for the top 20 percent of reads), we have established a new approach for quantitative population diversity comparisons measuring power-law slopes (S) of circulating and alloreactive T cell populations, enabling the comparison of key repertoire information not captured in other analyses. Using S, we observe a significant increase in alloreactive repertoire diversity with increased HLA mismatches between responder and stimulator. In addition, we are able to quantitatively measure repertoire divergence, and have discovered that the alloreactive repertoire is highly allo-specific: the alloreactive repertoires generated by the same responder to two different stimulators are markedly distinct. However, when the stimulators share Class I HLA, we identify shared CD8 alloreactive clones. Perhaps most strikingly, we have found that although alloreactive clones are abundant, they circulate at such low frequencies that the majority are undetectable with deep sequencing. Cumulative CD4 and CD8 alloreactive clones by frequency detected in circulation account for 0.83% ± 0.73% and 0.76% ± 0.75%, respectively (mean ± SD). We have also developed a statistical model to estimate the frequency of undetected alloreactive clones, which added a total frequency of 1.5% ± 0.69% for CD4s and 0.53% ± 0.31% for CD8s. Lastly, we sought to identify clones known to be public viral-reactive clones to EBV, CMV, HSV, and Influenza A within our alloreactive T cell populations. Contrary to a common hypothesis that a large fraction of alloreactive clones are cross-reactive to viral antigens, very few public viral clones were found in our alloreactive repertoires in the presence of relevant responder HLA alleles. Taken together, high-throughput TCR sequencing yielded new insight into the alloimmune response, the key immune response in GVHD. Our approach has the potential to provide novel mechanistic information about the fate and frequency of alloreactive clones in hematopoietic stem cell transplantation, particularly in the haploidentical setting. Furthermore, our new analytic approaches including a model to extrapolate the circulating frequency of an population of “unseen” clones has a broad range of applications for TCR sequencing analysis in any setting in which only a sub-group of clones is identified within a circulating T cell population. DisclosuresDewolf:American Society of Hematology (HONORS award): Research Funding.

Next-Generation Sequencing Based Monitoring of Circulating-Tumor DNA in Untreated Peripheral T-Cell Lymphoma

Background: Peripheral T-cell lymphomas (PTCLs) are aggressive lymphomas with a wide range of clinical outcomes. Overall prognosis is worse for PTCL than B-cell lymphomas, but pts who achieve remission can be cured. After initial therapy, controversy exists regarding consolidation with autologous stem cell transplant, and clinical decisions are largely empirical. Circulating-tumor DNA (ctDNA) is emerging as a promising non-invasive biomarker that may aid clinical decision-making. Quantitative baseline levels of ctDNA and kinetics of ctDNA clearance during therapy may provide prognostic information. Further, novel methods that detect disease relapse prior to imaging may improve outcomes. We studied a high-throughput DNA sequencing method of detecting T-cell receptor (TCR) β and γ gene sequences in serum with a sensitivity of 1 lymphoma molecule per million leukocytes. Using this platform, we determined the predictive ability of monitoring ctDNA in pts undergoing frontline therapy for PTCL.Methods: All pts had PTCL undergoing frontline EPOCH-based chemotherapy between May 1993 through December 2015. Pre-treatment FFPE biopsy samples and/or baseline serum samples were sent for VDJ amplification and TCR sequencing calibration. High-throughput TCR sequencing of the CDR3s of TCRβ/TCRγ genes was employed to provide a comprehensive and quantitative analysis of distinct T-cell clones within each sample, the relative frequency of each clone, and the exact unique nucleotide sequences of each clone's CDR3 regions. Pts with a calibrating TCRβ/TCRγ sequence had all available serum samples sent for identification and quantification. Prospectively collected serum sample time points included baseline, start of each cycle, and each follow-up visit until disease progression. Clinic visits after therapy included paired contrast CT scans performed at 3, 4, 6, 12 and 12 mos. intervals for the first 5 years, respectively. All analyses for ctDNA were performed blinded to clinical outcomes.Results: Fifty PTCL pts had a pre-treatment FFPE or serum sample available for calibration. Nineteen of 23 (83%) pts with pre-treatment FFPE had a calibrating rearrangement; Fourteen of 27 (52%) pts with only pre-treatment serum available had a calibrating rearrangement; 1 pt who did not calibrate on FFPE had a dominant sequence identified in serum. Overall, tumor-specific clonotypes were identified in 34 (68%) of 50 pts tested. Ten of 18 (56%) pts with both FFPE and serum available had dominant clones in both compartments. Of the 44 calibrating pre-treatment samples, 1 calibrated for TCRβ only, 15 for TCRγ only, and 28 for both loci. Pt characteristics shown in Table 1. Of 34 study pts, 20 (59%) progressed and 14 (41%) were non-progressors. Quantitative baseline ctDNA levels were significantly higher in pts with stage IV disease compared to stages I-III (p=0.014) but did not correlate with LDH levels (p=0.53).We analyzed the predictive ability of ctDNA as a measure of minimal residual disease (MRD) within 1 month of completing therapy in 28 pts. We compared pts who became MRD- to those who remained MRD+ and identified a trend towards improved PFS in those who became MRD- (median, NR vs. 208 days, p=0.07) (Figure 1). In 20 pts who achieved remission, we studied the use of ctDNA monitoring as a surveillance tool. Of the 10 pts who progressed after remission, ctDNA was detected prior to clinical relapse in 6 cases with a median lead-time of 13.2 mos. (range, 2.7 to 88.5) and 4 with detectable ctDNA > 12 mos. prior to clinical relapse. Four pts progressed without detectable ctDNA prior to clinical relapse. Interestingly, of the 10 non-progressors followed in surveillance, 5 pts developed low-level ctDNA positivity without clinical progression. Results of interim ctDNA kinetics and TCRβ testing is ongoing and will be presented at the meeting.Conclusions: Next-generation based sequencing of the TCR can identify tumor-specific clonotypic sequences in the majority of pts from both FFPE and serum. As a marker of MRD, monitoring TCR sequences in serum may identify pts who do not benefit from treatment intensification, but further studies are needed. Surveillance monitoring of ctDNA in serum identifies TCR sequences months before clinical progression but not all pts with clonotypic sequences progressed clinically in our series. Further studies are needed to investigate surveillance monitoring methods and optimize clinical utility. [Display omitted] DisclosuresJacob:Adaptive Biotechnologies: Employment, Equity Ownership. Chan:Adaptive Biotechnologies: Employment. Vignali:Adaptive Biotechnologies: Employment.

ICAT: diagnostic assessment tool of immunological history using high-throughput T-cell receptor sequencing

The pathogen exposure history of an individual is recorded in their T-cell repertoire and can be accessed through the study of T-cell receptors (TCRs) if the tools to identify them were available. For each T-cell, the TCR loci undergoes genetic rearrangement that creates a unique DNA sequence. In theory these unique sequences can be used as biomarkers for tracking T-cell responses and cataloging immunological history. We developed the immune Cell Analysis Tool (iCAT), an R software package that analyzes TCR sequencing data from exposed (positive) and unexposed (negative) samples to identify TCR sequences statistically associated with positive samples. The presence and absence of associated sequences in samples trains a classifier to diagnose pathogen-specific exposure. We demonstrate the high accuracy of iCAT by testing on three TCR sequencing datasets. First, iCAT successfully diagnosed smallpox vaccinated versus naïve samples in an independent cohort of mice with 95% accuracy. Second, iCAT displayed 100% accuracy classifying naïve and monkeypox vaccinated mice. Finally, we demonstrate the use of iCAT on human samples before and after exposure to SARS-CoV-2, the virus behind the COVID-19 global pandemic. We were able to correctly classify the exposed samples with perfect accuracy. These experimental results show that iCAT capitalizes on the power of TCR sequencing to simplify infection diagnostics. iCAT provides the option of a graphical, user-friendly interface on top of usual R interface allowing it to reach a wider audience.

Quantitative characterization of the T cell receptor repertoires of human immunized by rabies virus vaccine

ABSTRACT Cellular immunity is crucial for an efficient host immune response against rabies virus (RABV) infection. But the T cell receptor (TCR) repertoire in human after RABV vaccine immunization remained unclear. In this study, we conducted high-throughput sequencing of TCR β chain complementarity determining region 3(CDR3) repertoires in 4 healthy volunteers before and after immunization with RABV vaccine. Our data showed that RABV vaccination changed the TCR diversity and the usage of V/J gene segments, as well as V-J pairing. The high-frequency clonotypes that altered after vaccination were identified. These results may provide us with new insights into T cell receptor condition after RABV vaccination.

Identification and Phenotype of MAIT Cells in Cattle and Their Response to Bacterial Infections.

Mucosal-associated invariant T (MAIT) cells are a population of innate-like T cells that utilize a semi-invariant T cell receptor (TCR) α chain and are restricted by the highly conserved antigen presenting molecule MR1. MR1 presents microbial riboflavin biosynthesis derived metabolites produced by bacteria and fungi. Consistent with their ability to sense ligands derived from bacterial sources, MAIT cells have been associated with the immune response to a variety of bacterial infections, such as Mycobacterium spp., Salmonella spp. and Escherichia coli. To date, MAIT cells have been studied in humans, non-human primates and mice. However, they have only been putatively identified in cattle by PCR based methods; no phenotypic or functional analyses have been performed. Here, we identified a MAIT cell population in cattle utilizing MR1 tetramers and high-throughput TCR sequencing. Phenotypic analysis of cattle MAIT cells revealed features highly analogous to those of MAIT cells in humans and mice, including expression of an orthologous TRAV1-TRAJ33 TCR α chain, an effector memory phenotype irrespective of tissue localization, and expression of the transcription factors PLZF and EOMES. We determined the frequency of MAIT cells in peripheral blood and multiple tissues, finding that cattle MAIT cells are enriched in mucosal tissues as well as in the mesenteric lymph node. Cattle MAIT cells were responsive to stimulation by 5-OP-RU and riboflavin biosynthesis competent bacteria in vitro. Furthermore, MAIT cells in milk increased in frequency in cows with mastitis. Following challenge with virulent Mycobacterium bovis, a causative agent of bovine tuberculosis and a zoonosis, peripheral blood MAIT cells expressed higher levels of perforin. Thus, MAIT cells are implicated in the immune response to two major bacterial infections in cattle. These data suggest that MAIT cells are functionally highly conserved and that cattle are an excellent large animal model to study the role of MAIT cells in important zoonotic infections.

ICAT: diagnostic assessment tool of immunological history using high-throughput T-cell receptor sequencing.

The pathogen exposure history of an individual is recorded in their T-cell repertoire and can be accessed through the study of T-cell receptors (TCRs) if the tools to identify them were available.For each T-cell, the TCR loci undergoes genetic rearrangement that creates a unique DNA sequence. In theory these unique sequences can be used as biomarkers for tracking T-cell responses and cataloging immunological history. We developed the immune Cell Analysis Tool (iCAT), an R software package that analyzes TCR sequencing data from exposed (positive) and unexposed (negative) samples to identify TCR sequences statistically associated with positive samples. The presence and absence of associated sequences in samples trains a classifier to diagnose pathogen-specific exposure.We demonstrate the high accuracy of iCAT by testing on three TCR sequencing datasets.First, iCAT successfully diagnosed smallpox vaccinated versus naïve samples in an independent cohort of mice with 95% accuracy.Second, iCAT displayed 100% accuracy classifying naïve and monkeypox vaccinated mice. Finally, we demonstrate the use of iCAT on human samples before and after exposure to SARS-CoV-2, the virus behind the COVID-19 global pandemic.We were able to correctly classify the exposed samples with perfect accuracy.These experimental results show that iCAT capitalizes on the power of TCR sequencing to simplify infection diagnostics.iCAT provides the option of a graphical, user-friendly interface on top of usual R interface allowing it to reach a wider audience.

The enigmatic role of HLA-B*27 in spondyloarthritis pathogenesis.

Establishing a clear role for HLA-B*27 in the pathogenesis of spondyloarthritis continues to be challenging. Aberrant properties of the heavy chain as well as a potential role presenting arthritogenic peptides continue to be pursued as plausible mechanisms. Recent studies implicate HLA-B*27 in aberrant bone formation. An unanticipated cell surface interaction between HLA-B*27 and the bone morphogenetic protein pathway receptor subunit ALK2 may augment TGFβ superfamily signaling pathways, increasing responsiveness to Activin A and TGFβ. This has the potential to increase bone formation as well as Th17 T cell development, presenting an attractive model to explain several aspects of axial and peripheral spondyloarthritis. In a separate study, intracellular effects of misfolded HLA-B*27 implicate this mechanism in increased osteoblast mineralization and bone formation. HLA-B*27 expression in early osteoblasts activates unfolded protein response-mediated X-box binding protein-1 mRNA splicing and induction of the retinoic acid receptor-β gene, with downstream increases in expression of tissue non-specific alkaline phosphatase. Increased TNAP expression in osteoblasts was linked to increased mineralization in vitro and bone formation in vivo. In the ongoing search for evidence of arthritogenic peptides, high-throughput TCR (T cell receptor) sequencing has provided evidence for reduced clonal expansion and increased TCR diversity in ankylosing spondylitis. In addition to two common CD8+ TCR sequences identified in one study, similar CD8 and CD4 TCR motifs were found in another study. Further work will be needed to shed light on the nature of the peptide-HLA class I complex recognized by these T cells and its role in disease.

Diagnostic differentiation of Zika and dengue virus exposure by analyzing T cell receptor sequences from peripheral blood of infected HLA-A2 transgenic mice.

Zika virus (ZIKV) is a significant global health threat due to its potential for rapid emergence and association with severe congenital malformations during infection in pregnancy. Despite the urgent need, accurate diagnosis of ZIKV infection is still a major hurdle that must be overcome. Contributing to the inaccuracy of most serologically-based diagnostic assays for ZIKV, is the substantial geographic and antigenic overlap with other flaviviruses, including the four serotypes of dengue virus (DENV). Within this study, we have utilized a novel T cell receptor (TCR) sequencing platform to distinguish between ZIKV and DENV infections. Using high-throughput TCR sequencing of lymphocytes isolated from DENV and ZIKV infected mice, we were able to develop an algorithm which could identify virus-associated TCR sequences uniquely associated with either a prior ZIKV or DENV infection in mice. Using this algorithm, we were then able to separate mice that had been exposed to ZIKV or DENV infection with 97% accuracy. Overall this study serves as a proof-of-principle that T cell receptor sequencing can be used as a diagnostic tool capable of distinguishing between closely related viruses. Our results demonstrate the potential for this innovative platform to be used to accurately diagnose Zika virus infection and potentially the next emerging pathogen(s).

Impact of T Cell Repertoire Diversity on Mortality Following Cord Blood Transplantation.

IntroductionCord blood transplantation (CBT) recipients are at increased risk of mortality due to delayed immune recovery (IR). Prior studies in CBT patients have shown that recovery of absolute lymphocyte count is predictive of survival after transplant. However, there are no data on the association of T-cell receptor (TCR) and clinical outcomes after CBT. Here we retrospectively performed TCR beta chain sequencing on peripheral blood (PB) samples of 34 CBT patients.MethodsAll patients received a total body irradiation based conditioning regimen and cyclosporine and MMF were used for graft versus host disease (GvHD) prophylaxis. PB was collected pretransplant on days 28, 56, 80, 180, and 1-year posttransplant for retrospective analysis of IR utilizing high-throughput sequencing of TCRβ rearrangements from genomic DNA extracted from PB mononuclear cells. To test the association between TCR repertoire diversity and patient outcomes, we conducted a permutation test on median TCR repertoire diversity for patients who died within the first year posttransplant versus those who survived.ResultsMedian age was 27 (range 1–58 years) and most of the patients (n = 27) had acute leukemias. There were 15 deaths occurring between 34 to 335 days after transplant. Seven deaths were due to relapse. Rapid turnover of T cell clones was observed at each time point, with TCR repertoires stabilizing by 1-year posttransplant. TCR diversity values at day 100 for patients who died between 100 and 365 days posttransplant were significantly lower than those of the surviving patients (p = 0.01).ConclusionsUsing a fast high-throughput TCR sequencing assay we have demonstrated that high TCR diversity is associated with better patient outcomes following CBT. Importantly, this assay is easily performed on posttransplant PB samples, even as early as day 28 posttransplant, making it an excellent candidate for early identification of patients at high risk of death.

Histopathologic Characterization of Mogamulizumab-associated Rash.

Rash is one of the most common adverse events observed with mogamulizumab, an anti-C-C chemokine receptor 4 monoclonal antibody approved for previously treated mycosis fungoides (MF) and Sezary syndrome (SS). Given the nonspecific clinical presentations of this rash, histopathologic distinction from MF/SS is critical for informing clinical management. We performed a comprehensive characterization of the histopathologic findings in mogamulizumab-associated rash (MAR) with the integration of high-throughput sequencing of T-cell receptor (TCR) genes. Fifty-two biopsy specimens from 19 patients were evaluated retrospectively. Three major histologic reaction patterns were identified: spongiotic/psoriasiform dermatitis (33/52), interface dermatitis (11/52), and granulomatous dermatitis (8/52). Almost half of the specimens (21/52) showed at least 2 of these reaction patterns concurrently. Dermal eosinophils were not a consistent feature, being present in only half (27/52) of specimens and prominent in only 3. Features mimicking MF/SS, including lymphocyte exocytosis, lamellar fibroplasia, and adnexal involvement, were commonly seen but tended to be focal and mild. In 38/43 specimens with available immunohistochemistry, intraepidermal lymphocytes demonstrated a CD4:CD8 ratio ≤1 : 1. Low background levels of the patient's previously identified MF/SS-associated TCR sequence(s) were demonstrated in 20/46 specimens analyzed by high-throughput sequencing of TCR. We conclude that MAR may demonstrate diverse histologic features. Findings that may distinguish MAR from MF/SS include the inverted or normalized CD4:CD8 ratio within intraepidermal lymphocytes and demonstration of absent or nondominant levels of disease-associated TCR sequences. Correlation with the clinical findings and immunohistochemical and molecular characterization of the patient's MF/SS before mogamulizumab, when possible, may facilitate recognition of MAR.

Next-Generation Sequencing Technologies for Early-Stage Cutaneous T-Cell Lymphoma.

The diagnosis of early stage cutaneous T-cell lymphoma is often difficult, particularly in mycosis fungoides (MF), because the clinical presentation, histological findings, and laboratory findings of MF resemble those of inflammatory skin diseases such as atopic dermatitis, psoriasis, and parapsoriasis en plaque. Furthermore, MF sometimes occurs with or after these inflammatory skin diseases. The current diagnostic criteria heavily rely on clinical impressions along with assessments of T cell clonality. To make a diagnosis of early-stage MF, the detection of a malignant clone is critical. T cell receptor (TCR) gene rearrangements have been detected by southern blotting or polymerase chain reaction for this purpose, but the results of these methods are insufficient. High-throughput TCR sequencing has provided insights into the complexities of the immune repertoire. Accordingly, his technique is more sensitive and specific than current methods, making it useful for the detection of early lesions and monitoring responses to therapy.

Intrahepatic T-Cell Receptor β Immune Repertoire Is Essential for Liver Regeneration.

T lymphocytes synergize with the cellular immune system to promote hepatocyte regeneration. The T-cell receptor (TCR) immune repertoire is closely associated with the host immune response and regenerative proliferation. High-throughput sequencing of TCR provides deep insight into monitoring the immune microenvironment. Here, we aimed to determine the role of the TCRβ immune repertoire in liver regeneration (LR). We investigated hepatic regeneration in TCRβ chain-deficient (tcrb-/- ) mice by two-thirds partial hepatectomy (PHx) method. Our results demonstrated that tcrb-/- mice revealed a reduced capacity for LR, which was characterized by impaired hepatocyte proliferation and enhanced hepatocyte apoptosis. Dysregulation of inflammatory signaling activation and inflammatory factors was observed in regenerated tcrb-/- livers. Simultaneously, significantly altered immunocyte levels and aberrant cytokine levels were observed during hepatic regeneration. In addition, we first determined the profile of the TCRβ immune repertoire during LR, indicating that PHx resulted in remarkably lower TCRβ diversity in intrahepatic T lymphocytes. Conclusion: Taken together, our data suggest that TCRβ deficiency gives a rise to aberrant intrahepatic immune microenvironment that impairs LR, and the TCRβ reconstitution is required for hepatic immunocyte recruitment and activation during LR.

TCR repertoire intratumor heterogeneity of CD4+ and CD8+ T cells in centers and margins of localized lung adenocarcinomas.

Intratumor heterogeneity (ITH) of T cell receptor (TCR) repertoire in different T-cell subsets and locations in lung adenocarcinomas was unclear. Here, we investigated percentages and TCR repertoire of freshly isolated CD4+ and CD8+ tumor infiltrating lymphocytes (TILs) in tumor centers and margins by flow cytometry on 80 tumor samples from 20 patients and high-throughput TCR sequencing on 27 and 25 samples of CD4+ and CD8+ TILs from seven patients. Our results demonstrated that amount and TCR repertoire diversity of CD4+ TILs were significantly higher than those of CD8+ TILs and moreover substantial ITH regarding amount and TCR repertoire of CD4+ and CD8+ TILs were observed. Additionally, ITH of CD4/CD8 T-cell ratio and CD8+ TIL repertoire across center regions was lower than that across margin regions. The amount and TCR repertoire ITH of CD4+ and CD8+ TILs and mean clonality of CD8+ TILs in tumor centers were associated with relapse. Our study provides insights into amount and TCR repertoire ITH of CD4+ and CD8+ TILs in tumor centers and margins as well as corresponding association with prognosis in lung adenocarcinoma patients, suggesting potential clinical significance of TCR repertoire.

T cell receptor repertoires of mice and humans are clustered in similarity networks around conserved public CDR3 sequences.

Diversity of T cell receptor (TCR) repertoires, generated by somatic DNA rearrangements, is central to immune system function. However, the level of sequence similarity of TCR repertoires within and between species has not been characterized. Using network analysis of high-throughput TCR sequencing data, we found that abundant CDR3-TCRβ sequences were clustered within networks generated by sequence similarity. We discovered a substantial number of public CDR3-TCRβ segments that were identical in mice and humans. These conserved public sequences were central within TCR sequence-similarity networks. Annotated TCR sequences, previously associated with self-specificities such as autoimmunity and cancer, were linked to network clusters. Mechanistically, CDR3 networks were promoted by MHC-mediated selection, and were reduced following immunization, immune checkpoint blockade or aging. Our findings provide a new view of T cell repertoire organization and physiology, and suggest that the immune system distributes its TCR sequences unevenly, attending to specific foci of reactivity.

Diversity and divergence of the glioma-infiltrating T-cell receptor repertoire.

Although immune signaling has emerged as a defining feature of the glioma microenvironment, how the underlying structure of the glioma-infiltrating T-cell population differs from that of the blood from which it originates has been difficult to measure directly in patients. High-throughput sequencing of T-cell receptor (TCR) repertoires (TCRseq) provides a population-wide statistical description of how T cells respond to disease. We have defined immunophenotypes of whole repertoires based on TCRseq of the α- and β-chains from glioma tissue, nonneoplastic brain tissue, and peripheral blood from patients. Using information theory, we partitioned the diversity of these TCR repertoires into that from the distribution of VJ cassette combinations and diversity due to VJ-independent factors, such as selection due to antigen binding. Tumor-infiltrating lymphocytes (TILs) possessed higher VJ-independent diversity than nonneoplastic tissue, stratifying patients according to tumor grade. We found that the VJ-independent components of tumor-associated repertoires diverge more from their corresponding peripheral repertoires than T-cell populations in nonneoplastic brain tissue, particularly for low-grade gliomas. Finally, we identified a "signature" set of TCRs whose use in peripheral blood is associated with patients exhibiting low TIL divergence and is depleted in patients with highly divergent TIL repertoires. This signature is detectable in peripheral blood, and therefore accessible noninvasively. We anticipate that these immunophenotypes will be foundational to monitoring and predicting response to antiglioma vaccines and immunotherapy.

High-throughput T-cell receptor sequencing across chronic liver diseases reveals distinct disease-associated repertoires.

Hepatic T-cell infiltrates and a strong genetic human leukocyte antigen association represent characteristic features of various immune-mediated liver diseases. Conceptually the presence of disease-associated antigens is predicted to be reflected in T-cell receptor (TCR) repertoires. Here, we aimed to determine if disease-associated TCRs could be identified in the nonviral chronic liver diseases primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), and alcoholic liver disease (ALD). We performed high-throughput sequencing of the TCRβ chain complementarity-determining region 3 of liver-infiltrating T cells from PSC (n = 20), PBC (n = 10), and ALD (n = 10) patients, alongside genomic human leukocyte antigen typing. The frequency of TCRβ nucleotide sequences was significantly higher in PSC samples (2.53 ± 0.80, mean ± standard error of the mean) compared to PBC samples (1.13 ± 0.17, P < 0.0001) and ALD samples (0.62 ± 0.10, P < 0.0001). An average clonotype overlap of 0.85% was detected among PSC samples, significantly higher compared to the average overlap of 0.77% seen within the PBC (P = 0.024) and ALD groups (0.40%, P < 0.0001). From eight to 42 clonotypes were uniquely detected in each of the three disease groups (≥30% of the respective patient samples). Multiple, unique sequences using different variable family genes encoded the same amino acid clonotypes, providing additional support for antigen-driven selection. In PSC and PBC, disease-associated clonotypes were detected among patients with human leukocyte antigen susceptibility alleles. We demonstrate liver-infiltrating disease-associated clonotypes in all three diseases evaluated, and evidence for antigen-driven clonal expansions. Our findings indicate that differential TCR signatures, as determined by high-throughput sequencing, may represent an imprint of distinctive antigenic repertoires present in the different chronic liver diseases; this thereby opens up the prospect of studying disease-relevant T cells in order to better understand and treat liver disease.