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

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.

Tracking the Fate and Origin of Clinically Relevant Adoptively Transferred CD8 T Cells .

Adoptively transferred tumor-specific cells can mediate tumor regression in cancers refractory to conventional therapy. Autologous polyclonal tumor-specific cytotoxic T cells (CTL) generated from peripheral blood and infused into patients with metastatic melanoma show enhanced persistence, compared to equivalent numbers of more extensively expanded monoclonal CTL, and are associated with complete remissions (CR) in select patients. We applied high-throughput T cell receptor Vβ sequencing (HTTCS) to identify individual clonotypes within CTL products, track them post-infusion and then deduce the pre-adoptive transfer (endogenous) frequencies of cells ultimately responsible for tumor regression.

Identification of patient-specific and tumor-shared T cell receptor sequences in renal cell carcinoma patients.

A major requirement for cancer immunotherapy is the development of biomarkers for prognosis and for monitoring therapy response. In an attempt to evaluate the immune response of renal cell carcinoma (RCC) patients, tumor lesions and / or blood samples from 12 RCC patients underwent deep T cell receptor (TCR) sequencing. Despite the low number of samples, different TCR distribution patterns could be detected.

T-cell receptor repertoire variation may be associated with type 2 diabetes mellitus in humans.

Background: Recent work in Pima Indians, a population with high rates of obesity and type 2 diabetes mellitus (T2DM), demonstrated that human leukocyte antigen haplotype DRB1*02 carriers have an increased acute insulin response and decreased risk for the development of T2DM, implicating loss of self-tolerance in the pathogenesis of T2DM. Advances in genomic sequencing have made T-cell receptor repertoire analysis a practical mode of investigation.

Methods: High-throughput sequencing of T-cell receptor complementarity-determining region 3 was carried out in male Pima Indians with normal glucose regulation (n = 11; age = 31 ± 8 years; %fat = 30.

Intrathecal BCR transcriptome in multiple sclerosis versus other neuroinflammation: Equally diverse and compartmentalized, but more mutated, biased and overlapping with the proteome.

The mechanisms driving the intrathecal synthesis of IgG in multiple sclerosis (MS) are unknown. We combined high-throughput sequencing of transcribed immunoglobulin heavy-chain variable (IGHV) genes and mass spectrometry to chart the diversity and compartmentalization of IgG-producing B cells in the cerebrospinal fluid (CSF) of MS patients and controls with other neuroinflammatory diseases. In both groups, a few clones dominated the intrathecal IGHV transcriptome.

Common clonal origin of central and resident memory T cells following skin immunization.

Central memory T (TCM) cells in lymph nodes (LNs) and resident memory T (TRM) cells in peripheral tissues have distinct roles in protective immunity. Both are generated after primary infections, but their clonal origins have been unclear. To address this question, we immunized mice through the skin with a protein antigen, a chemical hapten, or a non-replicating poxvirus.

IgH-V(D)J NGS-MRD measurement pre- and early post-allotransplant defines very low- and very high-risk ALL patients.

Positive detection of minimal residual disease (MRD) by multichannel flow cytometry (MFC) prior to hematopoietic cell transplantation (HCT) of patients with acute lymphoblastic leukemia (ALL) identifies patients at high risk for relapse, but many pre-HCT MFC-MRD negative patients also relapse, and the predictive power MFC-MRD early post-HCT is poor. To test whether the increased sensitivity of next-generation sequencing (NGS)-MRD better identifies pre- and post-HCT relapse risk, we performed immunoglobulin heavy chain (IgH) variable, diversity, and joining (V[D]J) DNA sequences J NGS-MRD on 56 patients with B-cell ALL enrolled in Children's Oncology Group trial ASCT0431. NGS-MRD predicted relapse and survival more accurately than MFC-MRD (P < .

CMV reactivation drives posttransplant T-cell reconstitution and results in defects in the underlying TCRβ repertoire.

Although cytomegalovirus (CMV) reactivation has long been implicated in posttransplant immune dysfunction, the molecular mechanisms that drive this phenomenon remain undetermined. To address this, we combined multiparameter flow cytometric analysis and T-cell subpopulation sorting with high-throughput sequencing of the T-cell repertoire, to produce a thorough evaluation of the impact of CMV reactivation on T-cell reconstitution after unrelated-donor hematopoietic stem cell transplant. We observed that CMV reactivation drove a >50-fold specific expansion of Granzyme B(high)/CD28(low)/CD57(high)/CD8(+) effector memory T cells (Tem) and resulted in a linked contraction of all naive T cells, including CD31(+)/CD4(+) putative thymic emigrants.

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

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.

Immune activation and a 9-year ongoing complete remission following CD40 antibody therapy and metastasectomy in a patient with metastatic melanoma.

Direct immune activation via agonistic mAbs is a potentially complementary approach to therapeutic blockade of inhibitory immune receptors in cancer. Here, we provide genetic analysis of the immunologic consequences associated with the use of an agonistic CD40 mAb in a patient with metastatic melanoma who responded, underwent a single metastasectomy, and then achieved a complete remission ongoing for more than 9 years after starting therapy. Tumor microenvironment after immunotherapy was associated with proinflammatory modulations and emergence of a de novo T-cell repertoire as detected by next-generation sequencing of T-cell receptors (TCR) in the tumor and blood.

T cell repertoire following autologous stem cell transplantation for multiple sclerosis.

Autologous hematopoietic stem cell transplantation (HSCT) is commonly employed for hematologic and non-hematologic malignancies. In clinical trials, HSCT has been evaluated for severe autoimmunity as a method to "reset" the immune system and produce a new, non-autoimmune repertoire. While the feasibility of eliminating the vast majority of mature T cells is well established, accurate and quantitative determination of the relationship of regenerated T cells to the baseline repertoire has been difficult to assess.

Intramuscular therapeutic vaccination targeting HPV16 induces T cell responses that localize in mucosal lesions.

About 25% of high-grade cervical intraepithelial neoplasias (CIN2/3) caused by human papillomavirus serotype 16 (HPV16) undergo complete spontaneous regression. However, to date, therapeutic vaccination strategies for HPV disease have yielded limited success when measured by their ability to induce robust peripheral blood T cell responses to vaccine antigen. We report marked immunologic changes in the target lesion microenvironment after intramuscular therapeutic vaccination targeting HPV16 E6/E7 antigens, in subjects with CIN2/3 who had modest detectable responses in circulating T lymphocytes.

Regression of metastatic Merkel cell carcinoma following transfer of polyomavirus-specific T cells and therapies capable of re-inducing HLA class-I.

Merkel cell carcinoma (MCC) is an aggressive skin cancer that typically requires the persistent expression of Merkel cell polyomavirus (MCPyV) oncoproteins that can serve as ideal immunotherapeutic targets. Several immune evasion mechanisms are active in MCC including down-regulation of HLA class-I expression on tumor cells and dysfunctional endogenous MCPyV-specific CD8 T cell responses. To overcome these obstacles, we combined local and systemic immune therapies in a 67-year-old man, who developed metastatic MCPyV-expressing MCC.

Minimal residual disease monitoring with high-throughput sequencing of T cell receptors in cutaneous T cell lymphoma.

Mycosis fungoides (MF) and the leukemic presentation Sézary syndrome (SS) are clonal T cell lymphomas arising from the skin and are considered noncurable with standard therapies. To develop a specific and sensitive monitoring tool, we tested the ability of high-throughput sequencing (HTS) of T cell receptors (TCRB) to monitor minimal residual disease (MRD) after allogeneic hematopoietic cell transplantation. Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs) or skin samples.

Using synthetic templates to design an unbiased multiplex PCR assay.

T and B cell receptor loci undergo combinatorial rearrangement, generating a diverse immune receptor repertoire, which is vital for recognition of potential antigens. Here we use a multiplex PCR with a mixture of primers targeting the rearranged variable and joining segments to capture receptor diversity. Differential hybridization kinetics can introduce significant amplification biases that alter the composition of sequence libraries prepared by multiplex PCR.

Tumor-infiltrating lymphocytes in colorectal tumors display a diversity of T cell receptor sequences that differ from the T cells in adjacent mucosal tissue.

Tumors from colorectal cancer (CRC) are generally immunogenic and commonly infiltrated with T lymphocytes. However, the details of the adaptive immune reaction to these tumors are poorly understood. We have accrued both colon tumor samples and adjacent healthy mucosal samples from 15 CRC patients to study lymphocytes infiltrating these tissues.

The restricted DH gene reading frame usage in the expressed human antibody repertoire is selected based upon its amino acid content.

The Ab repertoire is not uniform. Some variable, diversity, and joining genes are used more frequently than others. Nonuniform usage can result from the rearrangement process, or from selection.

Estimating the ratio of CD4+ to CD8+ T cells using high-throughput sequence data.

Mature T cells express either CD8 or CD4, defining two physiologically distinct populations of T cells. CD8+ T cells, or killer T-cells, and CD4+ T cells, or helper T cells, effect different aspects of T cell mediated adaptive immunity. Currently, determining the ratio of CD4+ to CD8+ T cells requires flow cytometry or immunohistochemistry.

Fractal organization of the human T cell repertoire in health and after stem cell transplantation.

T cell repertoire diversity is generated in part by recombination of variable (V), diversity (D), and joining (J) segments in the T cell receptor β (TCR) locus. T cell clonal frequency distribution determined by high-throughput sequencing of TCR β in 10 stem cell transplantation (SCT) donors revealed a fractal, self-similar frequency distribution of unique TCR bearing clones with respect to V, D, and J segment usage in the T cell repertoire of these individuals. Further, ranking of T cell clones by frequency of gene segment usage in the observed sequences revealed an ordered distribution of dominant clones conforming to a power law, with a fractal dimension of 1.

Ultra-sensitive detection of rare T cell clones.

Advances in high-throughput sequencing have enabled technologies that probe the adaptive immune system with unprecedented depth. We have developed a multiplex PCR method to sequence tens of millions of T cell receptors (TCRs) from a single sample in a few days. A method is presented to test the precision, accuracy, and sensitivity of this assay.

Deep sequencing of the human TCRγ and TCRβ repertoires suggests that TCRβ rearranges after αβ and γδ T cell commitment.

T lymphocytes respond to a broad array of pathogens with the combinatorial diversity of the T cell receptor (TCR). This adaptive response is possible because of the unique structure of the TCR, which is composed of two chains, either αβ or γδ, that undergo genetic rearrangement in the thymus. αβ and γδ T cells are functionally distinct within the host but are derived from a common multipotent precursor.

Polymorphisms of the IL1-receptor antagonist gene (IL1RN) are associated with multiple markers of systemic inflammation.

Background: Circulating levels of acute phase reactant proteins such as plasma C-reactive protein (CRP) are likely influenced by multiple genes regulating the innate immune response.

Methods And Results: We screened a set of 16 inflammation-related genes for association with CRP in a large population-based study of healthy young adults (n=1627). Results were validated in 2 independent studies (n=1208 and n=4310), including a pooled analysis of all 3 studies.