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• W2136875369 abstract "Adoptive T cell therapy can be effective for Epstein–Barr virus (EBV)-associated posttransplant lymphoproliferative disease and melanoma. Transducing high-affinity TCR genes into T lymphocytes is an emerging method to improve potency and specificity of tumor-specific T cells. However, both methods necessitate in vitro lymphocyte proliferation, generating highly differentiated effector cells that display reduced survival and antitumor efficacy postinfusion. TCR-transduction of naive lymphocytes isolated from peripheral blood is reported to provide superior in vivo survival and function. We utilized cord blood (CB) lymphocytes, which comprise mainly naive cells, for transducing EBV-specific TCR. Comparable TCR expression was achieved in adult and CB cells, but the latter expressed an earlier differentiation profile. Further antigen-driven stimulation skewed adult lymphocytes to a late differentiation phenotype associated with immune exhaustion. In contrast, CB T cells retained a less differentiated phenotype after antigen stimulation, remaining CD57-negative but were still capable of antigen-specific polyfunctional cytokine expression and cytotoxicity in response to EBV antigen. CB T cells also retained longer telomeres and in general possessed higher telomerase activity indicative of greater proliferative potential. CB lymphocytes therefore have qualities indicating prolonged survival and effector function favorable to immunotherapy, especially in settings where donor lymphocytes are unavailable such as in solid organ and CB transplantation. Adoptive T cell therapy can be effective for Epstein–Barr virus (EBV)-associated posttransplant lymphoproliferative disease and melanoma. Transducing high-affinity TCR genes into T lymphocytes is an emerging method to improve potency and specificity of tumor-specific T cells. However, both methods necessitate in vitro lymphocyte proliferation, generating highly differentiated effector cells that display reduced survival and antitumor efficacy postinfusion. TCR-transduction of naive lymphocytes isolated from peripheral blood is reported to provide superior in vivo survival and function. We utilized cord blood (CB) lymphocytes, which comprise mainly naive cells, for transducing EBV-specific TCR. Comparable TCR expression was achieved in adult and CB cells, but the latter expressed an earlier differentiation profile. Further antigen-driven stimulation skewed adult lymphocytes to a late differentiation phenotype associated with immune exhaustion. In contrast, CB T cells retained a less differentiated phenotype after antigen stimulation, remaining CD57-negative but were still capable of antigen-specific polyfunctional cytokine expression and cytotoxicity in response to EBV antigen. CB T cells also retained longer telomeres and in general possessed higher telomerase activity indicative of greater proliferative potential. CB lymphocytes therefore have qualities indicating prolonged survival and effector function favorable to immunotherapy, especially in settings where donor lymphocytes are unavailable such as in solid organ and CB transplantation. Epstein–Barr virus (EBV)-associated posttransplant lymphoproliferative disease (PTLD) complicates up to 13% of solid organ transplantation (SOT) (1Manlhiot C Pollock-Barziv SM Holmes C et al.Past-transplant lymphoproliferative disorder in pediatric heart transplant recipients.J Heart Lung Transplant. 2010; 29: 648-657Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Anti-CD20 immunotherapy is an effective first line treatment but around 40% of cases remain refractory. Cord blood (CB) transplantation is also associated with significant EBV and cytomegalovirus (CMV) reactivation and PTLD. Adoptive cellular immunotherapy (ACT) using third-party partially HLA-matched EBV-specific T cells is an effective treatment for PTLD in both settings (2Haque T Wilkie GM Jones MM et al.Allogeneic cytotoxic T-cell therapy for EBV-positive posttransplantation lymphoproliferative disease: Results of a phase 2 multicenter clinical trial.Blood. 2007; 110: 1123-1131Crossref PubMed Scopus (493) Google Scholar, 3Barker JN Doubrovina E Sauter C et al.Successful treatment of EBV-associated posttransplantation lymphoma after cord blood transplantation using third-party EBV-specific cytotoxic T lymphocytes.Blood. 2010; 116: 5045-5049Crossref PubMed Scopus (181) Google Scholar, 4Heslop HE Slobod KS Pule MA et al.Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients.Blood. 2010; 115: 925-935Crossref PubMed Scopus (650) Google Scholar). However, for PTLD following SOT only 50% respond to ACT (2Haque T Wilkie GM Jones MM et al.Allogeneic cytotoxic T-cell therapy for EBV-positive posttransplantation lymphoproliferative disease: Results of a phase 2 multicenter clinical trial.Blood. 2007; 110: 1123-1131Crossref PubMed Scopus (493) Google Scholar) possibly because the polyspecific T cells generated in vitro using an EBV-transformed B-lymphoblastoid cell line (LCL) contain too few effectors specific for the limited set of EBV antigens expressed by the tumor. An alternative approach to rapidly generate large numbers of potent and specific effectors is to engineer T cells to express an appropriate antigen-specific TCR or a chimeric antigen receptor (5Micklethwaite KP Savoldo B Hanley PJ et al.Derivation of human T lymphocytes from cord blood and peripheral blood with antiviral and antileukemic specificity from a single culture as protection against infection and relapse after stem cell transplantation.Blood. 2010; 115: 2695-2703Crossref PubMed Scopus (92) Google Scholar,6Gattinoni L Powell Jr., DJ Rosenberg SA Restifo NP Adoptive immunotherapy for cancer: building on success.Nat Rev Immunol. 2006; 6: 383-393Crossref PubMed Scopus (732) Google Scholar). This has been used successfully to treat cancers such as melanoma (7Rosenberg SA Yang JC Sherry RM et al.Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy.Clin Cancer Res. 2011; 17: 4550-4557Crossref PubMed Scopus (1578) Google Scholar), where naturally occurring tumor-specific T cells are rare and of low avidity (8Gilboa E The makings of a tumor rejection antigen.Immunity. 1999; 11: 263-270Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar, 9Goodyear O Piper K Khan N et al.CD8+ T cells specific for cancer germline gene antigens are found in many patients with multiple myeloma, and their frequency correlates with disease burden.Blood. 2005; 106: 4217-4224Crossref PubMed Scopus (71) Google Scholar, 10Stauss HJ Thomas S Cesco-Gaspere M et al.WT1-specific T cell receptor gene therapy: improving TCR function in transduced T cells.Blood Cells Mol Dis. 2008; 40: 113-116Crossref PubMed Scopus (46) Google Scholar). Using retroviral vectors, human T cells can be reliably transduced with TCR genes enabling them to recognize viral or tumor antigens. Adoptive transfer of engineered T cells is currently undergoing clinical trials with encouraging results (11Robbins PF Morgan RA Feldman SA et al.Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1.J Clin Oncol. 2011; 29: 917-924Crossref PubMed Scopus (1258) Google Scholar, 12Morgan RA Dudley ME Wunderlich JR et al.Cancer regression in patients after transfer of genetically engineered lymphocytes.Science. 2006; 314: 126-129Crossref PubMed Scopus (2141) Google Scholar, 13Porter DL Levine BL Kalos M Bagg A June CH Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia.N Engl J Med. 2011; 365: 725-733Crossref PubMed Scopus (2685) Google Scholar). ACT studies indicate that antitumor response is linked to long term in vivo persistence of infused cells (14Robbins PF Dudley ME Wunderlich J et al.Cutting edge: persistence of transferred lymphocyte clonotypes correlates with cancer regression in patients receiving cell transfer therapy.J Immunol. 2004; 173: 7125-7130Crossref PubMed Scopus (410) Google Scholar, 15Dudley ME Wunderlich JR Yang JC et al.Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma.J Clin Oncol. 2005; 23: 2346-2357Crossref PubMed Scopus (1379) Google Scholar, 16Zhou J Dudley ME Rosenberg SA Robbins PF Persistence of multiple tumor-specific T-cell clones is associated with complete tumor regression in a melanoma patient receiving adoptive cell transfer therapy.J Immunother. 2005; 28: 53-62Crossref PubMed Scopus (174) Google Scholar). The factors influencing in vivo persistence of lymphocytes are not fully understood, but evidence suggests that the differentiation status of the T cell is critical. Less differentiated naive (TN) and central memory (TCM) T cell subsets display superior proliferation, persistence and antitumor responses following infusion when compared to the more differentiated effector memory (TEM) subset (17Gattinoni L Klebanoff CA Palmer DC et al.Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8+ T cells.J Clin Invest. 2005; 115: 1616-1626Crossref PubMed Scopus (765) Google Scholar, 18Klebanoff CA Gattinoni L Torabi-Parizi P et al.Central memory self/tumor-reactive CD8+ T cells confer superior antitumor immunity compared with effector memory T cells.Proc Natl Acad Sci USA. 2005; 102: 9571-9576Crossref PubMed Scopus (732) Google Scholar, 19Berger C Jensen MC Lansdorp PM Gough M Elliott C Riddell SR Adoptive transfer of effector CD8 +T cells derived from central memory cells establishes persistent T cell memory in primates.J Clin Invest. 2008; 118: 294-305Crossref PubMed Scopus (673) Google Scholar). This raises an important issue for ACT using genetically engineered T cells because in vitro activation of adult lymphocytes, required for retroviral transduction, drives the majority of peripheral blood-derived T cells into highly differentiated effector. Thus current approaches using transduced T cells may be suboptimal because the majority of cells infused will be differentiated and may therefore be of limited efficacy in vivo (20Hinrichs CS Borman ZA Cassard L et al.Adoptively transferred effector cells derived from naive rather than central memory CD8+ T cells mediate superior antitumor immunity.Proc Natl Acad Sci USA. 2009; 106: 17469-17474Crossref PubMed Scopus (320) Google Scholar,21Hinrichs CS Borman ZA Gattinoni L et al.Human effector CD8+ T cells derived from naive rather than memory subsets possess superior traits for adoptive immunotherapy.Blood. 2010; 117: 808-814Crossref PubMed Scopus (237) Google Scholar). The challenge for ACT with genetically engineered T cells, or with any protocol involving cell expansion, is therefore to generate cells with a minimally differentiated phenotype. Recent studies (20Hinrichs CS Borman ZA Cassard L et al.Adoptively transferred effector cells derived from naive rather than central memory CD8+ T cells mediate superior antitumor immunity.Proc Natl Acad Sci USA. 2009; 106: 17469-17474Crossref PubMed Scopus (320) Google Scholar,21Hinrichs CS Borman ZA Gattinoni L et al.Human effector CD8+ T cells derived from naive rather than memory subsets possess superior traits for adoptive immunotherapy.Blood. 2010; 117: 808-814Crossref PubMed Scopus (237) Google Scholar) suggest that CD8 TN lymphocytes selected from adult peripheral blood (PB) are optimal for this purpose because, in contrast to TCM and TEM cells, they display minimal differentiation following TCR transduction. Human umbilical CB T cells, unlike adult-derived PB lymphocytes, are mostly TN. It is therefore reasonable to speculate whether CB might be an alternative source of T-lymphocytes for genetic engineering. As both solid organ and CB transplant recipients cannot access lymphocytes from the original donors, third-party allogeneic CB is a convenient alternate source of lymphocytes for ACT against EBV-PTLD. Such cells can also be used in lymphopenic cancer patients where autologous lymphopheresis is not possible. This study utilizes cryopreserved CB units from an unrelated cord blood bank and assesses the feasibility of using cord T cells to transduce EBV-specific TCR, and to analyze their functional capacity for in vivo use in immunotherapy. Frozen umbilical CB units, unsuitable for transplantation, were provided by NHS Cord Blood Bank, UK. Units were thawed in cold RPMI 1640 (Sigma-Aldrich, St. Louis, USA) plus 10% fetal calf serum (FCS) (PAA, Pasching, Austria). After washing, mononuclear cells were isolated using Ficoll. Adult PB mononuclear cells (PBMC) were isolated from aphereses cones (22Dietz AB Bulur PA Emery RL et al.A novel source of viable peripheral blood mononuclear cells from leukoreduction system chambers.Transfusion. 2006; 46: 2083-2089Crossref PubMed Scopus (98) Google Scholar) collected at the blood donor centre, where the mean donor age was 42 years. The study was approved by the West Midlands Research Ethics Committee (05/Q2706/91). Dendritic cells (DCs) were generated from adherent mononuclear cells after incubating in plates for 2 h. Adherent cells were cultured in the medium supplemented on days 0, 3 and 6 with 50 ng/mL GMCSF and 500 U/mL IL-4. On day 6, DCs were matured by adding 2 ng/mL IL-1β, 1000 U/mL IL-6 and 10 ng/mL TNFα (R&D Systems, Minneapolis, USA) (23Jonuleit H Kuhn U Muller G et al.Pro-inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum-free conditions.Eur J Immunol. 1997; 27: 3135-3142Crossref PubMed Scopus (1012) Google Scholar). DCs were recovered after a further 24/48 h. To confirm maturation, they were stained for CD14, CD83, CD86 and HLA class II antigens. DCs were pulsed with the SSCSSCPLSK (SSC) peptide epitope (24Lee SP Tierney RJ Thomas WA Brooks JM Rickinson AB Conserved CTL epitopes within EBV latent membrane protein 2: A potential target for CTL-based tumor therapy.J Immunol. 1997; 158: 3325-3334Crossref PubMed Google Scholar) at a concentration of 10 μg/mL for 2 h, washed and used together with 100 U/mL IL-2 to stimulate transduced T cells at a responder: stimulator ratio of 20: 1. Cell counts were performed using ABX-Pentra 60 (Horiba, Kyoto, Japan). CB mononuclear cells (CBMC) or PBMC were resuspended in RPMI containing 10% FCS, 1% pooled human AB serum (TCS Biosciences, Buckingham, UK), 2 mM L-glutamine, 100 mg/mL streptomycin and 100 IU/mL penicillin and activated with 30 ng/mL anti-CD3 antibody (OKT3) plus 600 U/mL IL-2 (Chiron, Emeryville, USA). Cells were transduced with retrovirus 48 h later. The retrovirus used was the pMP71-PRE vector (25Engels B Cam H Schuler T et al.Retroviral vectors for high-level transgene expression in T lymphocytes.Human Gene Therapy. 2003; 14: 1155-1168Crossref PubMed Scopus (147) Google Scholar) (provided by C. Baum, Hannover, Germany) into which we had inserted genes encoding TCR α and β chains isolated from an EBV-specific CD8+ T cell clone that targets the HLA A*1101-restricted epitope SSC derived from the viral protein LMP2 (manuscript in preparation, Zheng, Lee et al.). To generate the retrovirus, Phoenix amphotropic packaging cells (26Kinsella TM Nolan GP Episomal vectors rapidly and stably produce high-titer recombinant retrovirus.Human Gene Therapy. 1996; 7: 1405-1413Crossref PubMed Scopus (672) Google Scholar) were transfected with the pMP71-PRE vector using FuGENE HD (Roche, Basel, Switzerland). After 48 h the retroviral supernatant was recovered. Preactivated cells were seeded at 4–6 × 106 cells/well in 1 mL RPMI onto 6-well plates coated with retronectin (Takara, Shiga, Japan). Retroviral supernatant (1.5 mL/well) or medium alone (mock-transduced) was added to each well and centrifuged for 1 h × 800 g at 30°C. Medium supplemented with IL-2 (100 U/mL) was added three times weekly. Cells from six CB and six adult PB samples were assayed. Seven and 15 days after transduction, T cells were stimulated with SSC peptide-pulsed DCs. Sixteen days after transduction, CD8+ lymphocytes were isolated using immunomagnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Transduced lymphocytes were identified using HLA A*1101: SSC peptide-specific pentamers (Proimmune, Oxford, UK). Cells were also costained with fluorochrome-conjugated antibodies: anti-CCR7 (FITC; R&D), anti-CD4 (PerCP-Cy5.5 or PE-Cy7; eBioscience, San Diego, CA, USA), anti-CD3 (PE or APC-H7), anti-CD8 (APC or APC-H7), anti-CD27 (APC), anti-CD45RA (PE-Cy7), anti-CD62L (PE) (all from BD) and anti-CD57 (APC; BioLegend, San Diego, CA, USA). 7-aminoactinomycin D (7AAD from BD) was used as a viability marker. For intracellular staining, cells were fixed and permeabilized using the FIX&PERM kit (ADG, Kaumberg, Austria), followed by anti-Perforin FITC (eBioscience). For intracellular cytokine staining, cells were first stimulated with either anti-CD3 plus IL-2 or autologous peptide-pulsed DCs, and 1 h later Monensin (Golgi Stop; 1 μL/mL; BD) was added. After overnight incubation cells were then fixed and permeabilized, and stained with anti-IL-2 FITC, anti-IFNγ APC and anti-TNFα PE-Cy7 (BD). Proliferation was evaluated by staining cells for 2 min with 1 μM carboxyfluorescein succinimidyl ester (CFSE) prior to activation. The enumeration of cells in the different phases of cell cycle or in apoptosis was performed using propidium iodide (PI) (27Moore A Donahue CJ Bauer KD Mather JP Simultaneous measurement of cell cycle and apoptotic cell death.Methods Cell Biol. 1998; 57: 265-278Crossref PubMed Google Scholar). Briefly, the cell pellet was incubated for 30 sec in 200 μL 0.1% Triton ×100 in PBS. Afterward, the same volume of PBS plus PI 50 μg/mL and RNAse 500 μg/mL was added, and samples were analyzed after 10 min at RT. Data were acquired using a FACSCanto II flow cytometer (BD). Cytotoxicity of transduced T cells was assessed in a standard 5-h chromium release assay at known effector: target ratios using 2500 target cells/well. HLA A*1101-transduced T2 cells (28Masucci MG Zhang QJ Gavioli R et al.Immune escape by Epstein–Barr virus (EBV) carrying Burkitts lymphoma—in vitro reconstitution of sensitivity to EBV-specific cytotoxic T-cells.Int Immunol. 1992; 4: 1283-1292Crossref PubMed Scopus (22) Google Scholar) were used as targets and were pulsed with the SSC peptide or another A*1101-restricted peptide epitope (IVTDFSVIK) (29Gavioli R Kurilla MG de Campos-Lima PO et al.Multiple HLA A11-restricted cytotoxic T-lymphocyte epitopes of different immunogenicities in the Epstein-Barr virus-encoded nuclear antigen 4.J Virol. 1993; 67: 1572-1578Crossref PubMed Scopus (153) Google Scholar) as a control. The percentage-specific lysis and lytic units (LU) per 106 effector cells were calculated. One lytic unit was defined as the number of effectors required to achieve lysis of 50% of targets (30Cerottini JC Brunner KT In vitro assay of target cell lysis by sensitized lymphocytes.in: Bloom BR Glade PR In Vitro Methods in Cell-Mediated Immunity. Academic Press Inc., New York1971: 369Crossref Google Scholar). Telomere length measurements using automated multicolor flow-fluorescence in situ hybridization (flow FISH) was performed by Repeat Diagnostics Inc. (North Vancouver, Canada) as described by Baerlocher et al. (31Baerlocher GM Vulto I de Jong G Lansdorp PM Flow cytometry and FISH to measure the average length of telomeres (flow FISH).Nat Protoc. 2006; 1: 2365-2376Crossref PubMed Scopus (326) Google Scholar). Telomerase activity in extracts from cultured lymphocytes was measured using the telomerase detection assay kit (TRAPeze® telomerase Detection, Millipore) according to the manufacturer’s instructions. The statistical analyses were performed using the paired, two-tailed t-test. Initially we determined the relative proportions of naive and memory T cells within CB and compared this to blood from adult donors. T-lymphocytes were categorized into: TN: CCR7+CD27+CD45RA+CD62L+; TCM: CCR7+CD27+CD45RA-CD62L+; TEM: CCR7-CD27+CD45RA-CD62L-; or TEMRA: CCR7-CD27-CD45RA+CD62L- (6Gattinoni L Powell Jr., DJ Rosenberg SA Restifo NP Adoptive immunotherapy for cancer: building on success.Nat Rev Immunol. 2006; 6: 383-393Crossref PubMed Scopus (732) Google Scholar). CCR7 and CD45RA provided sufficient discrimination among the subsets and therefore results based on expression of these two markers are shown hereafter. Phenotypic analysis confirmed that a mean of 81% of T cells in CB samples (n = 6) displayed a TN phenotype in contrast to healthy adult blood donors (n = 6) where TN constituted a mean of 29% (Figure 1A). The memory cell subset within CB comprised predominantly TCM with very few TEM. The proliferation prior to retroviral transduction was monitored by the dilution of CFSE over the next 3 days. This showed an increased proliferation rate within CB cells compared to adult T cells (Figure 1B) and was confirmed with PI staining 48 h after activation which showed more than twice as many CB cells in the S to M phases of cell cycle compared to cells from adult donors. Importantly, this difference was not due to cell death as the proportion of apoptotic cells was the same in both cultures (Figure 1C) For the transduction of EBV-specific TCR genes, CBMC and adult PBMC were activated for 2 days and then infected with the retroviral vector encoding the TCR. Expression of the transduced TCR was determined using HLA-A*1101: SSC pentamers (Figure 2A). The TCR transduction efficiency was similar for both cord and adult lymphocytes (Figure 2B). Pentamer staining of CB CD8 and CD4 T cells showed mean transduction efficiencies of 9.8% and 5.6% respectively whereas for adult CD8 and CD4 cells they were 10.7% and 6.1%, respectively. There was no difference in the mean fluorescence intensity of pentamer staining of T cells from cord or adult blood, indicating comparable levels of surface TCR expression (Figure 2C). We then explored how retroviral transduction affected the differentiation status of CB and adult T cells. Among transduced CB cells, 60% were TCM and most of the remaining cells were TN (Figures 3A and B). In contrast, the majority of transduced adult T cells had a TEM phenotype. This pattern was similar for both CD4 and CD8 T cells and for those T cells that failed to express the TCR (Figure 3A and B). The ultimate aim of these studies would be to use TCR-transduced CB T cells in ACT. We therefore studied the proliferation, differentiation phenotype and cytokine production of such cells after prolonged in vitro culture and exposure to antigen. TCR-transduced CB and adult T cells were expanded using two rounds of stimulation with SSC peptide-pulsed autologous DCs. After 23 days of culture, both CB and adult T cells had expanded 50-fold with cell numbers continuing to rise (Figure 4A). Preferential expansion of transduced cells occurred so that by day 23 they comprised 50% of the CD8+ T cell population (Figure 4B). Comparing the differentiation status of transduced CB and adult T cells after stimulation with peptide-pulsed DCs we observed consistent differences especially within the CD8+ T cell subset (Figure 5A and B). Transduced CD4+ CB T cells were initially dominated by TN and TCM, the latter twice as common, while adult T cells were dominated by equal proportions of TCM and TEM with minimal TN. However, following stimulation with antigen the TEM expanded markedly in both populations while TCM proportions fell. After 16 days the final product was comparable in both cultures with means of 55–65% TEM cells. However, the differentiation status of CD8+ T cells during expansion was markedly different between cord and adult blood. CD8+ T cells in CB showed a memory cell transition that was similar to CD4+ CB T cells, with the proportion of TCM decreasing and TEM increasing until they dominated the population reaching a mean of 54.8% at the end of the culture period. Very few TEMRA were observed. In contrast, adult T cells showed a sharp decline in the proportion of both TCM and TEM subsets during expansion, and a marked increase in TEMRA which comprised a mean of 66.2% of the final product. At the end of the culture period, while 32.1% of CB CD8+ T cells retained the least differentiated TCM or TN phenotype, these two subsets accounted for only 5.4% of adult CD8+ cells. Note that only the transduced lymphocytes moved towards a more differentiated phenotype indicating that differentiation was driven by antigen-specific recognition (Figure 5A). Transduced CB CD8+ T cells therefore display a less differentiated phenotype compared to adult T cells throughout the in vitro culture period although this difference is not seen within the CD4 population. In addition, after 23 days of in vitro expansion, CD27, a marker of less differentiated T cells, was expressed on 85.5% of transduced CB CD8+ cells but only 39.1% of transduced adult CD8+ cells (Figure 5C). Similarly, after 13 days of in vitro expansion, CD57, a marker of replicative senescence and antigen-induced apoptotic death of T cells (32Brenchley JM Karandikar NJ Betts MR et al.Expression of CD57 defines replicative senescence and antigen-induced apoptotic death of CD8 +T cells.Blood. 2003; 101: 2711-2720Crossref PubMed Scopus (763) Google Scholar), was expressed on only 17.8% of transduced CD8+ CB T cells but 56.9% of adult CD8+ cells (Figure 5D). TCR-transduced adult and CB T cells were taken at day 0 (prestimulation) and day 21 (4 days after last stimulation) and assayed for telomere length (Figure 6A). Both adult and CB samples (with the exception of CB1) showed comparable rates of telomere length decline over 21 days of culture, consistent with expected shortening following cell proliferation, but telomeres were consistently longer in CB cells. Telomerase activity was assessed at day 3 after stimulation and again at day 21 (Figure 7B and C). Two out of three CB cells maintained relatively high levels of telomerase activity, indicative of greater proliferative potential, whereas all adult T cells showed a decline in telomerase activity to relatively low levels at day 21. Interestingly CB 1 lost telomerase activity by day 21, and was the only sample to show an increase in telomere length during the culture period.Figure 7:TCR-transduced cord blood T-lymphocytes can secrete cytokines and mediate cytolytic function. (A) Intracellular staining for IFNγ, IL-2 and TNFα was performed 24 h after activation with anti-CD3 (day 1) and 24 h after the first restimulation with autologous peptide-pulsed DCs (day 10). Mean percentage plus standard deviation of CD8 T lymphocytes staining for IFNγ and for the different combinations of cytokines are displayed for CBMC (n = 3, white symbols) and for PBMC (n = 3, gray symbols). The percentage of cells staining for all three cytokines was evaluated by first gating on double positive IFNγ/TNFα staining cells and then analyzed for IL-2 expression. (B) Intracellular perforin was assessed by flow cytometry on days 6 and 23 postactivation. The percentages of perforin-positive cells are indicated. One representative experiment of three is shown. (C) CD8 T-lymphocytes from CBMC (left panel) and from PBMC (right panel) were assayed for specific cell lysis activity against HLA A*1101-transduced T2 cells at the effector to target (E:T) ratios indicated. The 51Cr release assay was performed in the presence of an irrelevant peptide, DMSO or different concentrations of the cognate SSC peptide. Results show the mean% specific lysis plus standard deviation from three separate experiments. (D) The same data are expressed as mean lytic units with gray bars for PBMC and white bars for CBMC.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To investigate the effector function of the transduced cells we determined their cytokine production profile. T cells which secrete multiple cytokines are considered polyfunctional and this correlates with optimally functioning effector cells (33Kannanganat S Ibegbu C Chennareddi L Robinson HL Amara RR Multiple-cytokine-producing antiviral CD4 T cells are functionally superior to single-cytokine-producing cells.J Virol. 2007; 81: 8468-8476Crossref PubMed Scopus (255) Google Scholar,34Darrah PA Patel DT De Luca PM et al.Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major.Nat Med. 2007; 13: 843-850Crossref PubMed Scopus (1111) Google Scholar). As such, the cultured cells were analyzed for production of IFNγ, IL-2 and TNFα on day 1 (24 h after stimulation with anti-CD3) and on day 10 (24 h after stimulation with peptide-pulsed DCs) (Figure 7A). On day 1 IFNγ secretion was negligible in CBMC but detectable in PBMC. However, after restimulation, the proportion of IFNγ−secreting CD8 cells in both CBMC and PBMC increased to approximately half of both populations. Polyfunctional CBMC secreting more than one cytokine also increased several fold upon restimulation, exceeding multicytokine secretion by PBMC. Compared to PBMC, the proportion of CD8+ CBMC secreting all three cytokines was significantly greater (p = 0.025). This pattern likely reflects the earlier differentiation phenotype of CB T cells and the need for antigen rechallenge before full effector function is activated. Antigenic stimulation of transduced CD8 T cells from both cord and adult blood led to increased expression of intracellular perforin (Figure 7B) a marker of cytotoxic potential. Cytotoxic function was assessed using a chromium release assay. Lysis of HLA-A*1101-transfecte" @default.
• W2136875369 created "2016-06-24" @default.
• W2136875369 creator A5020948141 @default.
• W2136875369 creator A5032956379 @default.
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• W2136875369 date "2013-01-01" @default.
• W2136875369 modified "2023-09-23" @default.
• W2136875369 title "Cord Blood T Cells Retain Early Differentiation Phenotype Suitable for Immunotherapy After TCR Gene Transfer to Confer EBV Specificity" @default.
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