Profiling the T Cell Receptor Alpha/Delta Locus in Salmonids.

In jawed vertebrates, two major T cell populations have been characterized. They are defined as α/ß or γ/δ T cells, based on the expressed T cell receptor. Salmonids (family Salmonidae) include two key teleost species for aquaculture, rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) which constitute important models for fish immunology and important targets for vaccine development. The growing interest to decipher the dynamics of adaptive immune responses against pathogens or vaccines has resulted in recent efforts to sequence the immunoglobulin (IG) or antibodies and T cell receptor (TR) repertoire in these species. In this context, establishing a comprehensive and coherent locus annotation is the fundamental basis for the analysis of high-throughput repertoire sequencing data. We therefore decided to revisit the description and annotation of TRA/TRD locus in Atlantic salmon and two strains of rainbow trout (Swanson and Arlee) using the now available high-quality genome assemblies. Phylogenetic analysis of functional TRA/TRD V genes from these three genomes led to the definition of 25 subgroups shared by both species, some with particular feature. A total of 128 TRAJ genes were identified in Salmo, the majority with a close counterpart in Oncorhynchus. Analysis of expressed TRA repertoire indicates that most TRAV gene subgroups are expressed at mucosal and systemic level. The present work on TRA/TRD locus annotation along with the analysis of TRA repertoire sequencing data show the feasibility and advantages of a common salmonid TRA/TRD nomenclature that allows an accurate annotation and analysis of high-throughput sequencing results, across salmonid T cell subsets.

Exploring the role of the multiple sclerosis susceptibility gene CLEC16A in T cells.

C-type lectin-like domain family 16 member A (CLEC16A) is associated with autoimmune disorders, including multiple sclerosis (MS), but its functional relevance is not completely understood. CLEC16A is expressed in several immune cells, where it affects autophagic processes and receptor expression. Recently, we reported that the risk genotype of an MS-associated single nucleotide polymorphism in CLEC16A intron 19 is associated with higher expression of CLEC16A in CD4+ T cells. Here, we show that CLEC16A expression is induced in CD4+ T cells upon T cell activation. By the use of imaging flow cytometry and confocal microscopy, we demonstrate that CLEC16A is located in Rab4a-positive recycling endosomes in Jurkat TAg T cells. CLEC16A knock-down in Jurkat cells resulted in lower cell surface expression of the T cell receptor, however, this did not have a major impact on T cell activation response in vitro in Jurkat nor in human, primary CD4+ T cells.

Ibrutinib for improved chimeric antigen receptor T-cell production for chronic lymphocytic leukemia patients.

Chimeric antigen receptor T (CART) cells targeting CD19 have shown promising results in the treatment of chronic lymphocytic leukemia (CLL). However, efficacy seems to be inferior compared to diffuse large B-cell lymphoma or acute lymphoblastic leukemia. Impaired T-cell fitness of CLL patients may be involved in treatment failure. Less-differentiated naïve-like T cells play an important role in CART expansion and long-term persistence in vivo. These cells are sparse in CLL patients. Therefore, optimization of CART cell production protocols enriching less differentiated T cell subsets may overcome treatment resistance. The B-cell receptor inhibitor ibrutinib targeting Bruton's tyrosine kinase (BTK) is approved for the treatment of CLL. Besides BTK, ibrutinib additionally inhibits interleukin-2-inducible T-cell kinase (ITK) which is involved in T-cell differentiation. To evaluate the effect of ibrutinib on CART cell production, peripheral blood mononuclear cells from nine healthy donors and eight CLL patients were used to generate CART cells. T-cell expansion and phenotype, expression of homing and exhaustion makers as well as functionality of CART cells were evaluated. CART cell generation in the presence of ibrutinib resulted in increased cell viability and expansion of CLL patient-derived CART cells. Furthermore, ibrutinib enriched CART cells with less-differentiated naïve-like phenotype and decreased expression of exhaustion markers including PD-1, TIM-3 and LAG-3. In addition, ibrutinib increased the cytokine release capacity of CLL patient-derived CART cells. In summary, BTK/ITK inhibition with ibrutinib during CART cell culture can improve yield and function of CLL patient-derived CART cell products.

Idelalisib for optimized CD19-specific chimeric antigen receptor T cells in chronic lymphocytic leukemia patients.

Despite encouraging results with chimeric antigen receptor T (CART) cells, outcome can still be improved by optimization of the CART cell generation process. The proportion of less-differentiated T cells within the transfused product is linked to enhanced in vivo CART cell expansion and long-term persistence. The clinically approved PI3Kδ inhibitor idelalisib is well established in the treatment of B cell malignancies. Besides B cell receptor pathway inhibition, idelalisib can modulate T cell differentiation and function. Here, detailed longitudinal analysis of idelalisib-induced effects on T cell phenotype and function was performed during CART cell production. A third generation CD19.CAR.CD28.CD137zeta CAR vector system was used. CART cells were generated from peripheral blood mononuclear cells of healthy donors (HDs) and chronic lymphocytic leukemia (CLL) patients. Idelalisib-based CART cell generation resulted in an enrichment of less-differentiated naïve-like T cells (CD45RA+CCR7+), decreased expression of the exhaustion markers PD-1 and Tim-3, as well as upregulation of the lymph node homing marker CD62L. Idelalisib increased transduction efficiency, but did not impair viability and cell expansion. Strikingly, CD4:CD8 ratios that were altered in CART cells from CLL patients were approximated to ratios in HDs by idelalisib. Furthermore, in vivo efficacy of idelalisib-treated CART cells was validated in a xenograft mouse model. Intracellular TNF-α and IFN-γ production decreased in presence of idelalisib. This effect was reversible after resting CART cells without idelalisib. In summary, PI3Kδ inhibition with idelalisib can improve CART cell products, particularly when derived from CLL patients. Further studies with idelalisib-based CART cell generation protocols are warranted.

Challenges and prospects of chimeric antigen receptor T cell therapy in solid tumors.

Chimeric antigen receptor (CAR) T cell therapy is a novel and innovative immunotherapy. CAR-T cells are genetically engineered T cells, carrying MHC independent specific antigen receptor and co-stimulatory molecule which can activate an immune response to a cancer specific antigen. This therapy showed great results in hematological malignancies but were unable to prove their worth in solid tumors. Likely reasons for their failure are lack of antigens, poor trafficking, and hostile tumor microenvironment. Excessive amount of research is going on to improve the efficacy of CAR T cell therapy in solid tumors. In this article, we will discuss the challenges faced in improving the outcome of CAR T cell therapy in solid tumors and various strategies adopted to curb them.

Chimeric antigen receptor T cell therapy in pancreatic cancer: from research to practice.

Chimeric antigen receptor (CAR) T cell therapy is genetically engineered tumor antigen-specific anticancer immunotherapy, which after showing great success in hematological malignancies is currently being tried in advanced solid tumors like pancreatic cancer. Immunosuppressive tumor microenvironment and dense fibrous stroma are some of the limitation in the success of this novel therapy. However, genetic modifications and combination therapy is the topic of the research to improve its efficacy. In this article, we summarize the current state of knowledge, limitations, and future prospects for CAR T cell therapy in pancreatic cancer.

Prospects of chimeric antigen receptor T cell therapy in ovarian cancer.

Despite advances in various chemotherapy regimens, current therapeutic options are limited for ovarian cancer patients. Immunotherapy provides a promising and novel treatment option for ovarian cancer. Recently, chimeric antigen receptor (CAR) T cell therapy has shown promising results in hematological tumors and current research is going on in various solid tumors like ovarian cancer. CAR T cells are genetically engineered T cells with major histocompatibility complex-independent, tumor-specific, immune-mediated cytolytic actions against cancer cells. Initial studies of CAR T cell therapy have shown promising results in ovarian cancer, but there are some obstacles like impaired T cell trafficking, lack of antigenic targets, cytokine release syndrome and most important immunosuppressive tumor microenvironment. Optimization of design, improving tumor microenvironment and combinations with other therapies may help us in improving CAR T cell efficacy. In this review article, we highlight the current knowledge regarding CAR T cell therapy in ovarian cancer. We have discussed basic functioning of CAR T cells, their rationale and clinical outcome in ovarian cancer with limitations.

Invariant natural killer T infiltration in neuroblastoma with favorable outcome.

BACKGROUND: Tumor immunity has been suggested to play a key role in clinical and biological behavior of neuroblastomas. Given that CD1-restricted invariant natural killer T (iNKT) cells enhance both innate and acquired tumor immunity, we investigated the expression of the iNKT-cell-specific T-cell receptor Vα24-Jα18 in neuroblastoma tissues and its correlation with clinical and biological characteristics. METHODS: Using real- time quantitative PCR, we quantified the expression of Vα24-Jα18 in untreated tumor samples from 107 neuroblastoma cases followed in our institution and analyzed the correlation between the presence of infiltrated iNKT cells and clinical characteristics or patients' outcome. RESULTS: Vα24-Jα18 receptor was detected in 62 untreated cases (57.9%). The expression was significantly higher in stages 1, 2, 3, or 4S (P = 0.0099), in tumors with low or intermediate risk (P = 0.0050), with high TrkA expression (P = 0.0229), with favorable histology (P = 0.0026), with aneuploidy (P = 0.0348), and in younger patients (P = 0.0036). The overall survival rate was significantly higher in patients with iNKT-cell infiltration (log-rank; P = 0.0089). CONCLUSIONS: Since tumor-infiltrating iNKT cells were predominantly observed in neuroblastomas undergoing spontaneous differentiation and/or regression, we suggest that iNKT cells might play a key role in these processes.

Engineering CAR T Cells with Biomaterials.

Incorporating biomaterials into the design of CAR T cells may yield new, improved versions of this immunotherapy. Two preclinical studies indicate the potential of using biodegradable nanoparticles to program circulating T cells into CAR T cells in situ, and delivering these therapeutic cells directly to solid tumors via small dissolvable sponges.

Studying Immunoreceptor Signaling in Human T Cells Using Electroporation of In Vitro Transcribed mRNA.

The recognition bestowed upon T lymphocytes as key mediators of cellular immunity has been further attested by recent successful clinical studies using genetically modified T cells. With an ever-growing interest in the application of T cells to treat human malignancies, studying the molecular mechanisms of T cell activation, signaling, and function has become imperative. This, therefore, calls for the development of new easy-to-use and accurate models to investigate the biological phenomena that begin at the synaptic levels of T cell and antigen interactions to the ultimate exhaustion and death of the T cell. Here, we describe an approach to transiently express a chimeric molecule on the cell surface that permits activation and expansion of T cells, thereby providing a model to study T cell signaling.

Hepatitis C Virus-Specific T Cell Receptor mRNA-Engineered Human T Cells: Impact of Antigen Specificity on Functional Properties.

Therapy with genetically modified autologous T cells has shown great promise in cancer therapy. For an efficient control of hepatitis C virus (HCV) infection, cytotoxic T cells (CTL) are pivotal, but persistence of activated T cells may lead to liver toxicity. Here, anti-HCV T cell receptors (TCRs) recognizing the HCV nonstructural (NS) NS3 or NS5 viral peptide target were examined by mRNA transfection of human peripheral blood lymphocytes (PBLs) derived from healthy donors as well as chronically infected HCV patients. Immunological analysis shows that while the CTLs expressing the NS5-specific TCR reduced HCV RNA replication by a noncytotoxic mechanism, the NS3-specific TCR-redirected CTLs were polyfunctional and inhibited HCV RNA replication through antigen-specific cytotoxicity. Transcriptome signatures from these two types of CTL responses revealed uniquely expressed gene clusters upon encountering hepatoma target cells presenting endogenously expressed HCV proteins. The NS3 TCR induced a rapid expression of apoptotic signaling pathways and formation of embryonic gene clusters, whereas the NS5A TCR activation induced extended proliferative and metabolic pathways as the HCV target cells survived. Our results provide detailed insights into basic HCV T cell immunology and have clinical relevance for redirecting T cells to target virally infected hepatoma cells.IMPORTANCE Due to the protective ability of HCV-specific T cells and the hepatotoxic potential that they possess, there is a great need for the understanding of the functional aspects of HCV-specific T cells. To circumvent the low level of precursor frequency in patients, we engineered primary CD8+ T cells by mRNA TCR vectors to confer HCV specificity to new T cells. HCV TCRs that differ in antigen specificity and polyfunctionality were examined. mRNA TCR engineering of peripheral blood lymphocytes from healthy donors or chronically infected HCV patients resulted in strikingly high levels of HCV TCR expression and HCV-specific responses. While a cytotoxicity response from a polyfunctional T cell activation caused hepatotoxicity and the rapid induction of apoptotic signaling pathways, the noncytotoxic T cell activation showed extended proliferative, metabolic pathways and persistence of HCV target cells. Our results provide detailed insights into basic HCV T cell immunology and have clinical relevance for immune protection of HCV-associated diseases.

Intraperitoneal immunotherapy with T cells stably and transiently expressing anti-EpCAM CAR in xenograft models of peritoneal carcinomatosis.

The epithelial cell adhesion molecule (EpCAM) is overexpressed in a wide variety of tumor types, including peritoneal carcinomatosis (PC) from gastrointestinal and gynecological malignancies. To develop a chimeric antigen receptor T (CART) cell therapy approach to treat patients with end-stage PC, we constructed third generation CARs specific to EpCAM using the 4D5MOC-B single chain variable fragment. CART cells were generated with lentiviral transduction and exhibited specific in vitro killing activity against EpCAM-positive human ovarian and colorectal cancer cells. A single intraperitoneal injection of the CART cells eradicated established ovarian xenografts and resulted in significantly prolonged animal survival. Since EpCAM is also expressed on normal epithelium, anti-EpCAM CART cells were generated by mRNA electroporation that display a controlled cytolytic activity with a limited CAR expression duration. Multiple repeated infusions of these RNA CAR-modified T cells delayed disease progression in immunodeficient mice bearing well-established peritoneal ovarian and colorectal xenografts. Thus, our study demonstrates the effectiveness of using anti-EpCAM CAR-expressing T cells for local treatment of PC in mice. The possibility of using this approach for clinical treatment of EpCAM-positive gastrointestinal and gynecological malignancies warrants further validation.

An Optimized GD2-Targeting Retroviral Cassette for More Potent and Safer Cellular Therapy of Neuroblastoma and Other Cancers.

Neuroblastoma is the commonest extra cranial solid cancer of childhood. Despite escalation of treatment regimens, a significant minority of patients die of their disease. Disialoganglioside (GD2) is consistently expressed at high-levels in neuroblastoma tumors, which have been targeted with some success using therapeutic monoclonal antibodies. GD2 is also expressed in a range of other cancer but with the exception of some peripheral nerves is largely absent from non-transformed tissues. Chimeric Antigen Receptors (CARs) are artificial type I proteins which graft the specificity of a monoclonal antibody onto a T-cell. Clinical data with early CAR designs directed against GD2 have shown some promise in Neuroblastoma. Here, we describe a GD2-targeting CAR retroviral cassette, which has been optimized for CAR T-cell persistence, efficacy and safety.

Analysis of differential ß variable region of T cell receptor expression and NAV3/TNFRSF1B gene mutation in mycosis fungoides.

OBJECTIVE: This study aimed to analyze the predominant expression of the variable region of T cell receptor (TRBV) and determine whether NAV3 or TNFRSF1B gene mutation involved in the pathogenesis of MF. RESULTS: TRBV5-7 expression increased from the normal, early-stage to advanced-stage lesion in MF patient. By contrast, TRBV2 decreased with the lesion developed. We found no mutations of NAV3 or TNFRSF1B in the lesions from this study. MATERIALS AND METHODS: Real-time PCR were used to screen differential expression of TRBV in different lesions. Mutational analyses were used to validate genetic alterations in the skin lesions. CONCLUSIONS: The identification of TRBV gene expression differences between normal and different stages of MF lesions provide insight into promising new diagnostic and prognostic biomarkers. None of the reported genetic abnormalities suggests complexity of progress from a primary cytogenetic event to an advanced stage with poor prognosis in MF.

mRNA Cancer Vaccines.

mRNA cancer vaccines are a relatively new class of vaccines, which combine the potential of mRNA to encode for almost any protein with an excellent safety profile and a flexible production process. The most straightforward use of mRNA vaccines in oncologic settings is the immunization of patients with mRNA vaccines encoding tumor-associated antigens (TAAs). This is exemplified by the RNActive® technology, which induces balanced humoral and cellular immune responses in animal models and is currently evaluated in several clinical trials for oncologic indications. A second application of mRNA vaccines is the production of personalized vaccines. This is possible because mRNA vaccines are produced by a generic process, which can be used to quickly produce mRNA vaccines targeting patient-specific neoantigens that are identified by analyzing the tumor exome. Apart from being used directly to vaccinate patients, mRNAs can also be used in cellular therapies to transfect patient-derived cells in vitro and infuse the manipulated cells back into the patient. One such application is the transfection of patient-derived dendritic cells (DCs) with mRNAs encoding TAAs, which leads to the presentation of TAA-derived peptides on the DCs and an activation of antigen-specific T cells in vivo. A second application is the transfection of patient-derived T cells with mRNAs encoding chimeric antigen receptors, which allows the T cells to directly recognize a specific antigen expressed on the tumor. In this chapter, we will review preclinical and clinical data for the different approaches.

Retroviral Vectors for Cancer Gene Therapy.

Advances in molecular technologies have led to the discovery of many disease-related genetic mutations as well as elucidation of aberrant gene and protein expression patterns in several human diseases, including cancer. This information has driven the development of novel therapeutic strategies, such as the utilization of small molecules to target specific cellular pathways and the use of retroviral vectors to retarget immune cells to recognize and eliminate tumor cells. Retroviral-mediated gene transfer has allowed efficient production of T cells engineered with chimeric antigen receptors (CARs), which have demonstrated marked success in the treatment of hematological malignancies. As a safety point, these modified cells can be outfitted with suicide genes. Customized gene editing tools, such as clustered regularly interspaced short palindromic repeats-CRISPR-associated nucleases (CRISPR-Cas9), zinc-finger nucleases (ZFNs), or TAL-effector nucleases (TALENs), may also be combined with retroviral delivery to specifically delete oncogenes, inactivate oncogenic signaling pathways, or deliver wild-type genes. Additionally, the feasibility of retroviral gene transfer strategies to protect the hematopoietic stem cells (HSC) from the dose-limiting toxic effects of chemotherapy and radiotherapy was demonstrated. While some of these approaches have yet to be translated into clinical application, the potential implications for improved cellular replacement therapies to enhance and/or support the current treatment modalities are enormous.

Minicircle-Based Engineering of Chimeric Antigen Receptor (CAR) T Cells.

Plasmid DNA is being used as a pharmaceutical agent in vaccination, as well as a basic substance and starting material in gene and cell therapy, and viral vector production. Since the uncontrolled expression of backbone sequences present in such plasmids and the dissemination of antibiotic resistance genes may have profound detrimental effects, an important goal in vector development was to produce supercoiled DNA lacking bacterial backbone sequences: Minicircle (MC) DNA. The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform enabling a close-to-random profile of genomic integration. In combination, the MC platform greatly enhances SB transposition and transgene integration resulting in higher numbers of stably modified target cells. We have recently developed a strategy for MC-based SB transposition of chimeric antigen receptor (CAR) transgenes that enable improved transposition rates compared to conventional plasmids and rapid manufacturing of therapeutic CAR T cell doses (Monjezi et al. 2016). This advance enables manufacturing CAR T cells in a virus-free process that relies on SB-mediated transposition from MC DNA to accomplish gene-transfer. Advantages of this approach include a strong safety profile due to the nature of the MC itself and the genomic insertion pattern of MC-derived CAR transposons. In addition, stable transposition and high-level CAR transgene expression, as well as easy and reproducible handling, make MCs a preferred vector source for gene-transfer in advanced cellular and gene therapy. In this chapter, we will review our experience in MC-based CAR T cell engineering and discuss our recent advances in MC manufacturing to accelerate both pre-clinical and clinical implementation.

Pak2 Controls Acquisition of NKT Cell Fate by Regulating Expression of the Transcription Factors PLZF and Egr2.

NKT cells constitute a small population of T cells developed in the thymus that produce large amounts of cytokines and chemokines in response to lipid Ags. Signaling through the Vα14-Jα18 TCR instructs commitment to the NKT cell lineage, but the precise signaling mechanisms that instruct their lineage choice are unclear. In this article, we report that the cytoskeletal remodeling protein, p21-activated kinase 2 (Pak2), was essential for NKT cell development. Loss of Pak2 in T cells reduced stage III NKT cells in the thymus and periphery. Among different NKT cell subsets, Pak2 was necessary for the generation and function of NKT1 and NKT2 cells, but not NKT17 cells. Mechanistically, expression of Egr2 and promyelocytic leukemia zinc finger (PLZF), two key transcription factors for acquiring the NKT cell fate, were markedly diminished in the absence of Pak2. Diminished expression of Egr2 and PLZF were not caused by aberrant TCR signaling, as determined using a Nur77-GFP reporter, but were likely due to impaired induction and maintenance of signaling lymphocyte activation molecule 6 expression, a TCR costimulatory receptor required for NKT cell development. These data suggest that Pak2 controls thymic NKT cell development by providing a signal that links Egr2 to induce PLZF, in part by regulating signaling lymphocyte activation molecule 6 expression.

Therapeutic potential of CAR-T cell-derived exosomes: a cell-free modality for targeted cancer therapy.

Chimeric antigen receptor (CAR)-based T-cell adoptive immunotherapy is a distinctively promising therapy for cancer. The engineering of CARs into T cells provides T cells with tumor-targeting capabilities and intensifies their cytotoxic activity through stimulated cell expansion and enhanced cytokine production. As a novel and potent therapeutic modality, there exists some uncontrollable processes which are the potential sources of adverse events. As an extension of this impactful modality, CAR-T cell-derived exosomes may substitute CAR-T cells to act as ultimate attackers, thereby overcoming some limitations. Exosomes retain most characteristics of parent cells and play an essential role in intercellular communications via transmitting their cargo to recipient cells. The application of CAR-T cell-derived exosomes will make this cell-based therapy more clinically controllable as it also provides a cell-free platform to diversify anticancer mediators, which responds effectively to the complexity and volatility of cancer. It is believed that the appropriate application of both cellular and exosomal platforms will make this effective treatment more practicable.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays.

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the "hanging drop" method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3(+) cells and gave rise to the canonical γδ T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3(-/-) mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.