Dectin-1b activation by arabinoxylans induces trained immunity in human monocyte-derived macrophages.

Arabinoxylans of various structures and sources have shown to possess the ability to induce a range of immune responses in different cell types in vitro and in vivo. Although the underlying mechanisms remain to be fully established, several studies point towards the involvement of activation of pattern recognition receptors (PRRs). Activation of specific PRRs (i.e., Dectin-1 and CR3) has also been shown to play a key role in the induction of a non-specific memory response in innate immune cells, termed 'trained innate immunity'. In the current study, we assessed whether arabinoxylans are also able to induce trained innate immunity. To this end, a range of arabinoxylan preparations from different sources were tested for their physicochemical properties and their capacity to induce innate immune training and resilience. In human macrophages, rice and wheat-derived arabinoxylan preparations induced training and/or resilience effects, the extent depending on fiber particle size and solubility. Using a Dectin-1 antagonist or a CR3 antibody, it was demonstrated that arabinoxylan-induced trained immunity in macrophages is mainly dependent on Dectin-1b. These findings build on previous observations showing the immunomodulatory potential of arabinoxylans as biological response modifiers and open up promising avenues for their use as health promoting ingredients.

Clostridioides difficile toxin A-mediated Caco-2 cell barrier damage was attenuated by insect-derived fractions and corresponded to increased gene transcription of cell junctional and proliferation proteins.

Pathogenesis of C. difficile in the intestine is associated with the secretion of toxins which can damage the intestinal epithelial layer and result in diseases such as diarrhoea. Treatment for C. difficile infections consists of antibiotics which, however, have non-specific microbiocidal effects and may cause intestinal dysbiosis which results in subsequent health issues. Therefore, alternative treatments to C. difficile infections are required. We investigated whether different black soldier fly- and mealworm-derived fractions, after applying the INFOGEST digestion protocol, could counteract C. difficile toxin A-mediated barrier damage of small intestinal Caco-2 cells. Treatment and pre-treatment with insect-derived fractions significantly (p < 0.05) mitigated the decrease of the transepithelial electrical resistance (TEER) of Caco-2 cells induced by C. difficile toxin A. In relation to these effects, RNA sequencing data showed an increased transcription of cell junctional and proliferation protein genes in Caco-2 cells. Furthermore, the transcription of genes regulating immune signalling was also increased. To identify whether this resulted in immune activation we used a Caco-2/THP-1 co-culture model where the cells were only separated by a permeable membrane. However, the insect-derived fractions did not change the basolateral secreted IL-8 levels in this model. To conclude, our findings suggest that black soldier fly- and mealworm-derived fractions can attenuate C. difficile induced intestinal barrier disruption and they might be promising tools to reduce the symptoms of C. difficile infections.

Continuous Exposure to Non-Soluble ß-Glucans Induces Trained Immunity in M-CSF-Differentiated Macrophages.

Beta-glucans enable functional reprogramming of innate immune cells, a process defined as "trained immunity", which results in enhanced host responsiveness against primary (training) and/or secondary infections (resilience). Trained immunity holds great promise for promoting immune responses in groups that are at risk (e.g. elderly and patients). In this study, we modified an existing in vitro model for trained immunity by actively inducing monocyte-to-macrophage differentiation using M-CSF and applying continuous exposure. This model reflects mucosal exposure to ß-glucans and was used to study the training effects of a variety of soluble or non-soluble ß-glucans derived from different sources including oat, mushrooms and yeast. In addition, trained immunity effects were related to pattern recognition receptor usage, to which end, we analyzed ß-glucan-mediated Dectin-1 activation. We demonstrated that ß-glucans, with different sources and solubilities, induced training and/or resilience effects. Notably, trained immunity significantly correlated with Dectin-1 receptor activation, yet Dectin-1 receptor activation did not perform as a sole predictor for ß-glucan-mediated trained immunity. The model, as validated in this study, adds on to the existing in vitro model by specifically investigating macrophage responses and can be applied to select non-digestible dietary polysaccharides and other components for their potential to induce trained immunity.

Curdlan, zymosan and a yeast-derived ß-glucan reshape tumor-associated macrophages into producers of inflammatory chemo-attractants.

Anti-cancer T-cell responses are often halted due to the immune-suppressive micro-environment, in part related to tumor-associated macrophages. In the current study, we assessed indigestible ß-glucans (oatßG, curdlan, grifolan, schizophyllan, lentinan, yeast whole glucan particles (yWGP), zymosan and two additional yeast-derived ß-glucans a and b) for their physicochemical properties as well as their effects on the plasticity of human monocyte-derived macrophages that were polarized with IL-4 to immune-suppressive macrophages. Beta-glucans were LPS/LTA free, and tested for solubility, molecular masses, protein and monosaccharide contents. Curdlan, yeast-b and zymosan re-polarized M(IL-4) macrophages towards an M1-like phenotype, in particular showing enhanced gene expression of CCR7, ICAM1 and CD80, and secretion of TNF-α and IL-6. Notably, differential gene expression, pathway analysis as well as protein expressions demonstrated that M(IL-4) macrophages treated with curdlan, yeast-b or zymosan demonstrated enhanced production of chemo-attractants, such as CCL3, CCL4, and CXCL8, which contribute to recruitment of monocytes and neutrophils. The secretion of chemo-attractants was confirmed when using patient-derived melanoma-infiltrating immune cells. Taken together, the bacterial-derived curdlan as well as the yeast-derived ß-glucans yeast-b and zymosan have the unique ability to preferentially skew macrophages towards a chemo-attractant-producing phenotype that may aid in anti-cancer immune responses.

Hydrophobicity drives receptor-mediated uptake of heat-processed proteins by THP-1 macrophages and dendritic cells, but not cytokine responses.

Although an impact of processing on immunogenicity of food proteins has clearly been demonstrated, the underlying mechanisms are still unclear. We applied 3 different processing methods: wet heating (60 °C) and low- or high-temperature (50 °C or 130 °C, respectively) dry-heating in absence or presence of reducing sugars, to ß-lactoglobulin (BLG), lysozyme and thyroglobulin, which represent dietary proteins with different pI or molecular weight. Uptake of the soluble fraction of the samples was tested in two types of, genetically homogeneous, antigen-presenting cells (macrophages and dendritic cells derived from THP-1 monocytes). This revealed a strong correlation between the uptake of the different protein samples by macrophages and dendritic cells, and confirmed the key role of hydrophobicity, over aggregation, in determining the uptake. Several uptake routes were shown to contribute to the uptake of BLG by macrophages. However, cytokine responses following exposure of macrophages to BLG samples were not related to the levels of uptake. Together, our results demonstrate that heat-treatment-induced increased hydrophobicity is the prime driving factor in uptake, but not in cytokine production, by THP-1 macrophages.

Wheat-derived arabinoxylans reduced M2-macrophage functional activity, but enhanced monocyte-recruitment capacity.

The immunomodulatory properties of non-digestible polysaccharides (NDPs) have been recognized in in vitro and in vivo studies. The latter mostly demonstrated altered frequencies and inflammatory status of immune cells as clinical parameters. Most of the NDP activity will be exerted in the intestine where they can directly interact with macrophages. The predominant macrophage phenotype in the intestine is M2-like, with M1-like macrophages arising during inflammation. Here, we investigated transcriptional and functional impact on these macrophage phenotypes by NDP-treatment (i.e. yeast-derived soluble ß-glucan (yeast-ßG), apple-derived RG-I (apple-RGI), shiitake-derived ß-glucan (shiitake-ßG) or wheat-derived arabinoxylan (wheat-AX)). Wheat-AX, and to a lesser extent shiitake-ßG and apple-RGI but not yeast-ßG, reduced endocytosis and antigen processing capacity of M1- and M2-like macrophages. Moreover, the NDPs, and most notably wheat-AX, strongly induced transcription and secretion of a unique set of cytokines and chemokines. Conditioned medium from wheat-AX-treated M2-like macrophages subsequently demonstrated strongly increased monocyte recruitment capacity. These findings are in line with clinically observed immunomodulatory aspects of NDPs making it tempting to speculate that clinical activity of some NDPs is mediated through enhanced chemoattraction and modifying activity of intestinal immune cells.

Hydrophobicity and aggregation, but not glycation, are key determinants for uptake of thermally processed ß-lactoglobulin by THP-1 macrophages.

The aim of this study is to investigate the immunological relevance of modifications of food protein structure due to thermal processing. We investigated the uptake of ß-lactoglobulin, treated with 3 different processing methods, by THP-1 macrophages: wet heating (60 °C in solution) and high- or low-temperature (130 °C or 50 °C, respectively) dry heating, combined with either of 8 types of saccharides or without saccharide. The processing method that was applied significantly affected the uptake of BLG by THP-1 macrophages, while the type of saccharide only had an influence in high-temperature dry heated samples. A set of physicochemical parameters of processed samples was determined, to determine the samples' molecular weight, hydrophobicity, amyloid-like structure, surface charge and secondary structure. Analysis of protein structure alterations indicated the uptake to be linked to the wet heating processing method and percentage of α-helix structure, amyloid-like structures, polymers, and hydrophobicity. We hypothesize that both amyloid-like structures and molecular weight were related to the increased hydrophobicity and therefore postulate that the exposure of hydrophobic regions is the leading physicochemical characteristic for the observed uptake of wet heated BLG samples by THP-1 macrophages. This work demonstrates how differential thermal processing of foods, through protein modification, can have an impact on its interaction with the immune system.

Blood-brain barrier transport and neuroprotective potential of blackberry-digested polyphenols: an in vitro study.

PURPOSE: Epidemiological and intervention studies have attempted to link the health effects of a diet rich in fruits and vegetables with the consumption of polyphenols and their impact in neurodegenerative diseases. Studies have shown that polyphenols can cross the intestinal barrier and reach concentrations in the bloodstream able to exert effects in vivo. However, the effective uptake of polyphenols into the brain is still regarded with some reservations. Here we describe a combination of approaches to examine the putative transport of blackberry-digested polyphenols (BDP) across the blood-brain barrier (BBB) and ultimate evaluation of their neuroprotective effects. METHODS: BDP was obtained by in vitro digestion of blackberry extract and BDP major aglycones (hBDP) were obtained by enzymatic hydrolysis. Chemical characterization and BBB transport of extracts were evaluated by LC-MSn. BBB transport and cytoprotection of both extracts was assessed in HBMEC monolayers. Neuroprotective potential of BDP was assessed in NT2-derived 3D co-cultures of neurons and astrocytes and in primary mouse cerebellar granule cells. BDP-modulated genes were evaluated by microarray analysis. RESULTS: Components from BDP and hBDP were shown to be transported across the BBB. Physiologically relevant concentrations of both extracts were cytoprotective at endothelial level and BDP was neuroprotective in primary neurons and in an advanced 3D cell model. The major canonical pathways involved in the neuroprotective effect of BDP were unveiled, including mTOR signaling and the unfolded protein response pathway. Genes such as ASNS and ATF5 emerged as novel BDP-modulated targets. CONCLUSIONS: BBB transport of BDP and hBDP components reinforces the health benefits of a diet rich in polyphenols in neurodegenerative disorders. Our results suggest some novel pathways and genes that may be involved in the neuroprotective mechanism of the BDP polyphenol components.

Consumption of ß-glucans to spice up T cell treatment of tumors: a review.

INTRODUCTION: Adoptive T-cell treatments of solid cancers have evolved into a robust therapy with objective response rates surpassing those of standardized treatments. Unfortunately, only a limited fraction of patients shows durable responses, which is considered to be due to a T cell-suppressive tumor microenvironment (TME). Here we argue that naturally occurring ß-glucans can enable reversion of such T cell suppression by engaging innate immune cells and enhancing numbers and function of lymphocyte effectors. AREAS COVERED: This review summarizes timely reports with respect to absorption, trafficking and immune stimulatory effects of ß-glucans, particularly in relation to innate immune cells. Furthermore, we list effects toward well-being and immune functions in healthy subjects as well as cancer patients treated with orally administered ß-glucans, extended with effects of ß-glucan treatments in mouse cancer models. EXPERT OPINION: Beta-glucans, when present in food and following uptake in the proximal gut, stimulate immune cells present in gut-associated lymphoid tissue and initiate highly conserved pro-inflammatory pathways. When tested in mouse cancer models, ß-glucans result in better control of tumor growth and shift the TME toward a T cell-sensitive environment. Along these lines, we advocate that intake of ß-glucans provides an accessible and immune-potentiating adjuvant when combined with adoptive T-cell treatments of cancer.

The Effect of Tomatine on Gene Expression and Cell Monolayer Integrity in Caco-2.

More understanding of the risk-benefit effect of the glycoalkaloid tomatine is required to be able to estimate the role it might play in our diet. In this work, we focused on effects towards intestinal epithelial cells based on a Caco-2 model in order to analyze the influence on the cell monolayer integrity and on the expression levels of genes involved in cholesterol/sterol biosynthesis (LDLR), lipid metabolism (NR2F2), glucose and amino acid uptake (SGLT1, PAT1), cell cycle (PCNA, CDKN1A), apoptosis (CASP-3, BMF, KLF6), tight junctions (CLDN4, OCLN2) and cytokine-mediated signaling (IL-8, IL1ß, TSLP, TNF-α). Furthermore, since the bioactivity of the compound might vary in the presence of a food matrix and following digestion, the influence of both pure tomatine and in vitro digested tomatine with and without tomato fruit matrix was studied. The obtained results suggested that concentrations <20 µg/mL of tomatine, either undigested or in vitro digested, do not compromise the viability of Caco-2 cells and stimulate cytokine expression. This effect of tomatine, in vitro digested tomatine or in vitro digested tomatine with tomato matrix differs slightly, probably due to variations of bioactivity or bioavailability of the tomatine. The results lead to the hypothesis that tomatine acts as hormetic compound that can induce beneficial or risk toxic effects whether used in low or high dose.

Review of the health effects of berries and their phytochemicals on the digestive and immune systems.

Berries are generally considered beneficial to health. This health-promoting potential has mainly been ascribed to berries' phytochemical and vitamin content, and little attention has been paid to the potential benefits of berries for the digestive tract, despite this being the first point of contact. In vivo studies that described the health effects of berries on individual parts of the digestive tract (ie, the mouth, esophagus, stomach, small and large intestine, microbiome, and immune system) were reviewed. Immune effects were included because a large part of the immune system is located in the intestine. Beneficial health effects were mainly observed for whole berry extracts, not individual berry components. These effects ranged from support of the immune system and beneficial microbiota to reduction in the number and size of premalignant and malignant lesions. These results demonstrate the potency of berries and suggest berries can serve as a strong adjuvant to established treatments or therapies for a variety of gastrointestinal and immune-related illnesses.

Water-Soluble Polysaccharide Extracts from the Oyster Culinary-Medicinal Mushroom Pleurotus ostreatus (Agaricomycetes) with HMGCR Inhibitory Activity.

Water extracts from Pleurotus ostreatus containing no statins showed 3-hydroxy-3-methyl-glutaryl CoA reductase (HMGCR) inhibitory activity (in vitro) that might be due to specific water-soluble polysaccharides (WSPs); when isolated and deproteinized, increasing concentrations of the WSP extract induced higher inhibition. The WSP extract contained mainly ß-glucans, mannogalactans, and glycogen (e.g., α-glucans), although derivatives or fragments with lower molecular weights (between 14 and 3.5 kDa) were present and were able to induce the inhibitory activity. The extract contained more ß-(1→3)-glucans than ß-(11→3),(11→6)-glucans, and they partially survived digestion and managed to pass through Caco2 cell monolayers to the lower compartment after in vitro digestion and transport experiments. The WSP might also modulate Caco2 membrane integrity.

Optimized Triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins.

SCOPE: Investigations into the immunological response of proteins is often masked by lipopolysaccharide (LPS) contamination. We report an optimized Triton X-114 (TX-114) based LPS extraction method for ß-lactoglobulin (BLG) and soy protein extract suitable for cell-based immunological assays. METHODS AND RESULTS: Optimization of an existing TX-114 based phase LPS extraction method resulted in >99% reduction of LPS levels. However, remaining TX-114 was found to interfere with LPS and protein concentration assays and decreased viability of THP-1 macrophages and HEK-Blue 293 cells. Upon screening a range of TX-114 extraction procedures, TX-114-binding beads were found to most effectively lower TX-114 levels without affecting protein structural properties. LPS-purified proteins showed reduced capacity to activate TLR4 compared to non-treated proteins. LPS-purified BLG did not induce secretion of pro-inflammatory cytokines from THP-1 macrophages, as non-treated protein did, showing that LPS contamination masks the immunomodulatory effect of BLG. Both HEK293 cells expressing TLR4 and differentiated THP-1 macrophages were shown as a relevant model to screen the protein preparations for biological effects of LPS contamination. CONCLUSION: The reported TX-114 assisted LPS-removal from protein preparations followed by bead based removal of TX-114 allows evaluation of natively folded protein preparations for their immunological potential in cell-based studies.

The effects of Lactobacillus plantarum on small intestinal barrier function and mucosal gene transcription; a randomized double-blind placebo controlled trial.

The aim of this study was to investigate the effects of three Lactobacillus plantarum strains on in-vivo small intestinal barrier function and gut mucosal gene transcription in human subjects. The strains were selected for their differential effects on TLR signalling and tight junction protein rearrangement, which may lead to beneficial effects in a stressed human gut mucosa. Ten healthy volunteers participated in four different intervention periods: 7-day oral intake of either L. plantarum WCFS1, CIP104448, TIFN101 or placebo, proceeded by a 4 weeks wash-out period. Lactulose-rhamnose ratio (an indicator of small intestinal permeability) increased after intake of indomethacin, which was given as an artificial stressor of the gut mucosal barrier (mean ratio 0.06 ± 0.04 to 0.10 ± 0.06, p = 0.001), but was not significantly affected by the bacterial interventions. However, analysis in small intestinal biopsies, obtained by gastroduodenoscopy, demonstrated that particularly L. plantarum TIFN101 modulated gene transcription pathways related to cell-cell adhesion with high turnover of genes involved in tight- and adhesion junction protein synthesis and degradation (e.g. actinin alpha-4, metalloproteinase-2). These effects were less pronounced for L. plantarum WCFS1 and CIP104448. In conclusion, L. plantarum TIFN101 induced the most pronounced probiotic properties with specific gene transcriptional effects on repair processes in the compromised intestine of healthy subjects.

TCRs genetically linked to CD28 and CD3ε do not mispair with endogenous TCR chains and mediate enhanced T cell persistence and anti-melanoma activity.

Adoptive transfer of T cells that are gene engineered to express a defined TCR represents a feasible and promising therapy for patients with tumors. However, TCR gene therapy is hindered by the transient presence and effectiveness of transferred T cells, which are anticipated to be improved by adequate T cell costimulation. In this article, we report the identification and characterization of a novel two-chain TCR linked to CD28 and CD3ε (i.e., TCR:28ε). This modified TCR demonstrates enhanced binding of peptide-MHC and mediates enhanced T cell function following stimulation with peptide compared with wild-type TCR. Surface expression of TCR:28ε depends on the transmembrane domain of CD28, whereas T cell functions depend on the intracellular domains of both CD28 and CD3ε, with IL-2 production showing dependency on CD28:LCK binding. TCR:28ε, but not wild-type TCR, induces detectable immune synapses in primary human T cells, and such immune synapses show significantly enhanced accumulation of TCR transgenes and markers of early TCR signaling, such as phosphorylated LCK and ERK. Importantly, TCR:28ε does not show signs of off-target recognition, as evidenced by lack of TCR mispairing, as well as preserved specificity. Notably, when testing TCR:28ε in immune-competent mice, we observed a drastic increase in T cell survival, which was accompanied by regression of large melanomas with limited recurrence. Our data argue that TCR transgenes that contain CD28, and, thereby, may provide T cell costimulation in an immune-suppressive environment, represent candidate receptors to treat patients with tumors.

TCR-Engineered T Cells Meet New Challenges to Treat Solid Tumors: Choice of Antigen, T Cell Fitness, and Sensitization of Tumor Milieu.

Adoptive transfer of T cells gene-engineered with antigen-specific T cell receptors (TCRs) has proven its feasibility and therapeutic potential in the treatment of malignant tumors. To ensure further clinical development of TCR gene therapy, it is necessary to target immunogenic epitopes that are related to oncogenesis and selectively expressed by tumor tissue, and implement strategies that result in optimal T cell fitness. In addition, in particular for the treatment of solid tumors, it is equally necessary to include strategies that counteract the immune-suppressive nature of the tumor micro-environment. Here, we will provide an overview of the current status of TCR gene therapy, and redefine the following three challenges of improvement: "choice of target antigen"; "fitness of T cells"; and "sensitization of tumor milieu." We will categorize and discuss potential strategies to address each of these challenges, and argue that advancement of clinical TCR gene therapy critically depends on developments toward each of the three challenges.

Magnetic-activated cell sorting of TCR-engineered T cells, using tCD34 as a gene marker, but not peptide-MHC multimers, results in significant numbers of functional CD4+ and CD8+ T cells.

T cell-sorting technologies with peptide-MHC multimers or antibodies against gene markers enable enrichment of antigen-specific T cells and are expected to enhance the therapeutic efficacy of clinical T cell therapy. However, a direct comparison between sorting reagents for their ability to enrich T cells is lacking. Here, we compared the in vitro properties of primary human T cells gene-engineered with gp100(280-288)/HLA-A2-specific T cell receptor-αß (TCRαß) on magnetic-activated cell sorting (MACS) with various peptide-MHC multimers or an antibody against truncated CD34 (tCD34). With respect to peptide-MHC multimers, we observed that Streptamer(®), when compared with pentamers and tetramers, improved T cell yield as well as level and stability of enrichment, of TCR-engineered T cells (>65% of peptide-MHC-binding T cells, stable for at least 6 weeks). In agreement with these findings, Streptamer, the only detachable reagent, revealed significant T cell expansion in the first week after MACS. Sorting TCR and tCD34 gene-engineered T cells with CD34 monoclonal antibody (mAb) resulted in the most significant T cell yield and enrichment of T cells (>95% of tCD34 T cells, stable for at least 6 weeks). Notably, T cells sorted with CD34 mAb, when compared with Streptamer, bound about 2- to 3-fold less peptide-MHC but showed superior antigen-specific upregulated expression of CD107a and production of interferon (IFN)-γ. Multiparametric flow cytometry revealed that CD4(+) T cells, uniquely present in CD34 mAb-sorted T cells, contributed to enhanced IFN-γ production. Taken together, we postulate that CD34 mAb-based sorting of gene-marked T cells has benefits toward applications of T cell therapy, especially those that require CD4(+) T cells.

T-cell synapse formation depends on antigen recognition but not CD3 interaction: studies with TCR:ζ, a candidate transgene for TCR gene therapy.

T-cell receptors (TCRs) can be genetically modified to improve gene-engineered T-cell responses, a strategy considered critical for the success of clinical TCR gene therapy to treat cancers. TCR:ζ, which is a heterodimer of TCRα and ß chains each coupled to complete human CD3ζ, overcomes issues of mis-pairing with endogenous TCR chains, shows high surface expression and mediates antigen-specific T-cell functions in vitro. In the current study, we further characterized TCR:ζ in gene-engineered T cells and assessed whether this receptor is able to interact with surface molecules and drive correct synapse formation in Jurkat T cells. The results showed that TCR:ζ mediates the formation of synaptic areas with antigen-positive target cells, interacts closely with CD8α and MHC class I (MHCI), and co-localizes with CD28, CD45 and lipid rafts, similar to WT TCR. TCR:ζ did not closely associate with endogenous CD3ε, despite its co-presence in immune synapses, and TCR:ζ showed enhanced synaptic accumulation in T cells negative for surface-expressed TCR molecules. Notably, synaptic TCR:ζ demonstrated lowered densities when compared with TCR in dual TCR T cells, a phenomenon that was related to both extracellular and intracellular CD3ζ domains present in the TCR:ζ molecule and responsible for enlarged synapse areas.

T cell receptor (TCR) gene therapy to treat melanoma: lessons from clinical and preclinical studies.

IMPORTANCE OF THE FIELD: Adoptive T cell therapy (ACT) with tumour infiltrating lymphocytes is currently the best treatment option for metastatic melanoma. Despite its clinical successes, ACT has limitations in availability and generation of therapeutic T cells for a larger group of patients. Introduction of tumour-specific T cell receptors into T cells, termed TCR gene therapy, can provide an alternative for ACT that is more widely applicable and might be extended to other types of cancer. AREAS COVERED IN THIS REVIEW: The current status of TCR gene therapy studies including clinical challenges, such as on-target toxicity, compromised anti-tumour T cell responses, compromised T cell persistence and potential immunogenicity of receptor transgenes. Strategies to address these challenges are covered. WHAT THE READER WILL GAIN: A listing and discussion of strategies that aim at improving the efficacy and safety of TCR gene therapy. Such strategies address antigen choice, TCR mis-pairing, functional avidity and persistence of T cells, immune responses towards receptor transgenes, and combination of ACT with other therapies. TAKE HOME MESSAGE: To ensure further clinical development of TCR gene therapy, it is necessary to choose safe T cell target antigens, and implement (combinations of) strategies that enhance the correct pairing of TCR transgenes and the functional avidity and persistence of T cells.

T cell receptor gene therapy: strategies for optimizing transgenic TCR pairing.

T cell receptor (TCR) gene therapy provides patients with autologous T cells that are genetically engineered with TCRalphabeta chains and constitutes a promising approach for the treatment of tumors and virus infections. Among the current challenges of TCR gene therapy is the optimization of TCRalpha and beta transgene pairing to enhance the functional avidity of therapeutic T cells. Recently, various genetically modified TCRs have been developed that enhance TCR pairing and minimize mispairing, i.e. pairing between transgenic and endogenous TCR chains. Here, we classify such receptors according to their CD3-dependence for surface expression and review their abilities to address functional T cell avidity. In addition, we discuss the anticipated clinical value of these and other strategies to generate high-avidity T cells.