Publications by authors named "Marissa Vignali"

47 Publications

Antimalarial antibody repertoire defined by plasma IG proteomics and single B cell IG sequencing.

JCI Insight 2020 11 19;5(22). Epub 2020 Nov 19.

Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland, USA.

Plasma antimalarial Ab can mediate antiparasite immunity but has not previously been characterized at the molecular level. Here, we develop an innovative strategy to characterize humoral responses by integrating profiles of plasma immunoglobulins (IGs) or Abs with those expressed on B cells as part of the B cell receptor. We applied this strategy to define plasma IG and to determine variable (V) gene usage after vaccination with the Plasmodium falciparum zygote antigen Pfs25. Using proteomic tools coupled with bulk immunosequencing data, we determined human antigen-binding fragment [F(ab')2] peptide sequences from plasma IG of adults who received 4 doses of Pfs25-EPA/Alhydrogel. Specifically, Pfs25 antigen-specific F(ab')2 peptides (Pfs25-IG) were aligned to cDNA sequences of IG heavy (IGH) chain complementarity determining region 3 from a data set generated by total peripheral B cell immunosequencing of the entire vaccinated population. IGHV4 was the most commonly identified IGHV subgroup of Pfs25-IG, a pattern that was corroborated by V heavy/V light chain sequencing of Pfs25-specific single B cells from 5 vaccinees and by matching plasma Pfs25-IG peptides and V-(D)-J sequences of Pfs25-specific single B cells from the same donor. Among 13 recombinant human mAbs generated from IG sequences of Pfs25-specific single B cells, a single IGHV4 mAb displayed strong neutralizing activity, reducing the number of P. falciparum oocysts in infected mosquitoes by more than 80% at 100 μg/mL. Our approach characterizes the human plasma Ab repertoire in response to the Pfs25-EPA/Alhydrogel vaccine and will be useful for studying circulating Abs in response to other vaccines as well as those induced during infections or autoimmune disorders.
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http://dx.doi.org/10.1172/jci.insight.143471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710313PMC
November 2020

Lymphocytic infiltration in stage II microsatellite stable colorectal tumors: A retrospective prognosis biomarker analysis.

PLoS Med 2020 09 24;17(9):e1003292. Epub 2020 Sep 24.

Catalan Institute of Oncology (ICO), Hospitalet de Llobregat, Barcelona, Spain.

Background: Identifying stage II patients with colorectal cancer (CRC) at higher risk of progression is a clinical priority in order to optimize the advantages of adjuvant chemotherapy while avoiding unnecessary toxicity. Recently, the intensity and the quality of the host immune response in the tumor microenvironment have been reported to have an important role in tumorigenesis and an inverse association with tumor progression. This association is well established in microsatellite instable CRC. In this work, we aim to assess the usefulness of measures of T-cell infiltration as prognostic biomarkers in 640 stage II, CRC tumors, 582 of them confirmed microsatellite stable.

Methods And Findings: We measured both the quantity and clonality index of T cells by means of T-cell receptor (TCR) immunosequencing in a discovery dataset (95 patients with colon cancer diagnosed at stage II and microsatellite stable, median age 67, 30% women) and replicated the results in 3 additional series of stage II patients from 2 countries. Series 1 and 2 were recruited in Barcelona, Spain and included 112 fresh frozen (FF, median age 69, 44% women) and 163 formalin-fixed paraffin-embedded (FFPE, median age 67, 39% women) samples, respectively. Series 3 included 270 FFPE samples from patients recruited in Haifa, Northern Israel, as part of a large case-control study of CRC (median age 73, 46% women). Median follow-up time was 81.1 months. Cox regression models were fitted to evaluate the prognostic value of T-cell abundance and Simpson clonality of TCR variants adjusting by sex, age, tumor location, and stage (IIA and IIB). In the discovery dataset, higher TCR abundance was associated with better prognosis (hazard ratio [HR] for ≥Q1 = 0.25, 95% CI 0.10-0.63, P = 0.003). A functional analysis of gene expression on these tumors revealed enrichment in pathways related to immune response. Higher values of clonality index (lower diversity) were not associated with worse disease-free survival, though the HR for ≥Q3 was 2.32 (95% CI 0.90-5.97, P = 0.08). These results were replicated in an independent FF dataset (TCR abundance: HR = 0.30, 95% CI 0.12-0.72, P = 0.007; clonality: HR = 3.32, 95% CI 1.38-7.94, P = 0.007). Also, the association with prognosis was tested in 2 independent FFPE datasets. The same association was observed with TCR abundance (HR = 0.41, 95% CI 0.18-0.93, P = 0.03 and HR = 0.56, 95% CI 0.31-1, P = 0.042, respectively, for each FFPE dataset). However, the clonality index was associated with prognosis only in the FFPE dataset from Israel (HR = 2.45, 95% CI 1.39-4.32, P = 0.002). Finally, a combined analysis combining all microsatellite stable (MSS) samples demonstrated a clear prognosis value both for TCR abundance (HR = 0.39, 95% CI 0.26-0.57, P = 1.3e-06) and the clonality index (HR = 2.13, 95% CI 1.44-3.15, P = 0.0002). These associations were also observed when variables were considered continuous in the models (HR per log2 of TCR abundance = 0.85, 95% CI 0.78-0.93, P = 0.0002; HR per log2 or clonality index = 1.16, 95% CI 1.03-1.31, P = 0.016).

Limitations: This is a retrospective study, and samples had been preserved with different methods. Validation series lack complete information about microsatellite instability (MSI) status and pathology assessment. The Molecular Epidemiology of Colorectal Cancer (MECC) study had information about overall survival instead of progression-free survival.

Conclusion: Results from this study demonstrate that tumor lymphocytes, assessed by TCR repertoire quantification based on a sequencing method, are an independent prognostic factor in microsatellite stable stage II CRC.
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http://dx.doi.org/10.1371/journal.pmed.1003292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7514069PMC
September 2020

A large-scale database of T-cell receptor beta (TCRβ) sequences and binding associations from natural and synthetic exposure to SARS-CoV-2.

Res Sq 2020 Aug 4. Epub 2020 Aug 4.

We describe the establishment and current content of the ImmuneCODE™ database, which includes hundreds of millions of T-cell Receptor (TCR) sequences from over 1,400 subjects exposed to or infected with the SARS-CoV-2 virus, as well as over 135,000 high-confidence SARS-CoV-2-specific TCRs. This database is made freely available, and the data contained in it can be downloaded and analyzed online or offline to assist with the global efforts to understand the immune response to the SARS-CoV-2 virus and develop new interventions.
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http://dx.doi.org/10.21203/rs.3.rs-51964/v1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418738PMC
August 2020

Analytical evaluation of the clonoSEQ Assay for establishing measurable (minimal) residual disease in acute lymphoblastic leukemia, chronic lymphocytic leukemia, and multiple myeloma.

BMC Cancer 2020 Jun 30;20(1):612. Epub 2020 Jun 30.

Research and Development, Adaptive Biotechnologies Corporation, 1551 Eastlake Ave. E, Suite 200, Seattle, WA, 98102, USA.

Background: The clonoSEQ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) identifies and tracks unique disease-associated immunoglobulin (Ig) sequences by next-generation sequencing of IgH, IgK, and IgL rearrangements and IgH-BCL1/2 translocations in malignant B cells. Here, we describe studies to validate the analytical performance of the assay using patient samples and cell lines.

Methods: Sensitivity and specificity were established by defining the limit of detection (LoD), limit of quantitation (LoQ) and limit of blank (LoB) in genomic DNA (gDNA) from 66 patients with multiple myeloma (MM), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL), and three cell lines. Healthy donor gDNA was used as a diluent to contrive samples with specific DNA masses and malignant-cell frequencies. Precision was validated using a range of samples contrived from patient gDNA, healthy donor gDNA, and 9 cell lines to generate measurable residual disease (MRD) frequencies spanning clinically relevant thresholds. Linearity was determined using samples contrived from cell line gDNA spiked into healthy gDNA to generate 11 MRD frequencies for each DNA input, then confirmed using clinical samples. Quantitation accuracy was assessed by (1) comparing clonoSEQ and multiparametric flow cytometry (mpFC) measurements of ALL and MM cell lines diluted in healthy mononuclear cells, and (2) analyzing precision study data for bias between clonoSEQ MRD results in diluted gDNA and those expected from mpFC based on original, undiluted samples. Repeatability of nucleotide base calls was assessed via the assay's ability to recover malignant clonotype sequences across several replicates, process features, and MRD levels.

Results: LoD and LoQ were estimated at 1.903 cells and 2.390 malignant cells, respectively. LoB was zero in healthy donor gDNA. Precision ranged from 18% CV (coefficient of variation) at higher DNA inputs to 68% CV near the LoD. Variance component analysis showed MRD results were robust, with expected laboratory process variations contributing ≤3% CV. Linearity and accuracy were demonstrated for each disease across orders of magnitude of clonal frequencies. Nucleotide sequence error rates were extremely low.

Conclusions: These studies validate the analytical performance of the clonoSEQ Assay and demonstrate its potential as a highly sensitive diagnostic tool for selected lymphoid malignancies.
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http://dx.doi.org/10.1186/s12885-020-07077-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325652PMC
June 2020

Comprehensive T cell repertoire characterization of non-small cell lung cancer.

Nat Commun 2020 01 30;11(1):603. Epub 2020 Jan 30.

Department of Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe, Houston, TX, 77030, USA.

Immunotherapy targeting T cells is increasingly utilized to treat solid tumors including non-small cell lung cancer (NSCLC). This requires a better understanding of the T cells in the lungs of patients with NSCLC. Here, we report T cell repertoire analysis in a cohort of 236 early-stage NSCLC patients. T cell repertoire attributes are associated with clinicopathologic features, mutational and immune landscape. A considerable proportion of the most prevalent T cells in tumors are also prevalent in the uninvolved tumor-adjacent lungs and appear specific to shared background mutations or viral infections. Patients with higher T cell repertoire homology between the tumor and uninvolved tumor-adjacent lung, suggesting a less tumor-focused T cell response, exhibit inferior survival. These findings indicate that a concise understanding of antigens and T cells in NSCLC is needed to improve therapeutic efficacy and reduce toxicity with immunotherapy, particularly adoptive T cell therapy.
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http://dx.doi.org/10.1038/s41467-019-14273-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992630PMC
January 2020

The Utility of T-Cell Clonality in Differential Diagnostics of Acute Graft-versus-Host Disease from Drug Hypersensitivity Reaction.

J Invest Dermatol 2020 06 2;140(6):1282-1285. Epub 2019 Dec 2.

Department of Dermatology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA. Electronic address:

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http://dx.doi.org/10.1016/j.jid.2019.11.009DOI Listing
June 2020

T Cell Repertoire Dynamics during Pregnancy in Multiple Sclerosis.

Cell Rep 2019 10;29(4):810-815.e4

Institut für Neuroimmunologie und Multiple Sklerose (INIMS), Universitätsklinikum Hamburg-Eppendorf, Martinistraße 52, 20246 Hamburg, Germany; Charité - Universitätsmedizin Berlin, Klinik für Psychiatrie und Medizinische Klinik m.S. Psychosomatik, Campus Benjamin Franklin, Hindenburgdamm 30, 12203 Berlin, Germany. Electronic address:

Identifying T cell clones associated with human autoimmunity has remained challenging. Intriguingly, many autoimmune diseases, including multiple sclerosis (MS), show strongly diminished activity during pregnancy, providing a unique research paradigm to explore dynamics of immune repertoire changes during active and inactive disease. Here, we characterize immunomodulation at the single-clone level by sequencing the T cell repertoire in healthy women and female MS patients over the course of pregnancy. Clonality is significantly reduced from the first to third trimester in MS patients, indicating that the T cell repertoire becomes less dominated by expanded clones. However, only a few T cell clones are substantially modulated during pregnancy in each patient. Moreover, relapse-associated T cell clones identified in an individual patient contract during pregnancy and expand during a postpartum relapse. Our data provide evidence that profiling the T cell repertoire during pregnancy could serve as a tool to discover and track "private" T cell clones associated with disease activity in autoimmunity.
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http://dx.doi.org/10.1016/j.celrep.2019.09.025DOI Listing
October 2019

Mobilization of CD8 T Cells via CXCR4 Blockade Facilitates PD-1 Checkpoint Therapy in Human Pancreatic Cancer.

Clin Cancer Res 2019 Jul 2;25(13):3934-3945. Epub 2019 Apr 2.

Department of Surgery, University of Washington, Seattle, Washington.

Purpose: Pancreatic ductal adenocarcinoma (PDA) is rarely cured, and single-agent immune checkpoint inhibition has not demonstrated clinical benefit despite the presence of large numbers of CD8 T cells. We hypothesized that tumor-infiltrating CD8 T cells harbor latent antitumor activity that can be reactivated using combination immunotherapy.

Experimental Design: Preserved human PDA specimens were analyzed using multiplex IHC (mIHC) and T-cell receptor (TCR) sequencing. Fresh tumor was treated in organotypic slice culture to test the effects of combination PD-1 and CXCR4 blockade. Slices were analyzed using IHC, flow cytometry, and live fluorescent microscopy to assess tumor kill, in addition to T-cell expansion and mobilization.

Results: mIHC demonstrated fewer CD8 T cells in juxtatumoral stroma containing carcinoma cells than in stroma devoid of them. Using TCR sequencing, we found clonal expansion in each tumor; high-frequency clones had multiple DNA rearrangements coding for the same amino acid binding sequence, which suggests response to common tumor antigens. Treatment of fresh human PDA slices with combination PD-1 and CXCR4 blockade led to increased tumor cell death concomitant with lymphocyte expansion. Live microscopy after combination therapy demonstrated CD8 T-cell migration into the juxtatumoral compartment and rapid increase in tumor cell apoptosis.

Conclusions: Endogenous tumor-reactive T cells are present within the human PDA tumor microenvironment and can be reactivated by combined blockade of PD-1 and CXCR4. This provides a new basis for the rational selection of combination immunotherapy for PDA..
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http://dx.doi.org/10.1158/1078-0432.CCR-19-0081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606359PMC
July 2019

Association of Tumor Microenvironment T-cell Repertoire and Mutational Load with Clinical Outcome after Sequential Checkpoint Blockade in Melanoma.

Cancer Immunol Res 2019 03 11;7(3):458-465. Epub 2019 Jan 11.

Laura and Isaac Perlmutter Cancer Center at NYU Langone Medical Center, New York, New York.

To understand prognostic factors for outcome between differentially sequenced nivolumab and ipilimumab in a randomized phase II trial, we measured T-cell infiltration and PD-L1 by IHC, T-cell repertoire metrics, and mutational load within the tumor. We used next-generation sequencing (NGS) and assessed the association of those parameters with response and overall survival. Immunosequencing of the T-cell receptor β-chain locus (TCRβ) from DNA of 91 pretreatment tumor samples and an additional 22 pairs of matched pre- and posttreatment samples from patients who received nivolumab followed by ipilimumab (nivo/ipi), or the reverse (ipi/nivo), was performed to measure T-cell clonality and fraction. Mutational and neoantigen load were also assessed by NGS in 82 of the 91 patients. Tumors were stained using IHC for PD-L1 and CD8 T cells. Pretreatment tumor TCR clonality and neoantigen load were marginally associated with best response with nivo/ipi ( = 0.04 and 0.05, respectively), but not with ipi/nivo. Amalgamated pretreatment mutational load and tumor T-cell fraction were significantly associated with best response with nivo/ipi ( = 0.002). Pretreatment PD-L1 staining intensity and CD8 T-cell counts were correlated with T-cell fraction and clonality, but not mutational or neoantigen load. Patients with increased T-cell fraction posttreatment at week 13 had a 30-fold increased likelihood of survival ( = 0.002). Mutational and neoantigen load, and T-cell infiltrate within the tumor, were associated with outcome of sequential checkpoint inhibition using nivolumab then ipilimumab, but not when ipilimumab was administered before nivolumab.
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http://dx.doi.org/10.1158/2326-6066.CIR-18-0226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397694PMC
March 2019

Cytomegalovirus Exposure in the Elderly Does Not Reduce CD8 T Cell Repertoire Diversity.

J Immunol 2019 01 12;202(2):476-483. Epub 2018 Dec 12.

Herbold Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109;

With age, the immune system becomes less effective, causing increased susceptibility to infection. Chronic CMV infection further impairs immune function and is associated with increased mortality in the elderly. CMV exposure elicits massive CD8 T cell clonal expansions and diminishes the cytotoxic T cell response to subsequent infections, leading to the hypothesis that to maintain homeostasis, T cell clones are expelled from the repertoire, reducing T cell repertoire diversity and diminishing the ability to combat new infections. However, in humans, the impact of CMV infection on the structure and diversity of the underlying T cell repertoire remains uncharacterized. Using TCR β-chain immunosequencing, we observed that the proportion of the peripheral blood T cell repertoire composed of the most numerous 0.1% of clones is larger in the CMV seropositive and gradually increases with age. We found that the T cell repertoire in the elderly grows to accommodate CMV-driven clonal expansions while preserving its underlying diversity and clonal structure. Our observations suggest that the maintenance of large CMV-reactive T cell clones throughout life does not compromise the underlying repertoire. Alternatively, we propose that the diminished immunity in elderly individuals with CMV is due to alterations in cellular function rather than a reduction in CD8 T cell repertoire diversity.
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http://dx.doi.org/10.4049/jimmunol.1800217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321841PMC
January 2019

A Diverse Lipid Antigen-Specific TCR Repertoire Is Clonally Expanded during Active Tuberculosis.

J Immunol 2018 08 18;201(3):888-896. Epub 2018 Jun 18.

Department of Medicine, University of Washington, Seattle, WA 98195;

Human T cells that recognize lipid Ags presented by highly conserved CD1 proteins often express semi-invariant TCRs, but the true diversity of lipid Ag-specific TCRs remains unknown. We use CD1b tetramers and high-throughput immunosequencing to analyze thousands of TCRs from ex vivo-sorted or in vitro-expanded T cells specific for the mycobacterial lipid Ag, glucose monomycolate. Our results reveal a surprisingly diverse repertoire resulting from editing of germline-encoded gene rearrangements analogous to MHC-restricted TCRs. We used a distance-based metric (TCRDist) to show how this diverse TCR repertoire builds upon previously reported conserved motifs by including subject-specific TCRs. In a South African cohort, we show that TCRDist can identify clonal expansion of diverse glucose monomycolate-specific TCRs and accurately distinguish patients with active tuberculosis from control subjects. These data suggest that similar mechanisms govern the selection and expansion of peptide and lipid Ag-specific T cells despite the nonpolymorphic nature of CD1.
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http://dx.doi.org/10.4049/jimmunol.1800186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6057832PMC
August 2018

Role of high-throughput sequencing in the diagnosis of cutaneous T-cell lymphoma.

J Clin Pathol 2018 Sep 10;71(9):814-820. Epub 2018 Apr 10.

Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Aims: Substantial clinicopathological overlap exists between cutaneous T-cell lymphoma (CTCL) and benign conditions, leading to diagnostic difficulties. We sought to delineate the utility of high-throughput sequencing (HTS) across a spectrum of histological findings in CTCL and reactive mimics.

Methods: One hundred skin biopsies obtained for clinical concern for CTCL were identified, comprising 25 cases each from four histological categories: 'definitive CTCL', 'atypical lymphoid infiltrate, concerning for CTCL', 'atypical lymphoid infiltrate, favour reactive' or 'reactive lymphoid infiltrate'. T-cell receptor gamma chain gene (TRG) PCR and T-cell receptor beta chain gene HTS were performed on both skin biopsy and concurrently collected peripheral blood; most peripheral blood samples were also analysed by flow cytometry.

Results: Histologically defined CTCL specimens had significantly higher clonality scores and T-cell fractions via HTS than all other groups (all p<0.002 and p<0.03, respectively). HTS was more diagnostically specific than TRG PCR in skin (100% vs 88%), while diagnostic sensitivity (68% vs 72%) and accuracy (84% vs 80%) were similar. TRG PCR and flow cytometry performed on blood were the least diagnostically useful assays. Some identically sized peaks detected by TRG PCR in concurrent skin and peripheral blood specimens were non-identical by HTS analysis.

Conclusions: HTS, by assessing both clonality and T-cell fractions in skin biopsies, is a powerful tool to aid in the diagnosis of CTCL. It is more specific than TRG PCR in distinguishing definitive CTCL from reactive and indeterminate histology. Identically sized peaks by TRG PCR, typically interpreted to be clonally related, are not always clonally identical by sequencing.
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http://dx.doi.org/10.1136/jclinpath-2018-205004DOI Listing
September 2018

Alloreactive T Cell Receptor Diversity against Structurally Similar or Dissimilar HLA-DP Antigens Assessed by Deep Sequencing.

Front Immunol 2018 19;9:280. Epub 2018 Feb 19.

Institute for Experimental Cellular Therapy, University Hospital Essen, Essen, Germany.

T cell alloreactivity is mediated by a self-human leukocyte antigen (HLA)-restricted T cell receptor (TCR) repertoire able to recognize both structurally similar and dissimilar allogeneic HLA molecules (i.e., differing by a single or several amino acids in their peptide-binding groove). We hypothesized that thymic selection on self-HLA molecules could have an indirect impact on the size and diversity of the alloreactive response. To test this possibility, we used TCR Vβ immunophenotyping and immunosequencing technology in a model of alloreactivity between self-HLA selected T cells and allogeneic HLA-DPB1 (DPB1) differing from self-DPB1*04:02 by a single (DPB1*02:01) or several (DPB1*09:01) amino acids in the peptide-binding groove. CD4+ T cells from three different self-DPB1*04:01,*04:02 individuals were stimulated with HeLa cells stably transduced with the relevant peptide processing machinery, co-stimulatory molecules, and HLA-DP. Flow cytometric quantification of the DPB1-specific T cell response measured as upregulation of the activation marker CD137 revealed significantly lower levels of alloreactivity against DPB1*02:01 compared with DPB1*09:01 (mean CD4+CD137+ frequency 35.2 ± 9.9 vs. 61.5 ± 7.7%, respectively,  < 0.0001). These quantitative differences were, however, not reflected by differences in the breadth of the alloreactive response at the Vβ level, with both alloantigens eliciting specific responses from all TCR-Vβ specificities tested by flow cytometry, albeit with higher levels of reactivity from most Vβ specificities against DPB1*09:01. In line with these observations, -CDR3 immunosequencing showed no significant differences in mean clonality of sorted CD137+CD4+ cells alloreactive against DPB1*02:01 or DPB1*09:01 [0.39 (0.36-0.45) and 0.39 (0.30-0.46), respectively], or in the cumulative frequencies of the 10 most frequent responding clones (55-67 and 58-62%, respectively). Most of the clones alloreactive against DPB1*02:01 (68.3%) or DPB1*09:01 (75.3%) were characterized by low-abundance (i.e., they were not appreciable among the pre-culture T cells). Interestingly, however, their cumulative frequency was lower against DPB1*02:01 compared with DPB1*09:01 (mean cumulative frequency 35.3 vs. 50.6%, respectively). Our data show that, despite lower levels of alloreactivity, a similar clonal diversity can be elicited by structurally similar compared with structurally dissimilar HLA-DPB1 alloantigens and demonstrate the power of immunosequencing in unraveling subtle qualitative changes not appreciable by conventional methods.
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http://dx.doi.org/10.3389/fimmu.2018.00280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827552PMC
April 2019

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

Front Immunol 2018 20;9:291. Epub 2018 Feb 20.

Institut für Neuroimmunologie und Multiple Sklerose (INIMS), Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.

While a link between inflammation and the development of neuropsychiatric disorders, including major depressive disorder (MDD) is supported by a growing body of evidence, little is known about the contribution of aberrant adaptive immunity in this context. Here, we conducted in-depth characterization of T cell phenotype and T cell receptor (TCR) repertoire in MDD. For this cross-sectional case-control study, we recruited antidepressant-free patients with MDD without any somatic or psychiatric comorbidities ( = 20), who were individually matched for sex, age, body mass index, and smoking status to a non-depressed control subject ( = 20). T cell phenotype and repertoire were interrogated using a combination of flow cytometry, gene expression analysis, and next generation sequencing. T cells from MDD patients showed significantly lower surface expression of the chemokine receptors CXCR3 and CCR6, which are known to be central to T cell differentiation and trafficking. In addition, we observed a shift within the CD4 T cell compartment characterized by a higher frequency of CD4CD25CD127 cells and higher mRNA expression in purified CD4 T cells obtained from patients with MDD. Finally, flow cytometry-based TCR Vβ repertoire analysis indicated a less diverse CD4 T cell repertoire in MDD, which was corroborated by next generation sequencing of the TCR β chain CDR3 region. Overall, these results suggest that T cell phenotype and TCR utilization are skewed on several levels in patients with MDD. Our study identifies putative cellular and molecular signatures of dysregulated adaptive immunity and reinforces the notion that T cells are a pathophysiologically relevant cell population in this disorder.
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http://dx.doi.org/10.3389/fimmu.2018.00291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5826233PMC
March 2019

Robust Antitumor Responses Result from Local Chemotherapy and CTLA-4 Blockade.

Cancer Immunol Res 2018 02 16;6(2):189-200. Epub 2018 Jan 16.

Department of Immunology, MD Anderson Cancer Center, Houston, Texas.

Clinical responses to immunotherapy have been associated with augmentation of preexisting immune responses, manifested by heightened inflammation in the tumor microenvironment. However, many tumors have a noninflamed microenvironment, and response rates to immunotherapy in melanoma have been <50%. We approached this problem by utilizing immunotherapy (CTLA-4 blockade) combined with chemotherapy to induce local inflammation. In murine models of melanoma and prostate cancer, the combination of chemotherapy and CTLA-4 blockade induced a shift in the cellular composition of the tumor microenvironment, with infiltrating CD8 and CD4 T cells increasing the CD8/Foxp3 T-cell ratio. These changes were associated with improved survival of the mice. To translate these findings into a clinical setting, 26 patients with advanced melanoma were treated locally by isolated limb infusion with the nitrogen mustard alkylating agent melphalan followed by systemic administration of CTLA-4 blocking antibody (ipilimumab) in a phase II trial. This combination of local chemotherapy with systemic checkpoint blockade inhibitor resulted in a response rate of 85% at 3 months (62% complete and 23% partial response rate) and a 58% progression-free survival at 1 year. The clinical response was associated with increased T-cell infiltration, similar to that seen in the murine models. Together, our findings suggest that local chemotherapy combined with checkpoint blockade-based immunotherapy results in a durable response to cancer therapy. .
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http://dx.doi.org/10.1158/2326-6066.CIR-17-0356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6857638PMC
February 2018

TCR Repertoire Intratumor Heterogeneity in Localized Lung Adenocarcinomas: An Association with Predicted Neoantigen Heterogeneity and Postsurgical Recurrence.

Cancer Discov 2017 10 21;7(10):1088-1097. Epub 2017 Jul 21.

Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas.

Genomic intratumor heterogeneity (ITH) may be associated with postsurgical relapse of localized lung adenocarcinomas. Recently, mutations, through generation of neoantigens, were shown to alter tumor immunogenicity through T-cell responses. Here, we performed sequencing of the T-cell receptor (TCR) in 45 tumor regions from 11 localized lung adenocarcinomas and observed substantial intratumor differences in T-cell density and clonality with the majority of T-cell clones restricted to individual tumor regions. TCR ITH positively correlated with predicted neoantigen ITH, suggesting that spatial differences in the T-cell repertoire may be driven by distinct neoantigens in different tumor regions. Finally, a higher degree of TCR ITH was associated with an increased risk of postsurgical relapse and shorter disease-free survival, suggesting a potential clinical significance of T-cell repertoire heterogeneity. The present study provides insights into the ITH of the T-cell repertoire in localized lung adenocarcinomas and its potential biological and clinical impact. The results suggest that T-cell repertoire ITH may be tightly associated to genomic ITH and disease relapse. .
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http://dx.doi.org/10.1158/2159-8290.CD-17-0256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5628137PMC
October 2017

Contribution of systemic and somatic factors to clinical response and resistance to PD-L1 blockade in urothelial cancer: An exploratory multi-omic analysis.

PLoS Med 2017 05 26;14(5):e1002309. Epub 2017 May 26.

Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America.

Background: Inhibition of programmed death-ligand 1 (PD-L1) with atezolizumab can induce durable clinical benefit (DCB) in patients with metastatic urothelial cancers, including complete remissions in patients with chemotherapy refractory disease. Although mutation load and PD-L1 immune cell (IC) staining have been associated with response, they lack sufficient sensitivity and specificity for clinical use. Thus, there is a need to evaluate the peripheral blood immune environment and to conduct detailed analyses of mutation load, predicted neoantigens, and immune cellular infiltration in tumors to enhance our understanding of the biologic underpinnings of response and resistance.

Methods And Findings: The goals of this study were to (1) evaluate the association of mutation load and predicted neoantigen load with therapeutic benefit and (2) determine whether intratumoral and peripheral blood T cell receptor (TCR) clonality inform clinical outcomes in urothelial carcinoma treated with atezolizumab. We hypothesized that an elevated mutation load in combination with T cell clonal dominance among intratumoral lymphocytes prior to treatment or among peripheral T cells after treatment would be associated with effective tumor control upon treatment with anti-PD-L1 therapy. We performed whole exome sequencing (WES), RNA sequencing (RNA-seq), and T cell receptor sequencing (TCR-seq) of pretreatment tumor samples as well as TCR-seq of matched, serially collected peripheral blood, collected before and after treatment with atezolizumab. These parameters were assessed for correlation with DCB (defined as progression-free survival [PFS] >6 months), PFS, and overall survival (OS), both alone and in the context of clinical and intratumoral parameters known to be predictive of survival in this disease state. Patients with DCB displayed a higher proportion of tumor-infiltrating T lymphocytes (TIL) (n = 24, Mann-Whitney p = 0.047). Pretreatment peripheral blood TCR clonality below the median was associated with improved PFS (n = 29, log-rank p = 0.048) and OS (n = 29, log-rank p = 0.011). Patients with DCB also demonstrated more substantial expansion of tumor-associated TCR clones in the peripheral blood 3 weeks after starting treatment (n = 22, Mann-Whitney p = 0.022). The combination of high pretreatment peripheral blood TCR clonality with elevated PD-L1 IC staining in tumor tissue was strongly associated with poor clinical outcomes (n = 10, hazard ratio (HR) (mean) = 89.88, HR (median) = 23.41, 95% CI [2.43, 506.94], p(HR > 1) = 0.0014). Marked variations in mutation loads were seen with different somatic variant calling methodologies, which, in turn, impacted associations with clinical outcomes. Missense mutation load, predicted neoantigen load, and expressed neoantigen load did not demonstrate significant association with DCB (n = 25, Mann-Whitney p = 0.22, n = 25, Mann-Whitney p = 0.55, and n = 25, Mann-Whitney p = 0.29, respectively). Instead, we found evidence of time-varying effects of somatic mutation load on PFS in this cohort (n = 25, p = 0.044). A limitation of our study is its small sample size (n = 29), a subset of the patients treated on IMvigor 210 (NCT02108652). Given the number of exploratory analyses performed, we intend for these results to be hypothesis-generating.

Conclusions: These results demonstrate the complex nature of immune response to checkpoint blockade and the compelling need for greater interrogation and data integration of both host and tumor factors. Incorporating these variables in prospective studies will facilitate identification and treatment of resistant patients.
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http://dx.doi.org/10.1371/journal.pmed.1002309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5446110PMC
May 2017

Fractionated Radiation Therapy Stimulates Antitumor Immunity Mediated by Both Resident and Infiltrating Polyclonal T-cell Populations when Combined with PD-1 Blockade.

Clin Cancer Res 2017 Sep 22;23(18):5514-5526. Epub 2017 May 22.

Targeted Therapy Group, Division of Molecular and Clinical Cancer Sciences, Manchester Cancer Research Centre, Christie Hospital, Manchester Academic Health Sciences Centre, United Kingdom.

Radiotherapy is a highly effective anticancer treatment forming part of the standard of care for the majority of patients, but local and distal disease recurrence remains a major cause of mortality. Radiotherapy is known to enhance tumor immunogenicity; however, the contribution and mechanisms of radiotherapy-induced immune responses are unknown. The impact of low-dose fractionated radiotherapy (5 × 2 Gy) alone and in combination with αPD-1 mAb on the tumor microenvironment was evaluated by flow cytometry and next-generation sequencing of the T-cell receptor (TCR) repertoire. A dual-tumor model was used, with fractionated radiotherapy delivered to a single tumor site to enable evaluation of the local and systemic response to treatment and ability to induce abscopal responses outside the radiation field. We show that fractionated radiotherapy leads to T-cell infiltration at the irradiated site; however, the TCR landscape remains dominated by polyclonal expansion of preexisting T-cell clones. Adaptive resistance via the PD-1/PD-L1 pathway restricts the generation of systemic anticancer immunity following radiotherapy, which can be overcome through combination with αPD-1 mAb leading to improved local and distal tumor control. Moreover, we show that effective clearance of tumor following combination therapy is dependent on both T cells resident in the tumor at the time of radiotherapy and infiltrating T cells. These data provide evidence that radiotherapy can enhance T-cell trafficking to locally treated tumor sites and augment preexisting anticancer T-cell responses with the capacity to mediate regression of out-of-field tumor lesions when delivered in combination with αPD-1 mAb therapy. .
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http://dx.doi.org/10.1158/1078-0432.CCR-16-1673DOI Listing
September 2017

Innate Immune Landscape in Early Lung Adenocarcinoma by Paired Single-Cell Analyses.

Cell 2017 05;169(4):750-765.e17

Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; The Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Human Immune Monitoring Center, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

To guide the design of immunotherapy strategies for patients with early stage lung tumors, we developed a multiscale immune profiling strategy to map the immune landscape of early lung adenocarcinoma lesions to search for tumor-driven immune changes. Utilizing a barcoding method that allows a simultaneous single-cell analysis of the tumor, non-involved lung, and blood cells, we provide a detailed immune cell atlas of early lung tumors. We show that stage I lung adenocarcinoma lesions already harbor significantly altered T cell and NK cell compartments. Moreover, we identified changes in tumor-infiltrating myeloid cell (TIM) subsets that likely compromise anti-tumor T cell immunity. Paired single-cell analyses thus offer valuable knowledge of tumor-driven immune changes, providing a powerful tool for the rational design of immune therapies. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.cell.2017.04.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737939PMC
May 2017

T-cell infiltration and clonality correlate with programmed cell death protein 1 and programmed death-ligand 1 expression in patients with soft tissue sarcomas.

Cancer 2017 Sep 2;123(17):3291-3304. Epub 2017 May 2.

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington.

Background: Patients with metastatic sarcomas have poor outcomes and although the disease may be amenable to immunotherapies, information regarding the immunologic profiles of soft tissue sarcoma (STS) subtypes is limited.

Methods: The authors identified patients with the common STS subtypes: leiomyosarcoma, undifferentiated pleomorphic sarcoma (UPS), synovial sarcoma (SS), well-differentiated/dedifferentiated liposarcoma, and myxoid/round cell liposarcoma. Gene expression, immunohistochemistry for programmed cell death protein (PD-1) and programmed death-ligand 1 (PD-L1), and T-cell receptor Vβ gene sequencing were performed on formalin-fixed, paraffin-embedded tumors from 81 patients. Differences in liposarcoma subsets also were evaluated.

Results: UPS and leiomyosarcoma had high expression levels of genes related to antigen presentation and T-cell infiltration. UPS were found to have higher levels of PD-L1 (P≤.001) and PD-1 (P≤.05) on immunohistochemistry and had the highest T-cell infiltration based on T-cell receptor sequencing, significantly more than SS, which had the lowest (P≤.05). T-cell infiltrates in UPS also were more oligoclonal compared with SS and liposarcoma (P≤.05). A model adjusted for STS histologic subtype found that for all sarcomas, T-cell infiltration and clonality were highly correlated with PD-1 and PD-L1 expression levels (P≤.01).

Conclusions: In the current study, the authors provide the most detailed overview of the immune microenvironment in sarcoma subtypes to date. UPS, which is a more highly mutated STS subtype, provokes a substantial immune response, suggesting that it may be well suited to treatment with immune checkpoint inhibitors. The SS and liposarcoma subsets are less mutated but do express immunogenic self-antigens, and therefore strategies to improve antigen presentation and T-cell infiltration may allow for successful immunotherapy in patients with these diagnoses. Cancer 2017;123:3291-304. © 2017 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
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http://dx.doi.org/10.1002/cncr.30726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568958PMC
September 2017

Immunosequencing identifies signatures of cytomegalovirus exposure history and HLA-mediated effects on the T cell repertoire.

Nat Genet 2017 May 3;49(5):659-665. Epub 2017 Apr 3.

Adaptive Biotechnologies, Seattle, Washington, USA.

An individual's T cell repertoire dynamically encodes their pathogen exposure history. To determine whether pathogen exposure signatures can be identified by documenting public T cell receptors (TCRs), we profiled the T cell repertoire of 666 subjects with known cytomegalovirus (CMV) serostatus by immunosequencing. We developed a statistical classification framework that could diagnose CMV status from the resulting catalog of TCRβ sequences with high specificity and sensitivity in both the original cohort and a validation cohort of 120 different subjects. We also confirmed that three of the identified CMV-associated TCRβ molecules bind CMV in vitro, and, moreover, we used this approach to accurately predict the HLA-A and HLA-B alleles of most subjects in the first cohort. As all memory T cell responses are encoded in the common format of somatic TCR recombination, our approach could potentially be generalized to a wide variety of disease states, as well as other immunological phenotypes, as a highly parallelizable diagnostic strategy.
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http://dx.doi.org/10.1038/ng.3822DOI Listing
May 2017

Ibrutinib Therapy Increases T Cell Repertoire Diversity in Patients with Chronic Lymphocytic Leukemia.

J Immunol 2017 02 11;198(4):1740-1747. Epub 2017 Jan 11.

Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX 77230;

The Bruton's tyrosine kinase inhibitor ibrutinib is a highly effective, new targeted therapy for chronic lymphocytic leukemia (CLL) that thwarts leukemia cell survival, growth, and tissue homing. The effects of ibrutinib treatment on the T cell compartment, which is clonally expanded and thought to support the growth of malignant B cells in CLL, are not fully characterized. Using next-generation sequencing technology, we characterized the diversity of TCRβ-chains in peripheral blood T cells from 15 CLL patients before and after 1 y of ibrutinib therapy. We noted elevated CD4 and CD8 T cell numbers and a restricted TCRβ repertoire in all pretreatment samples. After 1 y of ibrutinib therapy, elevated peripheral blood T cell numbers and T cell-related cytokine levels had normalized, and T cell repertoire diversity increased significantly. Dominant TCRβ clones in pretreatment samples declined or became undetectable, and the number of productive unique clones increased significantly during ibrutinib therapy, with the emergence of large numbers of low-frequency TCRβ clones. Importantly, broader TCR repertoire diversity was associated with clinical efficacy and lower rates of infections during ibrutinib therapy. These data demonstrate that ibrutinib therapy increases diversification of the T cell compartment in CLL patients, which contributes to cellular immune reconstitution.
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http://dx.doi.org/10.4049/jimmunol.1601190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5296363PMC
February 2017

Discovery of T Cell Receptor β Motifs Specific to HLA-B27-Positive Ankylosing Spondylitis by Deep Repertoire Sequence Analysis.

Arthritis Rheumatol 2017 04;69(4):774-784

Toronto Western Hospital, University of Toronto, Toronto, Ontario, Canada.

Objective: Ankylosing spondylitis (AS), a chronic inflammatory disorder, has a notable association with HLA-B27. One hypothesis suggests that a common antigen that binds to HLA-B27 is important for AS disease pathogenesis. This study was undertaken to determine sequences and motifs that are shared among HLA-B27-positive AS patients, using T cell repertoire next-generation sequencing.

Methods: To identify motifs enriched among B27-positive AS patients, we performed T cell receptor β (TCRβ) repertoire sequencing on samples from 191 B27-positive AS patients, 43 B27-negative AS patients, and 227 controls, and we obtained >77 million TCRβ clonotype sequences. First, we assessed whether any of 50 previously published sequences were enriched in B27-positive AS patients. We then used training and test cohorts to identify discovered motifs that were enriched in B27-positive AS patients versus controls.

Results: Six previously published and 11 discovered motifs were enriched in the B27-positive AS samples as compared to controls. After combining motifs related by sequence, we identified a total of 15 independent motifs. Both the full set of 15 motifs and a set of 6 published motifs were enriched in the B27-positive AS patients as compared to B27-positive healthy individuals (P = 0.049 and P = 0.001, respectively). Using an independent cohort, we validated that at least some of these motifs were associated with AS, and not simply with B27-positive status.

Conclusion: We identified TCRβ motifs that are enriched in B27-positive AS patients as compared to B27-positive healthy controls. This suggests that a common antigen, presented by HLA-B27 and detected by CD8+ T cells, may be associated with AS disease pathogenesis.
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http://dx.doi.org/10.1002/art.40028DOI Listing
April 2017

Tissue distribution and clonal diversity of the T and B cell repertoire in type 1 diabetes.

JCI Insight 2016 12 8;1(20):e88242. Epub 2016 Dec 8.

Department of Pathology, Immunology and Laboratory Medicine, University of Florida Diabetes Institute, Gainesville, Florida, USA.

The adaptive immune repertoire plays a critical role in type 1 diabetes (T1D) pathogenesis. However, efforts to characterize B cell and T cell receptor (TCR) profiles in T1D subjects have been largely limited to peripheral blood sampling and restricted to known antigens. To address this, we collected pancreatic draining lymph nodes (pLN), "irrelevant" nonpancreatic draining lymph nodes, peripheral blood mononuclear cells (PBMC), and splenocytes from T1D subjects ( = 18) and control donors ( = 9) as well as pancreatic islets from 1 T1D patient; from these tissues, we collected purified CD4 conventional T cells (Tconv), CD4 Treg, CD8 T cells, and B cells. By conducting high-throughput immunosequencing of the TCR β chain () and B cell receptor (BCR) immunoglobulin heavy chain () on these samples, we sought to analyze the molecular signature of the lymphocyte populations within these tissues and of T1D. Ultimately, we observed a highly tissue-restricted CD4 repertoire, while up to 24% of CD8 clones were shared among tissues. We surveyed our data set for previously described proinsulin- and glutamic acid decarboxylase 65-reactive (GAD65-reactive) receptors, and interestingly, we observed a TRB with homology to a known GAD65-reactive TCR (clone GAD4.13) present in 7 T1D donors (38.9%), representing >25% of all productive TRB within Tconv isolated from the pLN of 1 T1D subject. These data demonstrate diverse receptor signatures at the nucleotide level and enriched autoreactive clones at the amino acid level, supporting the utility of coupling immunosequencing data with knowledge of characterized autoreactive receptors.
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http://dx.doi.org/10.1172/jci.insight.88242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5135280PMC
December 2016

Clonal expansion of CD8 T cells in the systemic circulation precedes development of ipilimumab-induced toxicities.

Proc Natl Acad Sci U S A 2016 10 3;113(42):11919-11924. Epub 2016 Oct 3.

Department of Genitourinary Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030; Immunotherapy Platform, The University of Texas MD Anderson Cancer Center, Houston, TX 77030; Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030

Immune checkpoint therapies, such as ipilimumab, induce dramatic antitumor responses in a subset of patients with advanced malignancies, but they may also induce inflammatory responses and toxicities termed immune-related adverse events (irAEs). These irAEs are often low grade and manageable, but severe irAEs may lead to prolonged hospitalizations or fatalities. Early intervention is necessary to minimize morbidities that occur with severe irAEs. However, correlative biomarkers are currently lacking. In a phase II clinical trial that treated 27 patients with metastatic prostate cancer, we aimed to test the safety and efficacy of androgen deprivation therapy plus ipilimumab. In this study, we observed grade 3 toxicities in >40% of treated patients, which led to early closure of the study. Because ipilimumab enhances T-cell responses, we hypothesized that increased clonal T-cell responses in the systemic circulation may contribute to irAEs. Sequencing of the T-cell receptor β-chains in purified T cells revealed clonal expansion of CD8 T cells, which occurred in blood samples collected before the onset of grade 2-3 irAEs. These initial results suggested that expansion of ≥55 CD8 T-cell clones preceded the development of severe irAEs. We further evaluated available blood samples from a second trial and determined that patients who experienced grade 2-3 irAEs also had expansion of ≥55 CD8 T-cell clones in blood samples collected before the onset of irAEs. We propose that CD8 T-cell clonal expansion may be a correlative biomarker to enable close monitoring and early intervention for patients receiving ipilimumab.
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http://dx.doi.org/10.1073/pnas.1611421113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5081579PMC
October 2016

A Public Database of Memory and Naive B-Cell Receptor Sequences.

PLoS One 2016 11;11(8):e0160853. Epub 2016 Aug 11.

Adaptive Biotechnologies, Seattle, United States of America.

The vast diversity of B-cell receptors (BCR) and secreted antibodies enables the recognition of, and response to, a wide range of epitopes, but this diversity has also limited our understanding of humoral immunity. We present a public database of more than 37 million unique BCR sequences from three healthy adult donors that is many fold deeper than any existing resource, together with a set of online tools designed to facilitate the visualization and analysis of the annotated data. We estimate the clonal diversity of the naive and memory B-cell repertoires of healthy individuals, and provide a set of examples that illustrate the utility of the database, including several views of the basic properties of immunoglobulin heavy chain sequences, such as rearrangement length, subunit usage, and somatic hypermutation positions and dynamics.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160853PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4981401PMC
August 2017

Annotation of pseudogenic gene segments by massively parallel sequencing of rearranged lymphocyte receptor loci.

Genome Med 2015 Nov 23;7:123. Epub 2015 Nov 23.

Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

Background: The adaptive immune system generates a remarkable range of antigen-specific T-cell receptors (TCRs), allowing the recognition of a diverse set of antigens. Most of this diversity is encoded in the complementarity determining region 3 (CDR3) of the β chain of the αβ TCR, which is generated by somatic recombination of noncontiguous variable (V), diversity (D), and joining (J) gene segments. Deletion and non-templated insertion of nucleotides at the D-J and V-DJ junctions further increases diversity. Many of these gene segments are annotated as non-functional owing to defects in their primary sequence, the absence of motifs necessary for rearrangement, or chromosomal locations outside the TCR locus.

Methods: We sought to utilize a novel method, based on high-throughput sequencing of rearranged TCR genes in a large cohort of individuals, to evaluate the use of functional and non-functional alleles. We amplified and sequenced genomic DNA from the peripheral blood of 587 healthy volunteers using a multiplexed polymerase chain reaction assay that targets the variable region of the rearranged TCRβ locus, and we determined the presence and the proportion of productive rearrangements for each TCRβ V gene segment in each individual. We then used this information to annotate the functional status of TCRβ V gene segments in this cohort.

Results: For most TCRβ V gene segments, our method agrees with previously reported functional annotations. However, we identified novel non-functional alleles for several gene segments, some of which were used exclusively in our cohort to the detriment of reported functional alleles. We also saw that some gene segments reported to have both functional and non-functional alleles consistently behaved in our cohort as either functional or non-functional, suggesting that some reported alleles were not present in the population studied.

Conclusions: In this proof-of-principle study, we used high-throughput sequencing of the TCRβ locus of a large cohort of healthy volunteers to evaluate the use of functional and non-functional alleles of individual TCRβ V gene segments. With some modifications, our method has the potential to be extended to gene segments in the α, γ, and δ TCR loci, as well as the genes encoding for B-cell receptor chains.
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http://dx.doi.org/10.1186/s13073-015-0238-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4657264PMC
November 2015

T-cell receptor profiling in cancer.

Mol Oncol 2015 Dec 15;9(10):2063-70. Epub 2015 Sep 15.

Adaptive Biotechnologies, Seattle, WA, USA; Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

Immunosequencing is a platform technology that allows the enumeration, specification and quantification of each and every B- and/or T-cell in any biologic sample of interest. Thus, it provides an assessment of the level and distribution of all the clonal lymphocytes in any sample, and allows "tracking" of a single clone or multiple clones of interest over time or from tissue to tissue within a given patient. It is based on bias-controlled multiplex PCR and high-throughput sequencing, and it is highly accurate, standardized, and sensitive. In this review, we provide evidence that immunosequencing is becoming an important analytic tool for the emerging field of immune-oncology, and describe several applications of this approach, including the assessment of residual disease post therapy in lymphoid malignancies, the prediction of response to immunotherapeutics of solid tumors containing tumor infiltrating lymphocytes, the identification of clonal responses in vaccination, infectious disease, bone marrow reconstitution, and autoimmunity, and the exploration of whether there are population-based stereotyped responses to certain exposures or interventions.
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http://dx.doi.org/10.1016/j.molonc.2015.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5528728PMC
December 2015

High-throughput pairing of T cell receptor α and β sequences.

Sci Transl Med 2015 Aug;7(301):301ra131

Adaptive Biotechnologies, Seattle, WA 98102, USA. Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

The T cell receptor (TCR) protein is a heterodimer composed of an α chain and a β chain. TCR genes undergo somatic DNA rearrangements to generate the diversity of T cell binding specificities needed for effective immunity. Recently, high-throughput immunosequencing methods have been developed to profile the TCR α (TCRA) and TCR β (TCRB) repertoires. However, these methods cannot determine which TCRA and TCRB chains combine to form a specific TCR, which is essential for many functional and therapeutic applications. We describe and validate a method called pairSEQ, which can leverage the diversity of TCR sequences to accurately pair hundreds of thousands of TCRA and TCRB sequences in a single experiment. Our TCR pairing method uses standard laboratory consumables and equipment without the need for single-cell technologies. We show that pairSEQ can be applied to T cells from both blood and solid tissues, such as tumors.
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http://dx.doi.org/10.1126/scitranslmed.aac5624DOI Listing
August 2015

Dynamics of the cytotoxic T cell response to a model of acute viral infection.

J Virol 2015 Apr 4;89(8):4517-26. Epub 2015 Feb 4.

Adaptive Biotechnologies, Seattle, Washington, USA Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

Unlabelled: A detailed characterization of the dynamics and breadth of the immune response to an acute viral infection, as well as the determinants of recruitment to immunological memory, can greatly contribute to our basic understanding of the mechanics of the human immune system and can ultimately guide the design of effective vaccines. In addition to neutralizing antibodies, T cells have been shown to be critical for the effective resolution of acute viral infections. We report the first in-depth analysis of the dynamics of the CD8(+) T cell repertoire at the level of individual T cell clonal lineages upon vaccination of human volunteers with a single dose of YF-17D. This live attenuated yellow fever virus vaccine yields sterile, long-term immunity and has been previously used as a model to understand the immune response to a controlled acute viral infection. We identified and enumerated unique CD8(+) T cell clones specifically induced by this vaccine through a combined experimental and statistical approach that included high-throughput sequencing of the CDR3 variable region of the T cell receptor β-chain and an algorithm that detected significantly expanded T cell clones. This allowed us to establish that (i) on average, ∼ 2,000 CD8(+) T cell clones were induced by YF-17D, (ii) 5 to 6% of the responding clones were recruited to long-term memory 3 months postvaccination, (iii) the most highly expanded effector clones were preferentially recruited to the memory compartment, and (iv) a fraction of the YF-17D-induced clones could be identified from peripheral blood lymphocytes solely by measuring clonal expansion.

Importance: The exhaustive investigation of pathogen-induced effector T cells is essential to accurately quantify the dynamics of the human immune response. The yellow fever vaccine (YFV) has been broadly used as a model to understand how a controlled, self-resolving acute viral infection induces an effective and long-term protective immune response. Here, we extend this previous work by reporting the identity of activated effector T cell clones that expand in response to the YFV 2 weeks postvaccination (as defined by their unique T cell receptor gene sequence) and by tracking clones that enter the memory compartment 3 months postvaccination. This is the first study to use high-throughput sequencing of immune cells to characterize the breadth of the antiviral effector cell response and to determine the contribution of unique virus-induced clones to the long-lived memory T cell repertoire. Thus, this study establishes a benchmark against which future vaccines can be compared to predict their efficacy.
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http://dx.doi.org/10.1128/JVI.03474-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4442358PMC
April 2015