Cutting Edge: Polycomb Repressive Complex 1 Subunit Cbx4 Positively Regulates Effector Responses in CD8 T Cells.

CTL differentiation is controlled by the crosstalk of various transcription factors and epigenetic modulators. Uncovering this process is fundamental to improving immunotherapy and designing novel therapeutic approaches. In this study, we show that polycomb repressive complex 1 subunit chromobox (Cbx)4 favors effector CTL differentiation in a murine model. Cbx4 deficiency in CTLs induced a transcriptional signature of memory cells and increased the memory CTL population during acute viral infection. It has previously been shown that besides binding to H3K27me3 through its chromodomain, Cbx4 functions as a small ubiquitin-like modifier (SUMO) E3 ligase in a SUMO-interacting motifs (SIM)-dependent way. Overexpression of Cbx4 mutants in distinct domains showed that this protein regulates CTL differentiation primarily in an SIM-dependent way and partially through its chromodomain. Our data suggest a novel role of a polycomb group protein Cbx4 controlling CTL differentiation and indicated SUMOylation as a key molecular mechanism connected to chromatin modification in this process.

Epigenetic regulation of T cells by Polycomb group proteins.

T cells are critical for pathogen elimination, tumor surveillance, and immunoregulation. The development, activation, and differentiation of CD8 and CD4 T lymphocytes are a set of complex and dynamically regulated events that require epigenetic control. The Polycomb group (PcG) proteins are a family of diverse and evolutionarily conserved epigenetic modulators fundamentally involved in several mechanisms of gene regulation. PcG proteins can assemble into distinct repressor complexes, the two most understood being the Polycomb Repressor Complex (PRC)1 and PRC2, which control chromatin structure mainly through posttranslational modifications of histones. In this review, we will summarize the most recent findings regarding the diverse roles performed by PcG proteins in T cell biology. We will focus on PRC1 and PRC2 contribution to the regulation of T cell development in the thymus, CD4 T cell differentiation in helper or regulatory phenotypes and CD8 T cell fate commitment in the context of infections and cancer, highlighting the known mechanisms and knowledge gaps that still need to be addressed.

IL-17 and IL-17C Signaling Protects the Intestinal Epithelium against Diisopropyl Fluorophosphate Exposure in an Acute Model of Gulf War Veterans' Illnesses.

Gulf War Veterans' Illnesses (GWI) encompasses a broad range of unexplained symptomology specific to Veterans of the Persian Gulf War. Gastrointestinal (GI) distress is prominent in veterans with GWI and often presents as irritable bowel syndrome (IBS). Neurotoxins, including organophosphorus pesticides and sarin gas, are believed to have contributed to the development of GWI, at least in a subset of Veterans. However, the effects of such agents have not been extensively studied for their potential impact to GI disorders and immunological stability. Here we utilized an established murine model of GWI to investigate deleterious effects of diisopropyl fluorophosphate (DFP) exposure on the mucosal epithelium in vivo and in vitro. In vivo, acute DFP exposure negatively impacts the mucosal epithelium by reducing tight junction proteins and antimicrobial peptides as well as altering intestinal microbiome composition. Furthermore, DFP treatment reduced the expression of IL-17 in the colonic epithelium. Conversely, both IL-17 and IL-17C treatment could combat the negative effects of DFP and other cholinesterase inhibitors in murine intestinal organoid cells. Our findings demonstrate that acute exposure to DFP can result in rapid deterioration of mechanisms protecting the GI tract from disease. These results are relevant to suspected GWI exposures and could help explain the propensity for GI disorders in GWI Veterans.

An Updated Model for the Epigenetic Regulation of Effector and Memory CD8+ T Cell Differentiation.

Naive CD8+ T cells, upon encountering their cognate Ag in vivo, clonally expand and differentiate into distinct cell fates, regulated by transcription factors and epigenetic modulators. Several models have been proposed to explain the differentiation of CTLs, although none fully recapitulate the experimental evidence. In this review article, we will summarize the latest research on the epigenetic regulation of CTL differentiation as well as provide a combined model that contemplates them.

Toll-like receptor 2 induces pathogenicity in Th17 cells and reveals a role for IPCEF in regulating Th17 cell migration.

Pathogenic Th17 cells drive inflammation in autoimmune disease, yet the molecular programming underlying Th17 cell pathogenicity remains insufficiently understood. Activation of Toll-like receptor 2 (TLR2) increases Th17 cell inflammatory potential, but little is known regarding the mechanistic outcomes of TLR2 signaling in Th17 cells. Here, we demonstrate that TLR2 is comparable to IL-23 in inducing pathogenicity and increasing the migratory capacity of Th17 cells. We perform RNA sequencing of Th17 cells stimulated though the TLR2 pathway and find differential expression of several genes linked with the Th17 genetic program as well as genes not previously associated with pathogenic Th17 cells, including Ipcef1. Enforced expression of Ipcef1 in Th17 cells abolishes the TLR2-dependent increases in migratory capacity and severely impairs the ability of Th17 cells to induce experimental autoimmune encephalomyelitis. This study establishes the importance of the TLR2 signaling pathway in inducing Th17 cell pathogenicity and driving autoimmune inflammation.

Kdm6b Regulates the Generation of Effector CD8+ T Cells by Inducing Chromatin Accessibility in Effector-Associated Genes.

The transcriptional and epigenetic regulation of CD8+ T cell differentiation is critical for balancing pathogen eradication and long-term immunity by effector and memory CTLs, respectively. In this study, we demonstrate that the lysine demethylase 6b (Kdm6b) is essential for the proper generation and function of effector CD8+ T cells during acute infection and tumor eradication. We found that cells lacking Kdm6b (by either T cell-specific knockout mice or knockdown using short hairpin RNA strategies) show an enhanced generation of memory precursor and early effector cells upon acute viral infection in a cell-intrinsic manner. We also demonstrate that Kdm6b is indispensable for proper effector functions and tumor protection, and that memory CD8+ T cells lacking Kdm6b displayed a defective recall response. Mechanistically, we identified that Kdm6b, through induction of chromatin accessibility in key effector-associated gene loci, allows for the proper generation of effector CTLs. Our results pinpoint the essential function of Kdm6b in allowing chromatin accessibility in effector-associated genes, and identify Kdm6b as a potential target for therapeutics in diseases with dysregulated effector responses.

Optimizing dialysis dose in the context of frailty: an exploratory study.

INTRODUCTION: Frailty is a multicausal syndrome characterized by a decrease in strength, resistance and physiological function, which makes the individual vulnerable and dependent, and increases his/her mortality. This syndrome is more prevalent among older individuals, and chronic kidney disease patients, particularly those on dialysis. Dialysis dose is currently standardized for hemodialysis (HD) patients regardless of their age and functional status. However, it has been postulated that the dialysis dose required in older patients, especially frail ones, should be lower, since it could increase their degree of frailty. Then, the purpose of this study was to evaluate if there would be a correlation between the dose of Kt/V and the degree of frailty in a population of adult patients on HD. MATERIALS AND METHODS: A cross-sectional study with 82 patients on HD in Barranquilla (Colombia) and Lobos (Argentina) was conducted. Socio-demographic and laboratory data, as well as dialysis doses (Kt/V) were recorded and scales of fragility, physical activity, gait and grip strength were applied. Then these data were correlated by a Spearman's correlation and a logistic regression. RESULTS: CFS, social isolation, physical activity, gait speed, and prehensile strength tests were outside the reference ranges in the studied group. No significant correlation was found between dialysis dose and all the above mentioned functional tests. However, a significant and inverse correlation between physical activity and CFS was documented (score - 1.41 (CI - 2.1 to - 0.7). CONCLUSION: No significant correlation was documented between Kt/V value and different parameters of the frailty status, but this status correlated significantly and inversely with physical activity in this group. Frailty status in hemodialysis patients was significantly higher in older individuals, although young individuals were not exempt from it.

NFAT1 and NFAT2 Differentially Regulate CTL Differentiation Upon Acute Viral Infection.

CD8+ T cell differentiation orchestrated by transcription regulators is critical for balancing pathogen eradication and long-term immunity by effector and memory CTLs, respectively. The transcription factor Nuclear Factor of Activated T cells (NFAT) family members are known for their roles in T cell development and activation but still largely undetermined in CD8+ T cell differentiation in vivo. Here, we interrogated the role of two NFAT family members, NFAT1 and NFAT2, in the effector and memory phase of CD8+ T cell differentiation using LCMVArm acute infection model. We found that NFAT1 is critical for effector population generation whereas NFAT2 is required for promoting memory CTLs in a cell intrinsic manner. Moreover, we found that mice lacking both NFAT1 and NFAT2 in T cells display a significant increase in KLRG1hi CD127hi population and are unable to clear an acute viral infection. NFAT-deficient CTLs showed different degrees of impaired IFN-γ and TNF-α expression with NFAT1 being mainly responsible for IFN-γ production upon ex-vivo stimulation as well as for antigen-specific cytotoxicity. Our results suggest that NFAT1 and NFAT2 have distinct roles in mediating CD8+ T cell differentiation and function.

Etnobotánica médica urbana y periurbana de la ciudad de Córdoba (Argentina) / Urban and peri-urban medical ethnobotany in the city of Córdoba (Argentina)

Following is submitted an ethnobotanic study on knowledge and practices on usage, role and meaning of plants and relatives used by practitioners of non-official medicine in urban and peri-urban places of the city of Córdoba, Argentina. In this study area, it can be observed a cultural structure of knowledge as well as local and foreign practices, which are typical of modern cultures. Diverse social actors perform as cultural connectors between urban and rural scenarios, which match traditional components with those of biomedicine as well as those that belong to a large chain of medical herbs marketing. Quantitative and qualitative methods were resorted to, through classical ethnobotanic techniques. An amount of 768 therapeutical usages were registered which correspond to 262 native and foreign medical taxons. These taxons belong to 95 family plants marketed within urban and peri-urban areas. It is also remarkable an urban pharmacopeia highly diverse as regards species and usages with a top level of exotic species (60%) as well as complementary alternative and global medicines in theses contexts.

Se presenta un estudio etnobotánico de los conocimientos y prácticas referidos al uso, rol y significado de los vegetales usados por practicantes de la medicina no oficial en poblaciones urbanas y periurbanas de la ciudad de Córdoba, Argentina. En este ámbito de estudio, se conforma un complejo cultural de saberes y prácticas locales y foráneas, típicas de culturas modernas. Se destaca el protagonismo de diversos actores sociales los que actúan como conectores culturales entre escenarios urbanos y rurales, los que combinan elementos tradicionales con los de la biomedicina, como así también los que forman parte de una larga cadena de comercialización de hierbas medicinales. Se recurrió a métodos cualitativos y cuantitativos mediante técnicas clásicas etnobotánicas. Se documentaron un total de 768 usos terapéuticos correspondientes a 262 taxones medicinales de estatus autóctonos y exóticos que pertenecen a 95 familias de plantas comercializadas en el ámbito urbano y periurbano. Se advierte una farmacopea urbana altamente diversificada en especies y aplicaciones con un predominio de especies exóticas (60%) como así también la presencia influyente de las medicinas alternativas complementarias y de medicinas globales en estos contextos.

Siglec-6 on Chronic Lymphocytic Leukemia Cells Is a Target for Post-Allogeneic Hematopoietic Stem Cell Transplantation Antibodies.

Although the 5-year survival rate of chronic lymphocytic leukemia (CLL) patients has risen to >80%, the only potentially curative treatment is allogeneic hematopoietic stem cell transplantation (alloHSCT). To identify possible new monoclonal antibody (mAb) drugs and targets for CLL, we previously developed a phage display-based human mAb platform to mine the antibody repertoire of patients who responded to alloHSCT. We had selected a group of highly homologous post-alloHSCT mAbs that bound to an unknown CLL cell surface antigen. Here, we show through next-generation sequencing of cDNAs encoding variable heavy-chain domains that these mAbs had a relative abundance of ∼0.1% in the post-alloHSCT antibody repertoire and were enriched ∼1,000-fold after three rounds of selection on primary CLL cells. Based on differential RNA-seq and a cell microarray screening technology for discovering human cell surface antigens, we now identify their antigen as Siglec-6. We verified this finding by flow cytometry, ELISA, siRNA knockdown, and surface plasmon resonance. Siglec-6 was broadly expressed in CLL and could be a potential target for antibody-based therapeutic interventions. Our study reaffirms the utility of post-alloHSCT antibody drug and target discovery. Cancer Immunol Res; 6(9); 1008-13. ©2018 AACR.

Trim33 mediates the proinflammatory function of Th17 cells.

Transforming growth factor-ß (TGF-ß) regulates reciprocal regulatory T cell (T reg) and T helper 17 (Th17) differentiation, the underlying mechanism of which is still not understood. Here, we report that tripartite motif-containing 33 (Trim33), a modulator of TGF-ß signaling that associates with Smad2, regulates the proinflammatory function of Th17 cells. Trim33 deficiency in T cells ameliorated an autoimmune disease in vivo. Trim33 was required for induction in vitro of Th17, but not T reg cells. Moreover, Smad4 and Trim33 play contrasting roles in the regulation of IL-10 expression; loss of Trim33 enhanced IL-10 production. Furthermore, Trim33 was recruited to the Il17a and Il10 gene loci, dependent on Smad2, and mediated their chromatin remodeling during Th17 differentiation. Trim33 thus promotes the proinflammatory function of Th17 cells by inducing IL-17 and suppressing IL-10 expression.

Regulation of Pathogenic T Helper 17 Cell Differentiation by Steroid Receptor Coactivator-3.

T helper 17 (Th17) cell development is programmed by the orphan nuclear receptor RORγt, but the underlying mechanism is not well understood. Nuclear receptor-mediated transcriptional activation depends on coactivators. Here, we show that steroid receptor coactivator-3 (SRC-3) critically regulates Th17 cell differentiation. Reduced incidence of experimental autoimmune encephalitis (EAE) associated with decreased Th17 cell generation in vivo was observed in mice with SRC-3 deletion specifically in T cells. In vitro, SRC-3 deficiency did not affect TGF-ß/IL-6-induced Th17 cell generation but severely impaired pathogenic Th17 differentiation induced by IL-1/IL-6/IL-23. Microarray analysis revealed that SRC-3 not only regulates IL-17A but also IL-1R1 expression. SRC-3 bound to Il17a and Il1r1 loci in a RORγt-dependent manner and was required for recruitment of the p300 acetyltransferase. Thus, SRC-3 is critical for RORγt-dependent gene expression in Th17 cell-driven autoimmune diseases.

Tumor Tolerance-Promoting Function of Regulatory T Cells Is Optimized by CD28, but Strictly Dependent on Calcineurin.

Regulatory T cells (Treg) restrain immune responses against malignant tumors, but their global depletion in cancer patients will likely be limited by systemic autoimmune toxicity. Instead, approaches to "tune" their activities may allow for preferential targeting of tumor-reactive Treg. Although Ag recognition regulates Treg function, the roles of individual TCR-dependent signaling pathways in enabling Treg to promote tumor tolerance are not well characterized. In this study, we examined in mouse tumor models the role of calcineurin, a key mediator of TCR signaling, and the role of the costimulatory receptor CD28 in the differentiation of resting central Treg into effector Treg endowed with tumor tropism. We find that calcineurin, although largely dispensable for suppressive activity in vitro, is essential for upregulation of ICOS and CTLA-4 in Treg, as well as for expression of chemokine receptors driving their accumulation in tumors. In contrast, CD28 is not critical, but optimizes the formation of tumor-homing Treg and their fitness in tumor tissue. Accordingly, although deletion of either CnB or CD28 strongly impairs Treg-mediated tumor tolerance, lack of CnB has an even more pronounced impact than lack of CD28. Hence, our studies reveal distinct roles for what has classically been defined as signal 1 and signal 2 of conventional T cell activation in the context of Treg-mediated tumor tolerance.

The Xenobiotic Transporter Mdr1 Enforces T Cell Homeostasis in the Presence of Intestinal Bile Acids.

CD4+ T cells are tightly regulated by microbiota in the intestine, but whether intestinal T cells interface with host-derived metabolites is less clear. Here, we show that CD4+ T effector (Teff) cells upregulated the xenobiotic transporter, Mdr1, in the ileum to maintain homeostasis in the presence of bile acids. Whereas wild-type Teff cells upregulated Mdr1 in the ileum, those lacking Mdr1 displayed mucosal dysfunction and induced Crohn's disease-like ileitis following transfer into Rag1-/- hosts. Mdr1 mitigated oxidative stress and enforced homeostasis in Teff cells exposed to conjugated bile acids (CBAs), a class of liver-derived emulsifying agents that actively circulate through the ileal mucosa. Blocking ileal CBA reabsorption in transferred Rag1-/- mice restored Mdr1-deficient Teff cell homeostasis and attenuated ileitis. Further, a subset of ileal Crohn's disease patients displayed MDR1 loss of function. Together, these results suggest that coordinated interaction between mucosal Teff cells and CBAs in the ileum regulate intestinal immune homeostasis.

Mining Naïve Rabbit Antibody Repertoires by Phage Display for Monoclonal Antibodies of Therapeutic Utility.

Owing to their high affinities and specificities, rabbit monoclonal antibodies (mAbs) have demonstrated value and potential primarily as basic research and diagnostic reagents, but, in some cases, also as therapeutics. To accelerate access to rabbit mAbs bypassing immunization, we generated a large naïve rabbit antibody repertoire represented by a phage display library encompassing >10 billion independent antibodies in chimeric rabbit/human Fab format and validated it by next-generation sequencing. Panels of rabbit mAbs selected from this library against two emerging cancer targets, ROR1 and ROR2, revealed high diversity, affinity, and specificity. Moreover, ROR1- and ROR2-targeting rabbit mAbs demonstrated therapeutic utility as components of chimeric antigen receptor-engineered T cells, further corroborating the value of the naïve rabbit antibody library as a rich and virtually unlimited source of rabbit mAbs.

Plantas de interés veterinario en la cultura campesina de la Sierra de Ancasti (Catamarca, Argentina) / Plants of veterinary interest in the peasant culture of Sierra de Ancasti (Catamarca, Argentina)

Se propone dar a conocer e interpretar, el uso y la significación de las plantas en la veterinaria tradicional de la Sierra de Ancasti (Catamarca, Argentina). Mediante el empleo de métodos y técnicas de investigación etnobotánica se documentaron los taxones de dolencias vernáculas, y un total de 62 usos medicinales correspondientes a 43 especies pertenecientes a 30 familias botánicas. Las principales aplicaciones medicinales son: cicatrizantes, digestivas, antiparasitarias y oxitócicas. Se analizó el rol de las especies tóxicas y peligrosas en la veterinaria local. Junto al repertorio de plantas, se presentan otras técnicas terapéuticas populares, así como prácticas culturales de tipo religioso-ritual. Finalmente, se analiza la tensión entre tradición y modernidad en este campo del saber. Se concluye que, a pesar de que el conocimiento etnoveterinario resulta relevante en cantidad de especies y usos, existe un creciente abandono de su praxis, lo que podría devenir en la extinción de la experiencia.

In this work we aim at presenting and interpreting the use and meaning of plants in traditional veterinary in Sierra de Ancasti (Catamarca, Argentina). By employing ethnobotanical research methods and techniques, we registered taxa of vernacular ailments and 62 medicinal uses corresponding to 43 species from 30 botanical families. They are used medicinally due to their healing, digestive, anti- parasitic and oxytocic properties. In addition, the role of toxic and dangerous species was analyzed in the local veterinary. Together with the repertoire of plants, we also described other popular therapeutic techniques and cultural practices of religious-ritualistic nature. Finally, tension was analyzed between tradition and modernity in this field of knowledge. It has been concluded that, despite ethnoveterinary knowledge is relevant for many species and uses, its practice is being gradually neglected, which could eventually lead to the extinction of experience.

Transcriptional and epigenetic regulation of T cell hyporesponsiveness.

Naive CD8+ T cells differentiate into effector and memory cytolytic T cells (CTLs) during an acute infection. In contrast, in scenarios of persistent antigen stimulation, such as chronic infections and cancer, antigen-specific CTLs show a gradual decrease in effector function, a phenomenon that has been termed CD8+ T cell "exhaustion" or "dysfunction." Another hyporesponsive state, termed "anergy", is observed when T cells are activated in the absence of positive costimulatory signals. Among the many negative regulators induced in hyporesponsive T cells are inhibitory cell-surface receptors, such as PD-1, LAG-3, CTLA-4, and TIM-3; "checkpoint blockade" therapies that involve treatment of patients with cancer with blocking antibodies to those receptors show considerable promise in the clinic because the blocking antibodies can mitigate hyporesponsiveness and promote tumor rejection. In this review, we describe recent advances in our molecular understanding of these hyporesponsive states. We review evidence for the involvement of diverse transcription factors, metabolic programs, and chromatin accessibility changes in hyporesponsive T cells, and we discuss how checkpoint blockade therapies affect the molecular program of CD8+ T cell exhaustion.