scholarly journals Single Cell Transcriptome Analysis Reveals Disease-Defining T Cell Subsets in the Tumor Microenvironment of Classic Hodgkin Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 547-547
Author(s):  
Tomohiro Aoki ◽  
Lauren C. Chong ◽  
Katsuyoshi Takata ◽  
Katy Milne ◽  
Monirath Hav ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Research Funding ◽  
Cell Clusters ◽  
Mhc Ii ◽  
Spatial Profiling ◽  
Classic Hodgkin Lymphoma ◽  
Cellular Components

INTRODUCTION: Classic Hodgkin lymphoma (cHL) is uniquely characterized by an extensively dominant microenvironment composed primarily of different types of non-cancerous immune cells with a rare population (~1%) of tumor cells. Detailed characterization of these cellular components and their spatial relationship is crucial to understand crosstalk and therapeutic targeting in the cellular ecosystem of the tumor microenvironment (TME). METHODS: In this study, we performed high dimensional and spatial profiling of immune cells in the TME of cHL. Single cell RNA sequencing (scRNA-seq) was performed with the 10x Genomics platform on cell suspensions collected from lymph nodes of 22 cHL patients, including 12 of nodular sclerosis subtype, 9 of mixed cellularity subtype and 1 of lymphocyte-rich subtype, with 5 reactive lymph nodes (RLNs) serving as normal controls. Illumina sequencing (HiSeq 2500) was performed to yield single-cell expression profiles for 127,786 cells. We also performed multicolor IHC and imaging mass cytometry (IMC) on TMA slides from the same patients. RESULTS: Unsupervised clustering using PhenoGraph identified 22 cell clusters including 12 T cell clusters, 7 B cell clusters and 1 macrophage cluster. While most immune cell populations were common between cHL and RLN, we observed an enrichment of cells from cHL in all 3 regulatory T cell (Treg) clusters. The most cHL-enriched cluster was characterized by high expression of LAG3, in addition to common Treg markers such as IL2RA (CD25) and TNFRSF18 (GITR), but lacked expression of FOXP3, consistent with a type 1 regulatory (Tr1) T cell population. LAG3+ T cells in cHL had high expression of immune-suppressive cytokines IL-10 and TGF-b . In vitro exposure of T cells to cHL cell line supernatant induced significantly higher levels of LAG3 in naïve T cells compared to co-culture with other lymphoma cell line supernatant or medium only. CD4+ LAG3+ T cells isolated by FACS also suppressed the proliferation of responder CD4+ T cells when co-cultured in vitro. Additionally, Luminex analysis revealed that cHL cell lines secrete substantial amounts of cytokines and chemokines that can promote Tr1 cell differentiation (e.g. IL-6). Our scRNA-seq analysis revealed that LAG3 expression was significantly higher in cHL cases with loss of major histocompatibility class II (MHC-II) expression on HRS cells as compared to MHC-II positive cases (P = 0.019), but was not correlated with EBV status or histological subtype. Strikingly, LAG3 was identified as the most up-regulated gene in cells from MHC-II negative cases compared to MHC-II positive cases. Topological analysis using multicolor IHC and IMC revealed that in MHC-II negative cases, HRS cells were surrounded by LAG3+ T cells. In these cases, the density of LAG3+ T cells in HRS cell-rich regions was significantly increased, and the average distance between an HRS cell and its closest LAG3+ T cell neighbor was significantly shorter. These associations were confirmed in an independent cohort of 166 cHL patients. Finally, we observed a trend towards an inferior disease-specific survival (DSS; P = 0.072) and overall survival (OS; P = 0.12) in cases with an increased number of LAG3+ T cells. A high proportion of LAG3+ T cells (> 20%) was identified as an independent prognostic factor for DSS by multivariate Cox regression. CONCLUSIONS: Our results reveal a diverse TME composition with inflammatory and immunosuppressive cellular components that are linked to MHC class II expression status on HRS cells (Figure). Unprecedented transcriptional and spatial profiling at the single cell level has established the pathogenic importance of HRS cell-induced CD4+ LAG3+ T cells as a mediator of immunosuppression in cHL, with potential implications for novel therapeutic approaches. Figure Disclosures Savage: Seattle Genetics, Inc.: Consultancy, Honoraria, Research Funding; BMS, Merck, Novartis, Verastem, Abbvie, Servier, and Seattle Genetics: Consultancy, Honoraria. Scott:Roche/Genentech: Research Funding; Celgene: Consultancy; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding. Steidl:Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Roche: Consultancy; Seattle Genetics: Consultancy; Bayer: Consultancy; Juno Therapeutics: Consultancy; Tioma: Research Funding.

scholarly journals Single-Cell Analysis of the Classical Hodgkin Lymphoma Immune Environment Reveals a Clonally-Expanded CD8+ T Cell Population with a Cytotoxic Phenotype

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 40-41
Author(s):  
Jovian Yu ◽  
Xiufen Chen ◽  
James Godfrey ◽  
Girish Venkataraman ◽  
Sonali M. Smith ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Immune Cell ◽  
Clonal Expansion ◽  
Advisory Committees ◽  
Cell Clusters

Introduction: Classical Hodgkin lymphoma (cHL) is characterized by a robust and complex immune cell infiltrate and the rare presence of malignant Hodgkin-Reed-Sternberg (HRS) cells. At the genetic level, HRS cells recurrently acquire alterations that lead to defective antigen presentation (β2 microglobulin mutations) and mediate T cell dysfunction (PD-L1 copy gains/amplifications) in order to subvert host immune surveillance. The clinical relevance of PD-L1 protein over-expression in cHL is clear, as response rates to PD-1 blockade therapy are extremely high among patients with relapsed/refractory (r/r) disease. Despite its remarkable efficacy, the cells that mediate response to anti-PD-1 therapy in cHL remain undefined. Recent analyses have highlighted a possible role for CD4+ T cells in mediating the clinical activity of anti-PD-1 therapy in cHL. CD4+ T cells significantly outnumber CD8+ T cells in cHL lesions and are more frequently juxtaposed to HRS cells in situ. Furthermore, HLA class II expression on HRS cells predicted higher complete response rates to PD-1 blockade therapy in r/r cHL patients. However, a candidate T cell population capable of specific reactivity to antigens expressed by HRS cells has yet to be identified. This information is critical as such T cells might be functionally reinvigorated to mediate HRS cell elimination following PD-1 blockade therapy. In order to address this key knowledge gap, we analyzed data at single cell (sc) resolution using paired RNA and T cell receptor (TCR) sequencing in 9 diagnostic cHL and 5 reactive lymph node (RLN) specimens. Methods: Sequencing was performed using the 10x Genomics Chromium Single Cell 5' Gene Expression and V(D)J workflows. B-cell depletion of each sample was achieved using CD19 microbeads and negative selection to enrich T cell populations. Reads were analyzed and aligned with CellRanger (v3.1.0) and Seurat (v3.2.0) was used to conduct clustering by a shared nearest neighbor (SNN) graph on scRNA data. TCR sequencing data was integrated using scRepertoire (v1.0.0). Results: A detailed map of the immune cell states in cHL was created using scRNA-seq (10X) data on 79,085 cells from 9 cHL (52,602 cells) and 5 RLN samples (26,484 cells) expressing a total of 21,421 genes (mean 5649 cells/sample; mean 2849 mRNA reads/cell). Dimensionality reduction and unsupervised graph-based clustering revealed 21 distinct cell type and activation state clusters, including T cells, NK cells, macrophages, and dendritic cells (Fig 1A-B). A cluster identifying HRS cells was not observed, consistent with a recently published report. Ten T cell clusters were identified (47,573 cells), including naive- and memory-like T cells, effector/cytotoxic CD8+ T cells, regulatory T cells, and T follicular helper cells. Unexpectedly, a putative exhausted T cell cluster was not clearly observed. The relative contributions of cHL and RLNs cases to these clusters are shown in Fig 1C. Paired TCR sequencing was available for 23,943 cells. Overall TCR diversity was lower among cHL samples compared to RLN specimens (Fig 1D). In cHL samples, modest clonal expansion within regulatory T cell and memory CD4+ T cell clusters was observed, but the most striking clonal expansion occurred among cells assigned to effector/cytotoxic CD8+ T cell clusters - a finding not observed in most RLN specimens (Fig 1E). Clonally-expanded effector/cytotoxic CD8+ T cells displayed high expression of granzymes (GZMA, GZMH, GZMK), cytokines (TNF, IFNG) and chemokines (CCL4/CCL5), and modest expression of exhaustion markers (PDCD1, ENTPD1, HAVCR2, ITGAE), contrasting with data from single-cell analyses of solid tumors. Clonal expansion of effector/cytotoxic CD8+ T cells was particularly robust in EBV-positive cHLs, likely due to recognition of viral-derived epitopes displayed on HRS cells (Fig 1F). Phenotypic and functional validation of key immune cell clusters in cHL specimens using spectral cytometry is underway and will be reported at the meeting. Conclusions: For the first time, our data have unveiled the nature of the T cell repertoire in cHL at single cell resolution. Our results reveal a recurrent pattern of clonal expansion within effector CD8+ cells, which may be the HRS antigen-specific T cells that mediate response to PD-1 blockade. This hypothesis requires confirmation through similar analyses of pre- and on-treatment biopsies of cHL patients receiving anti-PD-1 therapy. Disclosures Godfrey: Gilead: Research Funding; Merck: Research Funding; Verastem: Research Funding. Venkataraman:EUSA Pharma: Speakers Bureau. Smith:Janssen: Consultancy; BMS: Consultancy; TG Therapeutics: Consultancy, Research Funding; Genentech/Roche: Consultancy, Other: Support of parent study and funding of editorial support, Research Funding; Karyopharm: Consultancy, Research Funding; FortySeven: Research Funding; Pharmacyclics: Research Funding; Acerta: Research Funding; Celgene: Consultancy, Research Funding. Kline:Kite/Gilead: Speakers Bureau; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Aberrant Cathepsin S Induces a Supportive Immune Microenvironment in Follicular Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 657-657
Author(s):  
Johannes Adrian Hildebrand ◽  
Deepak Bararia ◽  
Sebastian Stolz ◽  
Sarah Haebe ◽  
Stefan Alig ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Clinical Course ◽  
Cd4 T Cell ◽  
Immune Microenvironment ◽  
Mhc Ii ◽  
Autocatalytic Cleavage

The highly variable clinical course of follicular lymphoma (FL) is determined by the molecular heterogeneity of the tumor cells and complex interactions with the microenvironment. Here, we provide biochemical, structural, functional and clinical evidence that aberrant Cathepsin S (CTSS) activity induces a supportive immune microenvironment in FL. By targeted DNA sequencing of 305 diagnostic FL biopsies, we identified somatic mutations of CTSS in 8% of cases (24/305), mostly clustered at Y132 (19/24) converting Y to D (16/19). A subset of CTSS Y132 mutations (N=5) occurred at lower variant allele frequencies (5-10%), indicating subclonality. Another 13% of FL had CTSS amplifications (37/286). CTSS Y132 mutations and CTSS amplifications were mutually exclusive. In a cohort of 51 FL, CTSS amplifications were associated with higher CTSS expression (P=0.05). Of note, a subset of FL without CTSS amplifications also had higher CTSS expression, suggesting additional mechanisms of transcriptional dysregulation. CTSS is a cysteine protease that is highly expressed in endolysosomes of antigen presenting cells and malignant B-cells. CTSS is involved in proteolytical processing of antigenic peptides for presentation on MHC-II to be recognized by antigen specific CD4+ T-cells. CTSS is synthesized as an inactive zymogen, which is converted to its active form by autocatalytic cleavage of the autoinhibitory propeptide (pro-CTSS). We used CRISPR/Cas9 to introduce CTSS Y132D into Karpas422, a B-cell lymphoma cell line that harbors the FL hallmark translocation t(14;18). Single-cell derived Y132D mutant clones showed >3-fold higher ratios of active CTSS to pro-CTSS (N=4, P=0.0003). Immunoprecipitated CTSS Y132D had >3-fold higher in vitro substrate cleavage activity compared to CTSS wild type (WT) (N=6, P=0.001). We purified pro-CTSS WT and Y132D and assayed their in vitro autocatalytic cleavage over time. The time required to convert 50% of pro-CTSS decreased from 17 minutes for WT to 11 minutes for Y132D (N=3, P=0.04). In contrast, purified active CTSS WT and Y132 had similar in vitro cleavage activities. Molecular dynamics simulations showed that the Y132D mutation shortens the distances by ~2Å between the catalytic triad of active CTSS (C139, H278, N298) and a stretch of amino acids from the proform (L80, G81, D82, S94), which may facilitate intramolecular cleavage. By mass spectrometry we could indeed detect novel intermediate-sized CTSS fragments. Thus, Y132D does not increase the activity of the mature enzyme but is a gain-of-function mutation by accelerating the conversion from pro-CTSS to catalytically active CTSS. CD74 (invariant chain) is a physiologic CTSS substrate that plays critical roles in the assembly, trafficking and stabilization of peptide-free MHC-II. CTSS cleaves CD74, thereby allowing binding and presentation of antigens on MHC-II to antigen specific CD4+ T-cells. We could show that CTSS Y132D enhanced CD74 cleavage in Karpas422 cells. We then tested the impact of CTSS on antigen specific CD4+ T-cell activation in co-culture assays. CTSS knock-out lymphoma cells were broadly incapable of activating CD4+ T-cells. Overexpression of CTSS WT activated CD4+ T-cells more efficiently compared to empty vector control. CTSS Y132 had the highest capacity to stimulate antigen specific CD4+ T-cell responses. Furthermore, in primary FL biopsies (N=51) CTSS Y132 mutations had gene expression profiles linked with antigen-processing and chemokine perturbation, including CXCL13, a B-cell chemoattractant produced by activated CD4+ T-follicular helper cells. Lastly, we aimed to correlate CTSS aberrations with clinical outcome in patients who received standard immunochemotherapy (R-CHOP) for advanced FL (N=51 with available CTSS mutation and gene expression data). Compared to all other patients (N=34), patients with CTSS Y132 mutations or CTSS overexpression (N=17) had longer failure free survival (P=0.012) and overall survival (P=0.041). In summary, we propose that aberrant CTSS activity - even if only present in a FL subclone - can elicit a CD4+ T-cell driven tumor-promoting immune response, which could be amplified within the microenvironment via pro-inflammatory and chemotactic cytokines and substantially impact the biology and clinical course of the disease. Thus, aberrant CTSS activity is a promising biomarker and therapeutic target in FL and potentially also other tumors. Disclosures Klapper: Roche, Takeda, Amgen, Regeneron: Honoraria, Research Funding. Hiddemann:Bayer: Research Funding; Roche: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Vector Therapeutics: Consultancy, Honoraria. Steidl:Juno Therapeutics: Consultancy; Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Roche: Consultancy; Tioma: Research Funding; Seattle Genetics: Consultancy; Bayer: Consultancy. Kridel:Gilead Sciences: Research Funding. Weinstock:Celgene: Research Funding; Verastem Oncology: Research Funding. Weigert:Novartis: Research Funding; Roche: Research Funding.

Single Cell Profiling Reveals Unique CXCL13 Positive T Cell Subsets in the Tumor Microenvironment of Lymphocyte Rich Classic Hodgkin Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-33
Author(s):  
Tomohiro Aoki ◽  
Lauren C. Chong ◽  
Katsuyoshi Takata ◽  
Katy Milne ◽  
Elizabeth Chavez ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Single Cell ◽  
Immune Cells ◽  
Research Funding ◽  
Immune Cell ◽  
Expression Patterns ◽  
The Other ◽  
Tfh Cells

Introduction: Classic Hodgkin lymphoma (CHL) features a unique crosstalk between malignant cells and different types of normal immune cells in the tumor-microenvironment (TME). On the basis of histomorphologic and immunophenotypic features of the malignant Hodgkin and Reed-Sternberg (HRS) cells and infiltrating immune cells, four histological subtypes of CHL are recognized: Nodular sclerosing (NS), Mixed cellularity, Lymphocyte-rich (LR) and Lymphocyte-depleted CHL. Recently, our group described the high abundance of various types of immunosuppressive CD4+ T cells including LAG3+ and/or CTLA4+ cells in the TME of CHL using single cell RNA sequencing (scRNAseq). However, the TME of LR-CHL has not been well characterized due to the rarity of the disease. In this study, we aimed at characterizing the immune cell profile of LR-CHL at single cell resolution. METHODS: We performed scRNAseq on cell suspensions collected from lymph nodes of 28 primary CHL patients, including 11 NS, 9 MC and 8 LR samples, with 5 reactive lymph nodes (RLN) serving as normal controls. We merged the expression data from all cells (CHL and RLN) and performed batch correction and normalization. We also performed single- and multi-color immunohistochemistry (IHC) on tissue microarray (TMA) slides from the same patients. In addition, an independent validation cohort of 31 pre-treatment LR-CHL samples assembled on a TMA, were also evaluated by IHC. Results: A total of 23 phenotypic cell clusters were identified using unsupervised clustering (PhenoGraph). We assigned each cluster to a cell type based on the expression of genes described in published transcriptome data of sorted immune cells and known canonical markers. While most immune cell phenotypes were present in all pathological subtypes, we observed a lower abundance of regulatory T cells (Tregs) in LR-CHL in comparison to the other CHL subtypes. Conversely, we found that B cells were enriched in LR-CHL when compared to the other subtypes and specifically, all four naïve B-cell clusters were quantitatively dominated by cells derived from the LR-CHL samples. T follicular helper (TFH) cells support antibody response and differentiation of B cells. Our data show the preferential enrichment of TFH in LR-CHL as compared to other CHL subtypes, but TFH cells were still less frequent compared to RLN. Of note, Chemokine C-X-C motif ligand 13 (CXCL13) was identified as the most up-regulated gene in LR compared to RLN. CXCL13, which is a ligand of C-X-C motif receptor 5 (CXCR5) is well known as a B-cell attractant via the CXCR5-CXCL13 axis. Analyzing co-expression patterns on the single cell level revealed that the majority of CXCL13+ T cells co-expressed PD-1 and ICOS, which is known as a universal TFH marker, but co-expression of CXCR5, another common TFH marker, was variable. Notably, classical TFH cells co-expressing CXCR5 and PD-1 were significantly enriched in RLN, whereas PD-1+ CXCL13+ CXCR5- CD4+ T cells were significantly enriched in LR-CHL. These co-expression patterns were validated using flow cytometry. Moreover, the expression of CXCR5 on naïve B cells in the TME was increased in LR-CHL compared to the other CHL subtypes We next sought to understand the spatial relationship between CXCL13+ T cells and malignant HRS cells. IHC of all cases revealed that CXCL13+ T cells were significantly enriched in the LR-CHL TME compared to other subtypes of CHL, and 46% of the LR-CHL cases showed CXCL13+ T cell rosettes closely surrounding HRS cells. Since PD-1+ T cell rosettes are known as a specific feature of LR-CHL, we confirmed co-expression of PD-1 in the rosetting cells by IHC in these cases. Conclusions: Our results reveal a unique TME composition in LR-CHL. LR-CHL seems to be distinctly characterized among the CHL subtypes by enrichment of CXCR5+ naïve B cells and CD4+ CXCL13+ PD-1+ T cells, indicating the importance of the CXCR5-CXCL13 axis in the pathogenesis of LR-CHL. Figure Disclosures Savage: BeiGene: Other: Steering Committee; Merck, BMS, Seattle Genetics, Gilead, AstraZeneca, AbbVie: Honoraria; Roche (institutional): Research Funding; Merck, BMS, Seattle Genetics, Gilead, AstraZeneca, AbbVie, Servier: Consultancy. Scott:Janssen: Consultancy, Research Funding; Celgene: Consultancy; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoString, Research Funding; NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; Roche/Genentech: Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy. Steidl:AbbVie: Consultancy; Roche: Consultancy; Curis Inc: Consultancy; Juno Therapeutics: Consultancy; Bayer: Consultancy; Seattle Genetics: Consultancy; Bristol-Myers Squibb: Research Funding.

scholarly journals Pre-Existing T Cell Subsets Determine Anti-PD1 Blockade Response in Richter's Transformation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 42-43
Author(s):  
Prajish Iyer ◽  
Lu Yang ◽  
Zhi-Zhang Yang ◽  
Charla R. Secreto ◽  
Sutapa Sinha ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Research Funding ◽  
Gene Signature ◽  
T Cell Subsets ◽  
Rna Seq ◽  
Advisory Committees

Despite recent developments in the therapy of chronic lymphocytic leukemia (CLL), Richter's transformation (RT), an aggressive lymphoma, remains a clinical challenge. Immune checkpoint inhibitor (ICI) therapy has shown promise in selective lymphoma types, however, only 30-40% RT patients respond to anti-PD1 pembrolizumab; while the underlying CLL failed to respond and 10% CLL patients progress rapidly within 2 months of treatment. Studies indicate pre-existing T cells in tumor biopsies are associated with a greater anti-PD1 response, hence we hypothesized that pre-existing T cell subset characteristics and regulation in anti-PD1 responders differed from those who progressed in CLL. We used mass cytometry (CyTOF) to analyze T cell subsets isolated from peripheral blood mononuclear cells (PBMCs) from 19 patients with who received pembrolizumab as a single agent. PBMCs were obtained baseline(pre-therapy) and within 3 months of therapy initiation. Among this cohort, 3 patients had complete or partial response (responders), 2 patients had rapid disease progression (progressors) (Fig. A), and 14 had stable disease (non-responders) within the first 3 months of therapy. CyTOF analysis revealed that Treg subsets in responders as compared with progressors or non-responders (MFI -55 vs.30, p=0.001) at both baseline and post-therapy were increased (Fig. B). This quantitative analysis indicated an existing difference in Tregs and distinct molecular dynamic changes in response to pembrolizumab between responders and progressors. To delineate the T cell characteristics in progressors and responders, we performed single-cell RNA-seq (SC-RNA-seq; 10X Genomics platform) using T (CD3+) cells enriched from PBMCs derived from three patients (1 responder: RS2; 2 progressors: CLL14, CLL17) before and after treatment. A total of ~10000 cells were captured and an average of 1215 genes was detected per cell. Using a clustering approach (Seurat V3.1.5), we identified 7 T cell clusters based on transcriptional signature (Fig.C). Responders had a larger fraction of Tregs (Cluster 5) as compared with progressors (p=0.03, Fig. D), and these Tregs showed an IFN-related gene signature (Fig. E). To determine any changes in the cellular circuitry in Tregs between responders and progressors, we used FOXP3, CD25, and CD127 as markers for Tregs in our SC-RNA-seq data. We saw a greater expression of FOXP3, CD25, CD127, in RS2 in comparison to CLL17 and CLL14. Gene set enrichment analysis (GSEA) revealed the upregulation of genes involved in lymphocyte activation and FOXP3-regulated Treg development-related pathways in the responder's Tregs (Fig.F). Together, the greater expression of genes involved in Treg activation may reduce the suppressive functions of Tregs, which led to the response to anti-PD1 treatment seen in RS2 consistent with Tregs in melanoma. To delineate any state changes in T cells between progressors and responder, we performed trajectory analysis using Monocle (R package tool) and identified enrichment of MYC/TNF/IFNG gene signature in state 1 and an effector T signature in state 3 For RS2 after treatment (p=0.003), indicating pembrolizumab induced proliferative and functional T cell signatures in the responder only. Further, our single-cell results were supported by the T cell receptor (TCR beta) repertoire analysis (Adaptive Biotechnology). As an inverse measure of TCR diversity, productive TCR clonality in CLL14 and CLL17 samples was 0.638 and 0.408 at baseline, respectively. Fifty percent of all peripheral blood T cells were represented by one large TCR clone in CLL14(progressor) suggesting tumor related T-cell clone expansion. In contrast, RS2(responder) contained a profile of diverse T cell clones with a clonality of 0.027 (Fig. H). Pembrolizumab therapy did not change the clonality of the three patients during the treatment course (data not shown). In summary, we identified enriched Treg signatures delineating responders from progressors on pembrolizumab treatment, paradoxical to the current understanding of T cell subsets in solid tumors. However, these data are consistent with the recent observation that the presence of Tregs suggests a better prognosis in Hodgkin lymphoma, Follicular lymphoma, and other hematological malignancies. Figure 1 Disclosures Kay: Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding; Sunesis: Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; MEI Pharma: Research Funding. Ansell:AI Therapeutics: Research Funding; Takeda: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Bristol Myers Squibb: Research Funding; Regeneron: Research Funding; Seattle Genetics: Research Funding; ADC Therapeutics: Research Funding. Ding:Astra Zeneca: Research Funding; Abbvie: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; DTRM: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: pembrolizumab

scholarly journals Characterizing T Cells in SCID Patients Presenting with Reactive or Residual T Lymphocytes

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Atar Lev ◽  
Amos J. Simon ◽  
Luba Trakhtenbrot ◽  
Itamar Goldstein ◽  
Meital Nagar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cell Function ◽  
Severe Combined Immunodeficiency ◽  
Cell Activity ◽  
Mhc Ii ◽  
Cell Functions ◽  
Circulating T Cells

Introduction. Patients with severe combined immunodeficiency (SCID) may present with residual circulating T cells. While all cells are functionally deficient, resulting in high susceptibility to infections, only some of these cells are causing autoimmune symptoms.Methods. Here we compared T-cell functions including the number of circulating CD3+T cells,in vitroresponses to mitogens, T-cell receptor (TCR) repertoire, TCR excision circles (TREC) levels, and regulatory T cells (Tregs) enumeration in several immunodeficinecy subtypes, clinically presenting with nonreactive residual cells (MHC-II deficiency) or reactive cells. The latter includes patients with autoreactive clonal expanded T cell and patients with alloreactive transplacentally maternal T cells.Results. MHC-II deficient patients had slightly reduced T-cell function, normal TRECs, TCR repertoires, and normal Tregs enumeration. In contrast, patients with reactive T cells exhibited poor T-cell differentiation and activity. While the autoreactive cells displayed significantly reduced Tregs numbers, the alloreactive transplacentally acquired maternal lymphocytes had high functional Tregs.Conclusion. SCID patients presenting with circulating T cells show different patterns of T-cell activity and regulatory T cells enumeration that dictates the immunodeficient and autoimmune manifestations. We suggest that a high-tolerance capacity of the alloreactive transplacentally acquired maternal lymphocytes represents a toleration advantage, yet still associated with severe immunodeficiency.

scholarly journals Single cell multiomic analysis of T cell exhaustion in vitro

2019 ◽  
Author(s):  
Mirko Corselli ◽  
Suraj Saksena ◽  
Margaret Nakamoto ◽  
Woodrow E. Lomas ◽  
Ian Taylor ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
T Cell Activation ◽  
T Cell Exhaustion ◽  
Chronic Stimulation ◽  
Cell Exhaustion ◽  
Car T ◽  
The Impact

AbstractA key step in the clinical production of CAR-T cells is the expansion of engineered T cells. To generate enough cells for a therapeutic product, cells must be chronically stimulated, which raises the risk of inducing T-cell exhaustion and reducing therapeutic efficacy. As protocols for T-cell expansion are being developed to optimize CAR T cell yield, function and persistence, fundamental questions about the impact of in vitro manipulation on T-cell identity are important to answer. Namely: 1) what types of cells are generated during chronic stimulation? 2) how many unique cell states can be defined during chronic stimulation? We sought to answer these fundamental questions by performing single-cell multiomic analysis to simultaneously measure expression of 39 proteins and 399 genes in human T cells expanded in vitro. This approach allowed us to study – with unprecedented depth - how T cells change over the course of chronic stimulation. Comprehensive immunophenotypic and transcriptomic analysis at day 0 enabled a refined characterization of T-cell maturational states (from naïve to TEMRA cells) and the identification of a donor-specific subset of terminally differentiated T-cells that would have been otherwise overlooked using canonical cell classification schema. As expected, T-cell activation induced downregulation of naïve-associated markers and upregulation of effector molecules, proliferation regulators, co-inhibitory and co-stimulatory receptors. Our deep kinetic analysis further revealed clusters of proteins and genes identifying unique states of activation defined by markers temporarily expressed upon 3 days of stimulation (PD-1, CD69, LTA), markers constitutively expressed throughout chronic activation (CD25, GITR, LGALS1), and markers uniquely up-regulated upon 14 days of stimulation (CD39, ENTPD1, TNFDF10). Notably, different ratios of cells expressing activation or exhaustion markers were measured at each time point. These data indicate high heterogeneity and plasticity of chronically stimulated T cells in response to different kinetics of activation. In this study, we demonstrate the power of a single-cell multiomic approach to comprehensively characterize T cells and to precisely monitor changes in differentiation, activation and exhaustion signatures in response to different activation protocols.

scholarly journals Ectonucleosidase CD39 Is Highly Expressed on ATLL Cells and Suppresses the Immune Response through the Adenosine Pathway

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3720-3720
Author(s):  
Yasuhiro Nagate ◽  
Sachiko Ezoe ◽  
Jiro Fujita ◽  
Takafumi Yokota ◽  
Michiko Ichii ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Tumor Cells ◽  
Research Funding ◽  
Poly I:C ◽  
Acute Type ◽  
Atll Cell

Abstract Background: Adult T-cell leukemia/lymphoma (ATLL) is a mature T-cell neoplasm, linked to the human T-cell lymphotropic virus, HTLV-1. Patients with ATLL are often at the risk of opportunistic infections. Some studies suggested that ATLL cells originate from HTLV-1-infected regulatory T cells (Tregs). It could be possible that this immunocompromised state is caused by the function of ATLL cells having similar phenotypes with Tregs. In this study, we examined the expression of immunosuppressive molecules associated with Tregs in ATLL cells, and analyzed their roles in the function of ATLL cells. Methods: The protocol of this study was approved by the Investigational Review Board of Osaka University Hospital. Peripheral blood mononuclear cells (PBMCs) were collected from 10 asymptomatic HTLV-1 carriers and 22 ATLL patients (1 with smoldering type, 5 with chronic type, 2 with lymphoma type, and 14 with acute type) after getting informed consent. PBMCs from 3 ATLL patients were separated into CD4+ CD7- CADM1+ATLL cells and adjacent CD4+CD7+ CADM1-normal T cells using Fluorescence-activated Cell Sorter (FACS), and cells in each fraction were subjected to total RNA sequencing experiments. Based on the results, we examined the expression patterns of CD39 and CD73 in HTLV-1 carriers or each type of ATLL patients, and also analyzed the immune functions of these molecules in ATLL tumor cells. Results: We compared whole transcriptome of ATLL cells and normal CD4+cells. Bioinformatic analyses showed that many genes associated with immunosuppressive functions were elevated or downregulated in ATLL cells. Among these genes we focused on CD39, CD73 and CD26, because they have recently been reported to be strongly associated with the functions of Tregs. CD39, expressed on normal Tregs, and extrinsic CD73 have immunosuppressive potential by catalyzing adenosine from extracellular ATP, and CD26 has opposite potential by resolving adenosine, which have a strong anti-inflammatory function and plays major role in Treg-mediated immunosuppression. We found that all of 4 ATLL cell lines (MJ, MT1, MT2, MT4) expressed CD39, but not CD73 just as human effector Tregs. Tumor cells from 12 acute ATLL patients (86%) and 2 chronic ATLL patients (40%) expressed CD39, but the expressions of CD73 were various. Also in asymptomatic carriers, we could detect CD39 and/or CD73 positive in CD7- CADM1+ abnormal fraction of CD4+cells. On the other hand, CD26, normally expressed on human CD4+Th cells other than effector Tregs, was negative in ATLL cell lines and primary ATLL cells except for cells in abnormal fraction of one asymptomatic carrier. CD39 negative cases in chronic/smoldering type tended to show slower disease progression after the blood collection. Next, the role of CD39 and/or CD73 in ATLL cells was assessed in vitro and in vivo. As expected, CD39+ ATLL cells converted significantly more extracellular ATP than CD39- ATLL cells, and mass spectrometry analysis of AMP/adenosine concentration identified the AMPase activity of CD73+ ATLL cells. Furthermore, we established CD39 knockout (KO) cells from ATL cell-line MJ using CRISPR/Cas9 system, and performed in vitro suppression assays for assessment of immunosuppressive function. Although wild type MJ suppressed the growth of normal CD4+ and CD8+ T cells, KO MJ did little. Next, we analyzed the role of CD39 in the progression of tumor cells in vivo. We transplanted mouse T-cell lymphoma cell-line EG7-OVA artificially expressing CD39 or mock into mice subcutaneously. The coinjection of immunoadjuvant poly(I:C) significantly suppressed the tumor growth of mock cells, but the tumor sizes of CD39 expressing cells were almost the same as those of mock cells without poly(I:C) injection (Figure). Conclusion: In this study, we reported that most of ATLL cells in acute type patients express CD39+ CD26- just as Tregs, and that CD39- KO of ATLL cell line cancelled its immunosuppressive effects, and forcibly expressed CD39 on tumor cells rejected the anti-tumor immunity in vivo. From these data, we clarified the pathological mechanism of immunosuppressive function in ATLL cells, and also showed that CD39 expression could be used as a prognostic clue and be a new therapeutic target of ATLL. Disclosures Ezoe: TAIHO Phamaceutical Co., Ltd.: Research Funding. Yokota:Celgene: Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer Inc.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; MSD K.K.: Research Funding. Ichii:Novartis Pharma K.K.: Speakers Bureau; Kowa Pharmaceutical Co.,LTD.: Speakers Bureau; Celgene K.K.: Speakers Bureau. Shibayama:Novartis Pharma K.K.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Fujimoto Pharmaceutical: Honoraria, Research Funding; Jansen Pharmaceutical K.K: Honoraria; Ono Pharmaceutical Co.,LTD: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Bristol-Meyer Squibb K.K: Honoraria, Research Funding. Oritani:Novartis Pharma: Speakers Bureau. Kanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.

scholarly journals Targeting the Membrane-Proximal C2-Set Domain of CD33 for Improved CD33-Directed CAR T Cell Therapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2776-2776
Author(s):  
Salvatore Fiorenza ◽  
George S. Laszlo ◽  
Tinh-Doan Phi ◽  
Margaret C. Lunn ◽  
Delaney R. Kirchmeier ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Set Domain ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Privately Held

Abstract Background: There is increasing interest in targeting CD33 in malignant and non-malignant disorders, but available drugs are ineffective in many patients. As one limitation, therapeutic CD33 antibodies typically recognize the membrane-distal V-set domain. Likewise, currently tested CD33-directed chimeric antigen receptor (CAR) T cells likewise target the V-set domain and have thus far shown limited clinical activity. We have recently demonstrated that binding closer to the cell membrane enhances the effector functions of CD33 antibodies. We therefore raised antibodies against the membrane-proximal C2-set domain of CD33 and identified antibodies that bound CD33 regardless of the presence/absence of the V-set domain ("CD33 PAN antibodies"). Here, we tested their properties as targeting moiety in CD33 PAN CAR T cell constructs, using a clinically validated lentiviral backbone. Methods: To generate CAR T cells, negatively selected CD8 + T cells were transduced with an epHIV7 lentivirus encoding the scFv from a CD33 PAN antibody (clone 1H7 or 9G2) linked to either a short (IgG 4 hinge only), intermediate (hinge plus IgG 4 CH3 domain), or long (hinge plus IgG 4 CH3 domain plus IgG 4 CH2 domain) spacer, the CD28-transmembrane domain, CD3zeta and 4-1BB intracellular signaling domains, and non-functional truncated CD19 (tCD19) as transduction marker. Similar constructs using scFvs from 2 different V-set domain-targeting CD33 antibodies, including hP67.6 (My96; used in gemtuzumab ozogamicin), were generated for comparison. CAR-T cells were sorted, expanded in IL-7 and IL-15, and used in vitro or in vivo against human AML cell lines endogenously expressing CD33 and cell lines engineered to lack CD33 (via CRISPR/Cas9) with/or without forced expression of different CD33 variants. Results: CD33 V-set-directed CAR T cells exerted significantly more cytolytic activity against AML cells expressing an artificial CD33 variant lacking the C2-set domain (CD33 ΔE3-4) than cells expressing full-length CD33 at similar or higher levels, consistent with the notion that CD33 CAR T cell efficacy is enhanced when targeting an epitope that is located closer to the cell membrane. CD33 PAN CAR T cells were highly potent against human AML cells in a strictly CD33-dependent fashion, with constructs containing the short and intermediate-length spacer demonstrating robust cytokine secretion, cell proliferation, and in vitro cytolytic activity, as determined by 51Cr release cytotoxicity assays. When compared to optimized CD33 V-set CAR T cells, optimized CD33 PAN CAR T cells were significantly more potent in cytotoxicity, proliferation, and cytokine production without appreciably increased acquisition of exhaustion markers. In vivo, CD33 PAN CAR T cells extended survival in immunodeficient NOD.SCID. IL2rg -/- (NSG) mice bearing significant leukemic burdens from various cell line-derived xenografts (HL-60, KG1α and MOLM14) with efficient tumor clearance demonstrated in a dose-dependent fashion. Conclusion: Targeting the membrane proximal domain of CD33 enhances the anti-leukemic potency of CAR T cells. Our data provide the rationale for the further development of CD33 PAN CAR T cells toward clinical testing. Disclosures Fiorenza: Link Immunotherapeutics: Consultancy; Bristol Myers Squibb: Research Funding. Godwin: Pfizer: Research Funding; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Turtle: Allogene: Consultancy; Amgen: Consultancy; Arsenal Bio: Consultancy; Asher bio: Consultancy; Astrazeneca: Consultancy, Research Funding; Caribou Biosciences: Consultancy, Current holder of individual stocks in a privately-held company; Century Therapeutics: Consultancy, Other; Eureka therapeutics: Current holder of individual stocks in a privately-held company, Other; Juno therapeutics/BMS: Patents & Royalties, Research Funding; Myeloid Therapeutics: Current holder of individual stocks in a privately-held company, Other; Nektar therapeutics: Consultancy, Research Funding; PACT Pharma: Consultancy; Precision Biosciences: Current holder of individual stocks in a privately-held company, Other; T-CURX: Other; TCR2 Therapeutics: Research Funding. Walter: Kite: Consultancy; Janssen: Consultancy; Genentech: Consultancy; BMS: Consultancy; Astellas: Consultancy; Agios: Consultancy; Amphivena: Consultancy, Other: ownership interests; Selvita: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Research Funding; Macrogenics: Consultancy, Research Funding; Immunogen: Research Funding; Celgene: Consultancy, Research Funding; Aptevo: Consultancy, Research Funding; Amgen: Research Funding.

scholarly journals Differential Activation of V γ4V δ1 and V γ9V δ2 Cord Blood Derived Gamma Delta T Cells upon Exposure to EBV-Lcl and K562 Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2790-2790
Author(s):  
Jeremy Wee Kiat Ng ◽  
Joey Lai ◽  
Tony Kiat Hon Lim ◽  
William YK Hwang ◽  
Shang Li ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Single Cell ◽  
Γδ T Cells ◽  
Rapid Expansion ◽  
Leukemia Cell Line ◽  
Γδ T Cell ◽  
Gamma Delta

Abstract Gamma-delta (γδ) T cells have emerged as a promising candidate for adoptive cellular immunotherapy. To harness and maximize the anti-leukemia properties of these cells, we sort to comprehensively profile the transcriptomic signatures and immune repertoire of in vitro expanded γδ T cell products. Given the reported diverse TCR γδ repertoire and naïve nature of γδ T cells found in human cord blood (CB γδ), we serially track the molecular and cellular changes in these cells upon activation in expansion cultures. Based on the established viral reactivities of γδ T cell as well as prior studies showing their cross reactivities against leukemia and cancer cells, we had previously shown that stimulating CB γδ with an irradiated EBV-LCL feeder cell-based rapid expansion protocol (REP) is capable of generating cell products with potent and specific cytotoxicity against human AML cells. In the present study, using single cell RNA sequencing (scRNA-seq) coupled with single cell TCR γδ repertoire analysis, we compared the transcription signatures between our REP expanded γδ T cell (REP γδ) and non-manipulated γδ T cells reported in literatures, showing the progressive acquisition of an adult PB derived γδ T cell (PB γδ)-like cell states. Time course analysis demonstrated complex T cell activation and maturation trajectories correlating with variable level of clonal induction throughout the course of in vitro expansion. At the end of expansion, the harvested REP γδ are predominantly of the V γ4V δ1 subtype. Nevertheless, upon exposing REP γδ to target leukemia cell line K562, outgrowth of other non-V γ4V δ1 as well as the semi-invariant V γ9V δ2 cells were observed. Taken together, our data shows that as CB γδ expand and differentiate in culture, they adopt an adult PB γδ-like program. More importantly, our data highlights the rich clonal composition of in vitro expanded CB γδ, with different clonotypes being variably activated upon exposure to different stimuli. Such characteristics can potentially overcome the challenges of cancer heterogeneity and cell persistence, with the potential of improving outcomes in cell immunotherapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals In Vitro Generation of Human Pluripotent Stem Cell-Derived T Cells for Immunotherapy

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 691-691
Author(s):  
Amélie Montel-Hagen ◽  
Christopher S. Seet ◽  
Suwen Li ◽  
Brent Chick ◽  
Patrick Chang ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Pluripotent Stem Cells ◽  
Research Funding ◽  
Cytokine Release ◽  
Highly Efficient ◽  
Car T ◽  
Engineered T Cells

Abstract Adoptive cell therapy using T cells engineered to express antigen-specific T cell receptors (TCR-T) or chimeric antigen receptors (CAR-T) offer targeted and potentially curative treatments for malignancy. Current approaches rely on the genetic modification and expansion of mature circulating T-cells. Such processes are limited to autologous T cells due to the risk of graft-versus-host (GvHD) disease from allogeneic T cells through endogenous TCR expression as well as rejection through MHC incompatibility. Furthermore, prolonged ex-vivo expansion of T cells may reduce in vivo efficacy and harvesting sufficient T cells from lymphopenic patients is challenging. Direct in vitro differentiation of engineered T cells from human pluripotent stem cells (HSPCs) may overcome these problems by providing an unlimited source of cells that can be genetically edited, permitting the suppression of endogenous TCR expression through allelic exclusion, and the de novo generation of naïve antigen-specific T cells. We have developed an in vitro Artificial Thymic Organoid (ATO) system that induces highly efficient and reproducible production of mature naïve T cells from human hematopoietic stem cells and progenitor cells (HSPC). Here, we report the preclinical development of a modified ATO system that supports highly efficient in vitro differentiation and positive selection of naive human T cells from at least 5 different lines of human pluripotent stem cells (PSC), including Embryonic stem cells (ESC) and induced Pluripotent Stem Cells (iPSC). T cell differentiation from PSC was very similar phenotypically to that from HSPC. As in normal human thymopoiesis, the first evidence for T cell commitment was expression of CD7 and CD5, followed by the CD3-CD8lo "ISP8" stage, then CD4+CD8+ "DP" stage and finally production of CD3+CD8+CD4- "CD8SP" and Cd3+CD4+CD8- "CD4SP". As is typical with both monolayer cultures and ATOs (and opposite to normal thymus), CD8SP predominated over CD4SP. Surprisingly, differentiation occurred more rapidly from PSC than with HSPC. As with HSPC-ATOs, CD8SP from PSC ATOs showed a mature naïve conventional T cell phenotype i.e. CD3+TCRab+CD4- CD45RA+CD62L+CD27+ and exhibited a diverse, thymic-like TCR repertoire, and robust TCR-dependent cytokine release and proliferation. The differentiation in ATOs of an ESC line that expresses an HLA-A*02:01-restricted αβ TCR specific for NY-ESO-1 resulted in a markedly increased cell yield with an enhanced generation of naïve CD3+TCRαβ+CD8αβ+ conventional T cells, the majority of which were antigen-specific by tetramer staining. TCR-engineered T cells produced from PSC in ATOs displayed a near complete lack of endogenous TCR Vβ expression, consistent with induction of allelic exclusion by the exogenous TCR during T cell development. The TCR engineered T cells underwent polyfunctional cytokine release, and proliferation in response to artificial APCs. Moreover, the differentiation in ATOs of an ESC line that expresses a CD19-specific 2nd generation (CD28/CD3zeta) CAR construct resulted in the production of CD5+CD7+ CD45RA+ CAR T cells. As reported previously, the ESC-derived CAR T cells did not express CD4, CD8 or CD3; however, they responded to PMA/ionomycin and underwent specific cytokine release and degranulation in response to target cells expressing CD19. PSC-derivedATOs thus present a highly efficient platform for the generation of clinically relevant mature naïve and potentially non-alloreactive TCR and CAR engineered T cells for adoptive immunotherapy. Disclosures Montel-Hagen: Kite Pharma: Research Funding. Seet: Kite Pharma: Research Funding. Crooks: Kite Pharma: Research Funding.

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