Activation of naïve CD4+ T cells re-tunes STAT1 signaling to deliver unique cytokine responses in memory CD4+ T cells.

The cytokine IL-6 controls the survival, proliferation and effector characteristics of lymphocytes through activation of the transcription factors STAT1 and STAT3. While STAT3 activity is an ever-present feature of IL-6 signaling in CD4+ T cells, prior activation via the T cell antigen receptor limits IL-6's control of STAT1 in effector and memory populations. Here we found that phosphorylation of STAT1 in response to IL-6 was regulated by the tyrosine phosphatases PTPN2 and PTPN22 expressed in response to the activation of naïve CD4+ T cells. Transcriptomics and chromatin immunoprecipitation-sequencing (ChIP-seq) of IL-6 responses in naïve and effector memory CD4+ T cells showed how the suppression of STAT1 activation shaped the functional identity and effector characteristics of memory CD4+ T cells. Thus, tyrosine phosphatases induced by the activation of naïve T cells determine the way activated or memory CD4+ T cells sense and interpret cytokine signals.

FOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy.

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of haematological malignancies such as acute lymphoblastic leukaemia, B cell lymphoma and multiple myeloma1-4, but the efficacy of CAR T cell therapy in solid tumours has been limited5. This is owing to a number of factors, including the immunosuppressive tumour microenvironment that gives rise to poorly persisting and metabolically dysfunctional T cells. Analysis of anti-CD19 CAR T cells used clinically has shown that positive treatment outcomes are associated with a more 'stem-like' phenotype and increased mitochondrial mass6-8. We therefore sought to identify transcription factors that could enhance CAR T cell fitness and efficacy against solid tumours. Here we show that overexpression of FOXO1 promotes a stem-like phenotype in CAR T cells derived from either healthy human donors or patients, which correlates with improved mitochondrial fitness, persistence and therapeutic efficacy in vivo. This work thus reveals an engineering approach to genetically enforce a favourable metabolic phenotype that has high translational potential to improve the efficacy of CAR T cells against solid tumours.

T Cell LEGO: Identifying the Master Builders and What They Do.

Understanding how cell fate decisions are made during cellular differentiation and the mechanisms that drive them is a holy grail of cell biology. In this issue of Immunity, Hu et al. (2018) and Johnson et al. (2018) demonstrate that key transcriptional regulators and global changes in nuclear architecture underlie differentiation decisions during T cell development.

Helminth Infection-Induced Increase in Virtual Memory CD8 T Cells Is Transient, Driven by IL-15, and Absent in Aged Mice.

CD8 virtual memory T (TVM) cells are Ag-naive CD8 T cells that have undergone partial differentiation in response to common γ-chain cytokines, particularly IL-15 and IL-4. TVM cells from young individuals are highly proliferative in response to TCR and cytokine stimulation but, with age, they lose TCR-mediated proliferative capacity and exhibit hallmarks of senescence. Helminth infection can drive an increase in TVM cells, which is associated with improved pathogen clearance during subsequent infectious challenge in young mice. Given the cytokine-dependent profile of TVM cells and their age-associated dysfunction, we traced proliferative and functional changes in TVM cells, compared with true naive CD8 T cells, after helminth infection of young and aged C57BL/6 mice. We show that IL-15 is essential for the helminth-induced increase in TVM cells, which is driven only by proliferation of existing TVM cells, with negligible contribution from true naive cell differentiation. Additionally, TVM cells showed the greatest proliferation in response to helminth infection and IL-15 compared with other CD8 T cells. Furthermore, TVM cells from aged mice did not undergo expansion after helminth infection due to both TVM cell-intrinsic and -extrinsic changes associated with aging.

Th1 Cells Alter the Inflammatory Signature of IL-6 by Channeling STAT Transcription Factors to Alu-like Retroelements.

Cytokines that signal via STAT1 and STAT3 transcription factors instruct decisions affecting tissue homeostasis, antimicrobial host defense, and inflammation-induced tissue injury. To understand the coordination of these activities, we applied RNA sequencing, chromatin immunoprecipitation sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing to identify the transcriptional output of STAT1 and STAT3 in peritoneal tissues from mice during acute resolving inflammation and inflammation primed to drive fibrosis. Bioinformatics focused on the transcriptional signature of the immunomodulatory cytokine IL-6 in both settings and examined how profibrotic IFN-γ-secreting CD4+ T cells altered the interpretation of STAT1 and STAT3 cytokine cues. In resolving inflammation, STAT1 and STAT3 cooperated to drive stromal gene expression affecting antimicrobial immunity and tissue homeostasis. The introduction of IFN-γ-secreting CD4+ T cells altered this transcriptional program and channeled STAT1 and STAT3 to a previously latent IFN-γ activation site motif in Alu-like elements. STAT1 and STAT3 binding to this conserved sequence revealed evidence of reciprocal cross-regulation and gene signatures relevant to pathophysiology. Thus, we propose that effector T cells retune the transcriptional output of IL-6 by shaping a regulatory interplay between STAT1 and STAT3 in inflammation.

Distinct epigenetic signatures delineate transcriptional programs during virus-specific CD8(+) T cell differentiation.

The molecular mechanisms that regulate the rapid transcriptional changes that occur during cytotoxic T lymphocyte (CTL) proliferation and differentiation in response to infection are poorly understood. We have utilized ChIP-seq to assess histone H3 methylation dynamics within naive, effector, and memory virus-specific T cells isolated directly ex vivo after influenza A virus infection. Our results show that within naive T cells, codeposition of the permissive H3K4me3 and repressive H3K27me3 modifications is a signature of gene loci associated with gene transcription, replication, and cellular differentiation. Upon differentiation into effector and/or memory CTLs, the majority of these gene loci lose repressive H3K27me3 while retaining the permissive H3K4me3 modification. In contrast, immune-related effector gene promoters within naive T cells lacked the permissive H3K4me3 modification, with acquisition of this modification occurring upon differentiation into effector/memory CTLs. Thus, coordinate transcriptional regulation of CTL genes with related functions is achieved via distinct epigenetic mechanisms.

Mucosal-Associated Invariant T Cell Effector Function Is an Intrinsic Cell Property That Can Be Augmented by the Metabolic Cofactor α-Ketoglutarate.

Mucosal-associated invariant T (MAIT) cells are an innate-like population of unconventional T cells that respond rapidly to microbial metabolite Ags or cytokine stimulation. Because of this reactivity and surface expression of CD45RO+, CD45RA-, and CD127+, they are described as effector memory cells. Yet, there is heterogeneity in MAIT cell effector response. It is unclear what factors control MAIT cell effector capacity, whether it is fixed or can be modified and if this differs based on whether activation is TCR dependent or independent. To address this, we have taken a systematic approach to examine human MAIT cell effector capacity across healthy individuals in response to ligand and cytokine stimulation. We demonstrate the heterogenous nature of MAIT cell effector capacity and that the ability to produce an effector response is not directly attributable to TCR clonotype or coreceptor expression. Global gene transcription analysis revealed that the MAIT cell effector capacity produced in response to TCR stimulation is associated with increased expression of the epigenetic regulator lysine demethylase 6B (KDM6B). Addition of a KDM6B inhibitor did not alter MAIT cell effector function to Ag or cytokine stimulation. However, addition of the KDM6B cofactor α-ketoglutarate greatly enhanced MAIT cell effector capacity to TCR-dependent stimulation in a partially KDM6B-dependent manner. These results demonstrate that the TCR-dependent effector response of MAIT cells is epigenetically regulated and dependent on the availability of metabolic cofactors.

The Impact of MHC Class I Dose on Development and Maintenance of the Polyclonal Naive CD8+ T Cell Repertoire.

Naive CD8+ T cell survival in the periphery is critically dependent on tonic TCR signaling through peptide + MHC class I (MHCI) recognition; however, little is known about how natural variation in MHCI levels impacts the naive CD8+ T cell repertoire. Using mice that are hemizygous or homozygous for a single MHCI allele, we showed that despite a reduction in peripheral CD8+ T cell numbers of ∼50% in MHCI hemizygous mice, MHCI levels had no notable impact on the rate of thymic generation or emigration of CD8 single-positive T cells. Moreover, the peripheral T cell repertoire in hemizygous mice showed selective retention of T cell clonotypes with a greater competitive advantage as evidenced by increased expression of CD5 and IL-7Rα. The qualitative superiority of CD8+ T cells retained in hemizygous mice was also seen during influenza A virus infection, in which epitope-specific CD8+ T cells from hemizygous mice had a higher avidity for pMHCI and increased cytokine polyfunctionality, despite a reduced response magnitude. Collectively, this study suggests that natural variation in MHCI expression levels has a notable and biologically relevant impact on the maintenance, but not generation, of the naive CD8+ T cell repertoire.

A Three-Dimensional Paper-Based Isoelectric Focusing Device for Direct Analysis of Proteins in Physiological Samples.

On-site protein analysis is crucial for disease diagnosis in community and family medicine in which microfluidic paper-based analytical devices (µPADs) have attracted growing attention. However, the practical applications of µPADs in protein analysis for physiological samples with high complexity is still limited. Herein, we developed a three-dimensional (3D) paper-based isoelectric focusing (IEF) platform, which is composed of power supply, reservoirs, and separation channel and made by the origami and stacking method, to simultaneously separate and enrich proteins in both low-salt and high-salt samples. Under the optimized experimental conditions, standard proteins (bovine hemoglobin (BHb) and phycocyanin (Phy)) were separated within 18 min under a 36 V power supply and obtained a 10-fold enrichment using the 3D paper-based IEF platform. Then, the capability of the 3D paper-based IEF platform for direct pretreatment of high-salt samples using a 12 V battery as power supply was measured through separating three standard proteins in saline (0.9% NaCl) with separation resolution (SR) > 1.29. Through further coupling with colorimetric and lateral flow strip measurements, the 3D paper-based IEF platform was applied to directly pretreat and quantitatively analyze microalbuminuria and C-reactive proteins in clinical urine and serum samples with analytical results with relative deviations of <8.4% and < 13.1%, respectively, to the clinical test results. This work proposes a new strategy to minimize the difficulty of directly processing high-salt samples with the traditional IEF system and provides a versatile, miniaturized, and low voltage demand analytical platform for on-site analysis of proteins in physiological samples.

Early T-BET Expression Ensures an Appropriate CD8+ Lineage-Specific Transcriptional Landscape after Influenza A Virus Infection.

Virus infection triggers large-scale changes in the phenotype and function of naive CD8+ T cells, resulting in the generation of effector and memory T cells that are then critical for immune clearance. The T-BOX family of transcription factors (TFs) are known to play a key role in T cell differentiation, with mice deficient for the TF T-BET (encoded by Tbx21) unable to generate optimal virus-specific effector responses. Although the importance of T-BET in directing optimal virus-specific T cell responses is accepted, the precise timing and molecular mechanism of action remains unclear. Using a mouse model of influenza A virus infection, we demonstrate that although T-BET is not required for early CD8+ T cell activation and cellular division, it is essential for early acquisition of virus-specific CD8+ T cell function and sustained differentiation and expansion. Whole transcriptome analysis at this early time point showed that Tbx21 deficiency resulted in global dysregulation in early programming events with inappropriate lineage-specific signatures apparent with alterations in the potential TF binding landscape. Assessment of histone posttranslational modifications within the Ifng locus demonstrated that Tbx21 -/- CD8+ T cells were unable to activate "poised" enhancer elements compared with wild-type CD8+ T cells, correlating with diminished Ifng transcription. In all, these data support a model whereby T-BET serves to promote appropriate chromatin remodeling at specific gene loci that underpins appropriate CD8+ T cell lineage-specific commitment and differentiation.

Liquid Plasticine Integrated with Isoelectric Focusing for Miniaturized Protein Analysis.

Developing miniaturized and rapid protein analytical platforms is urgently needed for on-site protein analysis, which is important for disease diagnosis and monitoring. Liquid marbles (LMs), a kind of particle-coated droplets, as ideal microreactors have been used in various fields. However, their application as analytical platforms is limited due to the difficulty of pretreating complex samples in simple LMs. Herein, inspired by the microfluidic chip, we propose a strategy through fabricating fluid channels using deformable LM, termed liquid plasticine (LP), to achieve sample pretreatment function. Through combining isoelectric focusing (IEF) with an LP channel, an LP-IEF platform with simultaneous protein separation and concentration functions is realized. The pretreatment capability of the LP-IEF system for proteins in physiological samples is proven using standard proteins and human serum with the results of a clear separation, 10-fold concentration, and a resolution of 0.03 pH. Through cutting the LP after IEF to LMs and transiting isolated LMs containing target proteins for further downstream colorimetric and mass spectrometry measurements, the quantitative analysis of clinical microalbuminuria and identification of α-1-microglobulin/bikunin precursor in clinical diabetic urine samples are achieved. This work proposes a strategy to develop LMs/LPs as a multifunctional integrated analytical platform and the miniaturized LP-IEF device as a rapid protein analytical platform.

Chromatin-associated protein kinase C-θ regulates an inducible gene expression program and microRNAs in human T lymphocytes.

Studies in yeast demonstrate that signaling kinases have a surprisingly active role in the nucleus, where they tether to chromatin and modulate gene expression programs. Despite these seminal studies, the nuclear mechanism of how signaling kinases control transcription of mammalian genes is in its infancy. Here, we provide evidence for a hitherto unknown function of protein kinase C-theta (PKC-θ), which physically associates with the regulatory regions of inducible immune response genes in human T cells. Chromatin-anchored PKC-θ forms an active nuclear complex by interacting with RNA polymerase II, the histone kinase MSK-1, and the adaptor molecule 14-3-3ζ. ChIP-on-chip reveals that PKC-θ binds to promoters and transcribed regions of genes, as well as to microRNA promoters that are crucial for cytokine regulation. Our results provide a molecular explanation for the role of PKC-θ not only in normal T cell function, but also in circumstances of its ectopic expression in cancer.

Nuclear PKC-θ facilitates rapid transcriptional responses in human memory CD4+ T cells through p65 and H2B phosphorylation.

Memory T cells are characterized by their rapid transcriptional programs upon re-stimulation. This transcriptional memory response is facilitated by permissive chromatin, but exactly how the permissive epigenetic landscape in memory T cells integrates incoming stimulatory signals remains poorly understood. By genome-wide ChIP-sequencing ex vivo human CD4(+) T cells, here, we show that the signaling enzyme, protein kinase C theta (PKC-θ) directly relays stimulatory signals to chromatin by binding to transcriptional-memory-responsive genes to induce transcriptional activation. Flanked by permissive histone modifications, these PKC-enriched regions are significantly enriched with NF-κB motifs in ex vivo bulk and vaccinia-responsive human memory CD4(+) T cells. Within the nucleus, PKC-θ catalytic activity maintains the Ser536 phosphorylation on the p65 subunit of NF-κB (also known as RelA) and can directly influence chromatin accessibility at transcriptional memory genes by regulating H2B deposition through Ser32 phosphorylation. Furthermore, using a cytoplasm-restricted PKC-θ mutant, we highlight that chromatin-anchored PKC-θ integrates activating signals at the chromatin template to elicit transcriptional memory responses in human memory T cells.

Dynamic regulation of permissive histone modifications and GATA3 binding underpin acquisition of granzyme A expression by virus-specific CD8(+) T cells.

Numerous studies have focused on the molecular regulation of perforin (PFP) and granzyme B (GZMB) expression by activated cytotoxic T lymphocytes (CTLs), but little is known about the molecular factors that underpin granzyme A (GZMA) expression. In vitro activation of naïve CD8(+) T cells, in the presence of IL-4, enhanced STAT6-dependent GZMA expression and was associated with GATA3 binding and enrichment of transcriptionally permissive histone posttranslational modifications (PTMs) across the Gzma gene locus. While GZMA expression by effector influenza A virus specific CTLs was also associated with a similar permissive epigenetic signature, memory CTL lacked enrichment of permissive histone PTMs at the Gzma locus, although this was restored within recalled secondary effector CTLs. Importantly, GZMA expression by virus-specific CTLs was associated with GATA3 binding at the Gzma locus, and independent of STAT6-mediated signaling. This suggests regulation of GZMA expression is underpinned by differentiation-dependent regulation of chromatin composition at the Gzma locus and that, given GATA3 is key for CTL differentiation in response to infection, GATA3 expression is regulated by a distinct, IL-4 independent, signaling pathway. Overall, this study provides insights into the molecular mechanisms that control transcription of Gzma during virus-induced CD8(+) T-cell differentiation.

The microRNA-9/B-lymphocyte-induced maturation protein-1/IL-2 axis is differentially regulated in progressive HIV infection.

The fine control of T-cell differentiation and its impact on HIV disease states is poorly understood. In this study, we demonstrate that B-lymphocyte-induced maturation protein-1 (Blimp-1/Prdm1) is highly expressed in CD4(+) T cells from chronically HIV-infected (CHI) patients compared to cells from long-term nonprogressors or healthy controls. Stimulation through the T-cell receptor in the presence of IL-2 induces Blimp-1 protein expression. We show here that Blimp-1 levels are translationally regulated by microRNA-9 (miR-9). Overexpression of miR-9 induces Blimp-1 repression, restoring IL-2 secretion in CD4(+) T cells via reduction in the binding of Blimp-1 to the il-2 promoter. In CHI patients where IL-2 expression is reduced and there is generalized T-cell dysfunction, we show differential expression of both miR-9 and Blimp-1 in CD4(+) cells compared with levels in long-term nonprogressors. These data identify a novel miR-9/Blimp-1/IL-2 axis that is dysregulated in progressive HIV infection.

Induction of autophagy by palmitic acid via protein kinase C-mediated signaling pathway independent of mTOR (mammalian target of rapamycin).

Lipotoxicity refers to the cytotoxic effects of excess fat accumulation in cells and has been implicated as one of the contributing factors to diseases like obesity, diabetes, and non-alcoholic fatty liver. In this study we sought to examine effects of palmitic acid (PA) and oleic acid, two of the common dietary fatty acids on the autophagic process. We found that PA, but not oleic acid, was able to cause an increase in autophagic flux, evidenced by LC3-II accumulation and formation of GFP-LC3 puncta. Notably, PA-induced autophagy was found to be independent of mTOR regulation. Next, in search of the mechanism mediating PA-induced autophagy, we found increased levels of diacylglycerol species and protein kinase C (PKC) activation in PA-treated cells. More importantly, inhibition of classical PKC isoforms (PKC-α) was able to effectively suppress PA-induced autophagy. Finally, we showed that inhibition of autophagy sensitized the cells to PA-induced apoptosis, suggesting the pro-survival function of autophagy induced by PA. Taken together, results from this study reveal a novel mechanism underlying free fatty acid-mediated autophagy. Furthermore, the pro-survival function of autophagy suggests modulation of autophagy as a potential therapeutic strategy in protection of cells against lipotoxicity and lipid-related metabolic diseases.

Epigenetic regulation of inducible gene expression in the immune system.

T cells are exquisitely poised to respond rapidly to pathogens and have proved an instructive model for exploring the regulation of inducible genes. Individual genes respond to antigenic stimulation in different ways, and it has become clear that the interplay between transcription factors and the chromatin platform of individual genes governs these responses. Our understanding of the complexity of the chromatin platform and the epigenetic mechanisms that contribute to transcriptional control has expanded dramatically in recent years. These mechanisms include the presence/absence of histone modification marks, which form an epigenetic signature to mark active or inactive genes. These signatures are dynamically added or removed by epigenetic enzymes, comprising an array of histone-modifying enzymes, including the more recently recognized chromatin-associated signalling kinases. In addition, chromatin-remodelling complexes physically alter the chromatin structure to regulate chromatin accessibility to transcriptional regulatory factors. The advent of genome-wide technologies has enabled characterization of the chromatin landscape of T cells in terms of histone occupancy, histone modification patterns and transcription factor association with specific genomic regulatory regions, generating a picture of the T-cell epigenome. Here, we discuss the multi-layered regulation of inducible gene expression in the immune system, focusing on the interplay between transcription factors, and the T-cell epigenome, including the role played by chromatin remodellers and epigenetic enzymes. We will also use IL2, a key inducible cytokine gene in T cells, as an example of how the different layers of epigenetic mechanisms regulate immune responsive genes during T-cell activation.

Machining water through laser cutting of nanoparticle-encased water pancakes.

Due to the inherent disorder and fluidity of water, precise machining of water through laser cutting are challenging. Herein we report a strategy that realizes the laser cutting machining of water through constructing hydrophobic silica nanoparticle-encased water pancakes with sub-millimeter depth. Through theoretical analysis, numerical simulation, and experimental studies, the developed process of nanoparticle-encased water pancake laser cutting and the parameters that affect cutting accuracy are verified and elucidated. We demonstrate that laser-fabricated water patterns can form diverse self-supporting chips (SSCs) with openness, transparency, breathability, liquid morphology, and liquid flow control properties. Applications of laser-fabricated SSCs to various fields, including chemical synthesis, biochemical sensing, liquid metal manipulation, patterned hydrogel synthesis, and drug screening, are also conceptually demonstrated. This work provides a strategy for precisely machining water using laser cutting, addressing existing laser machining challenges and holding significance for widespread fields involving fluid patterning and flow control in biological, chemical, materials and biomedical research.

Active maintenance of CD8+ T cell naïvety through regulation of global genome architecture.

The differentiation of naïve CD8+ cytotoxic T lymphocytes (CTLs) into effector and memory states results in large scale changes in transcriptional and phenotypic profiles. Little is known about how large-scale changes in genome organisation reflect or underpin these transcriptional programs. We utilised Hi-C to map changes in the spatial organisation of long-range genome contacts within naïve, effector and memory virus-specific CD8+ T cells. We observed that the architecture of the naive CD8+ T cell genome was distinct from effector and memory genome configurations with extensive changes within discrete functional chromatin domains. However, deletion of the BACH2 or SATB1 transcription factors was sufficient to remodel the naïve chromatin architecture and engage transcriptional programs characteristic of differentiated cells. This suggests that the chromatin architecture within naïve CD8+ T cells is preconfigured to undergo autonomous remodelling upon activation, with key transcription factors restraining differentiation by actively enforcing the unique naïve chromatin state.

Active maintenance of CD8+ T cell naivety through regulation of global genome architecture.

The differentiation of naive CD8+ T lymphocytes into cytotoxic effector and memory CTL results in large-scale changes in transcriptional and phenotypic profiles. Little is known about how large-scale changes in genome organization underpin these transcriptional programs. We use Hi-C to map changes in the spatial organization of long-range genome contacts within naive, effector, and memory virus-specific CD8+ T cells. We observe that the architecture of the naive CD8+ T cell genome is distinct from effector and memory genome configurations, with extensive changes within discrete functional chromatin domains associated with effector/memory differentiation. Deletion of BACH2, or to a lesser extent, reducing SATB1 DNA binding, within naive CD8+ T cells results in a chromatin architecture more reminiscent of effector/memory states. This suggests that key transcription factors within naive CD8+ T cells act to restrain T cell differentiation by actively enforcing a unique naive chromatin state.