FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1970867883> ?p ?o ?g. }

• W1970867883 endingPage "2017" @default.
• W1970867883 startingPage "2006" @default.
• W1970867883 abstract "Adoptive transfer of antigen-specific cytotoxic T lymphocytes has shown promise for the therapy of cancer. However, tumor-specific T cells are susceptible to diverse inhibitory signals from the tumor microenvironment. The Akt/protein kinase B plays a central role in T-cell proliferation, function, and survival and we hypothesized that expression of constitutively active Akt (caAkt) in T cells could provide resistance to many of these tumor-associated inhibitory mechanisms. caAkt expression in activated human T cells increased proliferation and cytokine production, a likely result of their sustained expression of nuclear factor-κB (NF-κB) and provided resistance to apoptosis by upregulating antiapoptotic molecules. caAkt expressing T cells (caAkt-T-cells) were also relatively resistant to suppression by and conversion into regulatory T cells (Tregs). These characteristics provided a survival advantage to T cells cocultured with tumor cells in vitro; CD3/28-stimulated T cells expressing a chimeric antigen receptor (CAR) specific for disialoganglioside (GD2) that redirected their activity to the immunosuppressive, GD2-expressing neuroblastoma cell line, LAN-1, resisted tumor-induced apoptosis when co-expressing transgenic caAkt. In conclusion, caAkt-transduced T cells showed resistance to several evasion strategies employed by tumors and may therefore enhance the antitumor activity of adoptively transferred T lymphocytes. Adoptive transfer of antigen-specific cytotoxic T lymphocytes has shown promise for the therapy of cancer. However, tumor-specific T cells are susceptible to diverse inhibitory signals from the tumor microenvironment. The Akt/protein kinase B plays a central role in T-cell proliferation, function, and survival and we hypothesized that expression of constitutively active Akt (caAkt) in T cells could provide resistance to many of these tumor-associated inhibitory mechanisms. caAkt expression in activated human T cells increased proliferation and cytokine production, a likely result of their sustained expression of nuclear factor-κB (NF-κB) and provided resistance to apoptosis by upregulating antiapoptotic molecules. caAkt expressing T cells (caAkt-T-cells) were also relatively resistant to suppression by and conversion into regulatory T cells (Tregs). These characteristics provided a survival advantage to T cells cocultured with tumor cells in vitro; CD3/28-stimulated T cells expressing a chimeric antigen receptor (CAR) specific for disialoganglioside (GD2) that redirected their activity to the immunosuppressive, GD2-expressing neuroblastoma cell line, LAN-1, resisted tumor-induced apoptosis when co-expressing transgenic caAkt. In conclusion, caAkt-transduced T cells showed resistance to several evasion strategies employed by tumors and may therefore enhance the antitumor activity of adoptively transferred T lymphocytes. T lymphocytes are major effector cells in adaptive immunity against pathogens and tumor cells. Their efficacy, however, is dampened by a wide range of immune evasion mechanisms that exist in a diversity of pathogenic circumstances including tumorigenesis, when the homeostatic balance between activation and suppression of immunity strongly favors immune suppression. This imbalance allows tumors to progress even in the presence of tumor-specific T cells. Adoptive T-cell therapy is an effective treatment for virus-associated diseases and malignancies after hematopoietic stem cell transplantation1Rooney CM Smith CA Ng CY Loftin S Li C Krance RA et al.Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr-virus-related lymphoproliferation.Lancet. 1995; 345: 9-13Abstract PubMed Scopus (990) Google Scholar,2Riddell SR Walter BA Gilbert MJ Greenberg PD Selective reconstitution of CD8+ cytotoxic T lymphocyte responses in immunodeficient bone marrow transplant recipients by the adoptive transfer of T cell clones.Bone Marrow Transplant. 1994; 14 (Suppl 4): S78-S84PubMed Google Scholar,3Rooney CM Smith CA Ng CY Loftin SK Sixbey JW Gan Y et al.Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients.Blood. 1998; 92: 1549-1555Crossref PubMed Google Scholar and for lymphoma and melanoma in immunocompetent patients.4Straathof KC Bollard CM Popat U Huls MH Lopez T Morriss MC et al.Treatment of nasopharyngeal carcinoma with Epstein-Barr virus–specific T lymphocytes.Blood. 2005; 105: 1898-1904Crossref PubMed Scopus (305) Google Scholar,5Dudley ME Wunderlich JR Yang JC Sherry RM Topalian SL Restifo NP 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,6Morgan RA Dudley ME Wunderlich JR Hughes MS Yang JC Sherry RM et al.Cancer regression in patients after transfer of genetically engineered lymphocytes.Science. 2006; 314: 126-129Crossref PubMed Scopus (2140) Google Scholar,7Yee C Thompson JA Byrd D Riddell SR Roche P Celis E et al.Adoptive T cell therapy using antigen-specific CD8+ T cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, migration, and antitumor effect of transferred T cells.Proc Natl Acad Sci USA. 2002; 99: 16168-16173Crossref PubMed Scopus (1008) Google Scholar T-cell therapy has high specificity and low toxicity compared with other conventional cancer therapies, but the survival and function of adoptively transferred T cells are limited by the same immune evasion mechanisms that impede endogenous antitumor immune responses, including ineffective presentation of tumor antigens, induction of effector T-cell apoptosis,8Hahne M Rimoldi D Schröter M Romero P Schreier M French LE et al.Melanoma cell expression of Fas(Apo-1/CD95) ligand: implications for tumor immune escape.Science. 1996; 274: 1363-1366Crossref PubMed Scopus (1202) Google Scholar production of T-cell inhibitory ligands such as transforming growth factor (TGF)-β and programmed death ligand-1, and recruitment of regulatory T cells (Tregs).9Thomas DA Massagué J TGF-beta directly targets cytotoxic T cell functions during tumor evasion of immune surveillance.Cancer Cell. 2005; 8: 369-380Abstract Full Text Full Text PDF PubMed Scopus (902) Google Scholar,10Gorelik L Flavell RA Immune-mediated eradication of tumors through the blockade of transforming growth factor-beta signaling in T cells.Nat Med. 2001; 7: 1118-1122Crossref PubMed Scopus (639) Google Scholar,11Dong H Strome SE Salomao DR Tamura H Hirano F Flies DB et al.Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion.Nat Med. 2002; 8: 793-800Crossref PubMed Scopus (0) Google Scholar,12Iwai Y Ishida M Tanaka Y Okazaki T Honjo T Minato N Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade.Proc Natl Acad Sci USA. 2002; 99: 12293-12297Crossref PubMed Scopus (2291) Google Scholar,13Curiel TJ Coukos G Zou L Alvarez X Cheng P Mottram P et al.Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival.Nat Med. 2004; 10: 942-949Crossref PubMed Scopus (4135) Google Scholar,14Yu P Lee Y Liu W Krausz T Chong A Schreiber H et al.Intratumor depletion of CD4+ cells unmasks tumor immunogenicity leading to the rejection of late-stage tumors.J Exp Med. 2005; 201: 779-791Crossref PubMed Scopus (391) Google Scholar The efficacy of adoptively transferred T cells therefore depends on their capacity to survive and function in the presence of multiple inhibitory signals. Investigators are exploring strategies to enable tumor-specific T cells to survive and function in an adverse tumor environment in both preclinical and clinical systems. For example, resistance to apoptosis can be accomplished by downregulation of receptors for inhibitory ligands like Fas-ligand and TGF-β15Dotti G Savoldo B Pule M Straathof KC Biagi E Yvon E et al.Human cytotoxic T lymphocytes with reduced sensitivity to Fas-induced apoptosis.Blood. 2005; 105: 4677-4684Crossref PubMed Scopus (104) Google Scholar,16Bollard CM Rössig C Calonge MJ Huls MH Wagner HJ Massague J et al.Adapting a transforming growth factor beta-related tumor protection strategy to enhance antitumor immunity.Blood. 2002; 99: 3179-3187Crossref PubMed Scopus (265) Google Scholar or by expression of immunostimulatory cytokines including interleukin-7 (IL-7) and IL-12.17Vera JF Hoyos V Savoldo B Quintarelli C Giordano Attianese GM Leen AM et al.Genetic manipulation of tumor-specific cytotoxic T lymphocytes to restore responsiveness to IL-7.Mol Ther. 2009; 17: 880-888Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar,18Mehrotra PT Grant AJ Siegel JP Synergistic effects of IL-7 and IL-12 on human T cell activation.J Immunol. 1995; 154: 5093-5102PubMed Google Scholar,19Foster AE Leen AM Lee T Okamura T Lu A Vera J et al.Autologous designer antigen-presenting cells by gene modification of T lymphocyte blasts with IL-7 and IL-12.J Immunother. 2007; 30: 506-516Crossref PubMed Scopus (21) Google Scholar Although each strategy has effectively circumvented the individual suppressive mechanism for which it was designed, avoidance of a single immune evasion strategy may be insufficient to ensure T-cell function, survival, and proliferation in a complex, multifactorial, inhibitory environment. We reasoned that a strategy that could render tumor-specific T cells resistant to a broader range of inhibitory mechanisms might prove more effective. The serine/threonine kinase Akt is a major component of the phosphatidylinositol 3-kinase (PI3K) family, and is critical for cell growth and survival.20Manning BD Cantley LC AKT/PKB signaling: navigating downstream.Cell. 2007; 129: 1261-1274Abstract Full Text Full Text PDF PubMed Scopus (4762) Google Scholar The PI3K/Akt pathway is central to T-cell activation, acting downstream of major T-cell-activating signals including the T-cell receptor, co-stimulatory receptors, and cytokine receptors.21Song J Lei FT Xiong X Haque R Intracellular signals of T cell costimulation.Cell Mol Immunol. 2008; 5: 239-247Crossref PubMed Scopus (58) Google Scholar Ligation of the T-cell co-stimulatory molecules, CD28 and ICOS, upregulates Akt activity by activation of tyrosine kinases,22Wu LX La Rose J Chen L Neale C Mak T Okkenhaug K et al.CD28 regulates the translation of Bcl-xL via the phosphatidylinositol 3-kinase/mammalian target of rapamycin pathway.J Immunol. 2005; 174: 180-194Crossref PubMed Scopus (53) Google Scholar leading to membrane recruitment of PI3K and Akt. Conversely, the Akt pathway is a central node downstream of multiple co-inhibitory receptors, including cytotoxic T lymphocyte antigen-4 and Programmed Death-1 that oppose the activation of Akt by counteracting tyrosine phosphatases.23Schneider H Prasad KV Shoelson SE Rudd CE CTLA-4 binding to the lipid kinase phosphatidylinositol 3-kinase in T cells.J Exp Med. 1995; 181: 351-355Crossref PubMed Scopus (147) Google Scholar PI3K/Akt activation is reported to suppress several apoptotic mechanisms in T cells24Su CC Lin YP Cheng YJ Huang JY Chuang WJ Shan YS et al.Phosphatidylinositol 3-kinase/Akt activation by integrin-tumor matrix interaction suppresses Fas-mediated apoptosis in T cells.J Immunol. 2007; 179: 4589-4597Crossref PubMed Scopus (25) Google Scholar,25Hähnel PS Thaler S Antunes E Huber C Theobald M Schuler M Targeting AKT signaling sensitizes cancer to cellular immunotherapy.Cancer Res. 2008; 68: 3899-3906Crossref PubMed Scopus (64) Google Scholar and the activation status of the PI3K/Akt pathway in T effector cells is a primary determinant of T effector sensitivity to a number of inhibitory factors, including TGF-β and Tregs.24Su CC Lin YP Cheng YJ Huang JY Chuang WJ Shan YS et al.Phosphatidylinositol 3-kinase/Akt activation by integrin-tumor matrix interaction suppresses Fas-mediated apoptosis in T cells.J Immunol. 2007; 179: 4589-4597Crossref PubMed Scopus (25) Google Scholar,25Hähnel PS Thaler S Antunes E Huber C Theobald M Schuler M Targeting AKT signaling sensitizes cancer to cellular immunotherapy.Cancer Res. 2008; 68: 3899-3906Crossref PubMed Scopus (64) Google Scholar,26Wohlfert EA Clark RB ‘Vive la Résistance!'–the PI3K-Akt pathway can determine target sensitivity to regulatory T cell suppression.Trends Immunol. 2007; 28: 154-160Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar Thus, Akt targeting could both enhance activating stimuli and provide resistance to inhibitory signals. Expression of a highly potent, constitutively active Akt (caAkt; originally called MF-ΔAkt) was previously shown to promote the survival of dendritic cells by upregulating Bcl-2 as well as promoting activation and maturation.23Schneider H Prasad KV Shoelson SE Rudd CE CTLA-4 binding to the lipid kinase phosphatidylinositol 3-kinase in T cells.J Exp Med. 1995; 181: 351-355Crossref PubMed Scopus (147) Google Scholar,27Park D Lapteva N Seethammagari M Slawin KM Spencer DM An essential role for Akt1 in dendritic cell function and tumor immunotherapy.Nat Biotechnol. 2006; 24: 1581-1590Crossref PubMed Scopus (89) Google Scholar Here, we show that caAkt increases nuclear factor-κB (NF-κB) activity in human T cells, and promotes their proliferation, survival, and resistance to multiple inhibitory factors commonly utilized by tumors. caAkt-transduced T cells were also resistant to suppression by Tregs and to conversion into Tregs by TGF-β. Finally, co-expression of caAkt with a chimeric antigen receptor (CAR) for disialoganglioside (GD2) expressed on neuroblastoma, enhanced T-cell activation, and proliferation in response to and cytotoxicity against this immunosuppressive tumor. We transduced CD3/CD28 antibody–stimulated T cells with the retrovirus vector MSCV-MF-hΔAkt-IRES-GFP (caAkt), encoding constitutively active Akt as illustrated in Figure 1a. As determined by co-expression of GFP, 38.7% (34.2–45.7%, n = 7) cells were transduced with caAkt. Representative histograms of caAkt- and GFP control (control)–transduced T cells are shown in Figure 1b. Both CD4 and CD8 T cells were efficiently transduced (data not shown). To confirm the activation state of caAkt, we used intracellular fluorescence-activated cell-sorting analysis with an antibody specifically binding to the Akt phosphorylation site (S473). The percentage of T cells expressing phosphorylated Akt (pAkt) was consistent with GFP expression in caAkt-transduced T cells as 35.3–45% of the caAkt was phosphorylated, compared to ~1% controls (Figure 1c). These results indicated that the transduced caAkt gene was expressed in its active (phosphorylated) state in transduced T cells. To determine whether caAkt influenced T-cell growth, we cultured caAkt-transduced T cells in the presence of IL-2 (50 U/ml) and measured cell numbers over time. T cells expressing caAkt demonstrated greater numerical expansion than control-transduced T cells after 3 weeks of culture (mean 47 × 106 versus 24 × 106 T cells from 1 × 106 starting T cells). Without additional stimulation, caAkt-T-cells did not proliferate further, but maintained their viability, whereas control-T-cell numbers and viability declined (Figure 2a). To examine whether caAkt gene expression was stable over time, we analyzed the frequency of GFP-expressing cells and found selective expansion of caAkt-transduced T cells from 35% (range 31–39%) to 83% (range 76–91%) over the first 2 weeks of culture, while there was no selection of GFP+ cells in control cultures (Figure 2b, P = 0.01). Hence, caAkt-transduced T cells had a selective growth/survival advantage in culture. The selective expansion of caAkt-T-cells may be due to an increased rate of T-cell division and/or reduced apoptosis. We measured proliferation and apoptosis of transduced T cells at early (7–14 days) and late (4–5 weeks) time points. Early on, there were few apoptotic cells in either group, while caAkt-T-cells demonstrated a significantly higher rate of cell division in the absence or presence of IL-2 as measured using tritiated thymidine uptake (Figure 2c,d). The difference was even more marked in the cultures without IL-2 supplementation (Figure 2c). Four to five weeks later, the proliferation of both groups of transduced T cells dropped to basal levels (data not shown) but caAkt-T-cells had superior survival. Apoptosis as determined by surface staining with 7-aminoactinomycin D and annexin V was markedly higher in control-T-cells than in caAkt-T-cells (35.6 ± 5.2% versus 11.1 ± 2.9%, n = 5, P < 0.01; Figure 2e,f). In addition to proliferation and apoptosis, we evaluated telomere length in caAkt-T-cells using the Q-FISH and qPCR methods (Supplementary Materials and Methods) and found that telomeres were longer than in control-T-cells (Supplementary Figure S1a,b). Consistent with increased telomere length, caAkt-T-cells possessed stronger telomerase activity than control-T-cells (Supplementary Figure S1c). In summary, caAkt-T-cells had increased proliferation shortly after activation, maintained cell survival for significantly longer than control-T-cells but did not proliferate autonomously in the absence of prosurvival cytokines (Supplementary Figure S2). To determine the mechanism underlying decreased apoptosis in caAkt-expressing T cells, we examined the expression of antiapoptotic members of the Bcl-2 family that act downstream of Akt. T cells were harvested 3–4 weeks after transduction and cultured without IL-2 for 5 days. As shown before, the percentage of apoptotic cells was significantly higher in control-T-cells than in caAkt-T-cells. Increased levels of Bcl-2, Bcl-xL, and Mcl-1 expression were detected by intracellular staining (Figure 3a), and confirmed by western blot analysis at multiple time points (Figure 3b). The antiapoptotic molecules (especially Mcl-1 and Bcl-xL) were maintained at higher levels in caAkt-T-cells than in control-T-cells. The upregulation of these antiapoptotic molecules likely contributes to the longevity of T cells transduced with caAkt. To evaluate the effects of caAkt on cytokine production, we measured production of various cytokines in response to T-cell receptor stimulation. Figure 4a,b shows that 2 weeks after transduction, caAkt-expressing T cells produced significantly more IL-2 and interferon-γ (IFN-γ) as measured by intracellular staining analysis. There were comparable low levels of IL-4, IL-10, IL-17 (<2%) in both caAkt-T-cells and control-T-cells (data not shown). Therefore, the production of Th1 cytokines dominates in caAkt-T-cells and should favor the cytotoxic function of Th1 cells. To characterize events downstream of Akt activation, we utilized Phosflow to detect the kinetics of NF-κB activation (Figure 4c). Both control-T-cells and caAkt-T-cells upregulated p65-NF-κB to comparable levels at 15 minutes to 6 hours after phorbol myristate acetate stimulation (from 15 minutes to 6 hours). However, p65-NF-κB expression was greater in caAkt-T-cells after 24 hours; and by 72 hours while there was no detectable p65-NF-κB activity in control-T-cells in the absence of cytokines, caAkt-T-cells maintained NF-κB levels. Thus, caAkt induced and maintained NF-κB activity, which likely contributes to T-cell survival and cytokine production. Tregs infiltrate most tumors and add their inhibitory effects to those of the tumor cells themselves. Because Tregs inhibit T effector cells through Akt-regulated pathways,28Kojima H Kanno Y Hase H Kobata T CD4+CD25+ regulatory T cells attenuate the phosphatidylinositol 3-kinase/Akt pathway in antigen-primed immature CD8+ CTLs during functional maturation.J Immunol. 2005; 174: 5959-5967Crossref PubMed Scopus (13) Google Scholar we determined whether T cells expressing caAkt could resist Treg inhibition. We transduced peripheral blood mononuclear cells (PBMCs) from four healthy donors with caAkt or GFP, as described previously.15Dotti G Savoldo B Pule M Straathof KC Biagi E Yvon E et al.Human cytotoxic T lymphocytes with reduced sensitivity to Fas-induced apoptosis.Blood. 2005; 105: 4677-4684Crossref PubMed Scopus (104) Google Scholar CD4+CD25+ Tregs were isolated from autologous fresh PBMC using a Treg selection kit (Miltenyi Biotec, Bisley, UK). At 10 days after transduction, caAkt- and control-T-cells were labeled with the lipophilic dye, PKH26 Red, to allow cell division to be measured by flow cytometry, and cultured with or without autologous CD4+CD25+ Treg cells at different ratios in the presence of allogeneic PBMC and OKT3 MAb (500 ng/ml). Five days later, the proliferation of responder T cells was assessed from their PKH dye intensity. Compared with GFP-T-cells, caAkt-T-cells were resistant to Treg-mediated suppression and continued to proliferate as illustrated in Figure 5a. At a 3:1 ratio of responder T cells to Tregs, GFP-T-cells were inhibited by 43.6% (range 42.2–49.1%), compared to only 9.6% (range 0–18.3%) in caAkt-T-cells (Figure 5b, P < 0.01). These data directly illustrate the ability of caAkt to control the sensitivity of effector T cells to Treg suppression. In addition to their susceptibility to suppression by Tregs, effector T cells are also susceptible to conversion into Treg cells at tumor sites by mechanisms involving TGF-β.29Liu VC Wong LY Jang T Shah AH Park I Yang X et al.Tumor evasion of the immune system by converting CD4+CD25- T cells into CD4+CD25+ T regulatory cells: role of tumor-derived TGF-beta.J Immunol. 2007; 178: 2883-2892Crossref PubMed Scopus (387) Google Scholar The Akt-mTOR axis has been reported to regulate FoxP3, the key transcription factor of Tregs.30Sauer S Bruno L Hertweck A Finlay D Leleu M Spivakov M et al.T cell receptor signaling controls Foxp3 expression via PI3K, Akt, and mTOR.Proc Natl Acad Sci USA. 2008; 105: 7797-7802Crossref PubMed Scopus (686) Google Scholar,31Haxhinasto S Mathis D Benoist C The AKT-mTOR axis regulates de novo differentiation of CD4+Foxp3+ cells.J Exp Med. 2008; 205: 565-574Crossref PubMed Scopus (609) Google Scholar We therefore investigated the effect of caAkt activity on the induction of Tregs by TGF-β. Transduced CD3/28-stimulated T cells were restimulated on day 14 with anti-CD3/CD28 in the presence of TGF-β (2.5 ng/ml) and IL-2 (50 U/ml). Two weeks after reactivation, we observed significant elevations of FoxP3+ T cells in both caAkt- and control-T-cells cultured with TGF-β. However, the frequency of FoxP3+ T cells was markedly lower in caAkt-T-cells than in control-T-cells (4.8 ± 2.1% versus 14.5 ± 4.3% without TGF-β and 17 ± 6.5% versus 46.9 ± 8.6% with TGF-β, n = 4, P < 0.01). A representative histogram is shown in Figure 5c. To determine whether the induced FoxP3+ T cells were suppressive, we used TGF-β-treated T cells as suppressor cells (as there is no appropriate selection marker for these FoxP3+ T cells) and autologous PBMC as responders. TGF-β-activated caAkt-T-cells showed decreased suppressive capacity relative to TGF-β-activated control-T-cells (Figure 5d), which is consistent with their reduced expression of FoxP3. These data indicate that caAkt provides resistance to FoxP3+ Treg induction/conversion so that TGF-β-cultured caAkt-T-cells are less suppressive. We also determined whether Akt overexpression inhibits natural Treg function and FoxP3 expression; CD4+CD25+ T cells were purified from buffy coats and stimulated with anti-CD3 plus allogeneic PBMCs, then transduced with MSCV-MF-hΔAkt-IRES-GFP or control vector MSCV-IRES-GFP. caAkt-T-cells expressed less FoxP3 (35.2 ± 9.4% versus 61.5 ± 10.7%, n = 4, P < 0.05) and elevated levels of IL-2 (68.3 ± 5.8% versus 41.6 ± 8.1%, P < 0.01) compared to control-T-cells. Representative data are shown in Supplementary Figures S3 and S4a. Consistent with these findings, caAkt-transduced Tregs lost their suppressive capacity (Supplementary Figure S4b). Thus, transgenic caAkt can both enhance effector T cells and suppress the function of pre-existing Tregs. To determine whether constitutive Akt expression increases survival of tumor-specific T cells in a tumor model, we redirected the antigen specificity of T cells to the GD2 that is expressed on neuroblastoma cells by expression of a CAR. The GD2-CAR was generated by joining the heavy- and light-chain variable regions of a GD2-specific monoclonal antibody expressed as a single-chain Fv molecule, to the cytoplasmic CD3-ζ of the T-cell receptor signaling domain.32Rossig C Bollard CM Nuchtern JG Merchant DA Brenner MK Targeting of G(D2)-positive tumor cells by human T lymphocytes engineered to express chimeric T-cell receptor genes.Int J Cancer. 2001; 94: 228-236Crossref PubMed Scopus (132) Google Scholar We used the LAN-1 neuroblastoma cell line that highly expresses GD2, but lacks co-stimulatory and MHC class II molecules (Figure 6a) and actively inhibits the proliferation of GD2-CAR expressing T cells.33Pulè MA Straathof KC Dotti G Heslop HE Rooney CM Brenner MK A chimeric T cell antigen receptor that augments cytokine release and supports clonal expansion of primary human T cells.Mol Ther. 2005; 12: 933-941Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar We cotransduced caAkt or control GFP-vector with the GD2-CAR into T cells and cocultured them with irradiated LAN-1 neuroblastoma cells. As shown in Figure 6b, GD2-CAR and caAkt (or GFP control) could be co-expressed in T cells at comparable levels. Coculture with LAN-1 cells at different ratios for 48 hours suppressed control-T-cell proliferation in a dose-dependent manner as measured by tritiated thymidine assays. This is likely due to stimulation through the ζ chain of the TCR in the absence of co-stimulation. Compared with control-T-cells, caAkt/GD2-CAR-cotransduced T cells showed greater proliferation and resisted LAN-1-mediated suppression (Figure 6c). To begin to understand the mechanism of T-cell suppression by LAN-1 and the resistance provided by caAkt, we measured GD2-CAR transduced T-cell apoptosis by 7-aminoactinomycin D and annexin V staining after weekly stimulation by LAN-1. caAkt GD2-CAR-cotransduced T cells had greater resistance to multiple LAN-1 stimulations than GFP/GD2-CAR-cotransduced T cells (21 ± 3.3% versus 54 ± 14.5% apoptosis after the 2nd stimulation, 46 ± 4.5% versus 77 ± 7.6% after 3rd stimulation, n = 5, P < 0.05) as illustrated in Figure 6d,e. These data indicate that co-expression of caAkt supports the survival of tumor-antigen-specific T cells in the presence of LAN-1 neuroblastoma cells. As caAkt-cotransduced GD2-specific T cells maintained higher viability during their encounters with tumor cells, we next investigated their effector functions after these tumor cocultures. We detected higher granzyme B production in caAkt-cotransduced T cells, particularly in the CD8+ subset (Figure 7a), as well as higher levels of IL-2 and IFN-γ (Figure 7b,c). To evaluate the cytotoxicity of caAkt-transduced T cells, we cocultured them with LAN-1. In control-T-cell cultures, LAN-1 cells represented over 40% of the total cell number after 5 days, but were only 8% in cocultures with caAkt-cotransduced T cells (Figure 7d). caAkt-cotransduced T cells eliminated LAN-1 cells more efficiently than GFP control cells (5.33 ± 1.4% versus 25.03 ± 6.7% LAN-1 remaining in the culture, n = 4, P < 0.05; Figure 7e). Altogether, caAkt-cotransduced tumor-specific T cells retained higher activity than control-transduced T cells on exposure to tumor. We have demonstrated that a constitutively active Akt provides T cells with resistance to multiple distinct tumor immune inhibitory strategies. Both proliferation and survival of caAkt-expressing T cells were increased, an effect that was particularly evident under conditions of cytokine deprivation, and on exposure to TGF-β and Tregs. These characteristics could be explained by increased NF-κB activity, which upregulated the antiapoptotic molecules, Bcl-2, Bcl-xL, and Mcl-1 and the Th1 cytokines IL-2 and IFN-γ after stimulation. Finally, T cells co-expressing caAkt with a chimeric tumor antigen receptor had superior survival and function in the presence of an immunosuppressive neuroblastoma cell line. Despite their enhanced proliferation and effector functions, T cells expressing caAkt did not grow autonomously, but required continued stimulation with cytokines and antigen. However, to ensure the safety of this strategy, it would be possible to co-express suicide genes34Straathof KC Spencer DM Sutton RE Rooney CM Suicide genes as safety switches in T lymphocytes.Cytotherapy. 2003; 5: 227-230Abstract Full Text PDF PubMed Scopus (26) Google Scholar,35Ramos CA Asgari Z Liu E Yvon E Heslop HE Rooney CM et al.An inducible caspase 9 suicide gene to improve the safety of mesenchymal stromal cell therapies.Stem Cells. 2010; 28: 1107-1115Crossref PubMed Scopus (72) Google Scholar or to use an inducible caAkt construct that is expressed only in presence of 4-hydroxytamoxifen (data not shown). Thus, forced expression of constitutively active Akt may replace a multiplicity of other manipulations of tumor-specific T cells intended as countermeasures for the broad range of immune evasion strategies employed by human tumors. Potentially immunogenic tumors have evolved an array of immune evasion strategies that may present tumor antigens in an inhibitory context or directly or indirectly modulate effector T-cell induction and function. Several strategies to overcome specific tumor-mediated effects have been developed, such as expression of dominant-negative receptors for inhibitory molecules like TGF-β, short-hairpin RNA for the apoptotic receptor Fas and prosurvival cytokines to maintain T-cell proliferation and disrupt the inhibitory tumor environment.15Dotti G Savoldo B Pule M Straathof KC Biagi E Yvon E et al.Human cytotoxic T lymphocytes with reduced sensitivity to Fas-in" @default.
• W1970867883 created "2016-06-24" @default.
• W1970867883 creator A5001523714 @default.
• W1970867883 creator A5016693322 @default.
• W1970867883 creator A5023649972 @default.
• W1970867883 creator A5048236963 @default.
• W1970867883 creator A5048367073 @default.
• W1970867883 creator A5057196933 @default.
• W1970867883 creator A5063335678 @default.
• W1970867883 creator A5065544223 @default.
• W1970867883 date "2010-11-01" @default.
• W1970867883 modified "2023-10-06" @default.
• W1970867883 title "T Cells Expressing Constitutively Active Akt Resist Multiple Tumor-associated Inhibitory Mechanisms" @default.
• W1970867883 cites W1444462873 @default.
• W1970867883 cites W1505221840 @default.
• W1970867883 cites W1514448256 @default.
• W1970867883 cites W1548372537 @default.
• W1970867883 cites W1550192924 @default.
• W1970867883 cites W1675058229 @default.
• W1970867883 cites W1737344958 @default.
• W1970867883 cites W1966203378 @default.
• W1970867883 cites W1973262582 @default.
• W1970867883 cites W1977830709 @default.
• W1970867883 cites W1984621921 @default.
• W1970867883 cites W1999843922 @default.
• W1970867883 cites W2008686731 @default.
• W1970867883 cites W2011934933 @default.
• W1970867883 cites W2016198585 @default.
• W1970867883 cites W2016671320 @default.
• W1970867883 cites W2016983859 @default.
• W1970867883 cites W2020099065 @default.
• W1970867883 cites W2024120152 @default.
• W1970867883 cites W2031835772 @default.
• W1970867883 cites W2037787006 @default.
• W1970867883 cites W2040074251 @default.
• W1970867883 cites W2045796964 @default.
• W1970867883 cites W2049794524 @default.
• W1970867883 cites W2050247437 @default.
• W1970867883 cites W2063264144 @default.
• W1970867883 cites W2074604431 @default.
• W1970867883 cites W2094652659 @default.
• W1970867883 cites W2104036678 @default.
• W1970867883 cites W2106030214 @default.
• W1970867883 cites W2112016055 @default.
• W1970867883 cites W2129589052 @default.
• W1970867883 cites W2131548419 @default.
• W1970867883 cites W2131621910 @default.
• W1970867883 cites W2132207502 @default.
• W1970867883 cites W2133972276 @default.
• W1970867883 cites W2135265711 @default.
• W1970867883 cites W2146409195 @default.
• W1970867883 cites W2148523692 @default.
• W1970867883 cites W2150678612 @default.
• W1970867883 cites W2152943305 @default.
• W1970867883 cites W2162979395 @default.
• W1970867883 cites W2163062545 @default.
• W1970867883 cites W2163240418 @default.
• W1970867883 cites W2168273245 @default.
• W1970867883 cites W2397388882 @default.
• W1970867883 cites W2408576065 @default.
• W1970867883 cites W4205811845 @default.
• W1970867883 doi "https://doi.org/10.1038/mt.2010.185" @default.
• W1970867883 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2990520" @default.
• W1970867883 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/20842106" @default.
• W1970867883 hasPublicationYear "2010" @default.
• W1970867883 type Work @default.
• W1970867883 sameAs 1970867883 @default.
• W1970867883 citedByCount "60" @default.
• W1970867883 countsByYear W19708678832012 @default.
• W1970867883 countsByYear W19708678832013 @default.
• W1970867883 countsByYear W19708678832014 @default.
• W1970867883 countsByYear W19708678832015 @default.
• W1970867883 countsByYear W19708678832016 @default.
• W1970867883 countsByYear W19708678832017 @default.
• W1970867883 countsByYear W19708678832018 @default.
• W1970867883 countsByYear W19708678832019 @default.
• W1970867883 countsByYear W19708678832020 @default.
• W1970867883 countsByYear W19708678832021 @default.
• W1970867883 countsByYear W19708678832022 @default.
• W1970867883 countsByYear W19708678832023 @default.
• W1970867883 crossrefType "journal-article" @default.
• W1970867883 hasAuthorship W1970867883A5001523714 @default.
• W1970867883 hasAuthorship W1970867883A5016693322 @default.
• W1970867883 hasAuthorship W1970867883A5023649972 @default.
• W1970867883 hasAuthorship W1970867883A5048236963 @default.
• W1970867883 hasAuthorship W1970867883A5048367073 @default.
• W1970867883 hasAuthorship W1970867883A5057196933 @default.
• W1970867883 hasAuthorship W1970867883A5063335678 @default.
• W1970867883 hasAuthorship W1970867883A5065544223 @default.
• W1970867883 hasBestOaLocation W19708678831 @default.
• W1970867883 hasConcept C169760540 @default.
• W1970867883 hasConcept C17077164 @default.
• W1970867883 hasConcept C178790620 @default.
• W1970867883 hasConcept C185592680 @default.
• W1970867883 hasConcept C2779227376 @default.
• W1970867883 hasConcept C502942594 @default.
• W1970867883 hasConcept C53524968 @default.
• W1970867883 hasConcept C62478195 @default.

• next