Cellular Immunotherapy Of Cancer Research Articles

Ethical considerations of cellular immunotherapy for cancer.

With the rapid development of immunology, molecular biology, and associated technologies such as next-generation sequencing, cellular immunotherapy has recently become the fourth major cancer treatment. Immunotherapies based on T cells, natural killer cells, and dendritic cells play key roles in cancer immunotherapy. However, their application in clinical practice raises several ethical issues. Thus, studies should focus on proper adherence to basic ethical principles that can effectively guide and solve related clinical problems in the course of treatment, improve treatment effects, and protect the rights and interests of patients. In this review, we discuss cellular immunotherapy-related ethical issues and highlight the ethical practices and current status of cellular immunotherapy in China. These considerations may supplement existing ethical standards in cancer immunotherapy.

Cellular Immunotherapy of Canine Cancer.

Infusions with immune cells, such as lymphocytes or natural killer (NK) cells, represent one of several modalities of immunotherapy. In human patients with advanced B-cell leukemia or lymphoma, infusions with chimeric antigen receptor (CAR) T-lymphocytes have shown promising responses. However, the scientific and clinical development of cell-based therapies for dogs, who get cancer of similar types as humans, is lagging behind. One reason is that immune cells and their functionality in dogs are less well characterized, largely due a lack of canine-specific reagents to detect surface markers, and specific cytokines to isolate and expand their immune cells. This review summarizes the current status of canine cancer immunotherapies, with focus on autologous and allogeneic T-lymphocytes, as well as NK cells, and discusses potential initiatives that would allow therapies with canine immune cells to “catch up” with the advances in humans.

Immunological analysis of phase II glioblastoma dendritic cell vaccine (Audencel) trial: immune system characteristics influence outcome and Audencel up-regulates Th1-related immunovariables

Audencel is a dendritic cell (DC)-based cellular cancer immunotherapy against glioblastoma multiforme (GBM). It is characterized by loading of DCs with autologous whole tumor lysate and in vitro maturation via “danger signals”. The recent phase II “GBM-Vax” trial showed no clinical efficacy for Audencel as assessed with progression-free and overall survival in all patients. Here we present immunological research accompanying the trial with a focus on immune system factors related to outcome and Audencel’s effect on the immune system. Methodologically, peripheral blood samples (from apheresis before Audencel or venipuncture during Audencel) were subjected to functional characterization via enzyme-linked immunospot (ELISPOT) assays connected with cytokine bead assays (CBAs) as well as phenotypical characterization via flow cytometry and mRNA quantification. GBM tissue samples (from surgery) were subjected to T cell receptor sequencing and immunohistochemistry. As results we found: Patients with favorable pre-existing anti-tumor characteristics lived longer under Audencel than Audencel patients without them. Pre-vaccination blood CD8+ T cell count and ELISPOT Granzyme B production capacity in vitro upon tumor antigen exposure were significantly correlated with overall survival. Despite Audencel’s general failure to induce a significant clinical response, it nevertheless seemed to have an effect on the immune system. For instance, Audencel led to a significant up-regulation of the Th1-related immunovariables ELISPOT IFNγ, the transcription factor T-bet in the blood and ELISPOT IL-2 in a dose-dependent manner upon vaccination. Post-vaccination levels of ELISPOT IFNγ and CD8+ cells in the blood were indicative of a significantly better survival. In summary, Audencel failed to reach an improvement of survival in the recent phase II clinical trial. No clinical efficacy was registered. Our concomitant immunological work presented here indicates that outcome under Audencel was influenced by the state of the immune system. On the other hand, Audencel also seemed to have stimulated the immune system. Overall, these immunological considerations suggest that DC immunotherapy against glioblastoma should be studied further – with the goal of translating an apparent immunological response into a clinical response. Future research should concentrate on investigating augmentation of immune reactions through combination therapies or on developing meaningful biomarkers.

Ex Vivo PD-L1/PD-1 Pathway Blockade Reverses Dysfunction of Circulating CEA-Specific T Cells in Pancreatic Cancer Patients.

Purpose: Carcinoembryonic antigen (CEA) is a candidate target for cellular immunotherapy of pancreatic cancer. In this study, we have characterized the antigen-specific function of autologous cytotoxic T lymphocytes (CTL) specific for the HLA-A2-restricted peptide, pCEA691-699, isolated from the peripheral T-cell repertoire of pancreatic cancer patients and sought to determine if ex vivo PD-L1 and TIM-3 blockade could enhance CTL function.Experimental Design: CD8+ T-cell lines were generated from peripheral blood mononuclear cells of 18 HLA-A2+ patients with pancreatic cancer and from 15 healthy controls. In vitro peptide-specific responses were evaluated by flow cytometry after staining for intracellular cytokine production and carboxy fluorescein succinimydyl ester cytotoxicity assays using pancreatic cancer cell lines as targets.Results: Cytokine-secreting functional CEA691-specific CTL lines were successfully generated from 10 of 18 pancreatic cancer patients, with two CTL lines able to recognize and kill both CEA691 peptide-loaded T2 cells and CEA+ HLA-A2+ pancreatic cancer cell lines. In the presence of ex vivo PD-L1 blockade, functional CEA691-specific CD8+ T-cell responses, including IFNγ secretion and proliferation, were enhanced, and this effect was more pronounced on Ag-specific T cells isolated from tumor draining lymph nodes.Conclusions: These data demonstrate that CEA691-specific CTL can be readily expanded from the self-restricted T-cell repertoire of pancreatic cancer patients and that their function can be enhanced by PD-L1 blockade. Clin Cancer Res; 23(20); 6178-89. ©2017 AACR.

More Haste, Less Speed: Could Public-Private Partnerships Advance Cellular Immunotherapies?

Cellular immunotherapies promise to transform cancer care. However, they must overcome serious challenges, including: (1) the need to identify and characterize novel cancer antigens to expand the range of therapeutic targets; (2) the need to develop strategies to minimize serious adverse events, such as cytokine release syndrome and treatment-related toxicities; and (3) the need to develop efficient production/manufacturing processes to reduce costs. Here, we discuss whether these challenges might better be addressed through forms of public–private research collaborations, including public–private partnerships (PPPs), or whether these challenges are best addressed by way of standard market transactions. We reviewed 14 public–private relationships and 25 underlying agreements for the clinical development of cancer cellular immunotherapies in the US. Most were based on bilateral research agreements and pure market transactions in the form of service contracts and technology licenses, which is representative of the commercialization focus of the field. We make the strategic case that multiparty PPPs may better advance cancer antigen discovery and characterization and improved cell processing/manufacturing and related activities. In the rush toward the competitive end of the translational continuum for cancer cellular immunotherapy and the attendant focus on commercialization, many gaps have appeared in our understanding of cellular biology, immunology, and bioengineering. We conclude that the model of bilateral agreements between leading research institutions and the private sector may be inadequate to efficiently harness the interdisciplinary skills and knowledge of the public and private sectors to bring these promising therapies to the clinic for the benefit of cancer patients.

Preclinical trial of the multi-targeted lenvatinib in combination with cellular immunotherapy for treatment of renal cell carcinoma.

Lenvatinib is an oral, multi-targeted tyrosine kinase inhibitor of vascular endothelial growth factor receptors 1-3, fibroblast growth factor receptors 1-4, platelet-derived growth factor receptor β, RET and KIT. Cellular immunotherapy has the potential to be a highly targeted treatment, with low toxicity to normal tissues and a high capacity to eradicate tumor tissue. The present study assessed the safety, maximum tolerated dose (MTD) and preliminary antitumor activity of lenvatinib and cellular immunotherapy in a murine model of renal cell carcinoma (RCC). The present study used a therapeutic dose of 0.12 mg lenvatinib and/or 104 rat uterine cancer adenocarcinoma (RuCa)-sensitized lymphocytes administered once daily continuously in 7-day cycles. Tumor regression was observed in mice with RCC following treatment with lenvatinib and 104 RuCa-sensitized lymphocytes. MTD was established as once daily administration of 0.18 mg lenvatinib and 106 RuCa-sensitized lymphocytes. The most common treatment-related adverse effects observed were fatigue (40%), mucosal inflammation (30%), proteinuria, diarrhea, vomiting, hypertension and nausea (all 40%). Combination therapy using lenvatinib and cellular immunotherapy enhanced the antitumor effect induced by single treatments and prolonged the survival of mice with RCC compared with either of the single treatments. Treatment with lenvatinib (0.12 mg) combined with 104 RuCa-sensitized lymphocytes was associated with manageable toxicity consistent with individual agents. Further evaluation of this combination therapy in mice with advanced RCC is required. In conclusion, cellular immunotherapy and oncolytic therapy for cancer may be improved by the synergistic effects of lenvatinib and sensitized lymphocytes. In the present study, the inherent antineoplastic and immune stimulatory properties of the two agents were enhanced when used in combination, which may provide a basis for clinical treatment of patients with RCC.

Abstract 3722: In vitro induced regulatory T cells for understanding the nature of peripherally induced regulatory cells in relation to cell mediated immunotherapy for cancer

Abstract Background: pTregs are appearing to be one of the major obstacles of cellular immunotherapy of cancer. Tumor specific in vivo induced Cytotoxic T lymphocytes (CTL) are rapidly eliminated with concomitant expansion of pTreg cells. In human melanoma in ex vivo condition, tumor antigen specific CTL generated with CD8+ cells persisted for months while ex vivo induced CTL with whole PBL stimulated with Mart-127-35 A2 (tumor antigen) pulsed autologousDC, died within three weeks. Whereas Influenza (Flu 58-66) antigen specific CTL continued to expand and remained functional beyond four weeks. Method: Peripheral blood and tumors were obtained from patients diagnosed with metastatic melanoma after informed consent. Blood derived dendritic cells, PBL, CD4+ and CD8+ cells were isolated for in vitro co-cultures with Mart-1 or Flu peptide pulsed autologous DC or autologous tumor cells. CD8+ CTL response and CD4+ positive Treg responses were measured. Freshly obtained tumor tissue derived lymphocytes (TIL) were also used for the study. Results: Our results show that CTL response against Flu survived longer in culture with total PBL than CTL response against Mart-1 in identical culture conditions. CTL response using autologous tumor cells with PBL in culture lasted for a shorter duration but growth of Treg cells were observed. Cytokine analysis with expanding Treg cells showed secretion of IL-10 upon re-stimulation with autologous DC or anti-CD3 ab. Similarly, CD4+ cells isolated from freshly obtained tumor tissue (TIL, representing in vivo condition), suppressed T cell activation and secreted high amount of IL-10 upon re-stimulation. Conclusion: These findings suggested that in vitro induced iTreg from cultures or pTreg from TILs (in vivo), work identical way, to negatively regulate effector immune response by secreting IL-10. Further analysis with iTregs for their use of metabolic pathways would be important to identify the specific signaling molecules used by autologous (person specific) pTregs , as target molecules, to block the expansion of such pTregs for the better outcome for CTL mediated immunotherapy for cancer. Citation Format: Upendra P. Hegde, Nitya G. Chakraborty. In vitro induced regulatory T cells for understanding the nature of peripherally induced regulatory cells in relation to cell mediated immunotherapy for cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3722. doi:10.1158/1538-7445.AM2017-3722

Cellular immunotherapy of cancer: an overview and future directions.

The clinical success of checkpoint inhibitors has led to a renaissance of interest in cancer immunotherapies. In particular, the possibility of ex vivo expanding autologous lymphocytes that specifically recognize tumor cells has attracted much research and clinical trial interest. In this review, we discuss the historical background of tumor immunotherapy using cell-based approaches, and provide some rationale for overcoming current barriers to success of autologous immunotherapy. An overview of adoptive transfer of lymphocytes, tumor infiltrating lymphocytes and dendritic cell therapies is provided. We conclude with discussing the possibility of gene-manipulating immune cells in order to augment therapeutic activity, including silencing of the immune-suppressive zinc finger orphan nuclear receptor, NR2F6, as an attractive means of overcoming tumor-associated immune suppression.

Prospective pilot study of adoptive immunotherapy with autologous αβT cells for five cases of advanced and/or recurrent esophageal squamous cell carcinoma

Although several clinical trials of vaccination and cellular immunotherapy for esophageal cancer have been reported, clinical trials of anti-CD3-activated autologous αβT cell therapy for patients with esophageal squamous cell carcinoma are limited. A total of five patients with recurrent esophageal squamous cell carcinoma were enrolled in this prospective pilot study. Peripheral blood mononuclear cells from the patients were cultured with immobilized anti-CD3 antibody and interleukin-2 for approximately 14 days, and 5 × 109 lymphocytes were harvested. Expanded lymphocytes were intravenously infused every 2 weeks for six courses. All patients were followed up until death. The treatment response was assessed by the number of lymphocytes, CD4/CD8 ratio, serum tumor markers, and computed tomography scan. A total of 23 courses of treatment were completed. Three of the five patients (cases 2, 3, and 4) completed six courses of cell therapy according to the protocol. However, case 1 quit treatment after three courses and case 5 quit treatment after two courses because of tumor progression. There were no adverse effects related to cell therapy. Although cases 1 and 5 did not show any treatment benefits, cases 2, 3, and 4 showed some treatment benefits. Cases 2 and 4 showed long-term survived for more than 1 year. These results might suggest the safety and benefits of anti-CD3-activated autologous αβT cell therapy for the part of the patients with esophageal squamous cell carcinoma.

IL-21 maintains CD62L expression during NKT-cell ex vivo expansion and enhances antitumor activity of NKT cell therapy in vivo

Abstract Vα24-invariant Natural Killer T cells (NKTs) have potent antitumor properties and are being developed for cellular immunotherapy of cancer. Such therapy requires extensive ex vivo expansion of NKTs while preserving their longevity and function. In a recent report from our group, CD62L+ subset of NKTs has been shown to have longer persistence in vivo and stronger antitumor activity than CD62L− counterpart. However, the requirements for the preservation of CD62L+ NKTs during ex vivo expansion remain largely unknown. The gene expression analysis comparing CD62L+ and CD62L− NKTs revealed a significantly higher expression level of IL-21R mRNA in the former. FACS analysis confirmed the finding at the cell surface protein level. These led us to hypothesize that IL-21 preferentially supports CD62L+ NKTs. To test this hypothesis, we expanded primary human peripheral blood NKTs using in vitro stimulation with their cognate antigen, α-galactosylceramide. The culture was supplemented with IL-2, IL-21, or both cytokines. We found that in contrast to IL-2, IL-21 alone failed to support NKT-cell expansion. A combination of IL-2 and IL-21 led to similar absolute numbers of NKTs compared with IL-2 alone. However, the proportion of CD62L+ NKT-cell subset was significantly higher in the culture with IL-2/IL-21 combination compared with IL-2 alone after both primary and secondary expansions. Furthermore, after transfer to NSG mice, IL2/IL-21-expanded NKTs persisted significantly longer and had higher therapeutic efficacy in a lymphoma model compared with IL-2-expanded NKTs. Our results suggest that inclusion of IL-21 in the NKT-cell expansion protocol, which currently uses IL-2 alone, would increase antitumor potential of NKT cell therapy.

Natural Killer Group 2D Ligand Depletion Reconstitutes Natural Killer Cell Immunosurveillance of Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous and aggressive tumor originating from the epithelial lining of the upper aero-digestive tract accounting for 300,000 annual deaths worldwide due to failure of current therapies. The natural killer group 2D (NKG2D) receptors on natural killer (NK) cells and several T cell subsets play an important role for immunosurveillance of HNSCC and are thus targeted by tumor immune evasion strategies in particular by shedding of various NKG2D ligands (NKG2DLs). Based on plasma and tumor samples of 44 HNSCC patients, we found that despite compositional heterogeneity the total plasma level of NKG2DLs correlates with NK cell inhibition and disease progression. Strikingly, based on tumor spheroids and primary tumors of HNSCC patients, we found that NK cells failed to infiltrate HNSCC tumors in the presence of high levels of NKG2DLs, demonstrating a novel mechanism of NKG2DL-dependent tumor immune escape. Therefore, the diagnostic acquisition of the plasma level of all NKG2DLs might be instrumental for prognosis and to decipher a patient cohort, which could benefit from restoration of NKG2D-dependent tumor immunosurveillance. Along these lines, we could show that removal of shed NKG2DLs (sNKG2DLs) from HNSCC patients’ plasma restored NK cell function in vitro and in individual patients following surgical removal of the primary tumor. In order to translate these findings into a therapeutic setting, we performed a proof-of-concept study to test the efficacy of adsorption apheresis of sNKG2DLs from plasma after infusion of human MICA in rhesus monkeys. Complete removal of MICA was achieved after three plasma volume exchanges. Therefore, we propose adsorption apheresis of sNKG2DLs as a future preconditioning strategy to improve the efficacy of autologous and adoptively transferred immune cells in cellular cancer immunotherapy.

Oncolysate-loaded Escherichia coli bacterial ghosts enhance the stimulatory capacity of human dendritic cells.

The natural adjuvant properties of bacterial ghosts (BGs) lie within the presence of intact pathogen-associated molecular patterns on their surface. BGs can improve the direct delivery, natural processing and presentation of target antigens within dendritic cells (DCs). Moreover, sensitization of human DCs by cancer cell lysate (oncolysate)-loaded BGs in the presence of IFN-α and GM-CSF enhanced DC maturation as indicated by an increased expression of maturation markers and co-stimulatory molecules, higher production of IL-12p70 and stimulation of significantly increased proliferation of both autologous CD4+ and CD8+ T cells compared to DCs matured in the presence of purified lipopolysaccharide. The induced T cells efficiently recognized oncolysate-derived tumor-associated antigens expressed by cancer cells used for the production of oncolysate. Our optimized one-step simultaneous antigen delivery and DC maturation-inducing method emerges as a promising tool for the development and implementation of next-generation cellular cancer immunotherapies.

Abstract B065: Use of simultaneous detection of externalized CD107a and CD137 for evaluation of specificity and effector functions of polyclonal T-cells produced for adoptive cellular immunotherapy of prostate cancer

Abstract The ex vivo production of T-cells that can recognize tumor-associated antigens (TAAs) and through this recognition eliminate cancer cells is the goal of T-cell-based adoptive cellular immunotherapy (ACI). The technological approaches that use whole cancer cells or their pertinent lysates as a source of TAAs for production of these T-cells lead to generation of polyclonal T-cells their antigen specificity of which is often unknown. Evaluation of such polyclonal T-cells is therefore challenging because their target and off-target performance might be difficult to predict. In our study we used human dendritic cells (DCs) pulsed with inactivated prostate cancer cell line LNCaP and matured with polyinosinic:polycytidylic acid (poly(I:C)) to induce expansion of autologous lymphocytes. The expanded lymphocytes were then challenged with live LNCaP cells and analyzed by simultaneous detection of a marker of T-cell cytotoxicity and antigen-induced T-cell stimulation, the surface externalization of, respectively, CD107a and CD137. Our data showed that lymphocytes expanded with LNCaP-pulsed and poly(I:C)-matured DCs contained a notable fraction of CD137 and/or CD107a positive T-cells after their challenge with live LNCaP cells. No such notable fraction was however observed in resting lymphocytes, or when the lymphocytes were challenged with ovarian cancer cell line SKOV-3. Importantly, autologous lymphocytes that were expanded with poly(I:C)-matured DCs not previously pulsed with the inactivated LNCaP cells did not show a notable population of the marker positive cells. Collectively, our data showed that simultaneous detection of externalized CD107a and CD137 on the surface of polyclonal T-cells may allow for evaluation of polyclonal T-cell specificity and effector functions after their challenge with complex antigens. Citation Format: Pavla Taborska, Dmitry Stakheev, Katerina Vavrova, Petra Vrabcova, Jirina Bartunkova, Daniel Smrz. Use of simultaneous detection of externalized CD107a and CD137 for evaluation of specificity and effector functions of polyclonal T-cells produced for adoptive cellular immunotherapy of prostate cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B065.

Vaccination Against Human Papilloma Viruses Leads to a Favorable Cytokine Profile of Specific T Cells.

Several human papilloma viruses (HPV) are known to cause malignant transformation. The high-risk type HPV 16 is associated with cervical carcinoma and head and neck squamous cell carcinoma. HPV 16-positive tumor cells exclusively carry the HPV 16 oncogenes E6 and E7. These oncogenes appear as excellent targets for an adoptive immunotherapy. We here addressed the question whether specific T cells from HPV-vaccinated healthy volunteers could be especially suitable for an HPV-specific cellular immunotherapy. Of note, vaccines contain HPV 16. To quantify HPV 16 E6-specific and E7-specific cells, enzyme-linked immunospot assays to measure interferon-γ (IFN-γ) and interleukin-10 (Th1-Th2 balance) and the secretion of the cytotoxic molecules granzyme B and perforin have been optimized. The frequency of peripheral blood mononuclear cells secreting IFN-γ and perforin was significantly (P<0.05) increased in HPV-vaccinated versus nonvaccinated volunteers. Overall, however, the median frequency of HPV 16-specific cells with a favorable secretion profile (Th1 balanced and cytotoxic) was low even in vaccinated volunteers (IFN-γ: 0.0018% and 0.0023%, perforin: 0.01% and 0.0087% for E6-specific and E7-specific cells, respectively). But some vaccinated volunteers showed up to 0.1% HPV-specific, IFN-γ or perforin-secreting cells. In conclusion, our data suggest that vaccinated volunteers are superior to nonvaccinated donors for HPV-specific cellular cancer immunotherapy.

Abstract 4890: Preclinical analysis of combination therapy using radiation and cellular immunotherapy

Abstract Purpose: To optimize the combination of ionizing radiation and cellular immunotherapy using a preclinical autochthonous model of prostate cancer. Methods and Materials: Transgenic mice expressing a model antigen under a prostate-specific promoter were treated using a platform that integrates cone-beam CT imaging with 3-dimensional conformal therapy. Using this technology we investigated the immunologic and therapeutic effects of combining ionizing radiation with granulocyte/macrophage colonystimulating factor-secreting cellular immunotherapy for prostate cancer in mice bearing autochthonous prostate tumors. Results: The combination of ionizing radiation and immunotherapy resulted in a significant decrease in pathologic tumor grade and gross tumor bulk that was not evident with either single-modality therapy. Furthermore, combinatorial therapy resulted in improved overall survival in a preventive metastasis model and in the setting of established micrometastases. Mechanistically, combined therapy resulted in an increase of the ratio of effector-to regulatory T cells for both CD4 and CD8 tumor-infiltrating lymphocytes. Conclusions: Our preclinical model establishes a potential role for the use of combined radiation-immunotherapy in locally advanced prostate cancer, which warrants further exploration in a clinical setting. Citation Format: Satoshi Wada. Preclinical analysis of combination therapy using radiation and cellular immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4890.

DNA methyltransferase inhibition increases efficacy of adoptive cellular immunotherapy of murine breast cancer.

Adoptive T cell immunotherapy is a promising approach to cancer treatment that currently has limited clinical applications. DNA methyltransferase inhibitors (DNAMTi) have known potential to affect the immune system through multiple mechanisms that could enhance the cytotoxic T cell responses, including: upregulation of tumor antigen expression, increased MHC class I expression, and blunting of myeloid derived suppressor cells (MDSCs) expansion. In this study, we have investigated the effect of combining the DNAMTi, decitabine, with adoptive T cell immunotherapy in the murine 4T1 mammary carcinoma model. We found that expression of neu, MHC class I molecules, and several murine cancer testis antigens (CTA) was increased by decitabine treatment of 4T1 cells in vitro. Decitabine also increased expression of multiple CTA in two human breast cancer cell lines. Decitabine-treated 4T1 cells stimulated greater IFN-gamma release from tumor-sensitized lymphocytes, implying increased immunogenicity. Expansion of CD11b+Gr1+MDSC in 4T1 tumor-bearing mice was significantly diminished by decitabine treatment. Decitabine treatment improved the efficacy of adoptive T cell immunotherapy in mice with established 4T1 tumors, with greater inhibition of tumor growth and an increased cure rate. Decitabine may have a role in combination with existing and emerging immunotherapies for breast cancer.

CD62L+ NKT cells have superior in vivo persistence and anti-tumor activity

Abstract Va24-invariant Natural Killer T cells (NKTs) have potent antitumor properties and are being developed for cellular immunotherapy of cancer. Such therapy requires extensive ex vivo expansion of primary NKTs while preserving their longevity and function. However, the mechanism responsible for NKT-cell maintenance remains poorly understood. In this study we analyzed the proliferative potential of human NKT-cell subsets and found that antigen-induced in vitro expansion of primary NKTs is associated with the accumulation of a CD62L-positive subset. Following magnetic sorting, only CD62L-positive cells survived and proliferated in response to TCR-stimulation in vitro. After transfer to NSG mice, CD62L-positive NKTs persisted 5 times longer and had higher therapeutic efficacy in a lymphoma model compared with CD62L-negative NKTs. Proliferating CD62L-positive cells downregulated or maintained CD62L expression when they were activated via TCR alone or in combination with co-stimulatory receptors, respectively. After testing 161 clones of K562 cells, genetically modified to express CD1d and various combinations of co-stimulatory molecules, we selected the B-8-2 clone (HLAnullCD1dmedCD86high4-1BBmedOX40Lmed) which induced the highest rate of NKT-cell expansion with the preservation of CD62L-positive cells. Compared with NKTs expanded with autologous PBMC, those expanded with B-8-2 exhibited a prolonged in vivo persistence and superior antitumor activity. Thus, our results reveal a previously unanticipated functional hierarchy in human NKTs that can be exploited for cancer immunotherapy.

Abstract A039: Caspase-2 and oxidative-ER stress crosstalk regulates the exposure of “eat me” signal calreticulin by high hydrostatic pressure treated cancer cells

Abstract Immunogenic cell death (ICD) is a type of cell death induced by various chemotherapeutics, physical modalities such as photodynamic therapy, radiotherapy and high hydrostatic pressure (HHP). HHP induces immunogenic cell death of cancer cell is currently tested in running II. and III. phase of clinical trial for preparation of active cellular cancer immunotherapy (DCVAC). Previous studies have shown that the immunogenicity of ICD mainly relies on surface exposed calreticulin (ecto-CRT); CRT is normally located in the lumen of the endoplasmic reticulum (ER) and may translocate to the cell surface within hours after treatment. Although ICD can be triggers by HHP, underlying danger signaling pathways remain unclear. Therefore ovarian (OV90) cancer cell line was treated by HHP, UV-B and anthracycline (Idarubicin). The kinetics of ICD markers and key components of ER stress-mediated apoptotic pathway were analyzed by flow cytometry, western blotting and confocal microscopy. In this work we described the ability of HHP triggers oxidative ER-stress (the production of reactive oxygen species (ROS) together with ER stress response) which results in phosphorylation of PERK and eIF2α. We also observed a rapid caspase-8 activation subsequently followed by BAP31 cleavage, Bid activation and caspase-3 activation. While caspase-8 activation was necessary for cell surface exposure of calreticulin (ecto-CRT) after HHP treatment, proapoptotic proteins Bax and Bak were dispensable for this process. Moreover, we identified that HHP treatment leads to caspase-2 activation. Whereas Idarubicin treatment has no effect on ecto-CRT exposure in Caspase-2 knockdown tumor cells, ecto-CRT was significantly decreased in these cells after HHP treatment. In addition, phagocytosis of HHP-treated Caspase-2 knockdown tumor cells was compromised in contrast to Idarubicin-treated knockout tumor cells. In conclusion, we characterized the molecular mechanism of ecto-CRT exposure pathway induced by HHP which is analogous to that induced by anthracycline treatment. For the first time we provide evidence that caspase-2 might play an important role in the HHP-mediated ecto-CRT exposure. Citation Format: Irena Kusova Moserova, Iva Truxova, Abhishek G, Garg, Patrizia Agostinis, Piere Francois Cartron, Sarka Vosahlikova, Radek Spisek, Jitka Fucikova. Caspase-2 and oxidative-ER stress crosstalk regulates the exposure of “eat me” signal calreticulin by high hydrostatic pressure treated cancer cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A039.

Human CD21low CD86pos B Cells Are Potent Antigen Presenting Cells, Which Are Expanded in Inflammatory Conditions

Accumulating evidence from animal models demonstrates that antigen presentation by B cells plays a crucial role during the natural immune response as well as in several important diseases. While resting B cells are poor antigen-presenting cells they can acquire strong immunostimulatory properties after activation via receptors such as CD40. Studies in animal models suggest that ex vivo generated CD40-actived B cells could serve a promising platform of cellular cancer immunotherapy. However, our understanding of the function of antigen presentation by B cells in human remains limited. This is partly due to the lack of well-defined cell surface markers that can be used to identify distinct immunostimulatory B cell subsets. We hypothesized that antigen-presenting B cells in humans would be characterized by a similar phenotype than in vitro generated CD40-activated B cells. Using a transcriptomic approach combined with flow cytometric phenotypic screening, we were able to show that CD40-activated human B cells can be distinguished from resting B cells by high expression of costimulatory receptors and low expression of BCR-associated coreceptors such as CD21 and FcγRIIB. Here, we report that CD21low CD86pos B cells represent a distinct B lymphocyte subpopulation with potent immunostimulatory capacity. This antigen-presenting B-cell subset possesses phenotypic and functional features of strong antigen-presenting cells, i.e. they express high levels of costimulatory molecules and inflammatory adhesion molecules and low levels of ITIM-bearing inhibitory receptors. In vitro, purified human CD21low CD86pos B cells induced stronger T cell proliferation than the other three subsets, which are defined by the markers CD21 and CD86. Since Epstein Barr Virus (EBV) transforms human B cells with the help of the viral protein LMP1, which mimics CD40-mediated activation of B cells, we first studied patients with replicative EBV infection. We found that CD21low CD86pos B cells were expanded in severely immunosuppressed patients with EBV-reactivation. Analysis of CD21low CD86pos B lymphocytes in blood samples of healthy volunteers and patients with rheumatoid arthritis revealed that this subpopulation was increased in inflammatory conditions. One week following vaccination there was an increase in the frequency of this B-cell subset. Furthermore, compared to healthy controls CD21low CD86pos B cells were significantly elevated in the blood of patients with rheumatoid arthritis (RA). Interestingly, the CD21low CD86pos B-cell subset made up the majority of the B lymphocytes in synovial fluid of RA patients with inflammatory knee effusion. In summary, we demonstrate that CD21low CD86pos B cells are a novel antigen-presenting B-cell subset, which is expanded in inflammatory conditions. Our findings suggest that this subset might represent a promising therapeutic target for the treatment of inflammatory diseases, such as rheumatoid arthritis, were antigen presentation by B cells plays a pathogenetic role. DisclosuresHallek:Mundipharma: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Celgene: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Roche: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Boehringher Ingelheim: Honoraria, Other: Speakers Bureau and/or Advisory Boards; Pharmacyclics: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; AbbVie: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Gilead: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Janssen: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding.

Synthetic immunobiology boosts the IQ of T cells

Engineered chimeric antigen receptors show promise