scholarly journals 645 Single-cell transcriptional and clonal characterization of CD4+ T cells across tissues in long-term melanoma survivors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A674-A674
Author(s):  
Jichang Han ◽  
Yanding Zhao ◽  
Keisuke Shirai ◽  
Tyler Searles ◽  
Nikhil Khatwani ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cd4 T Cells ◽  
Skin Tumor ◽  
Cd4 T Cell ◽  
Memory T Cell ◽  
The Core ◽  
Melanoma Patients

BackgroundMelanoma patients who develop immunotherapy-related adverse events often have durable responses to treatment. We previously identified that long-term melanoma survivors presenting with the autoimmune adverse event, vitiligo, developed long lived CD8+ resident memory T cell (TRM) responses in skin and tumor with circulating memory T cell (TCIRC) clonal counterparts in blood.1 Despite the focus on CD8+ T cells in prior studies, CD4+ T cell features remain largely in the background.MethodsUsing the same patient cohort, we performed parallel single-cell RNA sequencing (scRNAseq) and single-cell TCR sequencing (scTCRseq) on CD4+ T cells sorted from matched skin, tumor, and blood using the 10X Genomics platform. The UMI counts-based gene expression matrix was processed using the R package Seurat (v.3.0).ResultsEleven distinct CD4+ T cell clusters were identified. The FOXP3 expressing regulatory T cell (Treg) cluster was comprised of cells from skin, tumor, and blood, and could be further sub-clustered into 3 distinct populations with one having transcripts associated with Treg activation. Of the T helper-like clusters, we identified subsets with transcripts associated with cytotoxicity (GZMA, GNLY, CX3CR1; TCYTO), exhaustion (PDCD1, HAVCR2, TOX; TEX), and three clusters that were excluded from blood with clear resident memory characteristics (high CD69, low KLF2, S1PR1). These three clusters were differentiated by expression of IL2 (TRM-IL2); ID2 and CD40LG (TRM-ACTIVATED) and CD28 (TRM-CD28). Paired scTCRseq revealed a high level of clonal overlap between the TCYTO and the TRM-ACTIVATED clusters, with RNA velocity analysis supporting a potential differentiation trajectory from TRM-ACTIVATED to TCYTO. Integrating our previously published CD8+ TRM and TCIRC profiles, we identified a core TRM signature and core TCIRC signature from both the CD4+ and CD8+ TRM and TCIRC cells, respectively, in melanoma patients. The core TRM signature predicted better overall survival of advanced melanoma patients in TCGA, while the core TCIRC signature did not.ConclusionsThis study supports the crucial anti-tumor role of TRM in cancer patients and extends this important observation to CD4+ T cells.ReferenceHan JC, Zhao YD, Shirai K, Molodtsov A, Kolling FW, Fisher JL, Zhang PS, Yan SF, Searles TG, Bader JM, Gui J, Cheng C, Ernstoff MS, Turk MJ, Angeles CV. Resident and circulating memory T cells persist for years in melanoma patients with durable responses to immunotherapy. Nature Cancer 2021;2(3).Ethics ApprovalIRB-approved written informed consent was obtained from patients with advanced melanoma, to perform skin and tumor biopsies, draw blood, and to access historical banked tissue and blood samples for analysis. All human studies were performed in accordance with ethical regulation, and pre-approved by the Committee for the Protection of Human Subjects at Dartmouth-Hitchcock Medical Center IRB (#00029821).

scholarly journals HIV-1 Viremia Prevents the Establishment of Interleukin 2–producing HIV-specific Memory CD4+ T Cells Endowed with Proliferative Capacity

2003 ◽  
Vol 198 (12) ◽  
pp. 1909-1922 ◽  
Author(s):  
Souheil-Antoine Younes ◽  
Bader Yassine-Diab ◽  
Alain R. Dumont ◽  
Mohamed-Rachid Boulassel ◽  
Zvi Grossman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cd4 T Cell ◽  
Memory Cells ◽  
Cell Responses ◽  
Specific Memory ◽  
Ifn Γ ◽  
Memory Cd4 T Cells

CD4+ T cell responses are associated with disease control in chronic viral infections. We analyzed human immunodeficiency virus (HIV)-specific responses in ten aviremic and eight viremic patients treated during primary HIV-1 infection and for up to 6 yr thereafter. Using a highly sensitive 5-(and-6)-carboxyfluorescein diacetate-succinimidyl ester–based proliferation assay, we observed that proliferative Gag and Nef peptide-specific CD4+ T cell responses were 30-fold higher in the aviremic patients. Two subsets of HIV-specific memory CD4+ T cells were identified in aviremic patients, CD45RA− CCR7+ central memory cells (Tcm) producing exclusively interleukin (IL)-2, and CD45RA− CCR7− effector memory cells (Tem) that produced both IL-2 and interferon (IFN)-γ. In contrast, in viremic, therapy-failing patients, we found significant frequencies of Tem that unexpectedly produced exclusively IFN-γ. Longitudinal analysis of HIV epitope–specific CD4+ T cells revealed that only cells that had the capacity to produce IL-2 persisted as long-term memory cells. In viremic patients the presence of IFN-γ–producing cells was restricted to periods of elevated viremia. These findings suggest that long-term CD4+ T cell memory depends on IL-2–producing CD4+ T cells and that IFN-γ only–producing cells are short lived. Our data favor a model whereby competent HIV-specific Tcm continuously arise in small numbers but under persistent antigenemia are rapidly induced to differentiate into IFN-γ only–producing cells that lack self-renewal capacity.

scholarly journals Age-Associated Change in the Frequency of Memory CD4+ T Cells Impairs Long Term CD4+ T Cell Responses to Influenza Vaccine

2004 ◽  
Vol 173 (1) ◽  
pp. 673-681 ◽  
Author(s):  
Insoo Kang ◽  
Myung Sun Hong ◽  
Helena Nolasco ◽  
Sung Hwan Park ◽  
Jin Myung Dan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Influenza Vaccine ◽  
Cd4 T Cells ◽  
T Cell Responses ◽  
Cd4 T Cell ◽  
Cell Responses ◽  
Memory Cd4 T Cells

scholarly journals Comparison of the Development of SARS-Coronavirus-2-Specific Cellular Immunity, and Central Memory CD4+ T-Cell Responses Following Infection versus Vaccination

Vaccines ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 1439
Author(s):  
Kevin M. Dennehy ◽  
Eva Löll ◽  
Christine Dhillon ◽  
Johanna-Maria Classen ◽  
Tobias D. Warm ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
T Cell Responses ◽  
Central Memory ◽  
Cd4 T Cell ◽  
Memory T Cell ◽  
Cell Responses ◽  
Specific Effector ◽  
Memory Cd4 T Cells

Memory T-cell responses following infection with coronaviruses are reportedly long-lived and provide long-term protection against severe disease. Whether vaccination induces similar long-lived responses is not yet clear since, to date, there are limited data comparing memory CD4+ T-cell responses induced after SARS-CoV-2 infection versus following vaccination with BioNTech/Pfizer BNT162b2. We compared T-cell immune responses over time after infection or vaccination using ELISpot, and memory CD4+ T-cell responses three months after infection/vaccination using activation-induced marker flow cytometric assays. Levels of cytokine-producing T-cells were remarkably stable between three and twelve months after infection, and were comparable to IFNγ+ and IFNγ+IL-2+ T-cell responses but lower than IL-2+ T-cell responses at three months after vaccination. Consistent with this finding, vaccination and infection elicited comparable levels of SARS-CoV-2 specific CD4+ T-cells after three months in addition to comparable proportions of specific central memory CD4+ T-cells. By contrast, the proportions of specific effector memory CD4+ T-cells were significantly lower, whereas specific effector CD4+ T-cells were higher after infection than after vaccination. Our results suggest that T-cell responses—as measured by cytokine expression—and the frequencies of SARS-CoV-2-specific central memory CD4+T-cells—indicative of the formation of the long-lived memory T-cell compartment—are comparably induced after infection and vaccination.

scholarly journals Characterisation of CD4+ T-cell subtypes using single cell RNA sequencing and the impact of cell number and sequencing depth

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
James Ding ◽  
Samantha L. Smith ◽  
Gisela Orozco ◽  
Anne Barton ◽  
Steve Eyre ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Cd4 T Cells ◽  
Cell Number ◽  
Sequencing Depth ◽  
Cd4 T Cell ◽  
Specific Cell ◽  
The Impact

AbstractCD4+ T-cells represent a heterogeneous collection of specialised sub-types and are a key cell type in the pathogenesis of many diseases due to their role in the adaptive immune system. By investigating CD4+ T-cells at the single cell level, using RNA sequencing (scRNA-seq), there is the potential to identify specific cell states driving disease or treatment response. However, the impact of sequencing depth and cell numbers, two important factors in scRNA-seq, has not been determined for a complex cell population such as CD4+ T-cells. We therefore generated a high depth, high cell number dataset to determine the effect of reduced sequencing depth and cell number on the ability to accurately identify CD4+ T-cell subtypes. Furthermore, we investigated T-cell signatures under resting and stimulated conditions to assess cluster specific effects of stimulation. We found that firstly, cell number has a much more profound effect than sequencing depth on the ability to classify cells; secondly, this effect is greater when cells are unstimulated and finally, resting and stimulated samples can be combined to leverage additional power whilst still allowing differences between samples to be observed. While based on one individual, these results could inform future scRNA-seq studies to ensure the most efficient experimental design.

scholarly journals OP0003 AUTOREACTIVE CD4+ T CELLS AND THEIR TCR REPERTOIRE IN PR3-ANCA ASSOCIATED VASCULITIS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 1.3-1
Author(s):  
R. Kumar ◽  
N. Yoosuf ◽  
A. Bartoletti ◽  
A. Avik ◽  
B. Raposo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cd4 T Cells ◽  
Cell Receptor ◽  
Cd4 T Cell ◽  
Proteinase 3 ◽  
Anca Associated Vasculitis ◽  
Cell Clones ◽  
Tcr Repertoire

Background:ANCA-associated vasculitis (AAV) with proteinase 3 (PR3) ANCA is genetically associated with HLA-DP [1], is often relapsing in nature, and has a predisposition for kidneys, lungs and ear-nose-throat involvement [2]. Despite the presence of PR3+ANCA, indicating CD4+T-cell help in the disease, the knowledge about autoreactive CD4+T cells is scarce. Activated T cells have been shown at site of inflammation [3] and involvement of proinflammatory cytokines in circulation is also reported [4, 5].Objectives:Identification of autoreactive T cells may help to identify the drivers of the immune responses and chronicity. We therefore aimed to investigate PR3-specific CD4+T-cell responses in peripheral blood of AAV patients with a focus on both phenotype and T-cell receptor (TCR) repertoires.Methods:The study included sixty-six patients: 26 with active PR3 autoantibody+ AAV, 21 with inactive but PR3+ AAV and 19 with inactive PR3- AAV. In-vitro cultures with PR3 protein were established to assess antigen-specific cytokine responses in a 3-color fluorospot assay. Deep immunophenotyping was performed by flow cytometry. Antigen-responsive CD4+ T cells were isolated and single cell TCRαβ sequences were generated and analyzed from PR3+ AAV patients (n=5) using a previously published protocol [6].Results:PBMCs from AAV patients demonstrated an HLA-DP associated cytokine responses to PR3 stimulation including IFN-γ and IL-10, but not IL-17A. This T-cell autoreactivity was found to be confined to a highly differentiated CD4+ T cell population characterized by perforin and GPR56 expression, implicating a cytotoxic feature of the response. Active disease involved a reduction in expression of several markers associated with cytotoxicity amongst the CD4+GPR56+ T cells. Their frequency was also negatively associated with the doses of prednisolone. A similar phenotype was shared with T cells activated by human cytomegalovirus (HCMV) peptides in the same patient cohort. Single cell sequencing of paired alpha beta T-cell receptors (TCRs) revealed different patterns of gene usage between PR3 and HCMV reactive T cells. Moreover, we could identify shared (public) PR3-reactive T-cell clones between different HLA-DPB1*04:01+ patients.Conclusion:PR3 is an autoantigen which provokes ANCA responses in AAV patients. Our study identified PR3-reactive CD4+ T cells at the level of their phenotype and TCR repertoire. The autoreactive CD4+ T cells, present in both active and inactive disease, implicate chronic antigen exposure and the persistence of long-lived T-cell clones. The presence of public autoreactive clones between HLA-DPB1*04:01+ patients suggests an active role for these cells in pathogenesis of AAV and validates the link with predisposed genotype.References:[1]Lyons PA, Rayner TF, Trivedi S, Holle JU, Watts RA, Jayne DR, et al. Genetically distinct subsets within ANCA-associated vasculitis. New England Journal of Medicine. 2012; 367(3):214-223.[2]Kumar Sharma R, Lövström B, Gunnarsson I, Malmström V. Proteinase 3 autoreactivity in Anti-Neutrophil Cytoplasmic Antibody-associated vasculitis–immunological versus clinical features. Scandinavian Journal of Immunology. 2020:e12958.[3]Wilde B, Thewissen M, Damoiseaux J, van Paassen P, Witzke O, Tervaert JWCJAr, et al. T cells in ANCA-associated vasculitis: what can we learn from lesional versus circulating T cells? 2010; 12(1):204.[4]Hoffmann JC, Patschan D, Dihazi H, Müller C, Schwarze K, Henze E, et al. Cytokine profiling in anti neutrophil cytoplasmic antibody-associated vasculitis: a cross-sectional cohort study. Rheumatology international. 2019; 39(11):1907-1917.[5]Berti A, Warner R, Johnson K, Cornec D, Schroeder D, Kabat B, et al. Circulating Cytokine Profiles and ANCA Specificity in Patients with ANCA-Associated Vasculitis. Arthritis & rheumatology (Hoboken, NJ). 2018; 70(7):1114.[6]Han A, Glanville J, Hansmann L, Davis MM. Linking T-cell receptor sequence to functional phenotype at the single-cell level. Nature biotechnology. 2014; 32(7):684-692.Disclosure of Interests:None declared

scholarly journals Single-Cell RNA Sequencing Unveils the Hallmarks of Populations at Different Stages of HTLV-1 Infection

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3323-3323
Author(s):  
Yan Huang ◽  
Peifang Jiang ◽  
Jiazheng Li ◽  
Yanxin Chen ◽  
Zhengjun Wu ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cd4 T Cells ◽  
Treg Cells ◽  
Cd4 T Cell ◽  
Expression Level ◽  
Normal People ◽  
Healthy Donors

Abstract Background Adult T-cell leukemia-lymphoma (ATL) is an aggressive mature T-cell neoplasm caused by human T -cell leukemia virus type 1 (HTLV-1). Up to 5% of infected individuals develop to ATL. HTLV-1 preferentially infects CD4 + T cells, and stimulates cell proliferation and prevents cell death by apoptosis. The viral oncogene-encoded proteins, Tax and HBZ, play important roles in viral infection and cell immortalization. However, the host factor of developing from carrier to patient is not clear. Results To characterize the heterogeneity of ATL patients, we performed single-cell RNA-sequencing (10x Genomics) analysis on single cell suspensions isolated from PBMCs of nine samples, including three ATL patients, three HTLV-1 asymptomatic carriers as well as three healthy donors (HD). We acquired 82977 high-quality cells with a median of 1718 genes detected per cell. Unsupervised clustering using Seurat followed by visualization in t-Stochastic Neighbor Embedding (t-SNE) identified 29 distinct cell clusters (Figure 1A). The single cell profiling of ATL patients were significantly different from that of carriers and healthy donors, while the latter two had little difference (Figure 1B). Based on singleR packages and marker genes of each cluster, 4 major cell populations (T cells, NK cells, B cells and myeloid cells) and other rare cell types were annotated, such as erythrocyte cluster and eosinophils cluster. We observed an enrichment of CD4 + T cell from patients in 4 cluster (Figure 1C), which proportion of cells was higher than that of carriers and healthy donors. According to cell type annotation, cells from cluster 11 were CD4 + CD25 + Foxp3 + Treg cells. Based on the increasing proportion of cluster 11 in healthy people, carriers and patients, without significant statistical differences, we assumed that Foxp3 + Treg cells were involved in the evolution of ATL tumor cells. That was identical with published literatures. To investigate the differences between tumor and normal CD4 + T cell, the gene expression was compared among 7 clusters of CD4 + T cell from three groups. Using a threshold of p value < 0.05 and | fold change| >2. Through integrated analysis, we identified 26 commonly upregulated genes (gene expression level: patients > carriers > HD) and 9 downregulated genes (gene expression level: patients < carriers < HD. To further analyze the biological function of the common DEGs, gene ontology (GO) analysis showed that these genes could be mainly categorized into plasma membrane and protein binding. Subsequently, we validated the mRNA expression level of upregulated common DEGs among three groups by qRT-PCR. The isolated CD4 + T cell using CD4 microbeads of a total of 6 patients, 3 carriers and 9 normal specimens were included. The result showed that the mRNA expression levels of gene CADM1 and RGS13 in patients were higher than those in carriers and healthy donors, although there was no statistical difference between patients and carriers, and the expression levels of carriers tended to be higher than those in normal people (Figure 1D and E). Previously, CADM1 has been revealed to be highly expressed in HTLV-1-infected CD4 + T cells. Our study confirmed this result by single-cell profiling. RGS13, a member of the regulators of G protein signaling (RGS) family, participates in cellular communication. The role of RGS13 in ATL needs to be investigated. Conclusions This study is the first time to analyze the single-cell RNA level of ATL patients, HTLV-1 virus carriers and normal people. The peripheral blood cell composition of the patient is significantly different from that of the carriers and healthy donors, while it is similar between carriers and normal people. CD4 + T cells are the main cell population of patients. The proportion of CD4 + CD25 + Foxp3 + Treg cells increased gradually in healthy people, carriers and patients. DEGs analysis showed that CADM1 and RGS13 were highly expressed in CD4 + T cells of patients, followed by carriers, validated by 18 clinical samples. However, the molecular mechanism of RGS13 in the process from HTLV-1 infection to ATL needs to be further studied. Figure 1 Figure 1. Disclosures Hu: Astellas Pharma, Inc.: Research Funding.

scholarly journals Intranasal nanoparticle vaccination elicits a persistent, polyfunctional CD4 T cell response in the murine lung specific for a highly conserved influenza antigen that are sufficient to mediate protection from influenza virus challenge

2021 ◽  
Author(s):  
Sean A. Nelson ◽  
Thamotharampillai Dileepan ◽  
Amy Rasley ◽  
Marc K. Jenkins ◽  
Nicholas O. Fischer ◽  
...  
Keyword(s):  
T Cells ◽  
Influenza Virus ◽  
T Cell ◽  
Cd4 T Cells ◽  
Critical Role ◽  
Influenza Virus Infection ◽  
T Cell Response ◽  
Cd4 T Cell ◽  
Vaccine Platform

Lung-localized CD4 T cells play a critical role in the control of influenza virus infection and can provide broadly protective immunity. However, current influenza vaccination strategies primarily target influenza hemagglutinin (HA) and are administered peripherally to induce neutralizing antibodies. We have used an intranasal vaccination strategy targeting the highly conserved influenza nucleoprotein (NP) to elicit broadly protective lung localized CD4 T cell responses. The vaccine platform consists of a self-assembling nanolipoprotein particle (NLP) linked to NP with an adjuvant. We have evaluated the functionality, in vivo localization and persistence of T cells elicited. Our study revealed that intranasal vaccination elicits a polyfunctional subset of lung-localized CD4 T cells that persist long term. A subset of these lung CD4 T cells localize to the airway, where they can act as early responders following encounter with cognate antigen. Polyfunctional CD4 cells isolated from airway and lung tissue produce significantly more effector cytokines IFNγ and TNFα as well as cytotoxic functionality. When adoptively transferred to naïve recipients, CD4 T cells from NLP:NP immunized lung were sufficient to mediate 100% survival from lethal challenge with H1N1 influenza virus. Importance Exploiting new, more efficacious strategies to potentiate influenza-specific immune responses is important, particularly for at-risk populations. We have demonstrated the promise of direct intranasal protein vaccination to establish long-lived immunity in the lung with CD4 T cells that possess features and positioning in the lung that are associated with both immediate and long-term immunity, as well as demonstrating direct protective potential.

scholarly journals Dissecting the landscape of activated CMV-stimulated CD4+ T cells in human by linking single-cell RNA-seq with T-cell receptor sequencing

2020 ◽  
Author(s):  
Menghua Lyu ◽  
Shiyu Wang ◽  
Kai Gao ◽  
Longlong Wang ◽  
Bin Li ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
T Cell Receptor ◽  
Cd4 T Cells ◽  
Cell Receptor ◽  
T Cell Subsets ◽  
Cd4 T Cell ◽  
Rna Seq ◽  
Cmv Infection

AbstractCD4 T cell is crucial in CMV infection, but its role is still unclear during this process. Here, we present a single-cell RNA-seq together with T cell receptor (TCR) sequencing to screen the heterogenicity and potential function of CMV pp65 reactivated CD4+ T cell subsets from human peripheral blood, and unveil their potential interactions. Notably, Treg composed the major part of these reactivated cells. Treg gene expression data revealed multiple transcripts of both inflammatory and inhibitory functions. Additionally, we describe the detailed phenotypes of CMV-reactivated effector-memory (Tem), cytotoxic T (CTL), and naïve T cells at the single-cell resolution, and implied the direct derivation of CTL from naïve CD4+ T cells. By analyzing the TCR repertoire, we identified a clonality in stimulated Tem and CTLs, and a tight relationship of Tem and CTL showing a large share in TCR. This study provides clues for understanding the function of CD4+ T cells subsets and unveils their interaction in CMV infection, and may promote the development of CMV immunotherapy.

scholarly journals Single-Cell Transcriptomics of Human TET2 Knockout CD4 T-Cells and Their Clonal Evolution

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 22-23 ◽  
Author(s):  
Yuping Li ◽  
Xiaoqian Liu ◽  
Xuxiang Liu ◽  
Xiwei Wu ◽  
Alyssa Bouska ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cd4 T Cells ◽  
Transcriptome Sequencing ◽  
Cell Lymphoma ◽  
Clonal Evolution ◽  
Cd4 T Cell ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

Angioimmunoblastic T-cell lymphoma (AITL), the most frequent subtype of peripheral T-cell lymphoma (PTCL), is a neoplasm with characteristics of mature T follicular helper (TFH) cells. We and others have identified frequent (~75%) inactivating mutations in the TET2 (Ten-Eleven Translocation-2) gene in AITL. TET2 belongs to a 3 member family of TET dioxygenases that catalyze DNA demethylation by oxidation of 5-methyl-cytosine (5-mC) to 5-hydroxymethyl-cytosine (5-hmC) and further oxidative cytosine products. Thus, loss of function (LOF) of TET2 will cause aberrant genome hypermethylation and reduction in 5-hmC. Studies of the variant allele fraction (VAF) of TET2 mutants suggest that this mutation is a founding abnormality in AITL. However, how TET2 loss promotes the development of AITL is still unclear. To study LOF of TET2 in CD4 T-cell lymphomagenesis without the noise generated by other mutations in an established lymphoma, we generated a human TET2 knock-out (KO) CD4 T-cell model using CRISPR/Cas9 technology, which allows us to perform functional genomic studies by directly editing genes at their genomic loci. Whole transcriptome sequencing and single-cell transcriptome sequencing were used to study the cell evolution after KO. We generated multiple TET2 KO primary CD4 T-cell models using two different CRISPR/Cas9 methods. The first approach used the plasmid PX458-a, which expresses green fluorescent protein (GFP) fused Cas9 and guide RNA-a targeting TET2 exon 6, to electroporate CD4 T-cell from healthy donor F25. The second approach used homologous DNA repair (HDR) mediated knock-in (KI) of tandem GFP gene and a SV40 transcription stop signal to terminate TET2 expression at exon 3. Cas9/sgRNA-e RNP complex, along with a long DNA template (about 1.6 kb), was electroporated into CD4 T-cells from two healthy donors, F25 and M40. GFP-positive cells were sorted by FACS after electroporation and were considered to be edited cells. Edited CD4 T-cells were cultured in vitro with 50 U/ml IL-2, and stimulated regularly (every 7~10 days) with 1:1 ratio of anti-CD3/CD28 T activator beads. TET2 KO in these cells was confirmed by qRT-PCR, Sanger sequencing and Western blotting. Compared with wild-type (WT) CD4 T-cells under the same culture conditions, a lower level of 5-hmC in TET2 KO cells was observed, indicating successful editing of TET2. Compared to WT cells, KO cells had a higher growth rate, due to a lower apoptosis rate and a higher proliferation rate, by Annexin V staining, EdU staining, and MTS experiments. The growth of KO cells or WT cells was still dependent on IL-2 and T activator beads stimulation. All batches of KO cells, generated by different guide RNAs or from different donors, showed a much longer life span than WT cells, which usually lived for 3~4 months, but KO cells can keep proliferating longer than one year. We also performed TCR analysis on these cell samples. Both WT and KO cells demonstrated oligoclonality when examined at Day 40 (40D, early stage) and TET2 KO cells showed a dominant clone by Day 90 (90D, late stage). We performed single-cell transcriptome analysis on M40 KO vs. WT cells, at 40D and 90D. KO90D cells had a low TCR diversity with the dominant population representing ~88% of cells (TRAV9-2,TRBV5-1). From single-cell transcriptome analysis, cell clustering profiles were very distinctive in these 4 cell populations analyzed (Figure 1A) and these clusters had unique gene expression profiles (Figure 1B). Cluster 6 was prominent in KO90D but almost absent in WT90D, whereas the reverse was true for clusters 1 and 5. From pathway analysis, KO90D cells showed a higher expression of signatures associated with proliferation, cell cycle and chemokine signaling and lower histidine and tryptophan metabolism signatures. Sanger sequencing showed a 79 bp indel in addition to the GFP KI allele in KO90D cells, demonstrated the homozygous deletion of TET2 on these cells. Similar results were observed in F25 TET2 KO cells by plasmid PX458-a. This indicated the selection of homozygously deleted TET2 cells in long-term culture. However, clonal evolution is highly dynamic and a minor clone in KO40D cells may become the dominant clone in KO90D cells. Comparison of the 5-mC and 5-hmC profiles between KO and WT cells are being conducted to elucidate epigenetic alterations that are associated with the functional alterations and predisposition to AITL lymphomagenesis. Figure Disclosures No relevant conflicts of interest to declare.

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