scholarly journals Cytokine-Induced NK-Like T Cells: From Bench to Bedside

2010 ◽  
Vol 2010 ◽  
pp. 1-8 ◽  
Author(s):  
Yeh Ching Linn ◽  
Kam M. Hui
Keyword(s):  
Clinical Trials ◽  
T Cells ◽  
Tumor Cells ◽  
Randomized Clinical Trials ◽  
Therapeutic Application ◽  
Effector Cells ◽  
T Effector Cells ◽  
Cik Cells ◽  
The Past ◽  
Cytokine Stimulation

Cytokine-induced killer (CIK) cells are polyclonal T effector cells generated when cultured under cytokine stimulation. CIK cells exhibit potent, non-MHC-restricted cytolytic activities against susceptible tumor cells of both autologous and allogeneic origins. Over the past 20 years, CIK cells have evolved from experimental observations into early clinical studies with encouraging preliminary efficacy towards susceptible autologous and allogeneic tumor cells in both therapeutic and adjuvant settings. This paper is our attempt to summarize the available published literature related to CIK cells. Looking into the future, we anticipate that the continuous therapeutic application of CIK cells will likely be developed along two major directions: overcoming the challenge to organize large prospective randomized clinical trials to define the roles of CIK cells in cancer immunotherapy and expanding its spectrum of cytotoxicity towards resistant tumor cells through experimental manipulations.

scholarly journals Immunomodulating nano-adaptors potentiate antibody-based cancer immunotherapy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Cheng-Tao Jiang ◽  
Kai-Ge Chen ◽  
An Liu ◽  
Hua Huang ◽  
Ya-Nan Fan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Cancer Immunotherapy ◽  
Tumor Cells ◽  
Noncovalent Interactions ◽  
Killer Cell ◽  
Antibody Immobilization ◽  
Effector Cells ◽  
Nanoparticle Surface

AbstractModulating effector immune cells via monoclonal antibodies (mAbs) and facilitating the co-engagement of T cells and tumor cells via chimeric antigen receptor- T cells or bispecific T cell-engaging antibodies are two typical cancer immunotherapy approaches. We speculated that immobilizing two types of mAbs against effector cells and tumor cells on a single nanoparticle could integrate the functions of these two approaches, as the engineered formulation (immunomodulating nano-adaptor, imNA) could potentially associate with both cells and bridge them together like an ‘adaptor’ while maintaining the immunomodulatory properties of the parental mAbs. However, existing mAbs-immobilization strategies mainly rely on a chemical reaction, a process that is rough and difficult to control. Here, we build up a versatile antibody immobilization platform by conjugating anti-IgG (Fc specific) antibody (αFc) onto the nanoparticle surface (αFc-NP), and confirm that αFc-NP could conveniently and efficiently immobilize two types of mAbs through Fc-specific noncovalent interactions to form imNAs. Finally, we validate the superiority of imNAs over the mixture of parental mAbs in T cell-, natural killer cell- and macrophage-mediated antitumor immune responses in multiple murine tumor models.

scholarly journals Participation of the H-2 antigens of tumor cells in their lysis by syngeneic T cells.

1976 ◽  
Vol 143 (3) ◽  
pp. 601-614 ◽  
Author(s):  
J W Schrader ◽  
G M Edelman
Keyword(s):  
T Cells ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Target Cell ◽  
Cytotoxic T Cells ◽  
Hybrid Origin ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Effector Cells

Cytotoxic T lymphocytes were generated in vitro against H-2 compatible or syngeneic tumor cells. In vitro cytotoxic activity was inhibited by specific anti-H2 sera, suggesting that H-2 antigens are involved in cell lysis. Two observations directly demonstrated the participation of the H-2 antigens on the tumor cells in their lysis by H-2-compatible T cells. First, coating of the H-2 antigens on the target tumor cell reduced the number of cells lysed on subsequent exposure to cytotoxic T cells. Second, when cytotoxic T cells were activated against an H-2 compatible tumor and assayed against an H-2-incompatible tumor, anti-H-2 serum that could bind to the target cell, but not to the cytotoxic lymphocyte, inhibited lysis. H-2 antigens were also shown to be present on the cytotoxic lymphocytes. Specific antisera reacting with these H-2 antigens, but not those of the target cell, failed to inhibit lysis when small numbers of effector cells were assayed against H-2-incompatible target cells or when effector cells of F1-hybrid origin and bearing two H-2 haplotypes were assayed against a tumor cell of one of the parental strains. These findings suggest that it is the H-2 antigens on the tumor cell and not those on the cytotoxic lymphocytes that are important in cell-mediated lysis of H-2-compatible tumor cells.

scholarly journals 566 ATOR-1017, a second generation 4–1BB antibody with potential to enhance efficacy of PD-1 therapies

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A595-A595
Author(s):  
Karin Enell Smith ◽  
Anneli Nilsson ◽  
Peter Ellmark
Keyword(s):  
T Cells ◽  
Transgenic Mice ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Effector Cells ◽  
Costimulatory Receptor ◽  
T Effector Cells ◽  
Murine Colon Carcinoma ◽  
Activation Profile

BackgroundATOR-1017 is a Fcγ-receptor (FcγR) crosslinking dependent agonistic IgG4 antibody targeting the costimulatory receptor 4 1BB, designed for improved tolerability and efficacy. 4-1BB is highly expressed on tumor infiltrating CD8+ T effector cells (T effs) in several cancer indications. By binding to 4-1BB, ATOR-1017 enhances the activity of tumor reactive T effs and NK cells within the tumor and induces a potent anti-tumor response. 4-1BB is a promising candidate for immunotherapy and holds great potential for combination with other immunomodulatory antibodies, targeting e.g. the PD-1 pathway.MethodsHuman 4-1BB knock-in transgenic mice with established murine colon carcinoma MC38 tumors were used to demonstrate anti-tumor efficacy after systemic treatment with ATOR-1017 in combination with anti-PD-1. Further, the effect of combining ATOR-1017 with anti-PD-1 on T cell activation (measured as production of IFNγ) was evaluated in a mixed lymphocyte reaction (MLR) assay with human primary CD4+ T cells and mature monocyte-derived DCs (mDC) expressing endogenous levels of both 4-1BB and PD-1.ResultsATOR-1017 in combination with anti-PD-1 improved survival and reduced tumor growth signifcantly in human 4-1BB knock-in transgenic mice with established tumors compared with each monotherapy alone. The potential for combining ATOR-1017 and PD-1 was further supported by data from a MLR assay demonstrating that the combination of ATOR-1017 with anti-PD-1 induced a more potent CD4+ T cells activation than each monotherapy alone.The functional activation profile of ATOR-1017 is expected to minimize the risk of systemic immune activation and toxicity, by directing a potent immune response to immune cells in tumor tissue and tumor draining lymph nodes. This is supported by early data from the ongoing first-in-human phase I study where ATOR-1017 has been shown to be safe and tolerable.ConclusionsIn summary, these results support further clinical development of ATOR-1017 in combination with PD-1 antibodies. By combining ATOR-1017 with anti-PD-1, tumor infiltrating T cells can be more effectively activated and potentially increase the response rate in multiple indications.Ethics ApprovalAll animal procedures were in accordance to IACUC guidance

scholarly journals Phase 2 Study of Combination of Cytarabine, Idarubicin, and Nivolumab for Initial Therapy of Patients with Newly Diagnosed Acute Myeloid Leukemia

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 815-815
Author(s):  
Farhad Ravandi ◽  
Naval Daver ◽  
Guillermo Garcia-Manero ◽  
Christopher B Benton ◽  
Philip A Thompson ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
T Cell Subsets ◽  
Newly Diagnosed ◽  
Effector Cells ◽  
T Effector Cells ◽  
Grade 3

Abstract Background: Blocking PD-1/PD-L1 pathways enhances anti-leukemia responses by enabling T-cells in murine models of AML (Zhang et al, Blood 2009). PD-1 positive CD8 T-cells are increased in bone marrow (BM) of pts with AML (Daver et al, AACR 2016). PD1 inhibition has shown activity in AML (Berger et al, Clin Cancer Res 2008). We hypothesized that addition of nivolumab to an induction regimen of ara-C and idarubicin may prolong relapse-free survival (RFS) and overall survival (OS); this study was designed to determine the feasibility of this combination. Methods: Pts with newly diagnosed acute myeloid leukemia (by WHO criteria; ≥20% blasts) and high risk MDS (≥10% blasts) were eligible to participate if they were 18-65 yrs of age and had adequate performance status (ECOG ≤3) and organ function (LVEF ≥ 50%; creatinine ≤ 1.5 g mg/dL, bilirubin ≤ 1.5 mg/dL and transaminases ≤ 2.5 times upper limit of normal). Treatment included 1 or 2 induction cycles of ara-C 1.5 g/m2 over 24 hours (days 1-4) and Idarubicin 12 mg/m2 (days 1-3). Nivolumab 3 mg/kg was started on day 24 ± 2 days and was continued every 2 weeks for up to a year. For pts achieving complete response (CR) or CR with incomplete count recovery (CRi) up to 5 consolidation cycles of attenuated dose ara-C and idarubicin was administered at approximately monthly intervals. Eligible pts received an allogeneic stem cell transplant (alloSCT) at any time during the consolidation or thereafter. Results: 3 pts with relapsed AML were treated at a run-in phase with a dose of nivolumab 1 mg/kg without specific drug-related toxicity. Subsequently, 32 pts (median age 53 yrs; range, 26-65) were treated as above including 30 with AML (24 de novo AML, 2 therapy-related AML, 3 secondary AML and 1 therapy-related secondary AML) and 2 high risk MDS. Pre-treatment genetic risk by ELN criteria was 11 adverse, 16 intermediate, and 5 favorable, including 2 FLT3 -ITD mutated, 5 NPM1 mutated, and 7 TP53 mutated. All 32 pts were evaluable for response and 23 (72%) achieved CR/CRi (19 CR, 4 CRi). The 4-week and 8 week mortality was 6% and 6%. The median number of doses of nivolumab received was 6 (range, 0-13); one pt did not receive nivolumab due to insurance issues. 9 pts underwent an alloSCT. After a median follow-up of 8.3 mths (range, 1.5-17.0) the median RFS among the responding pts has not been reached (range, 0.1 - 15.8 mths) and the median OS has not been reached (range 0.5-17.0 mths). Grade 3/4 immune mediated toxicities have been observed in 5 pts and include rash, pancreatitis, and colitis. Other grade 3/4 toxicities thought to be potentially related to nivolumab include cholecystitis in one pt. 9 pts proceeded to an alloSCT. Donor source was matched related in 2, matched unrelated in 6 and haplo-identical in 1 pt. Conditioning regimen was Fludarabine plus busulfan-based in 8, and fludarabine plus melphalan in 1 pt. 4 pts developed graft versus host disease (GVHD)(grade I/II in 3, grade III/IV in 1), which responded to treatment in 3. Multicolor flow-cytometry studies are conducted by the Immunotherapy Platform on baseline (prior to first dose of nivolumab) and on-treatment BM aspirate and peripheral blood to assess the T-cell repertoire and expression of co-stimulatory receptors and ligands on T-cell subsets and leukemic blasts, respectively. The baseline BM was evaluated on 23 of the 32 evaluable pts, including 18 responders and 5 non-responders. Pts who achieved a CR/CRi had a trend of higher frequency of live CD3+ total T cell infiltrate as compared to non-responders in the baseline BM aspirates (Fig 1A). We evaluated expression of immune markers on T cell subsets: CD4 T effector cells [Teff]: CD3+CD4+CD127lo/+Foxp3-, CD4 T regulatory cells [Treg]: CD3+CD4+CD127-Foxp3+, and CD8 T cells. At baseline, BM of non-responders had significantly higher percentage of CD4 T effector cells co-expressing the inhibitory markers PD1 and TIM3 (p<0.05) and a trend towards higher percentage of CD4 T effector cells co-expressing PD1 and LAG3 compared to responders (Fig 1B). Co-expression of TIM3 or LAG3 on PD1+ T cells have been shown to be associated with an exhausted immune phenotype in AML (Zhou et al., Blood 2011). Conclusion: Addition of nivolumab to ara-C and anthracycline induction chemotherapy is feasible and safe in younger pts with AML. Among the pts proceeding to alloSCT the risk of GVHD is not significantly increased. Figure 1 Figure 1. Disclosures Daver: Pfizer Inc.: Consultancy, Research Funding; Otsuka America Pharmaceutical, Inc.: Consultancy; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Bristol-Myers Squibb Company: Consultancy, Research Funding; Kiromic: Research Funding; Karyopharm: Consultancy, Research Funding; Jazz: Consultancy; Immunogen: Research Funding; Daiichi-Sankyo: Research Funding; Incyte Corporation: Honoraria, Research Funding. Thompson: Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jabbour: Bristol-Myers Squibb: Consultancy. Takahashi: Symbio Pharmaceuticals: Consultancy. DiNardo: Novartis: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Sharma: Jounce: Consultancy, Other: stock, Patents & Royalties: Patent licensed to Jounce; Astellas: Consultancy; EMD Serono: Consultancy; Amgen: Consultancy; Astra Zeneca: Consultancy; GSK: Consultancy; Consetellation: Other: stock; Evelo: Consultancy, Other: stock; Neon: Consultancy, Other: stock; Kite Pharma: Consultancy, Other: stock; BMS: Consultancy. Cortes: BMS: Consultancy, Research Funding; Sun Pharma: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding; ImmunoGen: Consultancy, Research Funding; ARIAD: Consultancy, Research Funding. Kantarjian: Delta-Fly Pharma: Research Funding; Amgen: Research Funding; ARIAD: Research Funding; Novartis: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding.

Decreased LAG3 expression on T effector cells and regulatory T cells in SAA

2020 ◽  
Vol 112 (6) ◽  
pp. 757-763
Author(s):  
Yingying Sun ◽  
Chunyan Liu ◽  
Ting Jiao ◽  
Ning Xie ◽  
Huaquan Wang ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Effector Cells ◽  
T Effector Cells

Effective Prevention of Acute Graft-Verses-Host Disease by Targeting PKC Alpha and PKC Theta in Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1898-1898
Author(s):  
Kelley M.K. Haarberg ◽  
Crystina Bronk ◽  
Dapeng Wang ◽  
Amer Beg ◽  
Xue-Zhong Yu
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Acute Gvhd ◽  
T Cell Signaling ◽  
Effector Cells ◽  
T Effector Cells ◽  
Immunologic Synapse

Abstract Abstract 1898 Protein kinase C theta (PKCθ), a T cell signaling molecule, has been implicated as a therapeutic target for several autoimmune diseases as well as graft-versus-host disease (GVHD). PKCθ plays a vital role in stabilization of the immunologic synapse between T effector cells and antigen presenting cells (APC), but has been shown to be excluded from the immunologic synapse in T regulatory cells (T reg). PKCθ inhibition reduces the alloreactivity of donor T cells responsible for induction of GVHD while preserving graft-versus-leukemia (GVL) responses. The roles of PKCθ and the potential compensatory alpha isoform (PKCα) are not clearly defined with regard to alloresponses or T cell mediated responses in GVHD. In this context, we measured PKCθ and PKCα/θ gene deficient T cell activation upon TCR-ligation in vitro using [3H]-TdR incorporation and CSFE labeling assays. T cells from PKCθ and PKCα/θ gene deficient donor mice were utilized in vivo in a pre-clinical allogenic murine model of myeloablative bone marrow transplantation (BMT). The development of GVHD was monitored in recipient mice with or without injection of A20-luciferase cells to observe the progression of GVL in vivo. Combined blockade of PKCα and PKCθ causes a significant decrease in T cell proliferation compared to blocking PKCθ alone in vitro. Deficiency in PKCα and PKCθ had no effect on immune reconstitution following irradiation and BMT in vivo. Even with a high transplant load of 5×106 CD4+ and CD8+ T cells, PKCα/θ deficient (PKCα/θ−/−) T cells failed to induce acute GVHD. Our data suggest that the ability of double deficient T cells to induce GVHD was further reduced than PKCθ-deficient T cells. Additionally, a greater number and percentage of B220+ B cells and FoxP3+ T regs were isolated from the spleens of PKCα/θ−/− T cell recipient mice 120 after BMT than were isolated from wild type (WT) or PKCθ−/− T cell recipients. Fewer CD4+ or CD8+ T effector cells were isolated from the spleens of PKCα/θ−/− T cell recipient mice 120 after BMT than were isolated from wild type or PKCθ−/− T cell recipients. Importantly, the activity of B cells isolated from PKCα/θ−/− T cell recipient mice 120 after BMT was greater on a per cell basis, while the activity of T effector cells isolated from these mice was greatly reduced compared to WT or PKCθ−/− T cell recipients. While not absent, GVL was reduced in PKCα/θ−/− T cell recipient mice when compared to WT or PKCθ−/− T cell recipients. This work demonstrates the requirement of PKCα and θ for optimal activation and function of T cells in vitro. These experiments highlight a potential compensatory role for PKCα in the absence of PKCθ in T cell signaling and activation. Combined deficiency of PKCα and θ prevents induction of acute GVHD while improving the maintenance of splenic cellularity in PKCα/θ T cell recipient mice. Additionally, PKCα/θ dual deficient T cell transplant shifts the splenic balance toward a greater number and percentage of T reg and B cells and away from T effector cells following BMT. The reduced and sub-optimally active T effector cells isolated from PKCα/θ−/− T cell recipient mice in combination with reduced GVL stresses the importance of PKCα and θ molecules and their roles in T cell activity in the context of both GVHD and GVL. Dual deficiency of PKCα/θ is associated with a decline of T effector function that is optimal for the amelioration of GVHD, but is perhaps too reduced to substantially maintain effective GVL. Modulation of PKCα and θ signaling presents a valid avenue of investigation as a therapeutic option for GVHD. Disclosures: No relevant conflicts of interest to declare.

BK Virus Reactivation After Double Umbilical Cord Blood Transplantation in Adults Correlates with Tregs and Delayed Reconstitution of CD4+ and CD8+ T Effector Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4174-4174
Author(s):  
Gowri Satyanarayana ◽  
Sarah Hammond ◽  
Haesook T Kim ◽  
Sean McDonough ◽  
Julia Brown ◽  
...  
Keyword(s):  
T Cells ◽  
Cord Blood ◽  
Immune Reconstitution ◽  
Hemorrhagic Cystitis ◽  
Cord Blood Transplantation ◽  
Bk Virus ◽  
Cellular Mechanisms ◽  
Effector Cells ◽  
T Effector Cells ◽  
Blood Transplantation

Abstract Abstract 4174 Umbilical cord blood transplantation (UCBT) in adults is associated with impaired immune function and increased infection-related morbidity and mortality due to lack of antigen experienced cells and delayed immune reconstitution. BK virus (BKV) is a human polyomavirus that remains latent in renal epithelial cells and can be reactivated after hematopoietic stem cell transplantation (HSCT), leading to hemorrhagic cystitis. Data regarding BKV reactivation and its association with immune reconstitution after UCBT is lacking. We evaluated the status, cellular mechanisms, and clinical implications of immune reconstitution on BK viremia in adults with hematologic malignancies undergoing double unit cord blood transplantation. Thirty-two patients with a median age of 50 years with hematopoietic malignancies were treated with reduced intensity conditioning (Flu/Mel/rATG) followed by infusion of two sequential UCB grafts and GvHD prophylaxis with tacrolimus and sirolimus. The grafts were at least a 4/6 match with each other and the recipient. The results are based on 27 evaluable patients. Assessments were done prior to transplant and at 1, 2, 3, 6 and 12 months after UCBT. After UCBT, 15 patients had detectable serum BKV DNA, median 2.6×104 copies/ml (range, 2.5×102–7.9×106) with a median time to viremia of 40 days (range, 26–733). The cumulative probability of developing BK viremia by day 100 was 0.52 (95% CI, 0.33–0.71). In 9 of the 15 patients with detectable serum BKV DNA, urinary BKV PCR was also performed. All 9 tested patients had detectable urinary BKV and developed clinical symptoms ranging from dysuria to hemorrhagic cystitis. To determine whether development of BK viremia was related to the immunological status, we analyzed detection of serum BKV DNA in conjunction with parameters of immune reconstitution. At 6 and 12 months after transplantation development of BK viremia displayed a statistically significant inverse correlation with CD4+ and CD8+ T cells (p<0.05). Development of BK viremia at these time intervals also inversely correlated with CD4+CD45RO+ and CD8+CD45RO+ T cells (p<0.05), consistent with a potentially significant role of these effector populations in preventing and/or clearing BKV. Conversely, simultaneoulsy, there was a significant positive correlation of BK viremia with T regulatory cell numbers (p<0.05) suggesting that cellular mechanisms of Treg-mediated immune suppression were directly involved in regulating this clinical outcome. At 3 months after UCBT there was a significant positive correlation (p<0.05) between BK viremia and T cell receptor excision circles (TRECs), which are expressed in recent thymic emigrant T cells. BK viremia was not dependent on any other immune cell populations including CD20+ B cells, CD16+CD56+ NK cells and CD14+ monocytes. Furthermore, prevention and/or clearance of BK viremia was not dependent on naïve cell numbers as determined by lack of correlation between BK viremia -or absence thereof- with CD4+CD45RA+ T cells and CD8+CD45RA+ T cells. These observations were in complete contrast to our previous findings regarding CMV-specific immunity, which revealed that prevention and/or clearance of CMV viremia depend on reconstitution of thymopoiesis and increase of TRECs and naïve CD4+CD45RA+ cells. In addition, we found no correlation between development of CMV viremia and BK viremia in UCBT recipients. Our results indicate that reactivation of BK virus occurs with high frequency in adult UCBT recipients and is related to the inability of TREC positive cells to control BK viremia, the impaired and delayed reconstitution of CD4+ and CD8+ T effector cells, and the suppressive function of Treg. Furthermore, our results indicate that distinct immunological mechanisms govern CMV-specific and BK-specific anti-viral responses after UCBT. Disclosures: No relevant conflicts of interest to declare.

Targeting An Ancient Retrovirus In Tumor Using Engineered T Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4206-4206
Author(s):  
Janani Krishnamurthy ◽  
Brian Rabinovich ◽  
Simon Olivares ◽  
Mi Teijuan ◽  
Kirsten Switzer ◽  
...  
Keyword(s):  
Clinical Trials ◽  
T Cells ◽  
Tumor Cells ◽  
Treatment Strategy ◽  
Conflicts Of Interest ◽  
Sleeping Beauty ◽  
Normal Organ ◽  
Car T Cells ◽  
Car T ◽  
Env Protein

Abstract Human endogenous retroviruses (HERVs) are ancient viruses forming 8% of human genome. One subset of HERVs, the HERV-K has recently been found to be expressed on tumor cells including melanoma, breast cancer and lymphoma but not on normal body cells. Thus, targeting HERV-K protein as a tumor associated antigen (TAA) may be a potential treatment strategy for tumors that are resistant to conventional therapies. One approach to improve therapeutic outcome is by infusing T cells rendered specific for such TAAs preferentially expressed on tumor cells. Recognition of cell-surface TAAs independent of major histocompatibility complex can be achieved by introducing a chimeric antigen receptor (CAR) on T cells using gene therapy. This approach is currently being used in our clinical trials adoptively transferring CD19-specific CAR+ T cells into patients with B-lineage malignancies. Preliminary analysis of HERV-K env protein expression in 268 melanoma samples and 139 normal organ donor tissues using immunohistochemistry demonstrated antigen expression in tumor cells and absence of expression in normal organ tissues. The scFv region from a mouse monoclonal antibody to target HERV-K env was used to generate a CAR and cloned into Sleeping Beauty (SB) plasmid for stable expression in T cells. HERV-K-specific CAR+T cells were selectively propagated ex vivo on artificial antigen presenting cells (aAPC) using an approach already in our clinical trials. Indeed, after genetic modification of T cells and selection on HERV-K+ aAPC, over 95% of propagated T cells stably expressed the introduced HERV-K-specific CAR and exhibited redirected specificity for HERV-K+ melanoma (Figure 1). Further, the adoptive transfer of HERV-K-specific CAR+T cells killed metastatic melanoma in a mouse xenograph model. While we have chosen melanoma as our tumor model, this study has the potential to be applied to other malignancies, including lymphoma and myeloma due to restricted expression of HERV-K envelope (env) protein on these tumor cells. These data demonstrate that it is feasible to generate T cells expressing a HERV-K-specific CAR using a clinically-appealing approach as a treatment strategy for HERV-K env+ tumors. Disclosures: No relevant conflicts of interest to declare.

Quantitative and Qualitative Analysis Of Regulatory T Cells (Tregs) Derived From The Peripheral Blood Of Chronic Lymphocytic Leukemia Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5280-5280
Author(s):  
Eleni Dikaia Ioannidou ◽  
Vassiliki Mpakou ◽  
Myrofora Vikentiou ◽  
Eugenia Konsta ◽  
Frieda Kontsioti ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Annexin V ◽  
Treg Cells ◽  
Lymphocytic Leukemia ◽  
Effector Cells ◽  
T Effector Cells ◽  
Healthy Donors

Abstract Introduction T regulatory cells are immunosuppressive cells, which are considered to play an important role in the regulation of immune response to cancer, by restraining autoreactive lymphocytes. Several studies, mostly in solid tumors, revealed that the number of Treg cells increases as the disease progresses and that Treg cells act by suppressing anti-tumor immune response, through the targeting of other immune cells, such as T cells, B cells and dendritic cells. Chronic lymphocytic leukemia (CLL) is a lymphoid malignancy, characterized by both, immunodeficiency and autoimmune disorders. Accumulated data indicate the role of T cells in the pathogenesis and development of CLL and reveal an increased number of Treg cells in CLL patients. The scope of this study is the analysis of the functional role of Tregs derived from the peripheral blood of CLL patients, mainly on B-CLL cells, and its correlation with well known prognostic factors. Methods Treg cells derived from mononuclear cells of 28 untreated B-cell CLL patients with a median age 62 (44-88) and 17 healthy donors were analyzed through Flow cytometry. Patients were classified according to Rai classification as Rai I:19, Rai II:4, Rai III:5 and according to Binet as Binet A: 24, Binet B:3 and Binet C:1. The following antibodies were used for the fluorescence-activated cell sorter (FACS) analysis: 1. CD45Ro-FITC/CD45RA-PE/CD4-ECD/CD25-PC5/CD127-PC7 2. CD1a-FITC/CD137-PE/CD4-ECD/CD25-PC5/CD127-PC7 3. CD95-FITC/cyCD152-PE/CD4-ECD/CD25-PC5/CD127-PC7 4. beads/FoxP3-PE/CD4-ECD/CD25-PC5/CD127-PC7 5. Annexin V-FITC/CD4-ECD/CD25-PC5/CD127-PC7 Moreover, peripheral blood was obtained from 15 patients with B-cell CLL. Mononuclear cells were isolated using Ficoll-Paque gradient centrifugation. CD4+CD25+ (Treg cells), CD4+CD25- (T effectοr cells, Teff), CD5+CD19+ (B-CLL) and CD5+CD19- (Normal B, NB) cells were separated using magnetic antibody cell sorting. To test the functionality of the assayed Tregs, the isolated cell populations were cultured in a 96-well plate (Tregs, Teff, B-CLL, NB cells, Tregs:Teff in a 4:1 ratio, B-cll:Tregs in 1:20 ratio, B-cll:Teff in 1:20 ratio, NB cells:Tregs in 1:20 ratio, NB cells:Teff in 1:20 ratio) and their proliferative capacity was measured using the BrdU assay. Results FACS analysis of the Treg cells resulted at the following observations: (1) The co-expression of the CD45RA-CD45RO markers was significantly higher in patients’ samples than in controls (p=0.047). (2) No significant differences were observed between patients and controls, regarding the expression of the CD1α marker, as well as the expression of CD95 and CD152 markers. (3) The Treg absolute cell number (cells/μL), estimated either as the number of CD4+ CD25+ CD127- cells or as the number of CD4+ CD25+ FoxP3+ cells, was statistically significantly higher in patients’ samples than in controls (CD127- p=0.047, FoxP3+ p= 0.036). (4) Annexin V expression in Treg cells from B- CLL patients was significantly lower compared to controls (p=0.027). Following the purification and culturing of T and B cells from B-cell CLL patients’ samples, functional analysis of the different cell populations was performed using the BrdU proliferation assay. We observed that Tregs were able to significantly suppress the proliferation of the Teff cells (p=0.002). After the co-culturing of NB cells (CD5+CD19-)and Tregs (CD4+CD25+) we found that NB cells seemed to significantly increase the proliferation of Treg cells, compared to the proliferation capacity of the Tregs when cultured alone (p=0.047). Moreover, we observed that Teff (CD4+CD25-) were able to significantly suppress the proliferation of B-CLL cells (CD5+CD19+), when co-cultured (B-CLL: Teff, 1:20 ratio) (p=0.05). Conclusions In B-cell CLL patients, Treg cells are significantly higher and present with lower apoptotic levels compared to healthy donors’ samples. The functional analysis of Treg cells indicates that they can effectively suppress the proliferation of T effector cells. Moreover, T effector cells seem to suppress the proliferation of B-CLL cells, while NB cells increase the proliferation of Treg cells. These observations could probably indicate that at the early stages of the disease, where NB cells are more aberrant, Treg cells’ activity is induced, leading to Teff cells’ suppression and therefore, to an indirect induction of B-CLL cells’ proliferation. Disclosures: No relevant conflicts of interest to declare.

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