CD19 CAR–T cells of defined CD4+:CD8+ composition in adult B cell ALL patientsThe Journal of Clinical Investigation | April, 2016
T cells that have been modified to express a CD19-specific chimeric antigen receptor (CAR) have antitumor activity in B cell malignancies; however, identification of the factors that determine toxicity and efficacy of these T cells has been challenging in prior studies in which phenotypically heterogeneous CAR–T cell products were prepared from unselected T cells.
Immunotherapy with a CAR–T cell product of defined composition enabled identification of factors that correlated with CAR–T cell expansion, persistence, and toxicity and facilitated design of lymphodepletion and CAR–T cell dosing strategies that mitigated toxicity and improved disease-free survival.VIEW
TCR Sequencing Can Identify and Track Glioma-Infiltrating T Cells after DC VaccinationCancer Immunology Research | March, 2016
Although immunotherapeutic strategies are emerging as adjunctive treatments for cancer, sensitive methods of monitoring the immune response after treatment remain to be established. We used a novel next-generation sequencing approach to determine whether quantitative assessments of tumor-infiltrating lymphocyte (TIL) content and the degree of overlap of T-cell receptor (TCR) sequences in brain tumors and peripheral blood were predictors of immune response and overall survival in glioblastoma patients treated with autologous tumor lysate–pulsed dendritic cell immunotherapy. A statistically significant correlation was found between a higher estimated TIL content and increased time to progression and overall survival.VIEW
T-cell receptor profiling in cancerMolecular Oncology | September, 2015
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.VIEW
Multiplex Identification of Antigen-Specific T Cell Receptors Using a Combination of Immune Assays and Immune Receptor SequencingPLOS ONE | October, 2015
Monitoring antigen-specific T cells is critical for the study of immune responses and development of biomarkers and immunotherapeutics. We developed a novel multiplex assay that combines conventional immune monitoring techniques and immune receptor repertoire sequencing to enable identification of T cells specific to large numbers of antigens simultaneously. We multiplexed 30 different antigens and identified 427 antigen-specific clonotypes from 5 individuals with frequencies as low as 1 per million T cells. The clonotypes identified were validated several ways including repeatability, concordance with published clonotypes, and high correlation with ELISPOT.VIEW
High-throughput pairing of T cell receptor α and β sequencesScience Translational Medicine | August, 2015
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.VIEW
Common clonal origin of central and resident memory T cells following skin immunizationNature Medicine | May, 2015
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. We then analyzed antigen-activated T cells from different tissues using high-throughput sequencing (HTS) of the gene encoding the T cell receptor (TCR) β-chain (Trb, also known asTcrb) using CDR3 sequences to simultaneously track thousands of unique T cells.VIEW
Adult Low-Hypodiploid Acute B-Lymphoblastic Leukemia With IKZF3 Deletion and TP53 MutationComparison With Pediatric Patients
Chromosomal ploidy is a major risk stratification tool for acute B-cell lymphoblastic leukemia (B-ALL). Low hypodiploidy and near-haploidy are thought to be confined to pediatric B-ALL and associated with a poor prognosis. Doubling of either a low-hypodiploid or a near-haploid clone results in an apparently high-hyperdiploid karyotype, which is often misclassified for risk.
METHODS: We studied four patients with B-ALL who had chromosome genomic array testing (CGAT), along with fluorescence in situ hybridization and mutation testing.
RESULTS: We identified a unique case of adult B-ALL with masked low hypodiploidy (mLH) by genomic duplication, along with a somatic deletion of the IKZF3 gene and a somatic TP53 mutation. Three cases of pediatric B-ALL with mLH, two with TP53 mutations and one untested, were also identified and compared with the adult patient.
CONCLUSIONS: CGAT was critical in the genotype clarification of these cases through detection of copy-neutral loss of heterozygosity and should be considered performing for B-ALL with apparent hyperdiploidy for accurate prognostic risk stratification and treatment planning.
Detection and Tracking of NY-ESO-1-Specific CD8+ T Cells by High-Throughput T Cell Receptor β (TCRB) Gene Rearrangements Sequencing in a Peptide-Vaccinated Patient.
Comprehensive immunological evaluation is crucial for monitoring patients undergoing antigen-specific cancer immunotherapy. The identification and quantification of T cell responses is most important for the further development of such therapies. Using well-characterized clinical samples from a high responder patient (TK-f01) in an NY-ESO-1f peptide vaccine study, we performed high-throughput T cell receptor β-chain (TCRB) gene next generation sequencing (NGS) to monitor the frequency of NY-ESO-1-specific CD8+ T cells. We compared these results with those of conventional immunological assays, such as IFN-γ capture, tetramer binding and limiting dilution clonality assays. We sequenced human TCRB complementarity-determining region 3 (CDR3) rearrangements of two NY-ESO-1f-specific CD8+ T cell clones, 6-8L and 2F6, as well as PBMCs over the course of peptide vaccination. Clone 6-8L possessed the TCRB CDR3 gene TCRBV11-03*01 and BJ02-01*01 with amino acid sequence CASSLRGNEQFF, whereas 2F6 possessed TCRBV05-08*01 and BJ02-04*01 (CASSLVGTNIQYF). Using these two sequences as models, we evaluated the frequency of NY-ESO-1-specific CD8+ T cells in PBMCs ex vivo. The 6-8L CDR3 sequence was the second most frequent in PBMC and was present at high frequency (0.7133%) even prior to vaccination, and sustained over the course of vaccination. Despite a marked expansion of NY-ESO-1-specific CD8+ T cells detected from the first through 6th vaccination by tetramer staining and IFN-γ capture assays, as evaluated by CDR3 sequencing the frequency did not increase with increasing rounds of peptide vaccination. By clonal analysis using 12 day in vitro stimulation, the frequency of B*52:01-restricted NY-ESO-1f peptide-specific CD8+ T cells in PBMCs was estimated as only 0.0023%, far below the 0.7133% by NGS sequencing. Thus, assays requiring in vitro stimulation might be underestimating the frequency of clones with lower proliferation potential. High-throughput TCRB sequencing using NGS can potentially better estimate the actual frequency of antigen-specific T cells and thus provide more accurate patient monitoring.
Deep Sequencing of the T-cell Receptor Repertoire Demonstrates Polyclonal T-cell Infiltrates in Psoriasis
It is well known that infiltration of pathogenic T-cells plays an important role in psoriasis pathogenesis. However, the antigen specificity of these activated T-cells is relatively unknown. Previous studies using T-cell receptor polymerase chain reaction technology (TCR-PCR) have suggested there are expanded T-cell receptor (TCR) clones in psoriatic skin, suggesting a response to an unknown psoriatic antigen. Here we describe the results of high-throughput deep sequencing of the entire αβ- and γδ- TCR repertoire in normal healthy skin and psoriatic lesional and non-lesional skin. From this study, we were able to determine that there is a significant increase in the abundance of unique β- and γ- TCR sequences in psoriatic lesional skin compared to non-lesional and normal skin, and that the entire T-cell repertoire in psoriasis is polyclonal, with similar diversity to normal and non-lesional skin. Comparison of the αβ- and γδ- TCR repertoire in paired non-lesional and lesional samples showed many common clones within a patient, and these close were often equally abundant in non-lesional and lesional skin, again suggesting a diverse T-cell repertoire. Although there were similar (and low) amounts of shared β-chain sequences between different patient samples, there was significantly increased sequence sharing of the γ-chain in psoriatic skin from different individuals compared to those without psoriasis. This suggests that although the T-cell response in psoriasis is highly polyclonal, particular γδ- T-cell subsets may be associated with this disease. Overall, our findings present the feasibility of this technology to determine the entire αβ- and γδ- T-cell repertoire in skin, and that psoriasis contains polyclonal and diverse αβ- and γδ- T-cell populations.
High-throughput pairing of T cell receptor α and β sequences
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.
Minimal residual disease testing after stem cell transplantation for multiple myeloma
Increased use of novel agents and autologous stem cell transplantation has led to a significant improvement in PFS and overall survival in patients with multiple myeloma. Despite improved treatment strategies, most patients eventually relapse due to persistent low levels of disease in the bone marrow. Increasingly sensitive methods to measure or detect such disease have been evaluated, including multi-parametric flow cytometry, PCR, next-generation sequencing and imaging modalities. The following literature review examines current methods for detecting and monitoring minimal or measurable residual disease (MRD) in the post-transplant setting. Improved methods for detecting MRD will refine the current definitions of remission and could guide treatment approaches.
Immune monitoring technology primer: immunosequencing
BACKGROUND: Profiling of the immune receptor repertoire is becoming increasingly relevant to the understanding and clinical management of cancer, autoimmunity, aging, and infectious disease.
FINDINGS: A platform technology is described that provides comprehensive immune receptor profiling.
CONCLUSION: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. It is based on bias-controlled multiplex PCR and high throughput sequencing and is highly accurate, standardized, and sensitive.
A high density of tertiary lymphoid structure B cells in lung tumors is associated with increased CD4+ T cell receptor repertoire clonality
Human CD45RA− FoxP3hi Memory-Type Regulatory T Cells Show Distinct TCR Repertoires With Conventional T Cells and Play an Important Role in Controlling Early Immune Activation
Adoptive immunotherapy with regulatory T cells (Treg) is a new option to promote immune tolerance following solid organ transplantation (SOT). However, Treg from elderly patients awaiting transplantation are dominated by the CD45RA-CD62L+ central memory type Treg subset (TregCM), and the yield of well- characterized and stable na€ıve Treg (TregN) is low. It is, therefore, important to determine whether these TregCM are derived from the thymus and express high stability, suppressive capacity and a broad antigen repertoire like TregN. In this study, we showed that TregCM use a different T cell receptor (TCR) repertoire from conventional T cells (Tconv), using next-generation sequencing of all 24 Vß families, with an average depth of 534 677 sequences. This showed almost no contamination with induced Treg. Further- more, TregCM showed enhanced suppressive activity on Tconv at early checkpoints of immune activation controlling activation markers expression and cytokine secretion, but comparable inhibition of proliferation. Following in vitro expansion under mTOR inhibition, TregCM expanded equally as well as TregN without losing their function. Despite relatively limited TCR repertoire, TregCM also showed specific alloresponse, although slightly reduced compared to TregN. These results support the therapeutic usefulness of manufacturing Treg products from CD45RA-CD62L+ Treg-enriched starting material to be applied for adoptive Treg therapy.
Abbreviations: CM, central memory; CTLA-4, cytotoxic T-lymphocyte antigen 4; EM, effector memory; iTreg, induced regulatory T cells; MH index, Morisita–Horn similarity; mTOR, mammalian target of rapamycin; NGS, next-generation sequencing; SE, Shannon entro- py; SOT, solid organ transplantation; Tconv, conven- tional T cells; TconvM, memory conventional T cells; TconvN, naïve conventional T cells; TCR, T cell receptor; TregM, memory regulatory T cells; TregN, naïve regulatory T cells; TSDR, Treg-specific demethylation region; tTreg, thymus-derived regulatory T cells .
Unexpected Role for Adaptive Alpha Beta Th17 Cells in Acute Respiratory Distress Syndrome
John T. Li, Andrew C. Melton, George Su, David E. Hamm, Michael LaFemina, James Howard, Xiaohui Fang, Sudarshan Bhat, Kieu-My Huynh, Cecilia M. O’Kane, Rebecca J. Ingram, Roshell R. Muir, Daniel F. McAuley, Michael A. Matthay, and Dean Sheppard
Acute respiratory distress syndrome (ARDS) is a devastating disorder characterized by increased alveolar permeability with no effective treatment beyond supportive care. Current mechanisms underlying ARDS focus on alveolar endothelial and epithelial injury caused by products of innate immune cells and platelets. However, the role of adaptive immune cells in ARDS remains largely unknown. In this study, we report that expansion of Ag-specific alpha beta Th17 cells contributes to ARDS by local secretion of IL-17A, which in turn directly increases alveolar epithelial permeability. Mice with a highly restrictive defect in Ag-specific alpha beta Th17 cells were protected from experimental ARDS induced by a single dose of endotracheal LPS. Loss of IL-17 receptor C or Ab blockade of IL-17A was similarly protective, further suggesting that IL-17A released by these cells was responsible for this effect. LPS induced a rapid and specific clonal expansion of alpha beta Th17 cells in the lung, as determined by deep sequencing of the hypervariable CD3RßVJ region of the TCR. Our findings could be relevant to ARDS in humans, because we found significant elevation of IL- 17A in bronchoalveolar lavage fluid from patients with ARDS, and rIL-17A directly increased permeability across cultured human alveolar epithelial monolayers. These results reveal a previously unexpected role for adaptive immune responses that increase alveolar permeability in ARDS and suggest that alpha beta Th17 cells and IL-17A could be novel therapeutic targets for this currently untreatable disease.
Analysis of Celiac Disease Autoreactive Gut Plasma Cells and Their Corresponding Memory Compartment in Peripheral Blood Using High-Throughput Sequencing
Omri Snir, Luka Mesin, Moriah Gidoni, Knut E. A. Lundin, Gur Yaari, and Ludvig M. Sollid
Autoreactive IgA plasma cells (PCs) specific for the enzyme transglutaminase 2 (TG2) are abundant in the small intestine of patients with active celiac disease (CD), and their number drops in patients treated by dietary gluten elimination. Little is known about their characteristics and their role in the disease. In this study, using high-throughput sequencing of the IgH V region (IGHV) genes, we have studied features of TG2-specific PCs and their related B cell clones in peripheral blood. We found that TG2-specific PCs from both untreated and treated patients have acquired lower number of somatic hypermutation and used focused IGHV repertoire with over- representation of the IGHV3-48, IGHV4-59, IGHV5-10-1, and IGHV5-51 gene segments. Furthermore, these PCs were clonally expanded and showed signs of affinity maturation. Lineage trees demonstrated shared clones between gut PCs and blood memory B cells, primarily IgAs. Some trees also involved IgG cells, suggesting that anti-TG2 IgA and IgG responses are related. Similarly to TG2-specific PCs, clonally related memory IgA B cells of blood showed lower mutation rates with biased usage of IGHV3-48 and IGHV5-51. Such memory cells were rare in peripheral blood, yet detectable in most patients assessed by production of anti-TG2 Abs in vitro following stimulation of cells from patients who had been on a long-term gluten-free diet. Thus, the Ab response to TG2 in CD, while maintaining its IGHV gene usage, is dynamically regulated in response to gluten exposure with a low degree of main- tenance at both PC and memory B cell levels in patients in remission.
TCR ITAM multiplicity is required for the generation of follicular helper T-cells
The T-cell antigen receptor (TCR) complex contains 10 copies of a di-tyrosine Immunoreceptor-Tyrosine-based-Activation-Motif (ITAM) that initiates TCR signalling by recruiting protein tyrosine kinases. ITAM multiplicity amplifies TCR signals, but the importance of this capability for T-cell responses remains undefined. Most TCR ITAMs (6 of 10) are contributed by the CD3z subunits. We generated ‘knock-in’ mice that express non-signalling CD3z chains in lieu of wild-type CD3z. Here we demonstrate that ITAM multiplicity is important for the development of innate-like T-cells and follicular helper T-cells, events that are known to require strong/sustained TCR–ligand interactions, but is not essential for ‘general’ T-cell responses including proliferation and cytokine production or for the generation of a diverse antigen-reactive TCR repertoire.
Longitudinal analysis of peripheral blood T cell receptor diversity in patients with systemic lupus erythematosus by next-generation sequencing
T cells play an important role in the pathogenesis of systemic lupus erythematosus (SLE). Clonal expansion of T cells correlating with disease activity has been observed in peripheral blood (PB) of SLE subjects. Recently, next-generation sequencing (NGS) of the T cell receptor (TCR) β loci has emerged as a sensitive way to measure the T cell repertoire. In this study we utilized NGS to assess whether changes in T cell repertoire diversity in PB of SLE patients correlate with or predict changes in disease activity.
Total RNA was isolated from the PB of 11 SLE patients. Each subject had three samples, collected at periods of clinical quiescence and at a flare. 12 age-matched healthy controls (HC) were used for reference. NGS was used to profile the complementarity determining region 3 (CDR3) of the rearranged TCR β loci.
Relative to the HC, SLE patients (at quiescence) demonstrated a 2.2 fold reduction in repertoire diversity in a given PB volume (p<0.0002), a more uneven distribution of the repertoire (Gini coefficient, HC vs SLE, p=0.015), and a trend towards increased percentage of expanded clones in the repertoire (clone size >1.0%, HC vs SLE, p=0.078). No significant correlation between the overall repertoire diversity and clinical disease activity was observed for most SLE patients with only 2 of 11 SLE patients showing a decreasing trend in repertoire diversity approaching the flare time point. We did not observe any overlap of CDR3 amino acid sequences or a preferential Vβ or Jβ gene usage amongst the top 100 expanded clones from all SLE patients. In both HC and SLE, the majority of the expanded clones were remarkably stable over time (HC=5.5 ±0.5 months, SLE=7.2 ±2.4 months).
A significant decrease in T cell repertoire diversity was observed in PB of SLE patients compared to HC. However, in most SLE patients, repertoire diversity did not change significantly with increases in disease activity to a flare. Thus, without a priori knowledge of disease specific clones, monitoring TCR repertoire in PB from SLE patients is not likely to be useful to predict changes in disease activity.
Human and Murine Clonal CD8+ T Cell Expansions Arise During Tuberculosis Because of TCR Selection
The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRβ bias. Using a retrogenic model of TB10.44-11-specific CD8+ T cells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.
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. We then analyzed antigen-activated T cells from different tissues using high-throughput sequencing (HTS) of the gene encoding the T cell receptor (TCR) β-chain (Trb, also known asTcrb) using CDR3 sequences to simultaneously track thousands of unique T cells. For every abundant TRM cell clone generated in the skin, an abundant TCM cell clone bearing the identical TCR was present in the LNs. Thus, antigen-reactive skin TRM and LN TCM cell clones were derived from a common naive T cell precursor after skin immunization, generating overlapping TCR repertoires. Although they bore the same TCR, TRM cells mediated rapid contact hypersensitivity responses, whereas TCM cells mediated delayed and attenuated responses. Studies in human subjects confirmed the generation of skin TRM cells in allergic contact dermatitis. Thus, immunization through skin simultaneously generates skin TRM and LN TCM cells in similar numbers from the same naive T cells.
CMV-specific T cells generated from naïve T cells recognize atypical epitopes and may be protective in vivo
Adoptive transfer of cytomegalovirus (CMV)–specific T cells derived from adult seropositive donors can effectively restore antiviral immunity after transplantation. However, CMV-seronegative donors lack CMV-specific memory T cells, which restricts the availability of virus-specific T cells for immunoprophylaxis. We demonstrate the feasibility of deriving CMV-specific T cells from naïve cells for T cell therapy. Naïve T cells primed to recognize CMV were restricted to different, atypical epitopes than T cells derived from CMV-seropositive individuals; however, these two cell populations had similar avidities. CMV-seropositive individuals also had T cells recognizing these atypical epitopes, but these cells had a lower avidity than those derived from the seronegative subjects, which suggests that high-avidity T cells to these epitopes may be lost over time. Indeed, recipients of cord blood (CB) grafts who did not develop CMV were found by clonotypic analysis to have T cells recognizing atypical CMVpp65 epitopes. Therefore, we examined unmanipulated CB units and found that T cells with T cell receptors restricted by atypical epitopes were the most common, which may explain why these T cells expanded. When infused to recipients, naïve donor–derived virus-specific T cells that recognized atypical epitopes were associated with prolonged periods of CMV-free survival and complete remission. These data suggest that naïve-derived T cells from seronegative patients may be an additional source of cells for CMV immunoprophylaxis.
Peripheral Blood-Derived Virus-Specific Memory Stem T cells Mature to Functional Effector Memory subsets with Self-Renewal Potency
Memory T cells expressing stem cell–like properties have been described recently. The capacity of self-renewal and differentiation into various memory/effector subsets make them attractive for adoptive T cell therapy to combat severe virus infections and tumors. The very few reports on human memory stem T cells (TSCM) are restricted to analyses on polyclonal T cells, but extensive data on Ag-specific TSCM are missing. This might be due to their very low frequency limiting their enrichment and characterization. In this article, we provide functional and phenotypic data on human viral-specific TSCM, defined as CD8+CD45RA+CCR7+CD127+CD95+. Whereas <1% of total T cells express the TSCM phenotype, human CMV–specific TSCM can be detected at frequencies similar to those seen in other subsets, resulting in ∼1/10,000 human CMV–specific TSCM. A new virus-specific expansion protocol of sort-purified TSCM reveals both upregulation of various T cell subset markers and preservation of their stem cell phenotype in a significant proportion, indicating both self-renewal and differentiation potency of virus-specific T cells sharing their TCR repertoire. Furthermore, we describe a simplified culture protocol that allows fast expansion of virus-specific TSCM starting from a mixed naive T/TSCM pool of PBLs. Due to the clinical-grade compatibility, this might be the basis for novel cell therapeutic options in life-threatening courses of viral and tumor disease.
Changes in peanut-specific T-cell clonotype with oral immunotherapy
Allergen-proliferation assays are widely used to determine allergen-specific changes in T-cell phenotype during immunotherapy, generally showing skewing of the pathological TH2 response toward a normal TH1 or regulatory T-cell response. Concerns have been raised, however, about the actual specificity of this approach, which often leads to the identification of very high rates of TH1 cells. Another question that is yet to be elucidated is whether the observed changes result from a reprogramming of existing allergen-specific clones (reeducation hypothesis) or from their replacement by different clones to determine the dominant response (replacement hypothesis).
Expanded CD8 T-cell sharing between periphery and CNS in multiple sclerosis
In multiple sclerosis (MS), central nervous system (CNS), cerebrospinal fluid (CSF), and blood display TCR clonal expansions of CD8+ T cells. These clones have been assumed – but never demonstrated – to be similar in the three compartments. Addressing this key question is essential to infer the implication of peripheral clonally expanded CD8+ T cells in the disease.
For the first time, TCR Vβ repertoire from paired blood (purified CD8+ and CD4+ T cells), CSF and CNS (22 lesions, various inflammatory and demyelination statuses) samples from three MS patients was studied using complementary determining region 3 (CDR3) spectratyping and high-throughput sequencing. In parallel, blood and CNS clonally expanded CD8+ T cells were characterized by fluorescent staining.
TCR Vβ repertoire analysis revealed strong sharing of predominant T-cell clones between CNS lesions, CSF, and blood CD8+ T cells. In parallel, we showed that blood oligoclonal CD8+ T cells exhibit characteristics of pathogenic cells, as they displayed a bias toward a memory phenotype in MS patients, with increased expression of CCR5, CD11a and Granzyme B (GZM-B) compared to non oligoclonal counterparts. CNS-infiltrating T cells were mainly CD8 expressing CD11a and GZM-B.
This study highlights the predominant implication of CD8+ T cells in MS pathophysiology and demonstrates that potentially aggressive CD8+ T cells can be easily identified and characterized from blood and CSF samples.
CMV reactivation drives post-transplant T cell reconstitution and results in defects in the underlying TCRβ repertoire
Although CMV reactivation has long been implicated in post-transplant 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 HSCT. We observed that CMV reactivation drove a >50-fold specific expansion of Granzyme Bhigh/CD28low/CD57high/CD8+ effector-memory T cells and resulted in a linked contraction of all naïve T cells, including CD31+/CD4+ putative thymic emigrants. TCRβ deep sequencing revealed a striking contraction of CD8+ Tem diversity due to CMV-specific clonal expansions in reactivating patients. In addition to querying the topography of the expanding CMV-specific T cell clones, deep sequencing allowed us, for the first time, to exhaustively evaluate the underlying TCR repertoire. Our results reveal new evidence for significant defects in underlying CD8 Tem TCR repertoire in patients who reactivate CMV, providing the first molecular evidence that in addition to driving expansion of virus-specific cells, CMV reactivation has a detrimental impact on the integrity and heterogeneity of the rest of the T cell repertoire. Registered to www.Clinicaltrials.gov as #NCT01012492.
A dendritic cell vaccine increases the breadth of diversity of melanoma neoantigen-specific T cells
T cell immunity directed against tumor-encoded amino acid substitutions (AAS) occurs in some melanoma patients. This implicates missense mutations (MM) as source of patient-specific neoantigens. However, a systematic evaluation of these putative neoantigens as validated targets of anti-tumor immunity is lacking. Moreover, whether vaccination can augment such responses is unknown. Here we show that a dendritic cell vaccine increased naturally occurring and revealed new HLA class I-restricted neoantigens in patients with advanced melanoma. the presentation of neoantigens by HLA-A*02:01 in human melanoma was confirmed by mass spectrometry. Vaccination promoted a diverse neoantigen-specific T cell receptor repertoire in terms of both TCRVβ usage and clonal composition. Our results demonstrate that vaccination directed at tumor AAS broadens the antigenic breadth and clonal diversity of anti-tumor immunity.
TCR Sequencing Facilitates Diagnosis and Identifies Mature T Cells as the Cell of Origin in CTCLScience Translational Medicine | October, 2015
Early diagnosis of cutaneous T cell lymphoma (CTCL) is difficult and takes on average 6 years after presentation, in part because the clinical appearance and histopathology of CTCL can resemble that of benign inflammatory skin diseases. Detection of a malignant T cell clone is critical in making the diagnosis of CTCL, but the T cell receptor g (TCRg) polymerase chain reaction (PCR) analysis in current clinical use detects clones in only a subset of patients. High-throughput TCR sequencing (HTS) detected T cell clones in 46 of 46 CTCL patients, was more sensitive and specific than TCRg PCR, and successfully discriminated CTCL from benign inflammatory diseases. HTS also accurately assessed responses to therapy and facilitated diagnosis of disease recurrence.VIEW
Prognostic Value of Deep Sequencing Method for Minimal Residual Disease Detection in Multiple MyelomaBlood | May, 2014
We assessed the prognostic value of minimal residual disease (MRD) detection in multiple myeloma (MM) patients using a sequencing-based platform in bone marrow samples from 133 MM patients in at least very good partial response (VGPR) after front-line therapy. Deep sequencing was carried out in patients in whom a high-frequency myeloma clone was identified and MRD was assessed using the IGH-VDJH, IGH-DJH, and IGK assays. The results were contrasted with those of multiparametric flow cytometry (MFC) and allele-specific oligonucleotide polymerase chain reaction (ASO-PCR). The applicability of deep sequencing was 91%. Concordance between sequencing and MFC and ASO-PCR was 83% and 85%, respectively.VIEW
Non-Invasive Monitoring of Diffuse Large B-Cell Lymphoma by Immunoglobulin High-Throughput SequencingBlood | June, 2015
Recent studies have shown limited utility of routine surveillance imaging for diffuse large B-cell lymphoma (DLBCL) patients achieving remission. Detection of molecular disease by immunoglobulin high-throughput sequencing (Ig-HTS) from peripheral blood provides an alternate strategy for surveillance. We prospectively evaluated the utility of Ig-HTS within 311 blood and 105 tumor samples from 75 patients with DLBCL, comparing Ig-HTS from the cellular (circulating leukocytes) and acellular (plasma cell-free DNA) compartments of peripheral blood to clinical outcomes and 18FDG PET/CT (n=173). Clonotypic immunoglobulin rearrangements were detected in 83% of patients with adequate tumor samples to enable subsequent monitoring in peripheral blood.VIEW
IgH-V(D)J NGS-MRD Measurement Pre- and Early Post- Allo-Transplant Defines Very Low and Very High Risk ALL PatientsBlood | May, 2015
Positive detection of minimal residual disease (MRD) by multichannel flow cytometry (MFC) prior to hematopoietic cell transplantation (HCT) of patients with 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 IgH V(D)J NGS-MRD on 56 patients with B-cell ALL enrolled in Children's Oncology Group (COG) trial ASCT0431. NGS-MRD predicted relapse and survival more accurately than MFC-MRD (p<0.0001), especially in the MRD negative cohort (relapse 0% vs. 16%; p=0.02, 2yr OS 96% vs. 77%; p=0.003).VIEW