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Featured Publications
Next-Generation Sequencing in Adult B Cell Acute Lymphoblastic Leukemia Patients
Biology of Blood and Marrow Transplantation | January, 2017We used next generation sequencing (NGS) of the immunoglobulin genes to evaluate residual disease in 153 specimens from 32 patients with adult B cell ALL enrolled in a single, multi-center study. The sequencing results were compared to multi-parameter flow cytometry (MFC) data in 66 specimens (25 patients) analyzed by both methods. There was a strong concordance (82%) between the methods in the qualitative determination of the presence of disease. However, in 17% of cases leukemia was detected by sequencing, but not by MFC. In 54 bone marrow (BM) and peripheral blood (PB) paired specimens, the burden of leukemia detected by NGS was lower in PB than BM, although still detectable in 68% of the 28 paired specimens with positive BM.
VIEW
Next-generation sequencing-based detection of circulating tumour DNA after allogeneic stem cell transplantation for lymphoma
British Journal of Haematology | December, 2016Next-generation sequencing (NGS)-based circulating tumour DNA (ctDNA) detection is a promising monitoring tool for lymphoid malignancies. We evaluated whether the presence of ctDNA was associated with outcome after allogeneic haematopoietic stem cell transplantation (HSCT) in lymphoma patients. We studied 88 patients drawn from a phase 3 clinical trial of reduced-intensity conditioning HSCT in lymphoma. Conventional restaging and collection of peripheral blood samples occurred at pre-specified time points before and after HSCT and were assayed for ctDNA by sequencing of the immunoglobulin or T-cell receptor genes. Tumour clonotypes were identified in 87% of patients with adequate tumour samples.
VIEW
CD19 CAR–T cells of defined CD4+:CD8+ composition in adult B cell ALL patients
The Journal of Clinical Investigation | April, 2016T 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.
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TCR Sequencing Can Identify and Track Glioma-Infiltrating T Cells after DC Vaccination
Cancer Immunology Research | March, 2016Although 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 cancer
Molecular Oncology | September, 2015Immunosequencing 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 Sequencing
PLOS ONE | October, 2015Monitoring 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
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
Objective
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.
Methods
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.
Results
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.
Interpretation
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.
Case study:
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.
Fatal autoimmunity in mice reconstituted with human hematopoietic stem cells encoding defective FOXP3
Mice reconstituted with a human immune system provide a tractable in vivo model to assess human immune cell function. To date, reconstitution of murine strains with human hematopoietic stem cells (HSCs) from patients with monogenic immune disorders have not been reported. One obstacle precluding the development of immune-disease specific "humanized" mice is that optimal adaptive immune responses in current strains have required implantation of autologous human thymic tissue. TO address this issue, we developed a mouse strain that lacks murine major histocompatibility complex class II (MHCII and instead expresses human MHCII DR1. These mice displayed improved adaptive immune responses when reconstituted with human HSCs including enhanced T cell reconstitution, delayed-type hypersensitivity response, and class-switch recombination. Following immune reconstitution of this novel strain with HSCs from a patient with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, associated with aberrant FOXP3 function, mice developed a lethal inflammatory disorder with multi-organ involvement and autoantibody production mimicking the pathology seen in affected humans. This humanized mouse model permits in vivo evaluation of immune response associated with genetically altered HSCs, including primary immunodeficiencies, and should facilitate the study of human immune pathobiology and the development of targeted therapeutics.
BTLA marks a less-differentiated tumor-infiltrating lymphocyte subset in melanoma with enhanced survival properties
In a recent adoptive cell therapy (ACT) clinical trial using autologous tumor-infiltrating lymphocytes (TILs) in patients with metastatic melanoma, we found an association between CD8+ T cells expressing the inhibitory receptor B- and T-lymphocyte attenuator (BTLA) and clinical response. Here, we further characterized this CD8+BTLA+ TIL subset and their CD8+BTLA- counterparts. We found that the CD8+ BTLA+ TILs had an increased response to IL-2, were less-differentiated effector-memory (TEM) cells, and persisted longer in vivo after infusion. In contrast, CD8+BTLA-TILs failed to proliferate and expressed genes associated with T-cell deletion/tolerance. Paradoxically, activation of BTLA signaling by its ligand, herpes virus entry mediator (HVEM), inhibited T-cell division and cytokine production, but also activated the Akt/PKB pathway thus protecting CD8+BTLA+ TILs from apoptosis. Our results point to a newrole of BTLA as a useful T-cell differentiation marker in ACT and a dual signaling molecule that curtails T-cell activation while also conferring a survival advantage for CD8+ T cells. These attributes may explain our previous observation that BTLA expression on CD8+ TILs correlates with clinical response to adoptive T-cell therapy in metastatic melanoma.
Generation of human memory stem T cells upon haploidentical T-replete hematopoietic stem cell transplantation
Memory stem T cells (TSCM) have been proposed as key determinants of immunological memory. However, their exact contribution to a mounting immune response as well as the mechanisms and timing of their in vivo generation are poorly understood. We longitudinally tracked TSCM dynamics in patients undergoing haploidentical hematopoietic stem cell transplantation (HSCT), thereby providing novel hints on the contribution of this subset to post transplant immune reconstitution in humans. We found that donor derived TSCM cells are highly enriched early after HSCT. We showed at the antigen-specific and clonal level that TSCM lymphocytes can differentiate directly from naïve precursors infused within the graft and that the extent of TSCM generation might correlate with IL-7 serum levels. In vivo fate mapping through TCR sequencing allowed defining the in vivo differentiation landscapes of human naïve T cells, supporting the notion that progenies of single naïve cells embrace disparate fates in vivo, and highlighting TSCM as relevant novel players in the diversification of immunological memory following allogeneic HSCT.
Radiation and dual checkpoint blockade activate non-redundant immune mechanisms in cancer
Immune checkpoint inhibitors result in impressive clinical responses, but optimal results will require combination with each other and other therapies. This raises fundamental questions about mechanisms of non-redundancy and resistance. Here we report major tumour regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation, and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumours, resistance was common. Unbiased analyses of mice revealed that resistance was due to upregulation of PD-L1 on melanoma cells and associated with T-cell exhaustion. Accordingly, optimal response in melanoma and other cancer types requires radiation, anti-CTLA4 and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T-regulatory cells (Treg cells), thereby increasing the CD8 T-cell to Treg (CD8/Treg) ratio. Radiation enhances the diversity of the T-cell receptor (TCR) repertoire of intratumoral T cells. Together, anti-CTLA4 promotes expansion of T cells, while radiation shapes the TCR repertoire of the expanded peripheral clones. Addition of PD-L1 blockade reverses T-cell exhaustion to mitigate depression in the CD8/Tregratio and further encourages oligoclonal T-cell expansion. Similarly to results from mice, patients on our clinical trial with melanoma showing high PD-L1 did not respond to radiation plus anti-CTLA4, demonstrated persistent T-cell exhaustion, and rapidly progressed. Thus, PD-L1 on melanoma cells allows tumours to escape anti-CTLA4-based therapy, and the combination of radiation, anti-CTLA4 and anti-PD-L1 promotes response and immunity through distinct mechanisms.
TLR7- and TLR9-Responsive Human B Cells Share Phenotypic and Genetic Characteristics
B cells activated by nucleic acid–sensing TLR7 and TLR9 proliferate and secrete immune globulins. Memory B cells are presumably more responsive due to higher TLR expression levels, but selectivity and differential outcomes remain largely unknown. In this study, peripheral blood human B cells were stimulated by TLR7 or TLR9 ligands, with or without IFN-a, and compared with activators CD40L plus IL-21, to identify differentially responsive cell populations, defined phenotypically and by BCR characteristics. Whereas all activators induced differentiation and Ab secretion, TLR stimulation expanded IgM+ memory and plasma cell lineage committed populations, and favored secretion of IgM, unlike CD40L/IL-21, which drove IgM and IgG more evenly. Patterns of proliferation similarly differed, with CD40L/IL-21 inducing proliferation of most memory and naive B cells, in contrast with TLRs that induced robust proliferation in a subset of these cells. On deep sequencing of the IgH locus, TLR-responsive B cells shared patterns of IgHV and IgHJ usage, clustering apart from CD40L/IL-21 and control conditions. TLR activators, but not CD40L/IL-21, similarly promoted increased sharing of CDR3 sequences. TLR-responsive B cells were characterized by more somatic hypermutation, shorter CDR3 segments, and less negative charges. TLR activation also induced long positively charged CDR3 segments, suggestive of autoreactive Abs. Testing this, we found culture supernatants from TLR-stimulated B cells to bind HEp-2 cells, whereas those from CD40L/IL-21–stimulated cells did not. Human B cells possess selective sensitivity to TLR stimulation, with distinctive phenotypic and genetic signatures.
Type II NKT-TFH cells against Gaucher lipids regulate B-cell immunity and inflammation
Chronic inflammation including B-cell activation is commonly observed in both inherited (Gaucher disease [GD]) and acquired disorders of lipid metabolism. However, the cellular mechanisms underlying B-cell activation in these settings remain to be elucidated. Here, we report that β-glucosylceramide 22:0 (βGL1-22) and glucosylsphingosine (LGL1), 2 major sphingolipids accumulated in GD, can be recognized by a distinct subset of CD1d-restricted human and murine type II natural killer T (NKT) cells. Human βGL1-22- and LGL1-reactive CD1d tetramer-positive T cells have a distinct T-cell receptor usage and genomic and cytokine profiles compared with the classical type I NKT cells. In contrast to type I NKT cells, βGL1-22- and LGL1-specific NKT cells constitutively express T-follicular helper (TFH) phenotype. Injection of these lipids leads to an increase in respective lipid-specific type II NKT cells in vivo and downstream induction of germinal center B cells, hypergammaglobulinemia, and production of antilipid antibodies. Human βGL1-22- and LGL1-specific NKT cells can provide efficient cognate help to B cells in vitro. Frequency of LGL1-specific T cells in GD mouse models and patients correlates with disease activity and therapeutic response. Our studies identify a novel type II NKT-mediated pathway for glucosphingolipid-mediated dysregulation of humoral immunity and increased risk of B-cell malignancy observed in metabolic lipid disorders.
High Expression of CD26 Accurately Identified Human Bacterial-reactive MR1-restricted MAIT cells
Mucosal associated invariant T (MAIT) cells express the semi-invariant T cell receptor TRAV1-2 and detect a range of bacteria and fungi through the MHC-like molecule MR1. Nonetheless at present, limited knowledge exists of the function and phenotype of bacterial-reactive MR1-restricted TRAV1-2+ MAIT cells from human blood. Here we broadly characterized the function of MR1-restricted MAIT cells in response to bacteria-infected targets and defined a phenotypic panel to identify these cells in the circulation. We demonstrated that bacterial-reactive MR1-restrcited T cells shared effector functions of cytolytic effector CD8+ T cells. By analyzing an extensive panel of phenotypic markers, we determined CD26, and CD161 were most strongly associated with these T cells. Using FACS to sort phenotypically defined CD8+ subsets we demonstrated that high expression of CD26 on CD8+ TRAV1-3+ cells identified with high specificity and sensitivity, bacterial-reactive MR1-restricted T cells from human blood. CD161hi was also specific for but lacked sensitivity in identifying all bacterial-reactive MR1-restricted T cells some of which were CD161dm. Using, cell surface expression of CD8, TRAV1-2, and CD26hi in the absence of stimulation we confirm that bacterial-reactive T cells are lacking in the blood of individuals with active tuberculosis (TB) and are restored in the blood of individuals undergoing treatment for TB.
Dynamics of the Cytotoxic T Cell Response to a Model of Acute Viral Infection
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 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 includes high throughput sequencing of the CDR3 variable region of the T cell receptor β chain and an algorithm that detects significantly expanded T cell clones. This allowed us to establish that: (a) on average ∼2,000 CD8+ T cell clones were induced by YF-17D, (b) 5-6% of the responding clones were recruited to long-term memory three months post-vaccination, (c) the most highly-expanded effector clones were preferentially recruited to the memory compartment, and (d) a fraction of the YF-17D-induced clones can be identified from peripheral blood lymphocytes solely by measuring clonal expansion.
Immune Reconstitution / Immunocompetence in Recipients of Kidney Plus Hematopoietic Stem/Facilitating Cell Transplants
Nineteen subjects have more than 18 months' follow-up in a phase IIb tolerance protocol in HLA-mismatched recipients of living donor kidney plus facilitating cell enriched hematopoietic stem cell allografts (FCRx). Reduced intensity conditioning preceded a kidney allograft, followed the next day by FCRx. Twelve have achieved stable donor chimerism and have been successfully taken off immunosuppression (IS). We prospectively evaluated immune reconstitution and immunocompetence. Return of CD4 and CD8 T central and effector memory cell populations was rapid. T-cell receptor (TCR) Excision Circle analysis showed a significant proportion of chimeric cells produced were being produced de novo. The TCR repertoires posttransplant in chimeric subjects were nearly as diverse as pretransplant donors and recipients, and were comparable to subjects with transient chimerism who underwent autologous reconstitution. Subjects with persistent chimerism developed few serious infections when off IS. The majority of infectious complications occurred while subjects were still on conventional IS. BK viruria and viremia resolved after cessation of IS and no tissue-invasive cytomegalovirus infections occurred. Notably, although 2 of 4 transiently or nonchimeric subjects experienced recurrence of their underlying autoimmune disorders, none of the chimeric subjects have, suggesting that self-tolerance is induced in addition to tolerance to alloantigen. No persistently chimeric subject has developed donor-specific antibody, and renal function has remained within normal limits. Patients were successfully vaccinated per The American Society for Blood and Marrow Transplantation guidelines without loss of chimerism or rejection. Memory for hepatitis vaccination persisted after transplantation. Chimeric subjects generated immune responses to pneumococcal vaccine. These data suggest that immune reconstitution and immunocompetence are maintained in persistently chimeric subjects.
Case study:
Clonal and constricted T cell repertoire in Common Variable Immune Deficiency
We used high throughput sequencing to examine the structure and composition of the T cell receptor β chain in Common Variable Immune Deficiency (CVID). TCRβ CDR3 regions were amplified and sequenced from genomic DNA of 44 adult CVID subjects and 22 healthy adults, using a high-throughput multiplex PCR. CVID TCRs had significantly less junctional diversity, fewer n-nucleotide insertions and deletions, and completely lacked a population of highly modified TCRs, with 13 or more V-gene nucleotide deletions, seen in healthy controls. The CVID CDR3 sequences were significantly more clonal than control DNA, and displayed unique V gene usage. Despite reduced junctional diversity, increased clonality and similar infectious exposures, DNA of CVID subjects shared fewer TCR sequences as compared to controls. These abnormalities are pervasive, found in out-of-frame sequences and thus independent of selection and were not associated with specific clinical complications. These data support an inherent T cell defect in CVID.
Case study:
The Analysis Of Clonal Diversity And Therapy Responses Using STAT3 Mutations As A Molecular Marker In Large Granular Lymphocytic Leukemia
T-cell large granular lymphocytic leukemia and chronic lymphoproliferative disorder of natural killer cells are intriguing entities between benign and malignant lymphoproliferation. The molecular pathogenesis has partly been uncovered by the recent discovery of somatic activating STAT3 and STAT5b mutations. Here we show that 43% (75/174) of patients with T-cell large granular lymphocytic leukemia and 18% (7/39) with chronic lymphoproliferative disorder of natural killer cells harbor STAT3 mutations when analyzed by quantitative deep amplicon sequencing. Surprisingly, 17% of the STAT3-mutated patients carried multiple STAT3 mutations, which were located in different lymphocyte clones. The size of the mutated clone correlated well with the degree of clonal expansion of the T-cell repertoire analyzed by T-cell receptor beta chain deep sequencing. The analysis of sequential samples suggested that current immunosuppressive therapy is not able to reduce the level of the mutated clone in most cases, thus warranting the search for novel targeted therapies. Our findings imply that the clonal landscape of large granular lymphocytic leukemia is more complex than considered before, and a substantial number of patients have multiple lymphocyte subclones harboring different STAT3 mutations, thus mimicking the situation in acute leukemia.
Polyclonal, newly derived T cells with low expression of inhibitory molecule PD-1 in tonsils define the phenotype of lymphocytes in children with Periodic Fever, Aphtous Stomatitis, Pharyngitis and Adenitis (PFAPA) syndrome
Purpose: PFAPA syndrome is a benign, recurrent inflammatory disease of childhood. Tonsillectomy is one of the therapeutic options with a yet unexplained biological mechanism. We tested whether specific lymphocyte subsets recruited from blood to human tonsils participate in PFAPA pathogenesis.
Methods: Paired tonsils/peripheral blood (PB) samples were investigated (a) from children with PFAPA that successfully resolved after tonsillectomy (n = 10) (b) from children with obstructive sleep apnoea syndrome as controls (n = 10). The lymphocyte profiles were analysed using 8-colour flow cytometry, immunoglobulin (IGH) and T-cell receptor (TCR) gene rearrangements via PCR and next generation sequencing; a TREC/KREC analysis was performed using qPCR.
Results: The PFAPA tonsils in the asymptomatic phase had a lower percentage of B-lymphocytes than controls; T-lymphocyte counts were significantly higher in PB. The percentages of cytotoxic CD8pos T-lymphocytes were approximately 2-fold higher in PFAPA tonsils; the transitional B cells and naïve stages of both the CD4pos and CD8pos T-lymphocytes with a low expression of PD-1 molecule and high numbers of TREC were also increased. With the exception of elevated plasmablasts, no other differences were significant in PB. The expression levels of CXCL10, CXCL9 and CCL19 genes were significantly higher in PFAPA tonsils. The IGH/TCR pattern showed no clonal/oligoclonal expansion. DNA from the Epstein-Barr virus, Human Herpervirus-6 or adenovirus was detected in 7 of 10 PFAPA tonsils but also in 7 of 9 controls.
Conclusions: Our findings suggest that the uninhibited, polyclonal response of newly derived lymphocytes participate in the pathogenesis of PFAPA. Because most of the observed changes were restricted to tonsils and were not present in PB, they partly explain the therapeutic success of tonsillectomy in PFAPA syndrome.
Tracking donor-reactive T cells: Evidence for clonal deletion in tolerant kidney transplant patients
T cell responses to allogeneic major histocompatibility complex antigens present a formidable barrier to organ transplantation, necessitating long-term immunosuppression to minimize rejection. Chronic rejection and drug-induced morbidities are major limitations that could be overcome by allograft tolerance induction. Tolerance was first intentionally induced in humans via combined kidney and bone marrow transplantation (CKBMT), but the mechanisms of tolerance in these patients are incompletely understood. We now establish an assay to identify donor-reactive T cells and test the role of deletion in tolerance after CKBMT. Using high-throughput sequencing of the T cell receptor B chain CDR3 region, we define a fingerprint of the donor-reactive T cell repertoire before transplantation and track those clones after transplant. We observed posttransplant reductions in donor-reactive T cell clones in three tolerant CKBMT patients; such reductions were not observed in a fourth, nontolerant, CKBMT patient or in two conventional kidney transplant recipients on standard immunosuppressive regimens. T cell repertoire turnover due to lymphocyte-depleting conditioning only partially accounted for the observed reductions in tolerant patients; in fact, conventional transplant recipients showed expansion of circulating donor-reactive clones, despite extensive repertoire turnover. Moreover, loss of donor-reactive T cell clones more closely associated with tolerance induction than in vitro functional assays. Our analysis supports clonal deletion as a mechanism of allograft tolerance in CKBMT patients. The results validate the contribution of donor-reactive T cell clones identified before transplant by our method, supporting further exploration as a potential biomarker of transplant outcomes.
T-Cell Receptor Sequencing Reveals the Clonal Diversity and Overlap of Colonic Effector and FOXP3+ T Cells in Ulcerative Colitis
BACKGROUND: FOXP3 regulatory T cell prevent inflammation but are paradoxically increased in ulcerative colitis (UC). Local T-cell activation has been hypothesized to account for increased FOXP3 expression in colon lamina propria (LP) T cells.
METHODS: To see if human FOXP3 LP T cells are an activated fraction of otherwise FOXP3 effector T cells and explore their clonal diversity in health and disease, we deep sequenced clonally unique T-cell receptor hypervariable regions of FOXP3 and FOXP3CD4 T-cell subpopulations from inflamed versus noninflamed colon LP or mesenteric lymph nodes of patients with or without UC.
RESULTS: The clonal diversity of each LP T-cell population was not different between patients with versus without UC. Repertoire overlap was only seen between a minority of FOXP3+ and FOXP3- cells, including recently activated CD38 cells and Th17-like CD161 effector T cells, but this repertoire overlap was not different between patients with versus without UC and was no larger than the overlap between Helios and Helios FOXP3 cells.
CONCLUSIONS: Thus, at steady state, only a minority of FOXP3, and particularly Helios, T cells share a T-cell receptor sequence with FOXP3 effector populations in the colon LP, even in UC, revealing distinct clonal origins for LP regulatory T cell and effector T cells in humans.
Polysaccharide A from the Capsule of Bacteroides Fragilis Induces Clonal CD4+ T Cell Expansion
For three decades, the view of class II major histocompatibility complex (MHCII)-dependent antigen presentation has been completely dominated by peptide antigens despite our 2004 discovery in which MHCII was shown to present processed fragments of zwitterionic capsular polysaccharides to T cells. Published findings further demonstrate that the polysaccharide PSA (polysaccharide A) from the capsule of Bacteroides fragilis is a potent activator of CD4+ T cells, and that these T cells have important biological functions, especially in the maintenance of immunologic homeostasis. However, little is known about the nature of T cell recognition of the polysaccharide-MHCII complex, or the phenotype of the resulting activated cells. Here, we use next generation sequencing of the αβT cell receptor (TCR) of CD4+ T cells from mice stimulated with PSA in comparison to protein antigen simulation and non-immunized controls and found that PSA immunization induced clonal expansion of a small subset of suppressive CD45RBlowCD4+ effector memory T cells. Moreover, the sequences of the complementarity determining region loop 3 (CDR3) from top clones indicate a lack of specific Vβ and J region use and average CDR3 length. There was also a preference for a zwitterionic motif within the CDR3 sequences, aligning well with the known requirement for a similar motif within PSA to enable T cell activation. These data support a model in which PSA, and possibly other T cell-dependent polysaccharide antigens, elicits a clonal, and therefore specific, CD4+ T cell response often characterized by pairing dual charged CDR3 sequences with dual charged PSA.
Featured Publications
IgH-V(D)J NGS-MRD Measurement Pre- and Early Post- Allo-Transplant Defines Very Low and Very High Risk ALL Patients
Blood | May, 2015Positive 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
Next-generation sequencing-based detection of circulating tumour DNA after allogeneic stem cell transplantation for lymphoma
British Journal of Haematology | December, 2016Next-generation sequencing (NGS)-based circulating tumour DNA (ctDNA) detection is a promising monitoring tool for lymphoid malignancies. We evaluated whether the presence of ctDNA was associated with outcome after allogeneic haematopoietic stem cell transplantation (HSCT) in lymphoma patients. We studied 88 patients drawn from a phase 3 clinical trial of reduced-intensity conditioning HSCT in lymphoma. Conventional restaging and collection of peripheral blood samples occurred at pre-specified time points before and after HSCT and were assayed for ctDNA by sequencing of the immunoglobulin or T-cell receptor genes. Tumour clonotypes were identified in 87% of patients with adequate tumour samples.
VIEW
Immunoglobulin and T-cell Receptor Gene High-Throughput Sequencing Quantifies Minimal Residual Disease in Acute Lymphoblastic Leukemia and Predicts Post-Transplant Relapse and Survival
Biology of Blood and Marrow Transplantation |Minimal residual disease (MRD) quantification is an important predictor of outcome after treatment for acute lymphoblastic leukemia (ALL). Bone marrow ALL burden ≥ 10−4 after induction predicts subsequent relapse. Likewise, MRD ≥ 10−4 in bone marrow before initiation of conditioning for allogeneic (allo) hematopoietic cell transplantation (HCT) predicts transplantation failure. Current methods for MRD quantification in ALL are not sufficiently sensitive for use with peripheral blood specimens and have not been broadly implemented in the management of adults with ALL.
VIEW
Prognostic Value of Deep Sequencing Method for Minimal Residual Disease Detection in Multiple Myeloma
Blood | May, 2014We 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.
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Treatment With Carfilzomib-Lenalidomide-Dexamethasone With Lenalidomide Extension in Patients With Smoldering or Newly Diagnosed Multiple Myeloma
JAMA Oncology | September, 2015Importance: Carfilzomib-lenalidomide-dexamethasone therapy yields deep responses in patients with newly diagnosed multiple myeloma (NDMM). It is important to gain an understanding of this combination’s tolerability and impact on minimal residual disease (MRD) negativity because this end point has been associated with improved survival.
Objective: To assess the safety and efficacy of carfilzomib-lenalidomide-dexamethasone therapy in NDMM and high-risk smoldering multiple myeloma (SMM).
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Next-Generation Sequencing in Adult B Cell Acute Lymphoblastic Leukemia Patients
Biology of Blood and Marrow Transplantation | January, 2017We used next generation sequencing (NGS) of the immunoglobulin genes to evaluate residual disease in 153 specimens from 32 patients with adult B cell ALL enrolled in a single, multi-center study. The sequencing results were compared to multi-parameter flow cytometry (MFC) data in 66 specimens (25 patients) analyzed by both methods. There was a strong concordance (82%) between the methods in the qualitative determination of the presence of disease. However, in 17% of cases leukemia was detected by sequencing, but not by MFC. In 54 bone marrow (BM) and peripheral blood (PB) paired specimens, the burden of leukemia detected by NGS was lower in PB than BM, although still detectable in 68% of the 28 paired specimens with positive BM.
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