scholarly journals Chromatin Accessibility Profiling to Increase Diagnostic Accuracy and Refine Cell-of-Origin Classification of Mature T-Cell Lymphomas

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 809-809
Author(s):  
Edith Julia ◽  
Sylvain Mareschal ◽  
Amel Chebel ◽  
Camille Golfier ◽  
Tony Andreas Müller ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Single Cells ◽  
Patent Application ◽  
Chromatin Accessibility ◽  
Open Chromatin ◽  
Cell Of Origin ◽  
T Cell Lymphomas ◽  
Origin Identification

Abstract Background: Mature T-cell lymphomas and leukemias (MTCL) are heterogeneous diseases with dismal prognosis. Differentiating between the numerous entities requires specialized pathology expertise and studies show up to 20% change in diagnosis after expert review of cases (Laurent, JCO, 2017). Assay for transposase accessible chromatin sequencing (ATAC-seq) is a simple technique to profile open chromatin regions (OCR) proven to be highly discriminant for cell-of-origin identification regardless of cell activation status (Shih, Cell, 2016). We applied ATAC-seq to MTCL in order to explore the epigenetic landscape of these diverse entities, compared them to normal T-cell subtypes and built a predictive model to help diagnosis. Method: Ten-thousand FACS-sorted single cells from primary MTCL samples and 50µm section of frozen tumoral tissue from the TENOMIC French T-cell Lymphoma Consortium were processed according to the previously published FAST-ATAC and OMNI-ATAC protocols respectively (Corces, Nat Genetics, 2016 & Nat. Methods, 2017). Concurrently we applied FAST ATAC to different normal T- and NK-cell subsets sorted from healthy donor PBMC or lymph node suspensions. Sequencing data were processed by an adapted version of ENCODE ATAC-seq pipeline. Matrix of insertion events in peaks by sample was obtained, normalized and most variant peaks were selected for UMAP projection. Results: In total, 678 normal and tumoral samples were sequenced to provide a comprehensive landscape of chromatin accessibility in MTCL. Epigenetic profiling by ATAC-seq of FACS-sorted tumoral samples resulted in a complete segregation of the known MTCL entities (AITL, TFH-PTCL, ALK+ and ALK- ALCL, HSTL, CTCL, ATLL, LGL and T-PLL). Most PTCL-NOS (13/17) clustered with a pre-defined MTCL subtype (mainly AITL/TFH-phenotype PTCL, CTCL and lymphomas exhibiting cytotoxic features). All but one discordant diagnosis between pathology and ATAC-seq (1/11) led to revised diagnosis after pathology review. Unsupervised clustering of normal NK- and T-cell subtypes (N=49) and sorted tumoral lymphoma cells (N=104) confirmed that AITL derive from TFH cells. HSTL and LGL closely segregated with NK- and gamma-delta T cells, in line with their known innate-like phenotype. Surprisingly, the cell-of-origin of T-PLL seems to be naïve T cells despite the known expression of central memory markers on leukemic cells. Beyond epigenetic classification, background reads from ATAC-seq profiles were used to detect copy number variation (CNV), such as isochromosome 7q in HSTL. In addition, HTLV1 and EBV viral sequence detection in ATAC-seq reads strengthened identification of ATLL and NKTCL cases. Finally, using unsupervised deconvolution approaches, we were able to discriminate different MTCL subtypes from 223 processed bulk frozen samples. All known MTCL subtypes were differentiated (AITL/PTCL-TFH, HSTL, NKTCL, ATLL, ALK- and ALK+ ALCL, MEITL, EATL). A subgroup of PTCL-NOS harboring GATA3 OCRs and a distinctly high CNV number was isolated that might correspond to previously described PTCL-GATA3 subtypes (Iqbal, Blood, 2019). A random forest model was trained to predict diagnosis based on chromatin-accessibility clusters defined in the discovery cohort of patients. The model showed accurate prediction performance by cross-validation. External validation on 172 samples collected from 5 tertiary care centers will be presented at the meeting. Conclusion: ATAC-seq is a fast and cost-effective technique to help and refine MTCL pathological classification and allows for putative cell-of-origin identification in lymphoma. Training of a machine learning model to predict MTCL entity diagnosis based on ATAC-seq analysis of fresh or frozen samples shows promising results. Figure 1 Figure 1. Disclosures Sibon: Janssen: Consultancy; Abbvie: Consultancy; iQone: Consultancy; Takeda: Consultancy; Roche: Consultancy. Drieux: Genexpath: Patents & Royalties: The author is a potential inventor on a patent application for the LymphoSign, which has been licensed for by Genexpath Patents & Royalties.. Ruminy: Genexpath: Patents & Royalties: The author is a potential inventor on a patent application for the LymphoSign, which has been licensed for by Genexpath Patents & Royalties. . Salles: Takeda: Consultancy; Velosbio: Consultancy; Ipsen: Consultancy; Allogene: Consultancy; Miltneiy: Consultancy; Genentech/Roche: Consultancy; Genmab: Consultancy; Janssen: Consultancy; Loxo: Consultancy; Kite/Gilead: Consultancy; Regeneron: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria; Novartis: Consultancy; Incyte: Consultancy; Rapt: Consultancy; Epizyme: Consultancy, Honoraria; Debiopharm: Consultancy; BMS/Celgene: Consultancy; Beigene: Consultancy; Abbvie: Consultancy, Honoraria; Bayer: Honoraria. Gaulard: Alderaan: Research Funding; Sanofi: Research Funding; Innate Pharma: Research Funding; Gilead: Consultancy; Takeda: Consultancy, Honoraria. Bachy: Kite, a Gilead Company: Honoraria; Novartis: Honoraria; Daiishi: Research Funding; Roche: Consultancy; Takeda: Consultancy; Incyte: Consultancy.

Molecular Heterogeneity in Peripheral T-Cell Lymphoma Not Otherwise Specified Revealed By Comprehensive Mutational Profiling

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2927-2927 ◽  
Author(s):  
Yosaku Watatani ◽  
Yasuharu Sato ◽  
Kenji Nishida ◽  
Hiroaki Miyoshi ◽  
Yuichi Shiraishi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Genetic Alterations ◽  
T Cell Lymphoma ◽  
Molecular Heterogeneity ◽  
Not Otherwise Specified ◽  
T Cell Lymphomas ◽  
Somatic Alterations

Abstract Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of lymphoproliferative disorders arising from mature T-cells. Among them, PTCL-not otherwise specified (PTCL-NOS) is a diagnosis of exclusion, comprising the largest fraction of PTCL with a diverse underlying pathogenesis. Recently, the concept of nodal T-cell lymphomas with T-follicular helper (TFH) phenotype, including angioimmunoblastic T-cell lymphoma (AITL) and PTCL-NOS that manifests a TFH phenotype, has been proposed, a distinguishing feature of which is the high frequency of TET2, IDH2, DNMT3A, and RHOA(G17V) mutations. Although recent large-scale genetic studies have uncovered mutational landscapes of several other subtypes of PTCLs, such as cutaneous T-cell lymphoma and adult T-cell leukemia/lymphoma (ATL), the entire picture of somatic alterations in PTCL-NOS still remains elusive. In addition, their similarities and differences among various histological subtypes in PTCLs have not been fully elucidated. To address this issue, we initially analyzed our and publicly available whole-exome/genome as well as transcriptome sequencing data from PTCL-NOS and other related PTCLs. Then, we carried out an extensive investigation of somatic mutations and structural variations (SVs) in PTCL-NOS using targeted-capture sequencing of 118 PTCL-NOS samples. Consistent with previous reports, TET2 (35%) was the most frequently mutated gene in PTCL-NOS with the majority (78%) affected by multiple mutations, followed by RHOA (25%), TP53 (16%), KMT2C (12%), PLCG1 (12%), and HLA-B (11%). Besides them, a considerable proportion of patients harbored mutations in components of T-cell receptor (TCR) /NF-κB pathway (such as PRKCB, CARD11, IRF4, and PRDM1), other signal transduction molecules (STAT3, NOTCH1, and SOCS1), chemokine receptors (CCR4 and CCR7), epigenetic modifiers (CREBBP, KDM6A, IDH2, and DNMT3A), transcriptional regulators (GATA3 and TBL1XR1), and molecules associated with immune evasion (HLA-A, HLA-B, FAS, B2M, and CD58). In addition to deteriorating SVs involving frequently affected genes (TP53, FAS, GATA3, and TBL1XR1), we discovered several genes almost exclusively affected by SVs, including TP73, IKZF2, and NFKB2, and CD274. Novel targets of recurrent mutation were also identified, including PDCD1, YTHDF2, and LRP1B, which were frequently targeted by nonsense and frameshift mutations distributed throughout the entire genes. Among them, PDCD1encodes PD-1 receptor transmitting an inhibitory signal from PD-L1 and PD-L2 ligands in T cells, and its loss of function seems to enable tumor cells to escape from the suppression by this negative signal. Although the roles of YTHDF2, a reader protein of N6-methyladenosine, and LRP1B, a member of the low density lipoprotein receptor family, in T cells are not immediately apparent, these findings shed light on a new biological function of these genes. Next, we investigated the co-existence relationship between frequently altered genes in PTCL-NOS. Interestingly, mutations characteristic of TFH lymphomas (TET2, RHOA, IDH2, and DNMT3A) tended to co-occur in a subset of PTCL-NOS cases, whereas they were almost mutually exclusive with mutations in TP53 and TCR/NF-κB pathway genes. This observation reveals the molecular distinction between TFH and non-TFH lymphomas in PTCL-NOS: the former is similar to AITL, although TET2 mutations did not show higher allelic burden than RHOA and IDH2mutations. In contrast, the latter is at least partly characterized by the genetic alterations shared with ATL. In summary, our findings illuminate the landscape of somatic alterations in PTCL-NOS and provide a novel insight into their genetic and molecular heterogeneity, which would help us to exploit a new therapeutic strategy to combat this disease. Disclosures Ohshima: CHUGAI PHARMACEUTICAL CO.,LTD.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau. Ogawa:Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding. Kataoka:Kyowa Hakko Kirin: Honoraria; Yakult: Honoraria; Boehringer Ingelheim: Honoraria.

scholarly journals Single Cell RNA-Seq Reveals Deranged Developmental Hierarchy with Coexisting Oncogenic States and Immune Evasion Programs in ETP T-ALL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3953-3953
Author(s):  
Amy Guillaumet-Adkins ◽  
Praveen Anand ◽  
Huiyoung Yun ◽  
Yotam Drier ◽  
Anna Rogers ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Single Cells ◽  
Pearson Correlation ◽  
Normal Donor ◽  
Developmental Hierarchy

Introduction: Early T-cell precursor acute lymphoblastic leukemia (ETP T-ALL) is a distinct subtype of T-ALL characterized by higher rates of relapse and induction failure. Large-scale genetic sequencing studies have identified frequently mutated oncogenes and gene fusions in ETP T-ALL, while bulk transcriptome analyses have revealed expression features resembling myeloid precursors and myeloid malignancies. However, the contributions of intra-tumoral functional heterogeneity and microenvironment to tumor biology and treatment failure remain unknown. Methods: We performed full-length single-cell RNA-sequencing of 5,077 malignant and normal immune cells from bone marrow or blood from five patients with relapsed/refractory ETP T-ALL (based on immunophenotyping, all with NOTCH1 mutations), before and after targeted therapy against NOTCH1. These patients were enrolled on a phase I trial with the γ-secretase inhibitor (GSI) BMS-906024 (NCT01363817). Expression of selected genes was validated by RT-PCR, flow cytometry and immunohistochemistry. Results: Single cell transcriptome analyses revealed a deranged developmental hierarchy characterized by co-expression of stemness programs in multiple malignant cells implying ineffectual commitment to either lymphoid or myeloid lineage. Most ETP T-ALL cells co-expressed HSC (hematopoietic stem cell), CMP (common myeloid progenitor) and CLP (common lymphoid progenitor) signatures simultaneously (Pearson correlation: CLP-CMP: R= 0.41, p < 2.2e-16; HSC-CLP: R= 0.53; p < 2.2e-16; HSC-CMP: R = 0.39, p <2.2e-16). Only a fraction of cells (less than 15%) demonstrated mutually exclusive CLP or HSC signatures. In contrast, CLP, CMP and HSC signatures were not co-expressed and always negatively correlated in normal bone marrow cells (CLP-CMP: R= -0.11, p < 2.2e-16; HSC-CLP: R= -0.38; p < 2.2e-16; HSC-CMP: R = -0.67, p <2.2e-16). Direct targeting of NOTCH1 as the driving oncogene has shown disappointing results in the clinical setting due to the rapid development of resistance. PI3K activation has been shown as a genetic mechanism of Notch resistance, however it is unclear if transcriptional rewiring can give rise to PI3K dependent cells after Notch inhibition. To address this question, we predicted the activity of signaling pathways in single cells after Notch inhibitor treatment using PROGENy. Most single cells demonstrated loss of Notch signaling. PI3K signaling activity was the most anti-correlated signaling pathway to Notch signaling (Pearson correlation: R= -0.51, p < 2.2e-16). Of note, this population preexisted at a frequency of ~30% in the untreated population, coexisting with cells with high Notch activation. Analysis of the immune microenvironment revealed an oligoclonal T-cell population in ETP T-ALL compared to normal donor T-cells. CD8+ T-cells from ETP patients expressed markers of T-cell exhaustion (PDCD1, TIGIT, LAG3, HAVCR2). Analyses of expression levels of the respective ligands on leukemic blasts and the predicted interaction with their receptors on endogenous CD8+ T-cells demonstrated the highest interaction score between HAVCR2 and its ligand LGALS9. LGALS9 was universally expressed in all leukemic cells, which was confirmed by flow cytometry staining in leukemic blasts and IHC staining in bone marrow of 8 patients with ETP T-ALL and 7 patients with T-ALL. T-ALL supernatant increased expression levels of the exhaustion markers HAVCR2,TIGIT and decreased effector marker GZMB in polyclonal activated normal donor CD8+ T-cells (RT-PCR). This effect was abrogated by neutralizing LGALS9 and could be rescued with recombinant LGALS9. Conclusion: We identified deranged developmental hierarchy characterized by co-expression of stemness programs in multiple malignant cell states and ineffectual commitment to either lymphoid or myeloid lineage in ETP T-ALL. Leukemic blasts demonstrate preexisting heterogeneity of diverse oncogenic states as evidenced by opposing PI3K and Notch activity, suggesting possible novel combination therapies. Notch inhibition abolishes the Notch high state without effecting the PI3K active state. Finally, we demonstrate a possible role for HAVCR2-LGALS9 interactions in causing CD8+ T-cell dysfunction in ETP T-ALL patients, which may provide a novel therapeutic strategy in this disease. Disclosures Silverman: Takeda: Consultancy; Servier: Consultancy, Research Funding. Lane:AbbVie: Research Funding; Stemline Therapeutics: Research Funding; N-of-One: Consultancy. DeAngelo:Glycomimetics: Research Funding; Amgen, Autolus, Celgene, Forty-seven, Incyte, Jazzs, Pfizer, Shire, Takeda: Consultancy; Blueprint: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Abbvie: Research Funding. Lohr:Celgene: Research Funding; T2 Biosystems: Honoraria.

scholarly journals Immune Profiling of Diagnostic DLBCL Biopsies Dramatically Improves upon Cell-of-Origin Risk Stratification

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 719-719
Author(s):  
Michael Nissen ◽  
Xuehai Wang ◽  
Clementine Sarkozy ◽  
Aixiang Jiang ◽  
Daisuke Ennishi ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Gene Expression Profiling ◽  
Patient Outcomes ◽  
Expression Profiling ◽  
Research Funding ◽  
Cell Of Origin ◽  
Discovery Cohort

Abstract Background: Diffuse large B cell lymphoma (DLBCL) is an aggressive malignancy of mature B cells. The disease has traditionally been subdivided into cell-of-origin (COO) subtypes - germinal centre B cell-like (GCB) or activated B cell-like (ABC) - as determined by expression profiling or immunohistochemistry of the tumor cells. However the role of the immune microenvironment, and how the tumor and immune system interact to influence patient outcomes, remains to be fully investigated. Methods: In this project, we used mass cytometry (CyTOF) to deeply profile both tumor cell phenotypes and the immune microenvironments, alongside ABC/GCB classification and mutation profiling, in a discovery cohort of 54 DLBCL cases. As well, a validation cohort of 129 DLBCL patients were immunologically profiled by high-dimensional conventional flow cytometry, and their immune profiles alongside ABC/GCB classification, mutation profiling, and RNAseq data, were correlated with patient outcomes as measured by progression-free survival (PFS). Results: Analysis of the CyTOF/discovery cohort demonstrated that DLBCL tumor cells are phenotypically unique to each patient, with a small number of samples displaying distinct sub-clonal structure, often distinguished by differential expression of immune-related proteins like MHC-II. ABC/GCB classifications could be recapitulated based on tumor cell phenotypes, demonstrating that while COO was a robust feature, a great deal of heterogeneity exists within these established subtypes. Immunological profiling of the CyTOF/discovery cohort revealed that DLBCL samples could be divided into three distinct groups which roughly correlated with abundances of naïve, activated, or terminally differentiated T cells, respectively. This profiling schema was extended to the validation cohort of 129 patients which in turn led to identification of a subset of patients with a very high risk of disease progression (5-year PFS; 30% high risk vs. 80% low risk, p&lt;0.0001). This final classifier was based on a combination of ABC-DLBCL designation, combined with the presence of an immune microenvironment dominated by terminally differentiated (CD57+) T cells. We performed a limited series of functional studies using primary DLBCL biopsy samples to characterize further these CD57+ T cells as clonally restricted and incapable of responding to antigenic challenge. Interestingly, traditional immune markers of T cell exhaustion such as PD-1, TIM3, LAG3 and TIGIT were not correlated with patient outcomes. Conclusions: Overall, this study demonstrates the utility of immune profiling in risk stratification based on initial diagnostic biopsy material and highlights a subset of DLBCL patients who may benefit from immune-based therapies to rejuvenate the anti-tumor T cell response. We conclude that T cell senescence, rather than exhaustion, is the more relevant feature in DLBCL disease biology and highlights an alternate target for immunomodulatory therapy. Figure 1 Figure 1. Disclosures Craig: Bayer: Consultancy. Slack: Seagen: Consultancy, Honoraria. Scott: Abbvie: Consultancy; AstraZeneca: Consultancy; Celgene: Consultancy; NanoString Technologies: Patents & Royalties: Patent describing measuring the proliferation signature in MCL using gene expression profiling.; BC Cancer: Patents & Royalties: Patent describing assigning DLBCL COO by gene expression profiling--licensed to NanoString Technologies. Patent describing measuring the proliferation signature in MCL using gene expression profiling. ; Rich/Genentech: Research Funding; Janssen: Consultancy, Research Funding; Incyte: Consultancy. Steidl: Epizyme: Research Funding; Bayer: Consultancy; Curis Inc.: Consultancy; Seattle Genetics: Consultancy; AbbVie: Consultancy; Trillium Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding.

scholarly journals Innate Immune Sensing in Anti-Tumor Immunity and Cancer Immunotherapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. SCI-27-SCI-27
Author(s):  
Thomas Gajewski
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Research Funding ◽  
Immune Escape ◽  
Patent Application ◽  
Type I ◽  
Inhibitory Mechanisms ◽  
Immune Sensing ◽  
Innate Immune Sensing

Abstract Most cancers express tumor antigens that can be recognized by T cells of the host. The fact that cancers that become clinically relevant grow, nonetheless implies that immune escape must occur to allow cancer outgrowth. We have observed two major subsets of human melanoma metastases based on gene expression profiling and confirmatory assays. One subgroup of patients has a T cell-inflamed phenotype that includes expression of chemokines, T cell markers, and a type I interferon (IFN) signature. In contrast, the other major subset lacks this phenotype and appears to display immune "exclusion". The mechanisms of immune escape are likely distinct in these two phenotypes, and therefore the optimal immunotherapeutic interventions necessary to promote clinical responses may be different. The T cell-inflamed tumor microenvironment subset shows the highest expression of negative regulatory factors, including PD-L1, IDO, and FoxP3+ Tregs, and evidence for T cell-intrinsic anergy has also emerged aided by a recently defined functional role of EGR2. In addition, the mechanism of induction of these inhibitory mechanisms has been elucidated-PD-L1 and IDO are induced by IFN-g, and Tregs are largely recruited by the chemokine CCL22, both being produced by activated CD8+ effector T cells. Preclinical experiments have confirmed a critical role for each of these mechanisms in limiting anti-tumor T cell efficacy in vivo, giving candidate treatment strategies for translation back into the clinic. These include anti-PD-1/PD-L1 mAbs, IDO inhibitors, and approaches to deplete CD25+ Tregs and/or reverse anergy. The presence of multiple inhibitory mechanisms in the same tumor microenvironment argues that combination therapies may be advantageous. Preclinical data have indicated synergy between anti-CTLA-4 +/- anti-PD-L1 +/- IDO inhibition. Clinical translation of multiple combination immunotherapies is promising and ongoing. In contrast to the T cell-inflamed melanomas, a new paradigm may be needed to promote de novo inflammation in cases of the non-T cell-infiltrated tumor microenvironment. Natural innate immune sensing of tumors appears to occur via the host STING pathway, type I IFN production, and cross-priming of T cells via CD8a+ dendritic cells. New strategies are being developed to engage or mimic this pathway as a therapeutic endeavor, including STING agonists. A phase I study of intratumoral injection of the first STING agonist is ongoing. As an environmental variable, recent work has implicated the commensal microbiota as a regulator of DC activation status and systemic anti-tumor immunity, and clinical analysis of microbiota sequencing in the context of checkpoint blockade is ongoing. Disclosures Gajewski: Evelo: Patents & Royalties: Patent application; Jounce: Consultancy; Merck: Consultancy, Research Funding; Aduro: Patents & Royalties: Patent application; Incyte: Consultancy, Research Funding; Celldex: Consultancy, Research Funding; BMS: Research Funding; Roche/Genentech: Consultancy, Research Funding; Bayer: Consultancy; Abbvie: Consultancy.

scholarly journals Identification of Previously Unrecognized CD1d-Restricted Peripheral T Cell Lymphomas (PTCLs) in Mouse and Human Reveals Blocking Anti-CD1d Monoclonal Antibodies As a New Therapeutic Possibility in PTCLs

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4485-4485
Author(s):  
Emmanuel Bachy ◽  
Mirjam Urb ◽  
Gabriel Bricard ◽  
Shilpi Jayaswal ◽  
Remy Robinot ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Profile Analysis ◽  
Nkt Cells ◽  
Type I ◽  
Type Ii ◽  
Cell Of Origin ◽  
Gamma Delta ◽  
Inkt Cells ◽  
T Cell Lymphomas

Abstract Background. Peripheral T-cell lymphomas (PTCLs) originate from post-thymic T cells but compared to B-cell lymphomas the exact cell of origin is usually unknown except for angioimmunoblastic T-cell lymphoma arising from a follicular helper T-cell. Furthermore, no recurrent cytogenetic or molecular abnormalities are identified in PTCLs. Recently, recurrent impairment of the p53 pathway has been pointed out in PTCLs. However, p53 knockout (KO) mice are known to develop immature thymic T-cell lymphomas and solid tumors but surprisingly PTCLs have not been reported for more than 20 years in those mice. NKT cells are a T-cell subset responsive to glycolipids presented by CD1d, a major histocompatibility complex (MHC) class I-like antigen-presenting molecule, in contrast to conventional T cells, which recognize peptide antigens. Two types of NKT cells have been described so far: type I or invariant NKT cells (iNKT) that express a Valpha14-Jalpha18 (in mice) or Valpha24-Jalpha18 (in humans) constant chain and type II NKT cells that express a variable TCR but are CD1d-dependent as well. Most type II NKT cells are of alpha/beta phenotype but CD1d-restricted gamma/delta T cells have also been described in mice and humans. Methods. The development of PTCLs in p53 KO mice (B6.129S2-Trp53tm1Tyj/J) was studied. Identification of PTCLs was made by immunohistochemistry and flow cytometry analysis. Gene expression profile analysis (GeneChip Mouse Genome 430 2.0 array, Affymetrix) was performed to characterize lymphomas developed in the mouse model. Transfer experiments were done by intravenously retro-orbital injection into syngeneic, immunocompetent C57Bl/6J WT animals or immunocompromised CD3ε-/- mice. Therapeutic trials in mice were performed with the use of blocking anti-CD1d monoclonal antibodies (mAb) (clone HB323; BioXcell). Results. We found that p53 KO mice developed well-known and characterized thymic T-cell lymphomas and solid tumors as previously described. However, about 20% of p53 KO mice spontaneously developed a previously unrecognized entity of PTCLs originating from CD1d-restricted iNKT cells (ie type I NKT cells) referred to as NKTLs for NKT lymphomas thereafter. Both alpha-galactosylceramide-CD1d tetramer staining and unique Valpha14-Jalpha18 TCR rearrangement confirmed the iNKT nature of these lymphomas. Chronic injection of Streptococcus pneumoniae (Spn), reported to express glycolipid antigens activating NKT cells, significantly increased the incidence of these NKTLs compared to a control group of p53 KO mice injected with PBS (P=0.03). Gene expression profile analysis indicated a significant down-regulation of genes in the TCR signaling pathway of NKTLs (false discovery rate q-value=0.01 by gene set enrichment analysis) suggesting an underlying antigenic chronic stimulation as previously reported in chronically activated T cells (Figure 1). Moreover, NKTLs were characterized by upregulation of PD-1 and loss of NK1.1 expression compared to resting NKT cells (P<0.01 for both), which are features of activated and anergic iNKT cells. Altogether, those data indicate that NKTLs in mice could arise from chronically activated iNKT cells by endogenous or exogenous glycolipids. Furthermore, in vivo TCR/CD1d interactions were required for NKTLs survival after transfer in recipient mice and the use of blocking anti-CD1d mAb significantly prolonged mice overall survival (logrank P<0.001, Figure 2). We did not identify human PTCLs arising from type I iNKT cells by using alphaGalCer-CD1d tetramer staining. However, using sulfatide-loaded CD1d tetramers (ie another type of glycolipid-CD1d tetramer identifying type II NKT cells), we identified CD1d-restricted human PTCLs among gamma/delta hepatosplenic T-cell lymphomas (HSTLs) and PTCL-not otherwise specified (PTCL-NOS) expressing the Vd1 TCR but not the Vd2 TCR (Figure 3). Conclusion. This demonstrates for the first time the existence of human PTCLs arising from gamma/delta CD1d-restricted type II NKT cells. These results refine the classification of PTCLs in humans by identifying a new cell of origin and pave the way for the development of blocking anti-CD1d antibodies for therapeutic purposes. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures No relevant conflicts of interest to declare.

scholarly journals Clonotype pattern in T-cell lymphomas map the cell of origin to immature lymphoid precursors

2021 ◽  
Author(s):  
Aishwarya Iyer ◽  
Dylan Hennessey ◽  
Robert Gniadecki
Keyword(s):  
T Cells ◽  
T Cell ◽  
Anaplastic Large Cell Lymphoma ◽  
Cell Lymphoma ◽  
Large Cell ◽  
T Cell Lymphoma ◽  
Cell Of Origin ◽  
Peripheral T Cell Lymphoma ◽  
T Cell Lymphomas ◽  
Large Cell Lymphoma

Background Mature T-cell lymphomas (TCLs) are rare, clinically heterogeneous hematologic cancers of high medical need. TCLs have inferior prognosis compared with their B-cell counterparts, which is attributed to poor understanding of their pathogenesis. Based on phenotypic similarities between normal and neoplastic lymphocytes it has been assumed that TCLs develop in the periphery, directly from various subtypes of normal T-cells. Methods and findings To address the debated question of the cell of origin in TCLs we analyzed to identify the highly variable complementarity determining regions (CDR3) regions of T-cell receptor (TCR) to trace the clonal history of the T-cells. We have collected previously published whole genome -exome, and -transcriptome sequencing data from 574 TCL patients comprising five nodal lymphomas [anaplastic large cell lymphoma (n=67), peripheral T-cell lymphoma (PTCL, n=55), adult T-cell lymphoma/leukemia (n=135), natural killer T-cell lymphoma (NKCL, n=25), not specified/other (n=30)] and three extranodal, cutaneous T-cell lymphomas [mycosis fungoides (n=122), Sezary syndrome (n=130), and subcutaneous panniculitis like T-cell lymphoma (n=10)]. TCR clonotypes contained in the tumor cell fraction, representing the clonotypes of malignant cells, were identified by de novo assembly of CDR3 regions of TCRγ, β and α. We have found that the vast majority of TCLs are clonotypically oligoclonal, although the pattern oligoclonality varied. Anaplastic large cell lymphoma was most diverse comprising multiple clonotypes of TCRγ, β and α whereas adult T-cell lymphoma/leukemia and peripheral T-cell lymphomas often showed monoclonality for TCRγ and β but had diverse TCRα clonotypes. These patterns of rearrangements were not compatible with the current mature T-cell precursor model and indicated that TCLs are initiated at the level of the lymphoid precursor. In keeping with this hypothesis, TCR rearrangements in TCLs resembled the pattern seen in the human thymus showing biased usage of V and J segments of high combinatorial probability resulting in recurrent, public CDR3 sequences shared between unrelated patients and across different clinical TCL entities. Frequencies of malignant clonotypes followed Zipf-Mandelbrot scaling law suggesting that TCLs comprise an interconnected system of expanding tumor clones. The major limitation of this study is that it is based on the analysis of the TCR clonotypes and does not directly inform about developmental trajectories of cellular clones. Conclusions Lymphoid precursors are the likely cells of origin for mature T-cell lymphomas. Anaplastic large cell lymphoma seems to be derived from the most immature precursors with germline TCR whereas peripheral T-cell lymphoma and adult T-cell lymphoma/leukemia map to the later stages after TCR lower case Greek beta rearrangement stage. Clonotypically diverse initiating cells may seed target tissues being responsible for disease relapses after therapy.

scholarly journals Detection of aberrant clones in nearly all cases of angioimmunoblastic lymphadenopathy with dysproteinemia-type T-cell lymphoma by combined interphase and metaphase cytogenetics

Blood ◽  
1994 ◽  
Vol 84 (8) ◽  
pp. 2640-2648 ◽  
Author(s):  
B Schlegelberger ◽  
Y Zhang ◽  
K Weber-Matthiesen ◽  
W Grote
Keyword(s):  
T Cell ◽  
High Frequency ◽  
Cell Lymphoma ◽  
Single Cells ◽  
T Cell Lymphoma ◽  
Neoplastic Disease ◽  
Cell Of Origin ◽  
Interphase Cytogenetics ◽  
Angioimmunoblastic Lymphadenopathy ◽  
T Cell Lymphomas

Abstract Trisomy 3, trisomy 5, and an X additional chromosome are the most frequent chromosome aberrations in angioimmunoblastic lymphadenopathy with proteinemia (AILD)-type T-cell lymphomas. To evaluate the frequency of +3 and +X clones, fluorescence in situ hybridization studies with centromere-specific probes for chromosome 3 and X were done in 41 patients with peripheral T-cell lymphomas (PTL). With this interphase cytogenetic approach, 32 of 41 patients (78%) showed +3 clones, and 14 patients (34%) +X clones. These frequencies far exceeded those observed with metaphase cytogenetics (+3, 41%; +X, 20%). Summing up the results of metaphase and interphase cytogenetics, aberrant clones were found in 37 of 41 patients with PTL (90%) and 32 of 36 patients with AILD-type T-cell lymphoma (89%). Although AILD-type T- cell lymphoma is considered a neoplastic disease, it is an exception in that it shows a high frequency of cytogenetically unrelated clones and single cells that cannot be derived from a common cell of origin because of their completely different karyotypes. In five patients, double hybridization with centromere-specific probes for chromosomes 3 and X showed that these aberrations occurred in different cells. When the results of metaphase and interphase cytogenetics were combined, 17 of 36 patients with AILD-type T-cell lymphoma (47%) had unrelated clones. This high frequency of oligoclonal proliferations may be caused by increased genetic instability and an immune defect resulting in impaired elimination of aberrant cells.

scholarly journals Mutation of the CD28 Co-Stimulatory Domain Confers Enhanced CAR T Cell Function

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 248-248 ◽  
Author(s):  
Justin C. Boucher ◽  
Gongbo Li ◽  
Hiroshi Kotani ◽  
Maria Cabral ◽  
Dylan Morrissey ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Signaling ◽  
Research Funding ◽  
Chromatin Accessibility ◽  
Target Cells ◽  
T Cell Signaling ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

An obstacle with continued clinical development of CAR T cells is the limited understanding of their biology and mechanisms of anti-tumor immunity. We and others have shown that CARs with a CD28 co-stimulatory domain drive high levels of T cell activation that also lead to exhaustion and shortened persistence. The CD28 domain includes 3 intracellular subdomains (YMNM, PRRP, and PYAP) that regulate signaling pathways post TCR-stimulation, but it is unknown how they modulate activation and/or exhaustion of CAR T cells. A detailed understanding of the mechanism of CD28-dependent exhaustion in CAR T cells will allow the design of a CAR less prone to exhaustion and reduce relapse rates. This led us to hypothesize that by incorporating null mutations of CD28 subdomains (Fig 1A) we could optimize CAR T cell signaling and reduce exhaustion. In vitro, we found mutated CAR T cells with only a functional PYAP (mut06) subdomain secrete significantly less IFNγ, IL6, and TNFα after 24hr stimulation compared to non-mutated CD28 CAR T cells, but greater than the 1st generation m19z CAR. Also, cytotoxicity was enhanced compared to non-mutated CARs (Fig 1B). Using a pre-clinical immunocompetent mouse tumor model, we found the mut06 CAR T cell treated mice had a significant survival advantage compared to non-mutated CD28 CAR T cells (Fig 1C). To examine exhaustion, we ex vivo stimulated CAR T cells with target cells expressing CD19 and PDL1 and found mut06 CAR T cells had increased IFNγ (42%), TNFα (62%) and IL2 (73%) secretion compared to exhausted non-mutated CD28 CAR T cells. This suggests that mut06 CAR T cells are more resistant to exhaustion. To find a mechanistic explanation for this observation we examined CAR T cell signaling. After 24hr stimulation with CD19 target cells mut06 CAR T cells had a significant reduction in pAkt compared to m1928z CAR T cells, which is a critical signaling mediator in the NFAT and NR4A1 transcription factor pathways. Additionally, mut06 had decreased p-NFAT compared to m1928z when examined by western blot. To determine how optimized CAR signaling affected T cell exhaustion we looked at 22 genes that are upregulated when NFAT is constitutively active and overlap with genes identified as important for T cell exhaustion. We found that most of the exhaustion related genes were upregulated in m1928z CAR T cells while they were decreased in m19hBBz. The mut06 CAR T cell gene expression pattern was more similar to m19hBBz with exhaustion related genes downregulated compared to m1928z (Fig 1D). To examine differences in the accessibility of exhaustion related genes we performed ATAC-seq and found NFAT (Nfatc1) and NR4A2 (Nr4a2) had lower chromatin accessibility profiles in mut06 compared to m1928z (Fig 1E). We also found that exhaustion related genes Havcr2 (TIM3), Pdcd1 (PD1), and Lag3 (LAG3) all had greatly reduced chromatin accessibility in mut06 CAR T cells compared m1928z. Overall, these genomic studies support our findings that mut06 optimizes CAR T cell signaling by lowering transcription factors that regulate exhaustion. Figure 1 Disclosures Li: ImmuneBro Therapeutics: Other: sole shareholder . Davila:Atara: Research Funding; Celgene: Research Funding; GlaxoSmithKline: Consultancy; Novartis: Research Funding; Anixa: Consultancy; Bellicum: Consultancy; Adaptive: Consultancy; Precision Biosciences: Consultancy.

scholarly journals Clonotype pattern in T-cell lymphomas map the cell of origin to immature lymphoid precursors

2022 ◽  
Author(s):  
Aishwarya Iyer ◽  
Dylan Christopher Hennessey ◽  
Robert Gniadecki
Keyword(s):  
T Cells ◽  
T Cell ◽  
De Novo ◽  
Cell Lymphoma ◽  
Large Cell ◽  
Cell Receptor ◽  
Cell Of Origin ◽  
Sequencing Data ◽  
T Cell Lymphomas ◽  
Complementarity Determining Regions

Mature T-cell lymphomas (TCLs) are rare, clinically heterogeneous hematologic cancers of high medical need. TCLs have inferior prognosis which is attributed to poor understanding of their pathogenesis. Based on phenotypic similarities between normal and neoplastic lymphocytes it has been assumed that TCLs develop in the periphery, directly from various subtypes of normal T-cells. To address the debated question of the cell of origin in TCLs we analyzed to identify the highly variable complementarity determining regions (CDR3) regions of T-cell receptor (TCR) to trace the clonal history of the T-cells. We have collected previously published whole genome -exome, and -transcriptome sequencing data from 574 TCL patients. TCR clonotypes were identified by de novo assembly of CDR3 regions of TCR γ, β and α. We have found that the vast majority of TCLs are clonotypically oligoclonal, although the pattern oligoclonality varied. Anaplastic large cell lymphoma was most diverse comprising multiple clonotypes of TCRγ, β and α whereas adult T-cell lymphoma/leukemia and peripheral T-cell lymphomas often showed monoclonality for TCRγ and β but had diverse TCRα clonotypes. These patterns of rearrangements indicated that TCLs are initiated at the level of the lymphoid precursor. In keeping with this hypothesis, TCR rearrangements in TCLs resembled the pattern seen in the human thymus showing biased usage of V and J segments of high combinatorial probability resulting in recurrent, "public" CDR3 sequences shared across unrelated patients and different clinical TCL entities. Clonotypically diverse initiating cells may seed target tissues being responsible for disease relapses after therapy.

scholarly journals Detection of aberrant clones in nearly all cases of angioimmunoblastic lymphadenopathy with dysproteinemia-type T-cell lymphoma by combined interphase and metaphase cytogenetics

Blood ◽  
1994 ◽  
Vol 84 (8) ◽  
pp. 2640-2648 ◽  
Author(s):  
B Schlegelberger ◽  
Y Zhang ◽  
K Weber-Matthiesen ◽  
W Grote
Keyword(s):  
T Cell ◽  
High Frequency ◽  
Cell Lymphoma ◽  
Single Cells ◽  
T Cell Lymphoma ◽  
Neoplastic Disease ◽  
Cell Of Origin ◽  
Interphase Cytogenetics ◽  
Angioimmunoblastic Lymphadenopathy ◽  
T Cell Lymphomas

Trisomy 3, trisomy 5, and an X additional chromosome are the most frequent chromosome aberrations in angioimmunoblastic lymphadenopathy with proteinemia (AILD)-type T-cell lymphomas. To evaluate the frequency of +3 and +X clones, fluorescence in situ hybridization studies with centromere-specific probes for chromosome 3 and X were done in 41 patients with peripheral T-cell lymphomas (PTL). With this interphase cytogenetic approach, 32 of 41 patients (78%) showed +3 clones, and 14 patients (34%) +X clones. These frequencies far exceeded those observed with metaphase cytogenetics (+3, 41%; +X, 20%). Summing up the results of metaphase and interphase cytogenetics, aberrant clones were found in 37 of 41 patients with PTL (90%) and 32 of 36 patients with AILD-type T-cell lymphoma (89%). Although AILD-type T- cell lymphoma is considered a neoplastic disease, it is an exception in that it shows a high frequency of cytogenetically unrelated clones and single cells that cannot be derived from a common cell of origin because of their completely different karyotypes. In five patients, double hybridization with centromere-specific probes for chromosomes 3 and X showed that these aberrations occurred in different cells. When the results of metaphase and interphase cytogenetics were combined, 17 of 36 patients with AILD-type T-cell lymphoma (47%) had unrelated clones. This high frequency of oligoclonal proliferations may be caused by increased genetic instability and an immune defect resulting in impaired elimination of aberrant cells.

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