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
Deep Sequencing of T-Cell Receptor DNA as a biomarker of clonally expanded TILs in breast cancer after immunotherapy
In early stage breast cancer, the degree of tumor-infiltrating lymphocytes (TILs) predicts response to chemotherapy and overall survival. Combination immunotherapy with immune checkpoint antibody plus tumor cryoablation can induce lymphocytic infiltrates and improve survival in mice. We used T-cell receptor (TCR) DNA sequencing to evaluate both the effect of cryo-immunotherapy in humans and the feasibility of TCR sequencing in early-stage breast cancer. In a pilot clinical trial, 18 women with early-stage breast cancer were treated preoperatively with cryoablation, single-dose anti-CTLA-4 (ipilimumab), or cryoablation + ipilimumab. TCRs within serially collected peripheral blood and tumor tissue were sequenced. In baseline tumor tissues, T-cell density as measured by TCR sequencing correlated with TIL scores obtained by hematoxylin and eosin (H&E) staining. However, tumors with little or no lymphocytes by H&E contained up to 3.6 x 106 TCR DNA sequences, highlighting the sensitivity of the ImmunoSEQ platform. In this dataset, ipilimumab increased intratumoral T-cell density over time, whereas cryoablation ± ipilimumab diversified and remodeled the intratumoral T-cell clonal repertoire. Compared to monotherapy, cryoablation plus ipilimumab was associated with numerically greater numbers of peripheral blood and intratumoral T-cell clones expanding robustly following therapy. In conclusion, TCR sequencing correlates with H&E lymphocyte scoring, and provides additional information on clonal diversity. These findings support further study of the use of TCR sequencing as a biomarker for T cell responses to therapy and for thestudy of cryo-immunotherapy in early-stage breast cancer.
Atezolizumab in combination with bevacizumab enhances antigen-specific T-cell migration in metastatic renal cell carcinoma
Anti-tumour immune activation by checkpoint inhibitors leads to durable responses in a variety of cancers, but combination approaches are required to extend this benefit beyond a subset of patients. In preclinical models tumour-derived VEGF limits immune cell activity while anti-VEGF augments intra-tumoral T-cell infiltration, potentially through vascular normalization and endothelial cell activation. This study investigates how VEGF blockade with bevacizumab could potentiate PD-L1 checkpoint inhibition with atezolizumab in mRCC. Tissue collections are before treatment, after bevacizumab and after the addition of atezolizumab. We discover that intra-tumoral CD8þ T cells increase following combination treatment. A related increase is found in intra-tumoral MHC-I, Th1 and T-effector markers, and chemokines, most notably CX3CL1 (fractalkine). We also discover that the fractalkine receptor increases on peripheral CD8þ T cells with treatment. Furthermore, trafficking lymphocyte increases are observed in tumors following bevacizumab and combination treatment. These data suggest that the anti-VEGF and anti-PD-L1 combination improves antigen-specific T-cell migration.
Human TRAV1-2-negative MR1-restricted T cells detect S. pyogenes and alternatives to MAIT riboflavin-based antigens
Mucosal-associated invariant T (MAIT) cells are thought to detect microbial antigens presented by the HLA-Ib molecule MR1 through the exclusive use of a TRAV1-2-containing TCRa. Here we use MR1 tetramer staining and ex vivo analysis with mycobacteria-infected MR1-deficient cells to demonstrate the presence of functional human MR1-restricted T cells that lack TRAV1-2. We characterize an MR1-restricted clone that expresses the TRAV12-2 TCRa, which lacks residues previously shown to be critical for MR1-antigen recognition. In contrast to TRAV1-2þ MAITcells, this TRAV12-2-expressing clone displays a distinct pattern of microbial recognition by detecting infection with the riboflavin auxotroph Streptococcus pyogenes. As known MAITantigens are derived from riboflavin metabolites, this suggests that TRAV12-2þ clone recognizes unique antigens. Thus, MR1-restricted T cells can discriminate between microbes in a TCR-dependent manner. We postulate that additional MR1-restricted T-cell subsets may play a unique role in defence against infection by broadening the recognition of microbial metabolites.
A Public Database of Memory and Naive B- Cell Receptor Sequences
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.
Accelerated Loss of TCR Repertoire Diversity in Common Variable Immunodeficiency
Although common variable immunodeficiency (CVID) has long been considered as a group of primary Ab deficiencies, growing experimental data now suggest a global disruption of the entire adaptive immune response in a segment of patients. Oligoclonality of the TCR repertoire was previously demonstrated; however, the manner in which it relates to other B cell and T cell findings reported in CVID remains unclear. Using a combination approach of high-throughput TCRb sequencing and multiparametric flow cytometry, we compared the TCR repertoire diversity between various subgroups of CVID patients according to their B cell immunophenotypes. Our data suggest that the reduction in repertoire diversity is predominantly restricted to those patients with severely reduced class-switched memory B cells and an elevated level of CD21 lo B cells (Freiburg 1a), and may be driven by a reduced number of naive T cells unmasking underlying memory clonality. Moreover, our data indicate that this loss in repertoire diversity progresses with advancing age far exceeding the expected physiological rate. Radiological evidence supports the loss in thymic volume, correlating with the decrease in repertoire diversity. Evidence now suggests that primary thymic failure along with other well-described B cell abnormalities play an important role in the pathophysiology in Freiburg group 1a patients. Clinically, our findings emphasize the integration of combined B and T cell testing to identify those patients at the greatest risk for infection. Future work should focus on investigating the link between thymic failure and the severe reduction in class-switched memory B cells, while gathering longitudinal laboratory data to examine the progressive nature of the disease. The Journal of Immunology, 2016, 197: 000–000.
Chemotherapy and radiation therapy elicits tumor specific T cell responses in a breast cancer patient
Background: Experimental evidence and clinical studies in breast cancer suggest that some anti-tumor therapy regimens generate stimulation of the immune system that accounts for tumor clinical responses, however, demonstration of the immunostimulatory power of these therapies on cancer patients continues to be a formidable challenge. Here we present experimental evidence from a breast cancer patient with complete clinical response after 7 years, associated with responsiveness of tumor specific T cells.
Methods: T cells were obtained before and after anti-tumor therapy from peripheral blood of a 63-years old woman diagnosed with ductal breast cancer (HER2/neu+++, ER-, PR-, HLA-A*02:01) treated with surgery, followed by paclitaxel, trastuzumab (suspended due to cardiac toxicity), and radiotherapy. We obtained a leukapheresis before surgery and after 8 months of treatment. Using in vitro cell cultures stimulated with autologous monocyte- derived dendritic cells (DCs) that produce high levels of IL-12, we characterize by flow cytometry the phenotype of tumor associated antigens (TAAs) HER2/neu and NY-ESO 1 specific T cells. The ex vivo analysis of the TCR-Vβ repertoire of TAA specific T cells in blood and Tumor Infiltrating Lymphocytes (TILs) were performed in order to correlate both repertoires prior and after therapy.
Results: We evidence a functional recovery of T cell responsiveness to polyclonal stimuli and expansion of TAAs specific CD8+ T cells using peptide pulsed DCs, with an increase of CTLA-4 and memory effector phenotype after anti-tumor therapy. The ex vivo analysis of the TCR-Vβ repertoire of TAA specific T cells in blood and TILs showed that whereas the TCR-Vβ04-02 clonotype is highly expressed in TILs the HER2/neu specific T cells are expressed mainly in blood after therapy, suggesting that this particular TCR was selectively enriched in blood after anti-tumor therapy.
Conclusions: Our results show the benefits of anti-tumor therapy in a breast cancer patient with clinical complete response in two ways, by restoring the responsiveness of T cells by increasing the frequency and activation in peripheral blood of tumor specific T cells present in the tumor before therapy.
T cell receptor diversity in the human thymus
Analyzing the CDR3 Repertoire with respect to TCR—Beta Chain V-D-J and V-J Rearrangements in Peripheral T Cells using HTS
V-D-J rearrangement of the TCR—beta chain follows the 12/23 rule and the beyond 12/23 restriction. Currently, the proportion and characteristics of TCR—beta chain V—J rearrangement is unclear. We used high-throughput sequencing to compare and analyze TCR—beta chain V-J rearrangement and V-D-J rearrangement in the CDR3 repertoires of T cells from the PBMCs of six volunteers and six BALB/c mice. The results showed that the percentage of V-J rearrangement of the volunteers was approximately 0.7%, whereas that of the mice was 2.2%. The clonality of mice V-J rearrangement was significantly reduced compared with the V-D-J rearrangement, whereas the clonality of human V-J rearrangement was slightly reduced compared with the V-D-J rearrangement. V-J rearrangement in CDR3 involved the significant usage of N, S, F and L, whereas V-D-J rearrangement in CDR3 involved the significant usage of R and G. The levels of V deletion and J deletion in V-J rearrangement were significantly reduced compared with V-D-J rearrangement. TRBD and TRBJ usage in V-J rearrangement differedfrom that of V-D-J rearrangement, including dominant usage of TRBV and TRBJ and their pairing. Taken together, these results provide new ideas and technology for studies of V-D-J rearrangement and V-J rearrangement in the CDR3 repertoire.
Definitive chemoradiation alters the immunologic landscape and immune checkpoints in head and neck cancer
Background: Preclinical and clinical studies suggest potential synergy between high dose per fraction focal radiation and immunotherapy. However, conventionally fractionated radiation regimens in combination with concurrent chemotherapy are more commonly administered to patients as definitive treatment and may have both immune-stimulating and -suppressive effects.
Methods: We prospectively collected longitudinal samples from head and neck squamous cell carcinoma patients receiving definitive radiation therapy. We quantified changes in populations of circulating immune cells and chemokines CXCL9, 10, and 16. Analyses of humoral and cellular immune responses were conducted in select patients via proteomic analysis and T-cell receptor sequencing.
Results: Treatment not only increased circulating CD-8þ T-effector cells, but also myeloid-derived suppressor cells, regulatory T cells, and checkpoint receptor-expressing T cells, particularly PD-1þ T cells. Significant decreases in CXCL10 and increases in CXLC16 were noted. Treatment also increased the percentage of unique and dominant TCR clones, and increased humoral responses as measured by proteomic array.
Conclusions: Our results suggest that fractionated chemoradiation leads to quantifiable effects in circulating immune mediators, including a balance of stimulatory and suppressive mechanisms. These results suggest future combinations with immune checkpoint blockade.
Tumor- and neoantigen-reactive T-cell receptors can be identified based on their frequency in fresh tumor
Adoptive transfer of T cells with engineered T-cell receptor (TCR) genes that target tumor-specific antigens can mediate cancer regression. Accumulating evidence suggests that the clinical success of many immunotherapies is mediated by T-cells targeting mutated neoantigens unique to the patient. We hypothesized that the most frequent TCR clonotypes infiltrating the tumor were reactive against tumor antigens. To test this, we developed a multi-step strategy that involved TCRB deep sequencing of the CD8 + PD-1 + T-cell subset, matching of TCRA-TCRB pairs by pairSEQ and single cell RT-PCR, followed by testing of the TCRs for tumor-antigen specificity. Analysis of 12 fresh metastatic melanomas revealed that in 11 samples, up to 5 tumor-reactive TCRs were present in the 5 most frequently occurring clonotypes, which included reactivity against neoantigens. These data demonstrate the feasibility of developing a rapid, personalized, TCR-gene therapy approach that targets the unique set of antigens presented by the autologous tumor without the need to identify their immunologic reactivity.
Redirecting T-Cell Specificity to EGFR Using mRNA to Self-limit Expression of Chimeric Antigen Receptor
Potential for on-target, but off-tissue toxicity limits therapeutic application of genetically modified T cells constitutively expressing chimeric antigen receptors (CARs) from tumor-associated antigens expressed in normal tissue, such as epidermal growth factor receptor (EGFR). Curtailing expression of CAR through modification of T cells by in vitro-transcribed mRNA species is one strategy to mitigate such toxicity. We evaluated expression of an EGFR-specific CAR coded from introduced mRNA in human T cells numerically expanded ex vivo to clinically significant numbers through coculture with activating and propagating cells (AaPC) derived from K562 preloaded with anti-CD3 antibody. The density of AaPC could be adjusted to affect phenotype of T cells such that reduced ratio of AaPC resulted in higher proportion of CD8+ and central memory T cells that were more conducive to electrotransfer of mRNA than T cells expanded with high ratios of AaPC. RNA-modified CAR+ T cells produced less cytokine, but demonstrated similar cytolytic capacity as DNA-modified CAR+ T cells in response to EGFR-expressing glioblastoma cells. Expression of CAR by mRNA transfer was transient and accelerated by stimulation with cytokine and antigen. Loss of CAR abrogated T-cell function in response to tumor and normal cells expressing EGFR. We describe a clinically applicable method to propagate and modify T cells to transiently express EGFR-specific CAR to target EGFR-expressing tumor cells that may be used to limit on-target, off-tissue toxicity to normal tissue.
Antigen-specificity of T-cell Infiltrates in Biopsies with T-cell Mediated Rejection and BK Polyomavirus Viremia: Analysis by Next Generation Sequencing
This study interrogates the antigen-specificity of inflammatory infiltrates in renal biopsies with BK polyomavirus (BKPyV) viremia (BKPyVM) with or without allograft nephropathy (BKPyVN). PBMC from 5 healthy HLA-A0101 subjects were stimulated by peptides derived from the BKPYV proteome or polymorphic regions of HLA. Next generation sequencing (NGS) of the T-cell receptor (TCR) cDNA was performed on peptide stimulated PBMC and 23 biopsies with T-cell mediated rejection (TCMR) or BKPyVN. Biopsies from patients with BKPyVM or BKVPyVN contained 7.7732 times more alloreactive than virus reactive clones. Biopsies with TCMR also contained BKPyV-specific clones, presumably a manifestation of heterologous immunity. The mean cumulative T-cell clonal frequency was 0.1378 for alloreactive clones and 0.0375 for BKPyV reactive clones. Samples with BKPyVN and TCMR clustered separately in dendrograms of V-family and J-gene utilization patterns. Dendrograms also revealed that V-gene, J-gene, and D-gene usage patterns were a function of HLA type. In conclusion, biopsies with BKPyVN contain abundant allospecific clones that exceed the number of virus reactive clones. The T-cell component of tissue injury in viral nephropathy appears to be mediated primarily by an 'innocent bystander' mechanism in which the principal element is secondary T-cell influx triggered by both anti-viral and anti-HLA immunity.
IL-15 promotes activation and expansion of CD8+ T cells in HIV-1 infection
In HIV-1–infected patients, increased numbers of circulating CD8+ T cells are linked to increased risk of morbidity and mortality. Here, we identified a bystander mechanism that promotes CD8 T cell activation and expansion in untreated HIV-1–infected patients. Compared with healthy controls, untreated HIV-1–infected patients have an increased population of proliferating, granzyme B+, CD8+ T cells in circulation. Vβ expression and deep sequencing of CDR3 revealed that in untreated HIV-1 infection, cycling memory CD8 T cells possess a broad T cell repertoire that reflects the repertoire of the resting population. This suggests that cycling is driven by bystander activation, rather than specific antigen exposure. Treatment of peripheral blood mononuclear cells with IL-15 induced a cycling, granzyme B+ phenotype in CD8+ T cells. Moreover, elevated IL-15 expression in the lymph nodes of untreated HIV-1–infected patients correlated with circulating CD8+ T cell counts and was normalized in these patients following antiretroviral therapy. Together, these results suggest that IL-15 drives bystander activation of CD8+ T cells, which predicts disease progression in untreated HIV-1–infected patients and suggests that elevated IL-15 may also drive CD8+ T cell expansion that is linked to increased morbidity and mortality in treated patients.
TCR repertoire sequencing identifies synovial Treg cell clonotypes in the bloodstream during active inflammation in human arthritis
Objectives: The imbalance between effector and regulatory T (Treg) cells is crucial in the pathogenesis of autoimmune arthritis. Immune responses are often investigated in the blood because of its accessibility, but circulating lymphocytes are not representative of those found in inflamed tissues. This disconnect hinders our understanding of the mechanisms underlying disease. Our goal was to identify Treg cells implicated in autoimmunity at the inflamed joints, and also readily detectable in the blood upon recirculation.
Methods: We compared Treg cells of patients with juvenile idiopathic arthritis responding or not to therapy by using: (i) T cell receptor (TCR) sequencing, to identify clonotypes shared between blood and synovial fluid; (ii) FOXP3 Treg cell-specific demethylated region DNA methylation assays, to investigate their stability and (iii) flow cytometry and suppression assays to probe their tolerogenic functions.
Results: We found a subset of synovial Treg cells that recirculated into the bloodstream of patients with juvenile idiopathic and adult rheumatoid arthritis. These inflammation-associated (ia)Treg cells, but not other blood Treg cells, expanded during active disease and proliferated in response to their cognate antigens. Despite the typical inflammatory-skewed balance of immune mechanisms in arthritis, iaTreg cells were stably committed to the regulatory lineage and fully suppressive. A fraction of iaTreg clonotypes were in common with pathogenic effector T cells.
Conclusions: Using an innovative antigen-agnostic approach, we uncovered a population ofbona fide synovial Treg cells readily accessible from the blood and selectively expanding during active disease, paving the way to non-invasive diagnostics and better understanding of the pathogenesis of autoimmunity.
High-throughput sequencing reveals restricted TCR VB usage and public TCRB clonotypes among pancreatic lymph node memory CD4+ T cells and their involvement in autoimmune diabetes
Islet-reactive memory CD4+ T cells are an essential feature of type 1 diabetes (T1D) as they are involved in both spontaneous disease and in its recurrence after islet transplantation. Expansion and enrichment of memory T cells have also been shown in the peripheral blood of diabetic patients. Here, using high-throughput sequencing, we investigated the clonal diversity of the TCRβ repertoire of memory CD4+ T cells in the pancreatic lymph nodes (PaLN) of non-obese diabetic (NOD) mice and examined their clonal overlap with islet-infiltrating memory CD4 T cells. Both prediabetic and diabetic NOD mice exhibited a restricted TCRβ repertoire dominated by clones expressing TRBV13-2, TRBV13-1 or TRBV5 gene segments. There is a limited degree of TCRβ overlap between the memory CD4 repertoire of PaLN and pancreas as well as between the prediabetic and diabetic group. However, public TCRβ clonotypes were identified across several individual animals, some of them with sequences similar to the TCRs from the islet-reactive T cells suggesting their antigen-driven expansion. Moreover, the majority of the public clonotypes expressed TRBV13-2 (Vβ8.2) gene segment. Nasal vaccination with an immunodominat peptide derived from the TCR Vβ8.2 chain led to protection from diabetes, suggesting a critical role for Vβ8.2+ CD4+ memory T cells in T1D. These results suggest that memory CD4+ T cells bearing limited dominant TRBV genes contribute to the autoimmune diabetes and can be potentially targeted for intervention in diabetes. Furthermore, our results have important implications for the identification of public T cell clonotypes as potential novel targets for immune manipulation in human T1D.
Identification of a CD4 T-cell epitope in the hemagglutinin stalk domain of pandemic H1N1 influenza virus and its antigen-driven TCR usage signature in BALB/c mice
The stalk region of the influenza virus hemagglutinin is relatively well conserved compared with the globular head domain, which makes it a potential target for use as a universal vaccine against influenza. However, the role of CD4 T cells in the hemagglutinin stalk-specific immune response is not clear. Here we identified a mouse CD4 T-cell epitope that encompasses residues HA2113-131 from the hemagglutinin stalk domain after a sub-lethal infection of influenza. In response to stimulation with the identified epitope, splenocytes derived from the infected mice showed significant polyfunctionality as shown by IL-2, TNF-α and IFN-γ production as well as degranulation. Moreover, mice immunized with the peptide corresponding to this CD4 T-cell epitope exhibited interindividual sharing of the CD4 T-cell receptor β sequences, and they had a higher survival rate following a challenge with a lethal dose of pandemic H1N1 influenza virus. Thus, our data demonstrated a crucial role of hemagglutinin stalk-specific CD4 T cells in the host immune response against influenza virus infection.
Immunodynamics: a cancer immunotherapy trials network review of immune monitoring in immuno-oncology clinical trials
The efficacy of PD-1/PD-L1 targeted therapies in addition to anti-CTLA-4 solidifies immunotherapyas a modality to add to the anticancer arsenal. Despite raising the bar of clinical efficacy, immunologically targeted agents raise new challenges to conventional drug development paradigms by highlighting the limited relevance of assessing standard pharmacokinetics (PK) and pharmacodynamics (PD). Specifically, systemic and intratumoral immune effects have not consistently correlated with standard relationships between systemic dose, toxicity, and efficacy for cytotoxic therapies. Hence, PK and PD paradigms remain inadequate to guide the selection of doses and schedules, both starting and recommended Phase 2 for immunotherapies. The promise of harnessing the immune response against cancer must also be considered in light of unique and potentially serious toxicities. Refining immune endpoints to better inform clinical trial design represents a high priority challenge. The Cancer Immunotherapy Trials Network investigators reviewthe immunodynamic effects of specific classes of immunotherapeutic agents to focus immuneassessment modalities and sites, both systemic and importantly intratumoral, which are critical to the success of the rapidly growing field of immuno-oncology.
Clonal expansion of CD4+ cytotoxic T lymphocytes in patients with IgG4-related disease
BACKGROUND: IgG4-related disease (IgG4-RD) is a systemic condition of unknown cause characterized by highly fibrotic lesions with dense lymphoplasmacytic infiltrates. CD4+ T cells constitute the major inflammatory cell population in IgG4-RD lesions.
OBJECTIVE: We used an unbiased approach to characterize CD4+ T-cell subsets in patients with IgG4-RD based on their clonal expansion and ability to infiltrate affected tissue sites.
METHODS: We used flow cytometry to identify CD4+ effector/memory T cells in a cohort of 101 patients with IgG4-RD. These expanded cells were characterized by means of gene expression analysis and flow cytometry. Next-generation sequencing of the T-cell receptor β chain gene was performed on CD4+SLAMF7+ cytotoxic T lymphocytes (CTLs) and CD4+GATA3+ TH2 cells in a subset of patients to identify their clonality. Tissue infiltration by specific T cells was examined by using quantitative multicolor imaging.
RESULTS: CD4+ effector/memory T cells with a cytolytic phenotype were expanded in patients with IgG4-RD. Next-generation sequencing revealed prominent clonal expansions of these CD4+ CTLs but not CD4+GATA3+ memory TH2 cells in patients with IgG4-RD. The dominant T cells infiltrating a range of inflamed IgG4-RD tissue sites were clonally expanded CD4+ CTLs that expressed SLAMF7, granzyme A, IL-1β, and TGF-β1. Clinical remission induced by rituximab-mediated B-cell depletion was associated with a reduction in numbers of disease-associated CD4+ CTLs.
CONCLUSIONS: IgG4-RD is prominently linked to clonally expanded IL-1β- and TGF-β1-secreting CD4+ CTLs in both peripheral blood and inflammatory tissue lesions. These active, terminally differentiated, cytokine-secreting effector CD4+ T cells are now linked to a human diseasecharacterized by chronic inflammation and fibrosis.
Novel technologies and emerging biomarkers for personalized cancer immunotherapy.
The culmination of over a century's work to understand the role of the immune system in tumor control has led to the recent advances in cancer immunotherapies that have resulted in durable clinical responses in patients with a variety of malignancies. Cancer immunotherapies are rapidly changing traditional treatment paradigms and expanding the therapeutic landscape for cancer patients. However, despite the current success of these therapies, not all patients respond to immunotherapy and even those that do often experience toxicities. Thus, there is a growing need to identify predictive and prognostic biomarkers that enhance our understanding of the mechanisms underlying the complex interactions between the immune system and cancer. Therefore, the Society for Immunotherapy of Cancer (SITC) reconvened an Immune Biomarkers Task Force to review state of the art technologies, identify current hurdlers, and make recommendations for the field. As a product of this task force, Working Group 2 (WG2), consisting of international experts from academia and industry, assembled to identify and discuss promising technologies for biomarker discovery and validation. Thus, this WG2 consensus paper will focus on the current status of emerging biomarkers for immune checkpoint blockade therapy and discuss novel technologies as well as high dimensional data analysis platforms that will be pivotal for future biomarker research. In addition, this paper will include a brief overview of the current challenges with recommendations for future biomarker discovery.
Daratumumab Depletes CD38 + Immune-regulatory Cells, Promotes T-cell Expansion, and Skews T-cell Repertoire in Multiple Myeloma
Daratumumab targets CD38-expressing myeloma cells through a variety of immune-mediated mechanisms (complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis) and direct apoptosis with cross- linking. These mechanisms may also target non-plasma cells that express CD38, which prompted evaluation of daratumumab’s effects on CD38-positive immune subpopulations. Peripheral blood (PB) and bone marrow (BM) from patients with relapsed/refractory myeloma from two daratumumab monotherapy studies were analyzed before and during therapy and at relapse. Regulatory B cells (B reg s) and myeloid-derived suppressor cells (MDSCs), previously shown to express CD38, were evaluated for immunosuppressive activity and daratumumab sensitivity in the myeloma setting. A novel subpopulation of regulatory T cells (T reg s) expressing CD38 was identified. These T reg s were more immunosuppressive in vitro than CD38-negative T reg s and were reduced in daratumumab-treated patients. In parallel, daratumumab induced robust increases in helper and cytotoxic T-cell absolute counts. In PB and BM, daratumumab induced significant increases in CD8 + :CD4 + and CD8 + :T reg ratios, and increased memory T cells while decreasing naïve T cells. The majority of patients demonstrated these broad T-cell changes, although patients with a partial response or better showed greater maximum effector and helper T cell increases, elevated antiviral and alloreactive functional responses, and significantly greater increases in T-cell clonality as measured by T-cell receptor (TCR) sequencing. Increased TCR clonality positively correlated with increased CD8 + PB T-cell counts. Depletion of CD38 + immune suppressive cells, which is associated with an increase in T-helper cells, cytotoxic T-cells, T-cell functional response, and TCR clonality, represent possible additional mechanisms of action for daratumumab and deserve further exploration.
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
Deep Sequencing Reveals Myeloma Cells in Peripheral Blood in Majority of Multiple Myeloma Patients
Introduction: The evaluation of myeloma cells in multiple myeloma (MM) patients has generally been limited to the assessment of bone marrow involvement because of the sensitivity limitations of traditional minimal-residual-disease–detection methods.
Materials and Methods: We developed a sequencing-based method to identify myeloma cells in bone marrow (BM) and peripheral blood (PB) samples, based on their unique immunoglobulin gene rearrangements, that can detect cancer clones at levels well below 1 in 1 million leukocytes (0.0001%). In this multisite study, we used this sequencing method to determine the fraction of patients with myeloma cells in their PB at diagnosis and posttreatment time points.
Results: Using this sequencing approach, we detected myeloma cells in the PB in the vast majority of MM patients (44/46, 96%). We demonstrated a clear correlation (R2 = 0.57) between myeloma clone levels in paired BM and PB samples, and noted that PB clone levels were approximately 100-fold lower than levels in BM samples. The sequencing assay demonstrated a clear sensitivity advantage in the BM compartment and at least equivalent sensitivity in the PB compared with that of monoclonal-protein results.
Conclusion: This study highlights the promise of a blood-based, sequencing minimal-residual-disease assay that can be used to measure MM disease burden at different time points and various disease stages.
Next-Generation Sequencing and Real-Time Quantitative PCR for Minimal Residual Disease Detection in B-Cell Disorders
In this study, we compared immunoglobulin heavy-chain-gene-based minimal residual disease (MRD) detection by real-time quantitative PCR (RQ-PCR) and next-generation sequencing (NGS) to assess whether NGS could overcome some limitations of RQ-PCR and further increase sensitivity, specificity, accuracy and reproducibility. In total, 378 samples from 55 patients with acute lymphoblastic leukemia (ALL), mantle cell lymphoma (MCL) or multiple myeloma (MM) were investigated for clonotype identification, clonotype identity and comparability of MRD results. Forty-five clonotypes were identified by RQ-PCR and 49 by NGS. Clonotypes identified by both tools were identical or >97% homologous in 96% of cases. Both tools were able to routinely reach a sensitivity level of 1 × E−05. A good correlation of MRD results was observed (R=0.791, P<0.001), with excellent concordance in 79.6% of cases. Few discordant cases were observed across all disease subtypes. NGS showed at least the same level of sensitivity as allele-specific oligonucleotides-PCR, without the need for patient-specific reagents. We conclude that NGS is an effective tool for MRD monitoring in ALL, MCL and MM. Prospective comparative analysis of unselected cases is required to validate the clinical impact of NGS-based MRD assessment.
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. The rearranged TCRβ loci were amplified using Vβ- and Jβ-specific primers, followed by HTS, to generate up to 1,000,000 reads spanning the CDR3 region of individual cells. Malignant clones were identified in diagnostic samples in all cases by a dominant CDR3 sequence. Before transplant, four patients had circulating Sézary cells by the routine flow cytometry, which was confirmed by TCRB HTS. Although the flow cytometry found no detectable Sézary cells, malignant clones were detected by TCRB HTS in all other six cases. Five patients achieved "molecular remission" in blood between +30 and +540 days after transplant. Four of these patients also achieved molecular clearance in skin after transplant. Experiments using blood samples spiked with purified Sézary cells demonstrated that TCRB HTS can detect Sézary cells at the level of 1 in 50,000 PBMCs, which is more sensitive than standard diagnostics. We have thus demonstrated the utility of TCRB HTS to assess MRD with increased sensitivity and specificity compared to other current methodologies, and to monitor response to therapy in this MF/SS patient population.
Detection of Circulating Tumour DNA in Patients with Aggressive B-cell Non-Hodgkin Lymphoma
Current methods for detecting the presence of disease in patients with diffuse large B cell lymphoma (DLBCL) or mediastinal large B-cell lymphoma (MLBCL) rely primarily on imaging methods, which are associated with significant cost and radiation exposure. Very few patients with DLBCL have evidence of circulating disease using flow cytometric assays (Mancuso et al, 2010). This has so far precluded the development of minimal residual disease (MRD) assessment tools in those diseases, in contrast to tumours with a circulating component, where MRD assays are emerging as important methods (Ferrero et al, 2011). The availability of high-throughput sequencing techniques now provides an opportunity to probe for the presence of very small amounts of circulating tumour genetic material in peripheral blood (PB). If sequencing-based methods can reliably detect circulating disease, they could eventually find a role in the treatment and monitoring of patients with those tumours. We present here a pilot study of a sequencing method designed to examine whether tumour DNA is detectable in patients with newly diagnosed DLBCL/MLBCL, and whether it becomes undetectable after therapy.
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. Using a synthetic immune receptor repertoire, we identify and minimize such biases and computationally remove residual bias after sequencing. We apply this method to a multiplex T cell receptor gamma sequencing assay. To demonstrate accuracy in a biological setting, we apply the method to monitor minimal residual disease in acute lymphoblastic leukaemia patients. A similar methodology can be extended to any adaptive immune locus.
Minimal Residual Disease Quantification Using Consensus Primers and High-Throughput IGH Sequencing Predicts Post-Transplant Relapse in Chronic Lymphocytic Leukemia
Quantification of minimal residual disease (MRD) following allogeneic hematopoietic cell transplantation (allo-HCT) predicts post-transplant relapse in patients with chronic lymphocytic leukemia (CLL). We utilized an MRD-quantification method that amplifies immunoglobulin heavy chain (IGH) loci using consensus V and J segment primers followed by high-throughput sequencing (HTS), enabling quantification with a detection limit of one CLL cell per million mononuclear cells. Using this IGH–HTS approach, we analyzed MRD patterns in over 400 samples from 40 CLL patients who underwent reduced-intensity allo-HCT. Nine patients relapsed within 12 months post-HCT. Of the 31 patients in remission at 12 months post-HCT, disease-free survival was 86% in patients with MRD <10−4 and 20% in those with MRD greater than or equal to10−4 (relapse hazard ratio (HR) 9.0; 95% confidence interval (CI) 2.5–32; P<0.0001), with median follow-up of 36 months. Additionally, MRD predicted relapse at other time points, including 9, 18 and 24 months post-HCT. MRD doubling time <12 months with disease burden greater than or equal to10−5 was associated with relapse within 12 months of MRD assessment in 50% of patients, and within 24 months in 90% of patients. This IGH–HTS method may facilitate routine MRD quantification in clinical trials.
Deep-Sequencing Approach for Minimal Residual Disease Detection in Acute Lymphoblastic Leukemia
The persistence of minimal residual disease (MRD) during therapy is the strongest adverse prognostic factor in acute lymphoblastic leukemia (ALL). We developed a high-throughput sequencing method that universally amplifies antigen-receptor gene segments and identifies all clonal gene rearrangements (ie, leukemia-specific sequences) at diagnosis, allowing monitoring of disease progression and clonal evolution during therapy. In the present study, the assay specifically detected 1 leukemic cell among greater than 1 million leukocytes in spike-in experiments. We compared this method with the gold-standard MRD assays multiparameter flow cytometry and allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) using diagnostic and follow-up samples from 106 patients with ALL. Sequencing detected MRD in all 28 samples shown to be positive by flow cytometry and in 35 of the 36 shown to be positive by ASO-PCR and revealed MRD in 10 and 3 additional samples that were negative by flow cytometry and ASO-PCR, respectively. We conclude that this new method allows monitoring of treatment response in ALL and other lymphoid malignancies with great sensitivity and precision. The www.clinicaltrials.gov identifier number for the Total XV study is NCT00137111.
Massive Evolution of the Immunoglobulin Heavy Chain Locus in Children with B Precursor Acute Lymphoblastic Leukemia
The ability to distinguish clonal B-cell populations based on the sequence of their rearranged immunoglobulin heavy chain (IgH) locus is an important tool for diagnosing B-cell neoplasms and monitoring treatment response. Leukemic precursor B cells may continue to undergo recombination of the IgH gene after malignant transformation; however, the magnitude of evolution at the IgH locus is currently unknown. We used next-generation sequencing to characterize the repertoire of IgH sequences in diagnostic samples of 51 children with B precursor acute lymphoblastic leukemia (B-ALL). We identified clonal IgH rearrangements in 43 of 51 (84%) cases and found that the number of evolved IgH sequences per patient ranged dramatically from 0 to 4024. We demonstrate that the evolved IgH sequences are not the result of amplification artifacts and are unique to leukemic precursor B cells. In addition, the evolution often follows an allelic exclusion pattern, where only 1 of 2 rearranged IgH loci exhibit ongoing recombination. Thus, precursor B-cell leukemias maintain evolution at the IgH locus at levels that were previously underappreciated. This finding sheds light on the mechanisms associated with leukemic clonal evolution and may fundamentally change approaches for monitoring minimal residual disease burden.
High-Throughput Sequencing Detects Minimal Residual Disease in Acute T Lymphoblastic Leukemia
High-throughput sequencing (HTS) of lymphoid receptor genes is an emerging technology that can comprehensively assess the diversity of the immune system. Here, we applied HTS to the diagnosis of T-lineage acute lymphoblastic leukemia/lymphoma. Using 43 paired patient samples, we then assessed minimal residual disease (MRD) at day 29 after treatment. The variable regions of TCRB and TCRG were sequenced using an Illumina HiSeq platform after performance of multiplexed polymerase chain reaction, which targeted all potential V-J rearrangement combinations. Pretreatment samples were used to define clonal T cell receptor (TCR) complementarity-determining region 3 (CDR3) sequences, and paired posttreatment samples were evaluated for MRD. Abnormal T lymphoblast identification by multiparametric flow cytometry was concurrently performed for comparison. We found that TCRB and TCRG HTS not only identified clonality at diagnosis in most cases (31 of 43 for TCRB and 27 of 43 for TCRG) but also detected subsequent MRD. As expected, HTS of TCRB and TCRG identified MRD that was not detected by flow cytometry in a subset of cases (25 of 35 HTS compared with 13 of 35, respectively), which highlights the potential of this technology to define lower detection thresholds for MRD that could affect clinical treatment decisions. Thus, next-generation sequencing of lymphoid receptor gene repertoire may improve clinical diagnosis and subsequent MRD monitoring of lymphoproliferative disorders.