Single-Cell Transcriptome of Early Hematopoiesis Guides Arterial Endothelium-Promoted Functional T Cell Generation from Human Pluripotent Stem Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 38-38
Author(s):  
Jun Shen ◽  
Yingxi Xu ◽  
Shuo Zhang ◽  
Shuzhen Lyu ◽  
Zack Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
Hematopoietic Differentiation ◽  
Cell Potential ◽  
Arterial Endothelium

Human pluripotent stem cells (hPSCs) provide a powerful platform for generating functional hematopoietic cells for blood disease modeling and therapeutic testing. However, the quantity and quality of hPSC-derived blood cells remain to be improved. Here, by performing extensive single-cell transcriptomic analyses to map fate choices and gene expression programs during hematopoietic differentiation of hPSCs, we construct the first hematopoietic landscape of hPSCs at the single-cell level and identify strategies to promote hematopoietic progenitor (HP) generation from hPSCs with functional T cell potential. By focusing specifically on cell populations and molecular events involved in endothelial-to-hematopoietic transition (EHT), we compared the difference of early hematopoiesis between hPSCs and human embryos (Yang Zeng et al. Cell Research. 2019) and found aerobic metabolism was dysregulated during in-vitro-directed differentiation. The decreased oxygen metabolism program was further deciphered as a key molecular event occurred during the EHT. Providing hypoxia at the stage of EHT enhanced hematopoietic differentiation of hPSCs via specifying arterial programs, including arterial hemogenic endothelium (AHE) and arterial endothelium cells (AE). To further determine the effect of AE on hematopoietic development, we isolated AE, venous endothelium and mesenchymal cells identified in our single-cell transcriptomic analyses and cocultured them with AHE respectively for HP generation. AE were finally validated as a critical regulator of definitive HP specification with more T cell potential. T cells generated from AE-primed HPs (AE-T) were highly functional and exhibited polyfunctional production of interferon (IFN)-γ, tumor necrosis factor alpha (TNF-α), and IL-2 in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin. To further evaluate the function of AE-T, we engineered T cells with CD19-CAR. The in vitro cytotoxicity of CAR-engineered AE-T was performed both in CD19+ cell lines (Nalm-6 and Raji) and human primary B-ALL samples. The efficacy of CAR-engineered AE-T in vivo was evaluated in a mouse xenograft model inoculated intravenously with luciferase-expressing Nalm-6 cells. Similar to CD19 CAR-transduced peripheral blood T cells, the AE-T potently inhibited tumor growth both in vitro and in vivo. Collectively, our study provides benchmark datasets to understand the origins of human hematopoiesis and presents an advance for guiding the generation of functional T cells in vitro for clinical applications. Disclosures No relevant conflicts of interest to declare.

scholarly journals High levels of interleukin 10 production in vivo are associated with tolerance in SCID patients transplanted with HLA mismatched hematopoietic stem cells.

1994 ◽  
Vol 179 (2) ◽  
pp. 493-502 ◽  
Author(s):  
R Bacchetta ◽  
M Bigler ◽  
J L Touraine ◽  
R Parkman ◽  
P A Tovo ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Mrna Expression ◽  
Fetal Liver ◽  
Hla Antigens ◽  
Interleukin 10 ◽  
Hematopoietic Stem

Transplantation of HLA mismatched hematopoietic stem cells in patients with severe combined immunodeficiency (SCID) can result in a selective engraftment of T cells of donor origin with complete immunologic reconstitution and in vivo tolerance. The latter may occur in the absence of clonal deletion of donor T lymphocytes able to recognize the host HLA antigens. The activity of these host-reactive T cells is suppressed in vivo, since no graft-vs. -host disease is observed in these human chimeras. Here it is shown that the CD4+ host-reactive T cell clones isolated from a SCID patient transplanted with fetal liver stem cells produce unusually high quantities of interleukin 10 (IL-10) and very low amounts of IL-2 after antigen-specific stimulation in vitro. The specific proliferative responses of the host-reactive T cell clones were considerably enhanced in the presence of neutralizing concentrations of an anti-IL-10 monoclonal antibody, suggesting that high levels of endogenous IL-10 suppress the activity of these cells. These in vitro data correlate with observations made in vivo. Semi-quantitative polymerase chain reaction analysis carried out on freshly isolated peripheral blood mononuclear cells (PBMC) of the patient indicated that the levels of IL-10 messenger RNA (mRNA) expression were strongly enhanced, whereas IL-2 mRNA expression was much lower than that in PBMC of healthy donors. In vivo IL-10 mRNA expression was not only high in the T cells, but also in the non-T cell fraction, indicating that host cells also contributed to the high levels of IL-10 in vivo. Patient-derived monocytes were found to be major IL-10 producers. Although no circulating IL-10 could be detected, freshly isolated monocytes of the patient showed a reduced expression of class II HLA antigens. However, their capacity to stimulate T cells of normal donors in primary mixed lymphocyte cultures was within the normal range. Interestingly, similar high in vivo IL-10 mRNA expressions in the T and non-T cell compartment were also observed in three SCID patients transplanted with fetal liver stem cells and in four SCID patients transplanted with T cell-depleted haploidentical bone marrow stem cells. Taken together, these data indicate that high endogenous IL-10 production is a general phenomenon in SCID patients in whom allogenic stem cell transplantation results in immunologic reconstitution and induction of tolerance. Both donor T cells and host accessory cells contribute to these high levels of IL-10, which would suppress the activity of host-reactive T cell in vivo.

Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1999-1999
Author(s):  
Annie L. Oh ◽  
Dolores Mahmud ◽  
Benedetta Nicolini ◽  
Nadim Mahmud ◽  
Elisa Bonetti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. 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.

scholarly journals Defining totipotency using criteria of increasing stringency

2020 ◽  
Author(s):  
Eszter Posfai ◽  
John Paul Schell ◽  
Adrian Janiszewski ◽  
Isidora Rovic ◽  
Alexander Murray ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Differentiated Cells ◽  
Totipotent Cell

AbstractTotipotency is the ability of a single cell to give rise to all the differentiated cells that build the conceptus, yet how to capture this property in vitro remains incompletely understood. Defining totipotency relies upon a variety of assays of variable stringency. Here we describe criteria to define totipotency. We illustrate how distinct criteria of increasing stringency can be used to judge totipotency by evaluating candidate totipotent cell types in the mouse, including early blastomeres and expanded or extended pluripotent stem cells. Our data challenge the notion that expanded or extended pluripotent states harbor increased totipotent potential relative to conventional embryonic stem cells under in vivo conditions.

scholarly journals DLL4 and VCAM1 enhance the emergence of T cell-competent hematopoietic progenitors from human pluripotent stem cells

2021 ◽  
Author(s):  
Yale S Michaels ◽  
John M Edgar ◽  
Matthew C Major ◽  
Elizabeth L Castle ◽  
Carla Zimmerman ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Pluripotent Stem Cells ◽  
Adaptive Immune Response ◽  
Hematopoietic Progenitors ◽  
Cell Potential ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
The Impact

T cells are key mediators of the adaptive immune response and show tremendous efficacy as cellular therapeutics. However, obtaining primary T cells from human donors is expensive and variable. Pluripotent stem cells (PSCs) have the potential to serve as a consistent and renewable source of T cells, but differentiating PSCs into hematopoietic progenitors with T cell potential remains a significant challenge. Here, we developed an efficient serum- and feeder-free protocol for differentiating human PSCs into hematopoietic progenitors and T cells. This defined method allowed us to study the impact of individual recombinant proteins on blood emergence and lineage potential. We demonstrate that the presence of DLL4 and VCAM1 during the endothelial-to-hematopoietic transition (EHT) enhances downstream progenitor T cell output by >80-fold. Using single cell transcriptomics, we showed that these two proteins synergise to drive strong notch signalling in nascent hematopoietic stem and progenitor cells and that VCAM1 additionally drives a pro-inflammatory transcriptional program. Finally, we applied this differentiation method to study the impact of cytokine concentration dynamics on T cell maturation. We established optimised media formulations that enabled efficient and chemically defined differentiation of CD8αβ+, CD4-, CD3+, TCRαβ+ T cells from PSCs.

scholarly journals Guiding T lymphopoiesis from pluripotent stem cells by defined transcription factors

Cell Research ◽  
2019 ◽  
Vol 30 (1) ◽  
pp. 21-33 ◽  
Author(s):  
Rongqun Guo ◽  
Fangxiao Hu ◽  
Qitong Weng ◽  
Cui Lv ◽  
Hongling Wu ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Pluripotent Stem Cells ◽  
Time Window ◽  
Immune Surveillance ◽  
Cell Stage ◽  
Immunodeficient Mice ◽  
T Lymphopoiesis

AbstractAchievement of immunocompetent and therapeutic T lymphopoiesis from pluripotent stem cells (PSCs) is a central aim in T cell regenerative medicine. To date, preferentially reconstituting T lymphopoiesis in vivo from PSCs remains a practical challenge. Here we documented that synergistic and transient expression of Runx1 and Hoxa9 restricted in the time window of endothelial-to-hematopoietic transition and hematopoietic maturation stages in a PSC differentiation scheme (iR9-PSC) in vitro induced preferential generation of engraftable hematopoietic progenitors capable of homing to thymus and developing into mature T cells in primary and secondary immunodeficient recipients. Single-cell transcriptome and functional analyses illustrated the cellular trajectory of T lineage induction from PSCs, unveiling the T-lineage specification determined at as early as hemogenic endothelial cell stage and identifying the bona fide pre-thymic progenitors. The induced T cells distributed normally in central and peripheral lymphoid organs and exhibited abundant TCRαβ repertoire. The regenerative T lymphopoiesis restored immune surveillance in immunodeficient mice. Furthermore, gene-edited iR9-PSCs produced tumor-specific T cells in vivo that effectively eradicated tumor cells. This study provides insight into universal generation of functional and therapeutic T cells from the unlimited and editable PSC source.

scholarly journals Mapping the biogenesis of forward programmed megakaryocytes from induced pluripotent stem cells

2021 ◽  
Author(s):  
Moyra Lawrence ◽  
Arash Shahsavari ◽  
Susanne Bornelöv ◽  
Thomas Moreau ◽  
Katarzyna Kania ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
Surface Markers ◽  
Single Cell Rna Sequencing ◽  
Induced Pluripotent ◽  
Insight Into ◽  
Megakaryocyte Progenitors

SummaryPlatelet deficiency, known as thrombocytopenia, can cause haemorrhage and is treated with platelet transfusions. We developed a system for the production of platelet precursor cells, megakaryocytes, from pluripotent stem cells. These cultures can be maintained for >100 days, implying culture renewal by megakaryocyte progenitors (MKPs). However, it is unclear whether the MKP state in vitro mirrors the state in vivo, and MKPs cannot be purified using conventional surface markers. We performed single cell RNA sequencing throughout in vitro differentiation and mapped each state to its equivalent in vivo. This enabled the identification of 5 surface markers which reproducibly purify MKPs, allowing us an insight into their transcriptional and epigenetic profiles. Finally, we performed culture optimisation, increasing MKP production. Altogether, this study has mapped parallels between the MKP states in vivo and in vitro and allowed the purification of MKPs, accelerating the progress of in vitro-derived transfusion products towards the clinic.

scholarly journals The Therapeutic Effect of ICAM-1-Overexpressing Mesenchymal Stem Cells on Acute Graft-Versus-Host Disease

2018 ◽  
Vol 46 (6) ◽  
pp. 2624-2635 ◽  
Author(s):  
Bo Tang ◽  
Xue Li ◽  
Yuanlin Liu ◽  
Xiuhui Chen ◽  
Ximei Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Mesenchymal Stem Cells ◽  
T Cell ◽  
Mouse Model ◽  
Graft Versus Host Disease ◽  
Culture Methods ◽  
Graft Versus Host

Background/Aims: Mesenchymal stem cells (MSCs) do not readily migrate to appropriate sites, and this creates a major obstacle for their use in the treatment of graft-versus-host disease (GVHD). Intercellular adhesion molecule-1 (ICAM-1) can guide the homing of various immune cells to the proper anatomical location within secondary lymphoid organs (SLOs), which are the major niches for generating immune responses or tolerance. MSCs rarely migrate to SLOs after intravenous infusion, and are constitutively low expression of ICAM-1. So in our previous work, ICAM-1 was engineered into a murine MSC line C3H10T1/2 by retrovirus transfection system (ICAM-1MSCs). Here, we hypothesized that ICAM-1highMSCs may significantly improve their immunomodulatory effect. Methods: We used different co-culture methods combined with real-time PCR and flow cytometry to evaluate ICAM-1highMSCs immunomodulatory effect on dendritic cells (DCs) and T cells in vitro and in vivo. MSCs were labeled with carboxyfluorescein diacetate succinimidylester (CFSE) to detect its distribution in mouse model. Results: Our in vitro analyses revealed ICAM-1 MSCs could suppress DCs maturation according to co-culture methods and suppress the T cell immune response according to the mixed lymphocyte response (MLR) and lymphoblast transformation test (LTT) tests. We found that infusion of ICAM-1highMSCs potently prolonged the survival of GVHD mouse model. The infused ICAM-1highMSCs migrate to SLOs in vivo, and suppressed DCs maturation, suppressed CD4+ T cell differentiation to Th1 cells, and increased the ratios of Treg cells. Conclusions: Taken together, these data demonstrate that ICAM-1highMSCs had an enhanced immunosuppressive effect on DCs and T cells, which may help explain the protective effect in a GVHD model. This exciting therapeutic strategy may improve the clinical efficacy of MSC-based therapy for GVHD.

In Vivo Tracking of T Cells in Humans Unveils Decade-Long Survival and Activity of Genetically Modified T Memory Stem Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 547-547
Author(s):  
Serena Scala ◽  
Luca Biasco ◽  
Luca Basso Ricci ◽  
Francesca Dionisio ◽  
Cristina Baricordi ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Future Application ◽  
Long Survival ◽  
Healthy Donors ◽  
Integration Sites

Abstract A deeper understanding of T lymphocytes survival and differentiation potential in humans is paramount for the development of effective gene/cell therapies based on T-cell engineering. We here performed a comprehensive study of T-cells dynamics and plasticity in humans by a unique combination of phenotypic/functional studies and high-throughput integration sites (IS) analyses. We analyzed samples from hematopoietic stem cells (HSC) (n=10) or mature lymphocytes (PBL) gene therapy (GT) (n=4) treated ADA (adenosine deaminase) deficient-SCID patients. For comparative analyses, we also collected data from pediatric (n=19) and adult (n=52) healthy donors (HD), and from bone marrow transplanted patients (BMT) with primary immunodeficiencies (n=10, 4 with ADA-SCID). We observed that vector-positive CD62L+/CD45RA+ putative T naïve cells were detectable 12 years after last infusion of gene-corrected lymphocytes in peripheral blood of PBL-GT patients that lack the support of transduced lymphocytes precursors. We then unveiled that the vast majority of these CD62L+/CD45RA+ cells (80.3%) in PBL-GT patients could be actually classified phenotypically (CD95, IL2Rβ and IL7Rα surface expression) and functionally (IFNγ production and aCD3/aCD28 in vitro differentiation) as active long-lasting T memory stem cells (Tscm). The peculiar Tscm frequency found in PBL-GT patients was most likely due to a combinatorial in vitro and in vivo effect. Indeed, by a series of in vitro assays, we showed that Tscm relative enrichment in CD45RA+CD62L+ compartment have occurred during the in vitro manipulation of T cells before infusion. Additionally, we found higher-then-normal Tscm contribution among CD45RA+/CD62L+ cells even in ADA-SCID patients receiving HSC-GT and BMT, suggesting a role of disease background on in vivo Tscm persistence. Analyzing our cohorts of healthy donors and treated individuals we were able to further correlate Tscm contribution in vivo with age, conditioning regimen, disease background, cell source, and long-term T-cell reconstitution. One unique aspect of our study consisted in the opportunity to track Tscm clonal dynamics in vivo in humans since each gene-corrected cell infused in our GT patients is univocally and permanently tagged by a retroviral integration site.To perform in vivo molecular tracing of individual T-cell clones we sorted T naïve, Tscm, central memory and effector memory subtypes. We then collected from these subpopulations, by LAM-PCR+Illumina-Miseq sequencing, 2.584.137 integration sites (IS) sequences mapped to 1.746 unique chromosomal positions, corresponding to 910 integrations from 5 HSC-GT patients in vivo, 79 integrations from 2 PBL-GT samples of transduced cell products prior to infusion and 754 integrations from 4 PBL-GT patients in vivo. Firstly, to establish a relationship between precursors and terminally differentiated T cells we searched for the presence of identical insertion sites detected in multiple T-cell subtypes, applying stringent analytical filters for cross-contaminations. Strikingly, the level of shared integrations in each subtype was directly correlated to its stage of differentiation with Tscm, isolated from PBL-GT patients, showing the highest proportion of integration sites shared with the other T-cell subsets. Importantly, the results of the same analysis performed on HSC-GT patients were outstandingly coherent with the progressive developmental model of memory T-cell differentiation. We then assessed the survival of individual Tscm clones by performing a longitudinal IS analysis of different T-cell subtypes isolated from 3 PBL-GT patients over a 2 to 5 years timeframe up to 12 years after last infusion. We were able to formally prove the persistence of individual Tscm by re-capturing identical IS tagging specific Tscm clones in two independent timepoints in a 5- years window. Importantly, the same IS were also detected in multiple T-cell subtypes, representing the best indirect evidence that these clones were endowed with long-term precursor activity. We also documented, by IS sequencing reads, the long-term polyclonal composition of each subtype and we did not observe enrichment for IS flanking proto-oncogenes. Overall, this study validates, for the first time in humans, the safe and functional decade-long survival of engineered Tscm, paving the way for their future application in clinical settings. Disclosures No relevant conflicts of interest to declare.

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