Specific Cytotoxic T Lymphocytes Research Articles

Genetic Modification of Multi Leukemia Antigen-Specific Cytotoxic T Lymphocytes (CTL) to Enhance In Vivo Safety and Persistency

Abstract 644Adoptive transfer of ex-vivo expanded leukemia-specific T cells has been clinically evaluated for its ability to produce anti tumor effects without causing graft-versus-host disease (GVHD). Previous protocols, however, allowed the expansion only of HLA class I-restricted CD8 T cells, which were short-lived and permitted tumor immune escape since they recognized only a single epitope/antigen target.To minimize tumor immune escape and extend this therapeutic alternative to more patients irrespective of HLA type we have developed a novel strategy to produce single cultures of in vitro generated T cell lines containing tumor-cytotoxic T cells from both CD4+ and CD8+ populations with specificity for a multiplicity of epitopes on several tumor associated antigens (TAAs) frequently expressed by myeloid leukemias.Tumor-directed T cells were activated in vitro using dendritic cells (DCs) loaded with peptide libraries (pepmixes) spanning Proteinase 3 (Pr3), PRAME, and WT1 and expanded in the presence of a cocktail of Th1-polarizing, pro-survival and pro-proliferative cytokines (IL7, 12, 15 and 27). This approach consistently generated TAA-specific cytotoxic T lymphocytes (CTLs) with simultaneous reactivity against PRAME (mean 651+/−69 spot forming units (SFU)/1×106 CTLs), WT1 (mean 156+/−20 SFU/1×106 CTLs) and PR3 (mean 52.3+/−8.8 SFU/1×106 CTLs) in IFNg ELIspot assays (n=10). These lines were polyclonal, comprising antigen-specific CD4+ (mean 32.5+/−2.3%) and CD8+ (mean 38.3+/−4%) T cells. Functionally, these CTLs killed autologous PHA blasts pulsed with PRAME (mean specific lysis 67+/−5% SEM), WT-1 (mean 54+/− 5%) and PR3 pepmixes (mean 16+/−6%, n=6) (E:T of 40:1). In addition, TAA CTLs killed the partially (1-3 alleles) HLA class I or HLA class II matched whole antigen-expressing AML cell line THP1 in coculture assays (n=5).The in vivo anti-tumor activity of multiTAA-CTLs was tested using a SCID mouse tumor model. After subcutaneous engraftment of FFLuc transduced THP-1 cells, mice were treated with either 1×107 multiTAA-CTLs or control EBV-CTLs generated from the same partially HLA-matched donor (sharing HLA-A2, DRB1-01,DQB1-05) plus 1000U of IL2 (5 times per week). Tumor signals as measured by bioluminescence using an in vivo imaging system rapidly increased in untreated and EBV-CTL treated groups, whereas mice treated with TAA specific CTLs were able to control tumor growth (p<0.05) and significantly improve overall survival (log rank p=0.008).Next, we transduced multiTAA-CTL with a retroviral vector (SFG-hIL-7R/iCasp9) encoding IL7Ralpha and the inducible suicide gene iCaspase9. This genetic modification improved the in vivo persistence, anti-tumor potency of multiTAA-CTL by restoring their response to homeostatic IL7. Furthermore we improve safety with the potential to control unwanted proliferation of the transgenic cells and to treat potentially occurring autoimmune disease caused by targeting self antigens through the administration of a small molecule chemical inducer of dimerization (CID) (AP20187) which activates the suicide gene.Transduction of multiTAA-CTLs did not adversely affect the antigen specificity of the product as evaluated by IFNg ELIspot (528.5 SFU/1×106 CTL transduced vs. 573.9 SFU/1×106 CTL non transduced, PRAME, 86.7 vs.46.5 SFU/1×106 CTL, Pr3 and 129.2 vs 130.2 SFU/1×106 CTL and WT1 transduced vs. non transduced); and cytolytic activity against peptide-pulsed PHA blasts as well as whole antigen presenting tumor cell lines was maintained. In the presence of IL-7, transgenic multiTAA-CTLs expanded significantly (transduced cells increased from 55%+/1% to 89%+/−1%). Such expansion was not observed when these cells were cultured in the presence of IL-15 (decrease 55%+/1% to 34.6%+/−3.5%) (n=8). Proliferation assays confirmed that only IL7/casp9 engineered multiTAA-CTLs were able to proliferate in the presence of IL-7 (84638 CPM vs. 14923 CPM transduced vs non-transduced). Upon activation of iCaspase9 by of CID (AP20187), the proliferation of multiTAA-CTLs was halted and a decrease of transgenic cells from 89% to 3% with was detected within 24 hrs (n=7).Hence CTLs can be prepared that simultaneously target multiple TAA antigens expressed by myeloid malignancies, reducing the risk of tumor immune escape. In addition these tumor CTLs can be genetically modified to safely improve their in vivo survival, and persistence. Disclosures:No relevant conflicts of interest to declare.

Preemptive Donor Lymphocyte Infusion Induces Polyspecific T-Cell Responses in a Patient with AML with NPM1 Mutation

Abstract 4311Delayed donor lymphocyte infusion (DLI) already plays a key role in supporting the graft-versus-tumor effect after allogeneic hematopoietic stem cell transplantation (HSCT) although the potency of DLI differs for each disease entitiy. The graft-versus-leukemia (GvL)-effect observed after DLI is based on the CTL-mediated immunity which is reactive against minor histocompatibility antigens (mHAg). To date, the role of leukemia-associated antigens (LAAs) in GvL is not clear.We therefore analysed peripheral blood of a 57-year old woman with AML with NPM1 mutation (NPM1mut) who developed molecular relapse without detection of myeloid blasts in the peripheral blood and bone marrow at d+171 after allo-HSCT and received preemptive DLI at d+ 260. In parallel to the molecular relapse, chimerism continuously decreased to 60 % in the polymorphonuclear and 75 % in the mononuclear cell fraction. Blood samples were taken before and after DLI and investigated for specific T-cell responses against several LAAs for which cytotoxic T-cell responses were already described. In addition, the immune status of the patient was determined by measuring the counts of CD8+ and CD4+ T-cells as well as B-cells in the peripheral blood at several time points. To test specific CD8+ T-cell responses, ELISpot analysis for interferon gamma and granzyme B were perfomed. Two epitopes derived from the NPM1mut peptide (NPM1-P3 and –P9) and PRAME (P300 and P435) were tested as well as one epitope obtained from Survivin, RHAMM, Proteinase 3 and WT-1.CD8+ T-cell responses against NPM1-P3, -P9, P300, Proteinase 3, Survivin and WT-1 were detected in blood samples after preemptive DLI but not in samples before DLI. In parallel, former detectable NPM1mut transcripts were no longer traceable – the patient achieved molecular complete remission - and former mixed chimerism became complete after DLI.Here, we could demonstrate for the first time polyspecific cytotoxic CD8+ T-cell responses against several known LAAs in a patient with AML with NPM1mut after preemptive DLI in molecular relapse. Interestingly, the immune responses against LAAs were associated with MRD negativity. Whether specific cytotoxic T-cell responses against epitopes derived from the NPM1mut peptide or the polyspecificity of the cytotoxic T-cells against several LAAs are decisive in the elimination of the myeloid blasts with NPM1mut remains to be determined in a larger cohort of patients. All in all it is of interest to perform preemptive DLI in patients with molecular relapse systematically. In addition, boostering of T-cells against specific LAAs could possibly be an approach to enhance GvL-effect with reducing GvHD-effect at the same time, but the most suitable LAA or combination of LAAs for each disease entitiy still has to be investigated. Disclosures:No relevant conflicts of interest to declare.

Identification of a Novel CD8+ T Cell Epitope Derived from Cancer-Testis Antigen MAGE-4 in Oesophageal Carcinoma

MAGE-4 is considered as an attractive cancer-testis (CT) antigen for tumour immunotherapy, and it is overexpressed in oesophageal carcinoma (EC). To identify MAGE-4-derived HLA-A2 restricted epitopes, native peptides and their analogues were predicted with prediction programs. The native peptide, p286 (KVLEHVVRV), and its analogues, p286-1Y2L and p286-1Y2L9L, showed potent binding affinity and stability towards HLA-A*0201 molecule. Cytotoxic T lymphocytes (CTLs) induced by p286-1Y2L9L could release IFN-γ in ELISPOT assay. In cytotoxicity assay, p286-1Y2L9L showed the capability to induce specific CTLs which could lyse the target cancer cells from both PBMCs of healthy donors and HLA-A2.1/K(b) transgenic mice. Our results indicated that the peptide p286-1Y2L9L could serve as a novel candidate epitope to develop peptide vaccines against oesophageal carcinoma.

Stepwise Maturation of Lytic Granules during Differentiation and Activation of Human CD8+ T Lymphocytes

During differentiation, cytotoxic T lymphocytes (CTL) acquire their killing potential through the biogenesis and maturation of lytic granules that are secreted upon target cell recognition. How lytic granule load in lytic molecules evolves during CTL differentiation and which subsets of lytic granules are secreted following activation remains to be investigated. We set up a flow cytometry approach to analyze single lytic granules isolated from primary human CTL according to their size and molecular content. During CTL in vitro differentiation, a relatively homogeneous population of lytic granules appeared through the progressive loading of Granzyme B, Perforin and Granzyme A within LAMP1+ lysosomes. PMA/ionomycin-induced lytic granule exocytosis was preceded by a rapid association of the docking molecule Rab27a to approximately half of the lytic granules. Activated CTL were found to limit exocytosis by sparing lytic granules including some associated to Rab27a. Our study provides a quantification of key steps of lytic granule biogenesis and highlights the potential of flow cytometry to study organelle composition and dynamics.

Summary of Presentations from the 11th Targeted Therapies for Lung Cancer Meeting; Immunotherapy and Vaccines for Treatment of Lung Cancer

Immune-based interventions are steadily broadening the range of choices among cancer prevention and treatment modalities. Recent major advances in the therapy of lymphoid malignancies and prevention of cervical cancer (to name a few) fuel the interest of investigators and clinicians who tried for decades to exploit the potential of the immune system to control the disease. Lung cancer presents a more challenging group of diseases, where major breakthroughs in immunotherapy or vaccination are yet to be seen. The following update may elicit cautious optimism for some areas of this endeavor. Dr. Scott Antonia summarized the essential processes by which the immune system recognizes and responses to tumor-associated antigens (TAAs) on tumor cells. From the MHC Class I antigen processing pathway, dendritic cellbased activation to cytotoxic T lymphocytes (CTLs) to the list of various types of tumor-associated antigens and evidence of the presence of specific CTLs for individual TAAs; experimental and clinical data provide the rationale for the development of tumor vaccines (Table 1). Nevertheless, once vaccines are tested in clinics, there is a “glass ceiling” with a tumor response rate of 5 to 10%. Efficacy can only be improved by developing combination immunotherapy with the use of (1) immunomodulatory agents (T-cell growth factors, anticheckpoint, or costimulatory agents); (2) elimination of suppressive cells; (3) inactivation of suppressive cytokines and factors in the tumor microenvironment; and (4) sequencing with chemotherapy.

Immunostimulatory CpG-DNA and PSA-peptide vaccination elicits profound cytotoxic T cell responses

ObjectiveNovel strategies for the treatment of advanced prostate cancer (CaP), including immunotherapy or gene therapy, are currently under evaluation with Sipuleucel-T as first FDA-approved immunotherapeutic. Here, we examine cytosine-phosphorothioate-guanine (CpG)-DNA oligonucleotides (ODN) to boost cytokine responses and costimulatory molecule expression on murine bone marrow-derived dendritic cells (mBMDC). Furthermore, we evaluate the potency of a PSA-peptide based vaccine in combination with CpG-DNA to elicit specific cytotoxic T cell (CTL) responses. Materials and methodsmBMDC were stimulated with CpG-DNA (1668: 5′-TCCATGACGTTCCTGATGCT-3′) or non-stimulatory control-ODN (1720: 5′-TCCATGAGCTTCCTGATGCT-3′). Subsequently, expression of the costimulatory molecules CD40 and CD86 and induction of proinflammatory cytokines (interleukin (IL)-6 and IL-12) were analyzed. For induction of PSA-peptide specific CTL, female C57BL/6 mice were immunized with PSA-peptide 65–73 (HCIRNKSVI) alone or in combination with 1668 or 1720-ODN. In vivo cytotoxicity assay determined PSA-peptide specific cytotoxicity 1 week after vaccination. ResultsTreatment of mBMDC with stimulatory CpG-DNA ODN resulted in pronounced up-regulation of costimulatory molecule expression on mBMDC in a dose-dependent manner. CpG-ODN significantly increased production of IL-6 and IL-12 in mBMDC (P < 0.001). Induction of PSA-peptide specific CTL responses in mice immunized with PSA-peptide and CpG-DNA were significantly greater than those of PSA-peptide and control-ODN immunized mice or PSA-peptide only vaccination. ConclusionsCpG-DNA acts as potent adjuvant for vaccination therapies and elicits profound PSA-peptide specific CTL responses in combination with an immunodominant PSA-peptide. CpG-ODN mediated immunotherapy represents a potentially inexpensive, safe, easy-to-produce, and easy-to-handle treatment alternative. Therefore, further evaluation of CpG-DNA in immunization therapies against CaP is warranted.

Clinical reagents of GM-CSF and IFN-α induce the generation of functional chronic myeloid leukemia dendritic cells in vitro

Dendritic cells (DCs) have been successfully induced in vitro from chronic myeloid leukemia (CML) cells, which may provide a promising immunotherapeutic protocol for CML. To facilitate the optimization of DCs-based vaccination protocols, we investigated the efficiency of in vitro generation of DCs from bone marrow mononuclear cells of CML patients by clinical reagents of GM-CSF and IFN-α. Bone marrow mononuclear cells were isolated from eight CML patients and CML-DCs were generated in the presence of different cytokines (Group A: GM-CSF for research and IL-4 for research; Group B: GM-CSF for injection and IFN-α for injection) in RMPI-1640 medium containing 10% human AB serum. After 8days, the morphologic features of CML-DCs were observed and their immunophenotypes were analyzed by flow cytometry. The activity of CML-DCs was determined by evaluating their ability to stimulate allogeneic mixed lymphocyte reaction (allo-MLR) and anti-leukemic cytotoxic T lymphocytes (CTLs). The culture protocols were successful in generating functional CML-DCs from all the CML patients as evidenced by the significant upregulation of CD80, CD86, CD83 HLA-DR and CD1a compared to pre-cultured (p<0.05), and increased allogeneic T cell stimulating proliferation capacity (p<0.05). CML-DCs could stimulate a specific anti-leukemia response. In summary, we demonstrate that the combination of clinical reagents GM-CSF and IFN-α induced the generation of DCs that have the ability to stimulate a specific anti-leukemia CTLs response in vitro, indicating their feasibility for clinical vaccination protocols for CML patients.

Human dendritic cells transfected with amplified MUC1 mRNA stimulate cytotoxic T lymphocyte responses against pancreatic cancer in vitro

Mucin (MUC) 1 is an epithelial cell glycoprotein that is aberrantly overexpressed in many adenocarcinomas, including pancreatic cancer (PC), providing an ideal tumor-associated antigen and target for immunotherapy. In this study, we investigated whether the cytotoxic T lymphocytes (CTLs) induced by dendritic cells (DCs) transfected with amplified MUC1 mRNA could respond against PC in vitro. Amplified mRNA encoding MUC1 were transfected into DCs using electroporation with an optimized setting and the MUC1 expression were evaluated by quantitative real-time polymerase chain reaction and Western blot. The MUC1 specific CTL responses were measured using the standard chromium 51 (51Cr)-release assays and the interferon-γ release assay. Dendritic cells could be transfected with amplified MUC1 mRNA efficiently. The transfected DCs were remarkably effective in stimulating MUC1-specific CTL responses in vitro. The function of MUC1 specific CTLs, induced by MUC1 mRNA-transfected DCs, was restricted by major histocompatibility complex (MHC) class I antigen presentation. The CTL responses stimulated by DCs transfected with MUC1 mRNA could only recognize and lyse HLA-A2+/MUC1+ PC and other target cells under restriction by MHC class I-specific antigen presentation, providing a preclinical rationale for using MUC1 as a target structure for immunotherapeutic strategies against PC.

Abstract B43: MART-1 and gp100 expressing and nonexpressing melanoma cells are equally proliferative in tumors and clonogenic in vitro

Abstract Background and objectives: Cutaneous melanoma (CM) is the neoplasia with the fastest growing incidence worldwide. When CM metastasizes, only a minority of patients may be cured. However, CM is an immunogenic tumor. MART-1 and gp100 are prototypical melanoma Ag, since in HLA-A0201 patients (40% Latin Americans), most tumor-infiltrating lymphocytes are directed against them. However, Ag-specific therapies, including vaccines or adoptive therapy with cytotoxic T lymphocytes (CTL), have achieved modest success. Therefore, mechanisms of tumor resistance still need to be addressed. One possibility is that clonogenic Ag-negative cells would be spared by immune effectors. Thus it is important to discern whether Ag heterogeneity is due to the presence of differentiated cells with limited proliferative potential (cancer stem cell, CSC model), or to Ag plasticity independent from proliferation (phenotypic plasticity model). Another possibility is that clonogenic cells would be intrinsically resistant to CTL. Therefore, we studied the relationship between MART-1 and gp100 expression and proliferation in CM cells in primary tumors and metastases. We analyzed Ag expression in clonogenic cells (CC), including early melanocyte differentiation Ag (MD-Ag) MART-1 and gp100, late MD-Ag tyrosinase, and CSC marker CD271; and CC sensitivity to specific CTL clones. Methods and Results: Analysis of MART-1/gp100 and the proliferation marker Ki-67 expression in 15 primary and seven metastatic CM biopsies by immunohistochemistry (IHC) revealed that MART-1/gp100 expressing and nonexpressing cells proliferated competitively, with no differences between primary and metastatic tumors (Wilcoxon tests, p&amp;lt;0.05). We studied colonies obtained from anchorage-independent growing clonogenic cells (CC) of two human CM cell lines, MEL-XY1 and MEL-XY3, as a model of in vitro proliferation. Two growth stages were analyzed: 7-day colonies, with most cells Ki-67+, and 14-day colonies, with colonies of different sizes, many of them with proliferative cells in the periphery and necrotic cores. MART-1, gp100, tyrosinase, and CD271 expression were studied in colonies by IHC. By 7 days, colonies displayed positive, negative, and mixed expression patterns. By 14 days, Ag were downregulated, suggesting Ag plasticity (t-test, p&amp;lt;0.05). Analysis of Ag expression in CC by FACS further supported these results (Kolgomorov-Smirnov test, p&amp;lt;0.05). Subcloning of MEL-XY1 colonies showed that MART-1 and gp100 expression varied with time without interfering with clonogenicity (t-test, p&amp;lt;0.05). We studied CpG methylation in Mart-1 promoter by MSP (methylation-specific PCR), and found that this mechanism would be involved in MART-1 heterogeneity in MEL-XY1 7-day CC (t-test, p&amp;lt;0.05). Finally MART-1/gp100 expressing CC were lysed by specific CTL (0.3% survived, t-test, p&amp;lt;0.05). Conclusions: The expression of MART-1 or gp100 is not associated to a diminished proliferative potential of CM cells. MART-1, gp100, tyrosinase, and CD271 expression do not interfere with clonogenic potential, and are endowed with considerable plasticity. Furthermore, CC expressing the proper Ag and HLA-class-l haplotype are not intrinsically resistant to lysis by CTL. Since MD-Ag-expressing and nonexpressing cells are proliferative and clonogenic, giving rise to colonies of several thousands of cells, both subpopulations should be taken into consideration as targets to eradicate tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B43.

Anti-gamma interferon antibodies enhance the immunogenicity of recombinant adenovirus vectors.

Vaccination for eliciting antigen-specific memory CD8(+) T cells may be facilitated by manipulating the pleiotropic effects of gamma interferon (IFN-γ). We assessed strategies for modulating the contribution of IFN-γ during the development of antigen-specific cytotoxic T lymphocyte (CTL) populations. We first showed that recombinant IFN-γ suppressed antigen expression in vitro from a recombinant adenovirus (rAd) vector in a dose-dependent manner and that addition of an anti-IFN-γ antibody (Ab) eliminated this suppression. Consistent with these in vitro findings, we found that HIV-1 envelope (Env)-specific CTL responses were higher in IFN-γ-knockout (GKO) mice than in wild-type mice following immunization with rAd. Since these observations suggested that IFN-γ might suppress rAd-induced CTL development, we assessed the ability of anti-IFN-γ Ab administration to augment rAd-elicited CTL in vivo. In fact, blockage of IFN-γ activity by monoclonal Ab administration was associated with elevated levels of interleukin 7 receptor alpha chain-positive (IL-7Rα(+)) Env-specific CTL populations postboost. These observations illustrate the utility of an anti-IFN-γ Ab for potentiating rAd immunizations to effect quantitative and qualitative changes in the effector and memory CTL populations.

6140 POSTER Cetuximab-mediated Immune-enhancing Effects in Vitro and in Metastatic Colorectal Cancer (mCRC) Patients

6140 POSTER Cetuximab-mediated Immune-enhancing Effects in Vitro and in Metastatic Colorectal Cancer (mCRC) Patients P. Correale1, C. Botta1, M.G. Cusi2, E. Bestoso1, S. Apollinari1, M. Caraglia3, M.T. Del Vecchio4, A. Abbruzzese3, A. Aquino5, P. Tagliaferri6. 1“S. Maria alle Scotte” Siena University Hospital, Department of Oncology, Siena, Italy; 2“S. Maria alle Scotte” Siena University Hospital, Department of Molecular Biology Microbiology Section, Siena, Italy; 3II University of Naples, Department of Biochemistry and Biophysics, Naples, Italy; 4“S. Maria alle Scotte” University Hospital of Siena, Section of Pathological Anatomy Department of Human Pathology and Oncology, Siena, Italy; 5“Tor Vergata” University, Department of Neurosciences Pharmacology Section, Rome, Italy; 6Magna Graecia University and Tommaso Campanella Cancer Center Catanzaro, Medical Oncology, Catanzaro, Italy Introduction: Cetuximab is a chimeric human-murine monoclonal IgG1 to the Epidermal-growth-factor-Receptor, approved for colorectal cancer treatment with chemotherapy. Its human constant fragment (Fc), throughout receptor binding, trigger additional immune-mediated effects, which may offer significant contribute to the final therapeutic effect. We investigated cetuximab ability in vitro to promote colon cancer cell phagocytosis and antigen-cross-priming by dendritic cells (DCs) to tumour-specific cytotoxic- T-cell (CTL) precursors. We also carried-out an immunological study in 26 mCRC patients enrolled in an on-going phase 2 trial, receiving an experimental biochemotherapy (GILFICet) regimen combining gemcitabine, irinotecan, fluoruracil, levofolinate, cetuximab, and metronomic sc. aldesleukine. Material and Methods: Transmission-electron-microscopy (TEM) and Flow cytometry were used to evaluate the susceptibility of multiple colon cancer cell lines to DC-mediated phagocytosis. Human DCs loaded with cetuximab-coated colon cancer cells, exposed or not to a combination of anti-cancer drugs, were used to in vitro sensitize human PBMCs from normal donors and cancer patients collected at baseline and after 3 GILFICet courses. T-cell cultures were characterized for immunephenotype and tumour-antigen specific CTL activity by Flow cytometry, LDH release and IFNg-ELISPOT. Results: ILF (irinotecan+ folinate+5-flurouracil) and GILF (gemcitabine+ ILF) poly-chemotherapies confirmed their ability to induce antigen remodelling and danger signals in colon cancer cells. After exposure to poly-chemotherapy and cetuximab these cells became highly susceptible to Fc-receptor-mediated phagocytosis/trogocytosis by human DCs, promoted their activation, and increased their ability to elicit a highly efficient CTL response on human PBMCs in vitro. Our study on the PBMCs of colon cancer patients enrolled in the GILFICet trial, revealed a significant treatment-related increase in na¨ive and central-memory-Tcells, activated-CTLs, NK/NKTs, mature-activated DCs and IFNg-releasing cells. Substantial differences were observed in T-cell lines generated from patients’ PBMCs taken before and after biochemotherapy. In the latter group there was in fact, a remarkable increase in proliferating CD8+Ki67+CTLs and tumour-antigen specific CTLs’ precursors. Discussion: These results suggest that cetuximab may exert immuneenhancing effects with potential antitumour activity

Cetuximab ± chemotherapy enhances dendritic cell‐mediated phagocytosis of colon cancer cells and ignites a highly efficient colon cancer antigen‐specific cytotoxic T‐cell response in vitro

Cetuximab is a human/mouse chimeric IgG1 monoclonal antibody (mAb) to epidermal growth factor receptor, approved for colorectal carcinoma treatment in combination with chemotherapy. The immune-mediated effects elicited by its human fraction of crystallization moiety might critically contribute to the overall anti-tumor effectiveness of the antibody. We therefore investigated cetuximab ability to promote colon cancer cell opsonization and phagocytosis by human dendritic cells (DCs) that are subsequently engaged in antigen-cross presentation to cytotoxic T-lymphocyte (CTL) precursors. Human colon cancer cell lines were evaluated for susceptibility to DC-mediated phagocytosis before and after treatment with chemotherapy ± cetuximab in vitro. Human DCs loaded with control or drug-treated cetuximab-coated colon cancer cells were used to in vitro generate cytotoxic T cell clones from peripheral blood mononuclear cells of human leucocyte antigen-A(*)02.01(+) donors. T-cell cultures were characterized for immune-phenotype and tumor-antigen specific CTL activity. The results confirmed that treatment of tumor cells with irinotecan + L-folinate + 5-flurouracil (ILF) or with gemcitabine + ILF increased tumor antigen expression. Moreover, malignant cells exposed to chemotherapy and cetuximab were highly susceptible to phagocytosis by human DCs and were able to promote their activation. The consequent DC-mediated cross-priming of antigens derived from mAb-covered/drug-treated cancer cells elicited a robust CTL anti-tumor response. On the basis of our data, we suggest a possible involvement of CTL-dependent immunity in cetuximab anti-cancer effects.

The study on the induction of specific immune cytotoxic T lymphocyte responses against pancreatic cancer by transfected dendritic cells with common tumor antigen survivin mRNAs in vitro

Objective To investigate the induction of specific anti-tumor immune response by transfected dendritic cells (DCs) with survivin mRNA of human pancreatic cancer, and to provide the experimental evidences for the treatment of human pancreatic cancer with DCs vaccine. Methods DCs were isolated and cultured from peripheral blood mononuclear cells (PBMCs). After being transcripted and amplified, survivin mRNA was transfected into DCs by electroporation. The expression of survivin in DCs at different time points was detected by quantitative real-time PCR. The survival rate of DCs before and after transfection was determined by MTT method. The induction of specific cytotoxic T lymphocyte (CTL) response by survivin mRNA transfected DCs was measured by 51Cr standard cytotoxicity test. The induction of specific CTL activation by survivin mRNA transfected DCs was evaluated through testing released IFN-γ by ELISA method. Results After survivin mRNA transfection for 48h, the expression of survivin mRNA in DCs reached the highest point (46.09±6.57). After transfection, the survival rate of DCs was stabilized around 80%. The DCs transfected with survivin mRNA could effectively induce HLA-A2+ / survivin+ specific CTL immune responses. Stimulated with pancreatic cancer cell line Capan-2 cells or SCL-1 cells as control group, the IFN-γ released in 24 hours by survivin specific CTL were (28.79±5.70) U/ml and (25.12±2.13) U/ml respectively, there was no significant difference (P=0.761). Conclusion The induction of CTLs by DCs transfected with human pancreatic cancer survivin mRNA could produce specific anti-tumor immunity. Key words: Dendritic cells; Microtubule-associated proteins; RNA, messenger; Transfection; Pancreatic neoplasms; T-lymphocytes, cytotoxic

Porcine major histocompatibility complex (MHC) class I molecules and analysis of their peptide-binding specificities

In all vertebrate animals, CD8+ cytotoxic T lymphocytes (CTLs) are controlled by major histocompatibility complex class I (MHC-I) molecules. These are highly polymorphic peptide receptors selecting and presenting endogenously derived epitopes to circulating CTLs. The polymorphism of the MHC effectively individualizes the immune response of each member of the species. We have recently developed efficient methods to generate recombinant human MHC-I (also known as human leukocyte antigen class I, HLA-I) molecules, accompanying peptide-binding assays and predictors, and HLA tetramers for specific CTL staining and manipulation. This has enabled a complete mapping of all HLA-I specificities (“the Human MHC Project”). Here, we demonstrate that these approaches can be applied to other species. We systematically transferred domains of the frequently expressed swine MHC-I molecule, SLA-1*0401, onto a HLA-I molecule (HLA-A*11:01), thereby generating recombinant human/swine chimeric MHC-I molecules as well as the intact SLA-1*0401 molecule. Biochemical peptide-binding assays and positional scanning combinatorial peptide libraries were used to analyze the peptide-binding motifs of these molecules. A pan-specific predictor of peptide–MHC-I binding, NetMHCpan, which was originally developed to cover the binding specificities of all known HLA-I molecules, was successfully used to predict the specificities of the SLA-1*0401 molecule as well as the porcine/human chimeric MHC-I molecules. These data indicate that it is possible to extend the biochemical and bioinformatics tools of the Human MHC Project to other vertebrate species.Electronic supplementary materialThe online version of this article (doi:10.1007/s00251-011-0555-3) contains supplementary material, which is available to authorized users.

A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro

Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparation of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin–biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.

B-cell polyclonal activation and Epstein-Barr viral abortive lytic cycle are two key features in acute infectious mononucleosis

Background Acute infectious mononucleosis (AIM) is generally associated with a large EBV B cell reservoir cells and an intense B-cell polyclonal activation whereas the number of quiescent EBV-infected memory B cells in chronically EBV-infected healthy controls is very low. Objectives To evaluate the extent and functionality of ex vivo B-cell polyclonal activation, quantify the EBV DNA integrated in B cells, enumerate the functional EBV DNA reservoir in B cells and circulating B cells spontaneously secreting EBV antigens in AIM. Study design Circulating B cells and B cells differentiating into plamablasts and plasma cells, early (BZLF1)- and late viral antigen (gp350)-secreting-cells (SCs) were enumerated in six AIM patients and seven healthy EBV carriers. Results In vitro B-cell polyclonal activation induced 8000–24,000 BZLF1- and 1000–3000 gp350-SCs/10 6 B cells, respectively. These data suggest that only 11.1–19.5% of cells expressing BZLF1 synthesized gp350 and so completed the EBV-lytic cycle. Furthermore, circulating spontaneous BZLF1- and gp350-SCs that reflect ongoing viral replication were rare (20–120 and 10–30/10 6 B cells, respectively), and their low numbers contrasted with the high levels of circulating plasma cells (1.1–10.2% of CD19 + B cells). Conclusion The in vivo terminal-B-cell differentiation into plasma cells could unmask EBV B-cell reservoir to specific cytotoxic T-cell response and combined with a predominant abortive functional-EBV-reservoir, strongly contribute to rapid decay of cellular EBV reservoir in AIM.

Identification of an HLA-A*0201-restricted cytotoxic T lymphocyte epitope from the lung carcinoma antigen, Lengsin

Lengsin is an eye lens protein with a glutamine synthetase domain. We previously identified this protein as a lung carcinoma antigen through cDNA microarray analysis. Lengsin protein is overexpressed irrespective of the histological type of lung carcinoma, but not in normal tissues other than the lens. Therefore, to significantly extend the use of Lengsin-based T-cell immunotherapies for the treatment of patients with lung carcinoma, we searched for HLA-A*0201-restricted epitopes from this protein by screening predicted Lengsin-derived candidate peptides for the induction of tumor-reactive CTLs. Four Lengsin-derived peptides were selected by computerized algorithm based on a permissive HLA-A*0201 binding motif, and were used to immunize HLA-A*0201 transgenic (HHD) mice. Two of the immunizing peptides, Lengsin(206-215)(FIYDFCIFGV) and Lengsin(270-279)(FLPEFGISSA), induced peptide-specific cytotoxic T lymphocytes (CTLs) in HHD mice, and thus were used to stimulate human peripheral blood lymphocytes invitro. Lengsin(206-215) and Lengsin (270-279) also induced human peptide-specific CTLs, and we were able to generate Lengsin(206-215)- and Lengsin(270-279)-specific CTL clones. The Lengsin(270-279)-specific CTL clone specifically recognized peptide-pulsed T2 cells, COS-7 cells expressing HLA-A*0201 and Lengsin, and HLA-A*0201+/Lengsin+ lung carcinoma cells in an HLA-A*0201-restricted manner. On the other hand, the Lengsin(206-215)-specific CTL clone failed to recognize HLA-A*0201+/Lengsin+ target cells in the absence of cognate peptide. These results suggest that Lengsin(270-279) is naturally processed and presented by HLA-A*0201 molecules on the surface of lung carcinoma cells and may be a new target for antigen-specific T-cell immunotherapy against lung cancer.

IL-7 receptor expression identifies suicide gene–modified allospecific CD8+ T cells capable of self-renewal and differentiation into antileukemia effectors

AbstractIn allogeneic hematopoietic cell transplantation (HSCT), donor T lymphocytes mediate the graft-versus-leukemia (GVL) effect, but induce graft-versus-host disease (GVHD). Suicide gene therapy—that is, the genetic induction of a conditional suicide phenotype into donor T cells—allows dissociating the GVL effect from GVHD. Genetic modification with retroviral vectors after CD3 activation reduces T-cell alloreactivity. We recently found that alloreactivity is maintained when CD28 costimulation, IL-7, and IL-15 are added. Herein, we used the minor histocompatibility (mH) antigens HA-1 and H-Y as model alloantigens to directly explore the antileukemia efficacy of human T cells modified with the prototypic suicide gene herpes simplex virus thymidine kinase (tk) after activation with different stimuli. Only in the case of CD28 costimulation, IL-7, and IL-15, the repertoire of tk+ T cells contained HA-1– and H-Y–specific CD8+ cytotoxic T cells (CTL) precursors. Thymidine kinase–positive HA-1– and H-Y–specific CTLs were capable of self-renewal and differentiation into potent antileukemia effectors in vitro, and in vivo in a humanized mouse model. Self-renewal and differentiation coincided with IL-7 receptor expression. These results pave the way to the clinical investigation of T cells modified with a suicide gene after CD28 costimulation, IL-7, and IL-15 for a safe and effective GVL effect.

Vaccination with immunoglobulin frame region-derived nonapeptide elicits cellular immune response against lymphoma in human leukocyte antigen-A2.1 transgenic mice

The epitope in the frame region (FR) of the immunoglobulin heavy chain variable region (IgHV) is a potential target for lymphoma immunotherapy. Our previous work identified a FR-derived nonapeptide (QLVQSGAEV) capable of in vitro eliciting anti-lymphoma specific cytotoxic T lymphocytes (CTLs) in lymphocytes from human leukocyte antigen (HLA)-A2.1 donors. Here we used HLA-A2.1 transgenic mice and SCID (severe combined immunodeficiency) mice to confirm the ability of these specific CTLs against lymphomas. We immunized transgenic mice with nonapeptide conjugated to the adjuvant of PADRE (pan HLA DR-binding epitope). The specificity of the elicited CTLs from the immunized transgenic mice was identified by enzyme-linked immunospot (ELISpot) assay and pentamer staining. The elicited CTLs specifically attacked lymphomas with surface IgHV1 in vitro. Adoptive transfer of the CTLs to SCID mice loaded with QLVQSGAEV(+)/HLA-A2.1(+) lymphoma cells effectively inhibited tumor growth. Our results indicate that the relatively constant epitope in the IgHV FR may be useful for immunotherapy of lymphomas with the same IgHV subfamily.

Neural bystander damage by infiltrating virus-infected T cells and the cytotoxic T lymphocytes in HTLV-I-associated neurological disease

We hypothesized that the cytotoxic T lymphocytes(CTLs) play a pivotal role in the pathogenesis of human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spasticparaparesis(HAM/TSP). One of the most striking features of the cellular immune response in the patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is the highly increased numbers of HTLV-I-specific cytotoxic T lymphocytes (CTLs) in the circulation of the blood and the cerebrospinal fluid, nevertheless HTLV-I proviral load in the PBMC remains high in the patients. To determine the CTL’s association with the pathogenesis of HAM/TSP in the CNS, we developed novel methods of in-situ detection for HTLV-I-specific CTL using HLA/antigen peptide tetramer and for the cells expressing HTLV-I viral protein. We visualized the HTLV-I specific CTLs and HTLV-I-infected cells in autopsied spinal cords of the patients with HAM/TSP. We demonstrated that HTLV-I-specific CTLs expressing cytotoxic molecules accumulated in the spinal cords from three patients with HAM/TSP and that HTLV-I exclusively infected CD4 positive T lymphocytes but neither resident cells nor macrophages. The phenotype of apoptotic cells was HTLV-I infected CD4+ T lymphocytes or HTLV-I non-infected oligodendrocytes. The findings suggest a unique pathogenesis for the neuroinflammatory disease that an inflammation of the central nervous system is attributed to the interaction between HTLV-I-infected CD4+ T cells and HTLV-I-specific CD8+ CTLs from the periphery.