Training of epitope-TCR prediction models with healthy donor-derived cancer-specific T cells.

Discovery of epitope-specific T-cell receptors (TCRs) for cancer therapies is a time consuming and expensive procedure that usually requires a large amount of patient cells. To maximize information from and minimize the need of precious samples in cancer research, prediction models have been developed to identify in silico epitope-specific TCRs. In this chapter, we provide a step-by-step protocol to train a prediction model using the user-friendly TCRex webtool for the nearly universal tumor-associated antigen Wilms' tumor 1 (WT1)-specific TCR repertoire. WT1 is a self-antigen overexpressed in numerous solid and hematological malignancies with a high clinical relevance. Training of computational models starts from a list of known epitope-specific TCRs which is often not available for new cancer epitopes. Therefore, we describe a workflow to assemble a training data set consisting of TCR sequences obtained from WT137-45-reactive CD8 T cell clones expanded and sorted from healthy donor peripheral blood mononuclear cells.

IL-15-secreting CAR natural killer cells directed toward the pan-cancer target CD70 eliminate both cancer cells and cancer-associated fibroblasts.

BACKGROUND: It remains challenging to obtain positive outcomes with chimeric antigen receptor (CAR)-engineered cell therapies in solid malignancies, like colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC). A major obstacle is the lack of targetable surface antigens that are not shared by healthy tissues. CD70 emerges as interesting target, due to its stringent expression pattern in healthy tissue and its apparent role in tumor progression in a considerable amount of malignancies. Moreover, CD70 is also expressed on cancer-associated fibroblasts (CAFs), another roadblock for treatment efficacy in CRC and PDAC. We explored the therapeutic potential of CD70 as target for CAR natural killer (NK) cell therapy in CRC, PDAC, focusing on tumor cells and CAFs, and lymphoma. METHODS: RNA-seq data and immunohistochemical analysis of patient samples were used to explore CD70 expression in CRC and PDAC patients. In addition, CD70-targeting CAR NK cells were developed to assess cytotoxic activity against CD70+ tumor cells and CAFs, and the effect of cytokine stimulation on their efficacy was evaluated. The in vitro functionality of CD70-CAR NK cells was investigated against a panel of tumor and CAF cell lines with varying CD70 expression. Lymphoma-bearing mice were used to validate in vivo potency of CD70-CAR NK cells. Lastly, to consider patient variability, CD70-CAR NK cells were tested on patient-derived organoids containing CAFs. RESULTS: In this study, we identified CD70 as a target for tumor cells and CAFs in CRC and PDAC patients. Functional evaluation of CD70-directed CAR NK cells indicated that IL-15 stimulation is essential to obtain effective elimination of CD70+ tumor cells and CAFs, and to improve tumor burden and survival of mice bearing CD70+ tumors. Mechanistically, IL-15 stimulation resulted in improved potency of CD70-CAR NK cells by upregulating CAR expression and increasing secretion of pro-inflammatory cytokines, in a mainly autocrine or intracellular manner. CONCLUSIONS: We disclose CD70 as an attractive target both in hematological and solid tumors. IL-15 armored CAR NK cells act as potent effectors to eliminate these CD70+ cells. They can target both tumor cells and CAFs in patients with CRC and PDAC, and potentially other desmoplastic solid tumors.

One-step CRISPR-Cas9-mediated knockout of native TCRαß genes in human T cells using RNA electroporation.

To avoid mispairing between native and introduced T cell receptors (TCRs) and to prevent graft-versus-host disease in allogeneic T cell therapies, TCRα and TCRß chains of native TCRs are knocked out via CRISPR-Cas9. We demonstrate the isolation and activation of CD8+ T cells followed by electroporation of T cells with in vitro transcribed eSpCas9(1.1)-P2A-EGFP mRNA and single-guide RNAs targeting the TCRα and TCRß constant regions. We then describe a flow cytometric analysis to determine TCR knockout efficiency.

In vitro expansion of Wilms' tumor protein 1 epitope-specific primary T cells from healthy human peripheral blood mononuclear cells.

Wilms' tumor protein 1 (WT1) is a tumor-associated antigen overexpressed in various cancers. As a self-antigen, negative selection reduces the number of WT1-specific T cell receptors (TCRs). Here, we provide a protocol to generate WT137-45-specific TCRs using healthy human peripheral blood mononuclear cells. We describe the expansion of WT1-specific T cell clones by two consecutive in vitro stimulations with autologous WT137-45-pulsed dendritic cells and peripheral blood lymphocytes. We then detail the detection with human leukocyte antigen/WT137-45 tetramers.

Anti-Tumor Potency of Short-Term Interleukin-15 Dendritic Cells Is Potentiated by In Situ Silencing of Programmed-Death Ligands.

Dendritic cell (DC) vaccines have proven to be a valuable tool in cancer immune therapy. With several DC vaccines being currently tested in clinical trials, knowledge about their therapeutic value has been significantly increased in the past decade. Despite their established safety, it has become clear that objective clinical responses are not yet robust enough, requiring further optimization. Improvements of this advanced therapy medicinal product encompass, among others, regulating their immune stimulating capacity by in situ gene engineering, in addition to their implementation in combination therapy regimens. Previously, we have reported on a superior monocyte-derived DC preparation, including interleukin-15, pro-inflammatory cytokines and immunological danger signals in the culture process. These so-called IL-15 DCs have already proven to exhibit several favorable properties as cancer vaccine. Evolving research into mechanisms that could further modulate the immune response towards cancer, points to programmed death-1 as an important player that dampens anti-tumor immunity. Aiming at leveraging the immunogenicity of DC vaccines, we hypothesized that additional implementation of the inhibitory immune checkpoint molecules programmed death-ligand (PD-L)1 and PD-L2 in IL-15 DC vaccines would exhibit superior stimulatory potential. In this paper, we successfully implemented PD-L silencing at the monocyte stage in the 3-day IL-15 DC culture protocol resulting in substantial downregulation of both PD-L1 and PD-L2 to levels below 30%. Additionally, we validated that these DCs retain their specific characteristics, both at the level of phenotype and interferon gamma secretion. Evaluating their functional characteristics, we demonstrate that PD-L silencing does not affect the capacity to induce allogeneic proliferation. Ultimately designed to induce a durable tumor antigen-specific immune response, PD-L silenced IL-15 DCs were capable of surpassing PD-1-mediated inhibition by antigen-specific T cells. Further corroborating the superior potency of short-term IL-15 DCs, the combination of immune stimulatory components during DC differentiation and maturation with in situ checkpoint inhibition supports further clinical translation.

Two for one: targeting BCMA and CD19 in B-cell malignancies with off-the-shelf dual-CAR NK-92 cells.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy has proven to be a valuable new treatment option for patients with B-cell malignancies. However, by applying selective pressure, outgrowth of antigen-negative tumor cells can occur, eventually resulting in relapse. Subsequent rescue by administration of CAR-T cells with different antigen-specificity indicates that those tumor cells are still sensitive to CAR-T treatment and points towards a multi-target strategy. Due to their natural tumor sensitivity and highly cytotoxic nature, natural killer (NK) cells are a compelling alternative to T cells, especially considering the availability of an off-the-shelf unlimited supply in the form of the clinically validated NK-92 cell line. METHODS: Given our goal to develop a flexible system whereby the CAR expression repertoire of the effector cells can be rapidly adapted to the changing antigen expression profile of the target cells, electrotransfection with CD19-/BCMA-CAR mRNA was chosen as CAR loading method in this study. We evaluated the functionality of mRNA-engineered dual-CAR NK-92 against tumor B-cell lines and primary patient samples. In order to test the clinical applicability of the proposed cell therapy product, the effect of irradiation on the proliferative rate and functionality of dual-CAR NK-92 cells was investigated. RESULTS: Co-electroporation of CD19 and BMCA CAR mRNA was highly efficient, resulting in 88.1% dual-CAR NK-92 cells. In terms of CD107a degranulation, and secretion of interferon (IFN)-γ and granzyme B, dual-CAR NK-92 significantly outperformed single-CAR NK-92. More importantly, the killing capacity of dual-CAR NK-92 exceeded 60% of single and dual antigen-expressing cell lines, as well as primary tumor cells, in a 4h co-culture assay at low effector to target ratios, matching that of single-CAR counterparts. Furthermore, our results confirm that dual-CAR NK-92 irradiated with 10 Gy cease to proliferate and are gradually cleared while maintaining their killing capacity. CONCLUSIONS: Here, using the clinically validated NK-92 cell line as a therapeutic cell source, we established a readily accessible and flexible platform for the generation of highly functional dual-targeted CAR-NK cells.

Engineering of regulatory T cells by means of mRNA electroporation in a GMP-compliant manner.

Regulatory T cells (Tregs) are crucial in inducing and maintaining tolerance. This unique capacity of Tregs, in combination with proof-of-principle in preclinical studies, highlights the potential clinical use of Tregs for the treatment of autoimmunity and transplant rejection. Although proven to be safe and well tolerated in the first clinical trials, only modest clinical results were observed. In this regard, it has been hypothesized that current challenges lie in the development of antigen-specific Tregs. Here, we present an innovative, good manufacturing practices (GMP)-compliant manufacturing protocol for Tregs applicable in a clinical-grade setting, allowing efficient and safe redirection of Treg specificity. First, a soluble polymer conjugated with antibodies to CD3 and CD28 and high amounts of exogenous IL-2 for in vitro Treg expansion resulted in a >70-fold and 185-fold increase of a pure population of CD4+CD127-CD25hi Tregs and CD4+CD127-CD25+CD45RA+ Tregs, respectively. Next, as a proof-of-principle, expanded Tregs were engineered by means of TCR-encoding mRNA electroporation to generate antigen-specific Tregs. This resulted in an expression of the newly introduced TCR in up to 85% of Tregs. Moreover, we did not observe a negative effect on the phenotype of Tregs, as demonstrated by the expression of FOXP3, Helios, CTLA-4 and CCR4, nor on the TSDR methylation status. Importantly, mRNA-engineered Tregs were still able to induce in vitro suppression of effector T cells and produced anti-inflammatory, but not pro-inflammatory, cytokines when activated. In conclusion, our findings demonstrate that high numbers of stable and functional Tregs can be obtained with high purity and successfully engineered for gain of function, in a GMP-compliant manner. We envisage that this clinical-grade protocol will provide solid basis for future clinical application of mRNA-engineered Tregs.

Trial Watch: Adoptive TCR-Engineered T-Cell Immunotherapy for Acute Myeloid Leukemia.

Despite the advent of novel therapies, acute myeloid leukemia (AML) remains associated with a grim prognosis. This is exemplified by 5-year overall survival rates not exceeding 30%. Even with frontline high-intensity chemotherapy regimens and allogeneic hematopoietic stem cell transplantation, the majority of patients with AML will relapse. For these patients, treatment options are few, and novel therapies are urgently needed. Adoptive T-cell therapies represent an attractive therapeutic avenue due to the intrinsic ability of T lymphocytes to recognize tumor cells with high specificity and efficiency. In particular, T-cell therapies focused on introducing T-cell receptors (TCRs) against tumor antigens have achieved objective clinical responses in solid tumors such as synovial sarcoma and melanoma. However, contrary to chimeric antigen receptor (CAR)-T cells with groundbreaking results in B-cell malignancies, the use of TCR-T cells for hematological malignancies is still in its infancy. In this review, we provide an overview of the status and clinical advances in adoptive TCR-T-cell therapy for the treatment of AML.

Novel Insights on MRGPRX2-Mediated Hypersensitivity to Neuromuscular Blocking Agents And Fluoroquinolones.

Neuromuscular blocking agents (NMBAs) like atracurium and rocuronium as well as fluoroquinolones (FQs) cause mast cell-mediated anaphylaxis by activating Mas-related G protein-coupled receptor X2 (MRGPRX2), but many questions remain unanswered. Here, we address three of them, namely whether primary human mast cells show similar activation by these drugs as murine mast cells and mast cell lines, how sugammadex protects from atracurium-induced MRGPRX2-mediated mast cell activation, and why some but not all patients treated with rocuronium develop anaphylaxis. We used peripheral blood-derived cultured mast cells from healthy donors and patients, assessed mast cell activation and degranulation by quantifying intracellular calcium and CD63 expression, respectively, and made use of MRGPRX2-silencing, via electroporation with Dicer-substrate small interfering RNAs, and single cell flow cytometric analyses. Atracurium, ciprofloxacin, and levofloxacin activated and degranulated primary human mast cells, but only MRGPRX2-positive and not MRGPRX2-negative or -silenced mast cells. Sugammadex attenuated the atracurium-induced and MRGPRX2-mediated activation and degranulation of human mast cells by reducing free atracurium levels. The mast cells of patients with IgE-independent anaphylaxis to rocuronium were similar, in their MRGPRX2 expression and function, to those of patients with IgE-mediated anaphylaxis. These findings further improve our understanding of the role and relevance of MRGPRX2-driven mast cell activation in anaphylactic reactions to NMBAs and FQs and may help to improve their prediction, prevention, and treatment.

The Ins and Outs of Messenger RNA Electroporation for Physical Gene Delivery in Immune Cell-Based Therapy.

Messenger RNA (mRNA) electroporation is a powerful tool for transient genetic modification of cells. This non-viral method of genetic engineering has been widely used in immunotherapy. Electroporation allows fine-tuning of transfection protocols for each cell type as well as introduction of multiple protein-coding mRNAs at once. As a pioneering group in mRNA electroporation, in this review, we provide an expert overview of the ins and outs of mRNA electroporation, discussing the different parameters involved in mRNA electroporation as well as the production of research-grade and production and application of clinical-grade mRNA for gene transfer in the context of cell-based immunotherapies.

Safety and clinical efficacy of BCMA CAR-T-cell therapy in multiple myeloma.

BACKGROUND: B-cell maturation antigen (BCMA)-targeted chimeric antigen receptor (CAR)-T-cell therapy is an emerging treatment option for multiple myeloma. The aim of this systematic review and meta-analysis was to determine its safety and clinical activity and to identify factors influencing these outcomes. METHODS: We performed a database search using the terms "BCMA," "CAR," and "multiple myeloma" for clinical studies published between 01/01/2015 and 01/01/2020. The methodology is further detailed in PROSPERO (CRD42020125332). RESULTS: Twenty-three different CAR-T-cell products have been used so far in 640 patients. Cytokine release syndrome was observed in 80.3% (69.0-88.2); 10.5% (6.8-16.0) had neurotoxicity. A higher neurotoxicity rate was reported in studies that included more heavily pretreated patients: 19.1% (13.3-26.7; I2 = 45%) versus 2.8% (1.3-6.1; I2 = 0%) (p < 0.0001). The pooled overall response rate was 80.5% (73.5-85.9); complete responses (CR) were observed in 44.8% (35.3-54.6). A pooled CR rate of 71.9% (62.8-79.6; I2 = 0%) was noted in studies using alpaca/llama-based constructs, whereas it was only 18.0% (6.5-41.1; I2 = 67%) in studies that used retroviral vectors for CAR transduction. Median progression-free survival (PFS) was 12.2 (11.4-17.4) months, which compared favorably to the expected PFS of 1.9 (1.5-3.7) months (HR 0.14; p < 0.0001). CONCLUSIONS: Although considerable toxicity was observed, BCMA-targeted CAR-T-cell therapy is highly efficacious even in advanced multiple myeloma. Subgroup analysis confirmed the anticipated inter-study heterogeneity and identified potential factors contributing to safety and efficacy. The results of this meta-analysis may assist the future design of CAR-T-cell studies and lead to optimized BCMA CAR-T-cell products.

The Quest for the Best: How TCR Affinity, Avidity, and Functional Avidity Affect TCR-Engineered T-Cell Antitumor Responses.

Over the past decades, adoptive transfer of T cells has revolutionized cancer immunotherapy. In particular, T-cell receptor (TCR) engineering of T cells has marked important milestones in developing more precise and personalized cancer immunotherapies. However, to get the most benefit out of this approach, understanding the role that TCR affinity, avidity, and functional avidity play on how TCRs and T cells function in the context of tumor-associated antigen (TAA) recognition is vital to keep generating improved adoptive T-cell therapies. Aside from TCR-related parameters, other critical factors that govern T-cell activation are the effect of TCR co-receptors on TCR-peptide-major histocompatibility complex (pMHC) stabilization and TCR signaling, tumor epitope density, and TCR expression levels in TCR-engineered T cells. In this review, we describe the key aspects governing TCR specificity, T-cell activation, and how these concepts can be applied to cancer-specific TCR redirection of T cells.

PD-L1 siRNA-mediated silencing in acute myeloid leukemia enhances anti-leukemic T cell reactivity.

Acute myeloid leukemia (AML) is an immune-susceptible malignancy, as demonstrated by its responsiveness to allogeneic stem cell transplantation (alloSCT). However, by employing inhibitory signaling pathways, including PD-1/PD-L1, leukemia cells suppress T cell-mediated immune attack. Notably, impressive clinical efficacy has been obtained with PD-1/PD-L1 blocking antibodies in cancer patients. Yet, these systemic treatments are often accompanied by severe toxicity, especially after alloSCT. Here, we investigated RNA interference technology as an alternative strategy to locally interfere with PD-1/PD-L1 signaling in AML. We demonstrated efficient siRNA-mediated PD-L1 silencing in HL-60 and patients' AML cells. Importantly, WT1-antigen T cell receptor+ PD-1+ 2D3 cells showed increased activation toward PD-L1 silenced WT1+ AML. Moreover, PD-L1 silenced AML cells significantly enhanced the activation, degranulation, and IFN-γ production of minor histocompatibility antigen-specific CD8+ T cells. Notably, PD-L1 silencing was equally effective as PD-1 antibody blockade. Together, our study demonstrates that PD-L1 silencing may be an effective strategy to augment AML immune-susceptibility. This provides rationale for further development of targeted approaches to locally interfere with immune escape mechanisms in AML, thereby minimizing severe toxicity. In combination with alloSCT and/or adoptive T cell transfer, this strategy could be very appealing to boost graft-versus-leukemia immunity and improve outcome in AML patients.

Rapid Assessment of Functional Avidity of Tumor-Specific T Cell Receptors Using an Antigen-Presenting Tumor Cell Line Electroporated with Full-Length Tumor Antigen mRNA.

The functional avidity of T-cell receptor (TCR)-engineered T cells towards their cognate epitope plays a crucial role in successfully targeting and killing tumor cells expressing the tumor-associated antigen (TAA). When evaluating in vitro functional T-cell avidity, an important aspect that is often neglected is the antigen-presenting cell (APC) used in the assay. Cell-based models for antigen-presentation, such as tumor cell lines, represent a valid alternative to autologous APCs due to their availability, off-the-shelf capabilities, and the broad range of possibilities for modification via DNA or messenger RNA (mRNA) transfection. To find a valuable model APC for in vitro validation of TAA Wilms' tumor 1 (WT1)-specific TCRs, we tested four different WT1 peptide-pulsed HLA-A2+ tumor cell lines commonly used in T-cell stimulation assays. We found the multiple myeloma cell line U266 to be a suitable model APC to evaluate differences in mean functional avidity (EC50) values of transgenic TCRs following transfection in 2D3 Jurkat T cells. Next, to assess the dose-dependent antigen-specific responsiveness of WT1 TCR-engineered 2D3 T cells to endogenously processed epitopes, we electroporated U266 cells with different amounts of full-length antigen WT1 mRNA. Finally, we analyzed the functional avidity of WT1 TCR-transfected primary CD8 T cells towards WT1 mRNA-electroporated U266 cells. In this study, we demonstrate that both the APC and the antigen loading method (peptide pulsing versus full-length mRNA transfection) to analyze T-cell functional avidity have a significant impact on the EC50 values of a given TCR. For rapid assessment of the functional avidity of a cloned TCR towards its endogenously processed MHC I-restricted epitope, we showcase that the TAA mRNA-transfected U266 cell line is a suitable and versatile model APC.

Chimeric Antigen Receptor-Modified T Cell Therapy in Multiple Myeloma: Beyond B Cell Maturation Antigen.

Chimeric antigen receptor (CAR)-modified T cell therapy is a rapidly emerging immunotherapeutic approach that is revolutionizing cancer treatment. The impressive clinical results obtained with CAR-T cell therapy in patients with acute lymphoblastic leukemia and lymphoma have fueled the development of CAR-T cells targeting other malignancies, including multiple myeloma (MM). The field of CAR-T cell therapy for MM is still in its infancy, but remains promising. To date, most studies have been performed with B cell maturation antigen (BCMA)-targeted CARs, for which high response rates have been obtained in early-phase clinical trials. However, responses are usually temporary, and relapses have frequently been observed. One of the major reasons for relapse is the loss or downregulation of BCMA expression following CAR-T therapy. This has fostered a search for alternative target antigens that are expressed on the MM cell surface. In this review, we provide an overview of myeloma target antigens other than BCMA that are currently being evaluated in pre-clinical and clinical studies.

Dendritic Cell-Based Immunotherapy of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a type of blood cancer characterized by the uncontrolled clonal proliferation of myeloid hematopoietic progenitor cells in the bone marrow. The outcome of AML is poor, with five-year overall survival rates of less than 10% for the predominant group of patients older than 65 years. One of the main reasons for this poor outcome is that the majority of AML patients will relapse, even after they have attained complete remission by chemotherapy. Chemotherapy, supplemented with allogeneic hematopoietic stem cell transplantation in patients at high risk of relapse, is still the cornerstone of current AML treatment. Both therapies are, however, associated with significant morbidity and mortality. These observations illustrate the need for more effective and less toxic treatment options, especially in elderly AML and have fostered the development of novel immune-based strategies to treat AML. One of these strategies involves the use of a special type of immune cells, the dendritic cells (DCs). As central orchestrators of the immune system, DCs are key to the induction of anti-leukemia immunity. In this review, we provide an update of the clinical experience that has been obtained so far with this form of immunotherapy in patients with AML.

Efficient and Non-genotoxic RNA-Based Engineering of Human T Cells Using Tumor-Specific T Cell Receptors With Minimal TCR Mispairing.

Genetic engineering of T cells with tumor specific T-cell receptors (TCR) is a promising strategy to redirect their specificity against cancer cells in adoptive T cell therapy protocols. Most studies are exploiting integrating retro- or lentiviral vectors to permanently introduce the therapeutic TCR, which can pose serious safety issues when treatment-related toxicities would occur. Therefore, we developed a versatile, non-genotoxic transfection method for human unstimulated CD8+ T cells. We describe an optimized double sequential electroporation platform whereby Dicer-substrate small interfering RNAs (DsiRNA) are first introduced to suppress endogenous TCR α and ß expression, followed by electroporation with DsiRNA-resistant tumor-specific TCR mRNA. We demonstrate that double sequential electroporation of human primary unstimulated T cells with DsiRNA and TCR mRNA leads to unprecedented levels of transgene TCR expression due to a strongly reduced degree of TCR mispairing. Importantly, superior transgenic TCR expression boosts epitope-specific CD8+ T cell activation and killing activity. Altogether, DsiRNA and TCR mRNA double sequential electroporation is a rapid, non-integrating and highly efficient approach with an enhanced biosafety profile to engineer T cells with antigen-specific TCRs for use in early phase clinical trials.

A versatile T cell-based assay to assess therapeutic antigen-specific PD-1-targeted approaches.

Blockade of programmed cell death protein 1 (PD-1) immune checkpoint receptor signaling is an established standard treatment for many types of cancer and indications are expanding. Successful clinical trials using monoclonal antibodies targeting PD-1 signaling have boosted preclinical research, encouraging development of novel therapeutics. Standardized assays to evaluate their bioactivity, however, remain restricted. The robust bioassays available all lack antigen-specificity. Here, we developed an antigen-specific, short-term and high-throughput T cell assay with versatile readout possibilities. A genetically modified T cell receptor (TCR)-deficient T cell line was stably transduced with PD-1. Transfection with messenger RNA encoding a TCR of interest and subsequent overnight stimulation with antigen-presenting cells, results in eGFP-positive and granzyme B-producing T cells for single cell or bulk analysis. Control antigen-presenting cells induced reproducible high antigen-specific eGFP and granzyme B expression. Upon PD-1 interaction, ligand-positive antigen-presenting immune or tumor cells elicited significantly lower eGFP and granzyme B expression, which could be restored by anti-PD-(L)1 blocking antibodies. This convenient cell-based assay shows a valuable tool for translational and clinical research on antigen-specific checkpoint-targeted therapy approaches.

The role of the common gamma-chain family cytokines in γδ T cell-based anti-cancer immunotherapy.

Cytokines of the common gamma-chain receptor family, comprising interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15 and IL-21, are vital with respect to organizing and sustaining healthy immune cell functions. Supporting the anti-cancer immune response, these cytokines inspire great interest for their use as vaccine adjuvants and cancer immunotherapies. It is against this background that gamma delta (γδ) T cells, as special-force soldiers and natural contributors of the tumor immunosurveillance, also received a lot of attention the last decade. As γδ T cell-based cancer trials are coming of age, this present review focusses on the effects of the different cytokines of the common gamma-chain receptor family on γδ T cells with respect to boosting γδ T cells as a therapeutic target in cancer immunotherapy. This review also gathers data that IL-15 in particular exhibits key features for augmenting the anti-tumor activity of effector killer γδ T cells whilst overcoming the myriad of immune escape mechanisms used by cancer cells.

An MIF Promoter Polymorphism Is Associated with Susceptibility to Pulmonary Arterial Hypertension in Diffuse Cutaneous Systemic Sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is a fibrotic immune-mediated disease of unknown etiology. Among its clinical manifestations, pulmonary involvement is the leading cause of mortality in patients with SSc. However, the genetic factors involved in lung complication are not well defined. We aimed to review the association of the MIF gene, which encodes a cytokine implicated in idiopathic pulmonary hypertension among other diseases, with the susceptibility and clinical expression of SSc, in addition to testing the association of this polymorphism with SSc-related pulmonary involvement. METHODS: A total of 4392 patients with SSc and 16,591 unaffected controls from 6 cohorts of European origin were genotyped for the MIF promoter variant rs755622. An inverse variance method was used to metaanalyze the data. RESULTS: A statistically significant increase of the MIF rs755622*C allele frequency compared with controls was observed in the subgroups of patients with diffuse cutaneous SSc (dcSSc) and with pulmonary arterial hypertension (PAH) independently (dcSSc: p = 3.20E-2, OR 1.13; PAH: p = 2.19E-02, OR 1.32). However, our data revealed a stronger effect size with the subset of patients with SSc showing both clinical manifestations (dcSSc with PAH: p = 6.91E-3, OR 2.05). CONCLUSION: We reviewed the association of the MIF rs755622*C allele with SSc and described a phenotype-specific association of this variant with the susceptibility to develop PAH in patients with dcSSc.