IL-33 induces thymic involution-associated naive T cell aging and impairs host control of severe infection.

Severe infection commonly results in immunosuppression, which leads to impaired pathogen clearance or increased secondary infection in both humans and animals. However, the exact mechanisms remain poorly understood. Here, we demonstrate that IL-33 results in immunosuppression by inducing thymic involution-associated naive T cell dysfunction with aberrant expression of aging-associated genes and impairs host control of infection in mouse disease models of schistosomiasis or sepsis. Furthermore, we illustrate that IL-33 triggers the excessive generation of medullary thymic epithelial cell (mTEC) IV (thymic tuft cells) in a Pou2f3-dependent manner, as a consequence, disturbs mTEC/cortical TEC (cTEC) compartment and causes thymic involution during severe infection. More importantly, IL-33 deficiency, the anti-IL-33 neutralizing antibody treatment, or IL-33 receptor ST2 deficient thymus transplantation rescues T cell immunity to better control infection in mice. Our findings not only uncover a link between severe infection-induced IL-33 and thymic involution-mediated naive T cell aging, but also suggest that targeting IL-33 or ST2 is a promising strategy to rejuvenate T cell immunity to better control severe infection.

DMGV Is a Rheostat of T Cell Survival and a Potential Therapeutic for Inflammatory Diseases and Cancers.

Activated effector T cells (Teff) and/or compromised regulatory T cells (Treg) underlie many chronic inflammatory diseases. We discovered a novel pathway to regulate survival and expansion of Teff without compromising Treg survival and a potential therapeutic to treat these diseases. We found dimethylguanidino valeric acid (DMGV) as a rheostat for Teff survival: while cell-intrinsic DMGV generated by Alanine-Glyoxylate Aminotransferase 2 (AGXT2) is essential for survival and expansion by inducing mitochondrial ROS and regulation of glycolysis, an excessive (or exogenous) DMGV level inhibits activated Teff survival, thereby the AGXT2-DMGV-ROS axis functioning as a switch to turn on and off Teff expansion. DMGV-induced ROS is essential for glycolysis in Teff, and paradoxically DMGV induces ROS only when glycolysis is active. Mechanistically, DMGV rapidly activates mitochondrial calcium uniporter (MCU), causing a surge in mitochondrial Ca2+ without provoking calcium influx to the cytosol. The mitochondrial Ca2+ surge in turn triggers the mitochondrial Na+/Ca2+ exchanger (NCLX) and the subsequent mitochondrial Na+ import induces ROS by uncoupling the Coenzyme Q cycle in Complex III of the electron transport chain. In preclinical studies, DMGV administration significantly diminished the number of inflammatory T cells, effectively suppressing chronic inflammation in mouse models of colitis and rheumatoid arthritis. DMGV also suppressed expansion of cancer cells in vitro and in a mouse T cell leukemic model by the same mechanism. Our data provide a new pathway regulating T cell survival and a novel mode to treat autoimmune diseases and cancers.

Does Aging Activate T-cells to Reduce Bone Mass and Quality?

PURPOSE OF REVIEW: Aging leads to decline in bone mass and quality starting at age 30 in humans. All mammals undergo a basal age-dependent decline in bone mass. Osteoporosis is characterized by low bone mass and changes in bone microarchitecture that increases the risk of fracture. About a third of men over the age of 50 years are osteoporotic because they have higher than basal bone loss. In women, there is an additional acute decrement in bone mass, atop the basal rate, associated with loss of ovarian function (menopause) causing osteoporosis in about half of the women. Both genetics and environmental factors such as smoking, chronic infections, diet, microbiome, and metabolic disease can modulate basal age-dependent bone loss and eventual osteoporosis. Here, we review recent studies on the etiology of age-dependent decline in bone mass and propose a mechanism that integrates both genetic and environmental factors. RECENT FINDINGS: Recent findings support that aging and menopause dysregulate the immune system leading to sterile low-grade inflammation. Both animal models and human studies demonstrate that certain kinds of inflammation, in both men and women, mediate bone loss. Senolytics, meant to block a wide array of age-induced effects by preventing cellular senescence, have been shown to improve bone mass in aged mice. Based on a synthesis of the recent data, we propose that aging activates long-lived tissue resident memory T-cells to become senescent and proinflammatory, leading to bone loss. Targeting this population may represent a promising osteoporosis therapy. Emerging data indicates that there are several mechanisms that lead to sterile low-grade chronic inflammation, inflammaging, that cause age- and estrogen-loss dependent osteoporosis in men and women.

MCL1 alternative polyadenylation is essential for cell survival and mitochondria morphology.

Alternative polyadenylation in the 3' UTR (3' UTR-APA) is a mode of gene expression regulation, fundamental for mRNA stability, translation and localization. In the immune system, it was shown that upon T cell activation, there is an increase in the relative expression of mRNA isoforms with short 3' UTRs resulting from 3' UTR-APA. However, the functional significance of 3' UTR-APA remains largely unknown. Here, we studied the physiological function of 3' UTR-APA in the regulation of Myeloid Cell Leukemia 1 (MCL1), an anti-apoptotic member of the Bcl-2 family essential for T cell survival. We found that T cells produce two MCL1 mRNA isoforms (pA1 and pA2) by 3' UTR-APA. We show that upon T cell activation, there is an increase in both the shorter pA1 mRNA isoform and MCL1 protein levels. Moreover, the less efficiently translated pA2 isoform is downregulated by miR-17, which is also more expressed upon T cell activation. Therefore, by increasing the expression of the more efficiently translated pA1 mRNA isoform, which escapes regulation by miR-17, 3' UTR-APA fine tunes MCL1 protein levels, critical for activated T cells' survival. Furthermore, using CRISPR/Cas9-edited cells, we show that depletion of either pA1 or pA2 mRNA isoforms causes severe defects in mitochondria morphology, increases apoptosis and impacts cell proliferation. Collectively, our results show that MCL1 alternative polyadenylation has a key role in the regulation of MCL1 protein levels upon T cell activation and reveal an essential function for MCL1 3' UTR-APA in cell viability and mitochondria dynamics.

Requirement of Xk and Vps13a for the P2X7-mediated phospholipid scrambling and cell lysis in mouse T cells.

A high extracellular adenosine triphosphate (ATP) concentration rapidly and reversibly exposes phosphatidylserine (PtdSer) in T cells by binding to the P2X7 receptor, which ultimately leads to necrosis. Using mouse T cell transformants expressing P2X7, we herein performed CRISPR/Cas9 screening for the molecules responsible for P2X7-mediated PtdSer exposure. In addition to Eros, which is required for the localization of P2X7 to the plasma membrane, this screening identified Xk and Vps13a as essential components for this process. Xk is present at the plasma membrane, and its paralogue, Xkr8, functions as a phospholipid scramblase. Vps13a is a lipid transporter in the cytoplasm. Blue-native polyacrylamide gel electrophoresis indicated that Xk and Vps13a interacted at the membrane. A null mutation in Xk or Vps13a blocked P2X7-mediated PtdSer exposure, the internalization of phosphatidylcholine, and cytolysis. Xk and Vps13a formed a complex in mouse splenic T cells, and Xk was crucial for ATP-induced PtdSer exposure and cytolysis in CD25+CD4+ T cells. XK and VPS13A are responsible for McLeod syndrome and chorea-acanthocytosis, both characterized by a progressive movement disorder and cognitive and behavior changes. Our results suggest that the phospholipid scrambling activity mediated by XK and VPS13A is essential for maintaining homeostasis in the immune and nerve systems.

A rational approach to assess off-target reactivity of a dual-signal integrator for T cell therapy.

Cell therapy is an emerging therapeutic modality with the power to exploit new cancer targets and potentially achieve positive outcomes for patients with few other options. Like all synthetic treatments, cell therapy has the risk of toxicity via unpredicted off-target behavior. We describe an empirical method to model off-tumor, off-target reactivity of receptors used for investigational T cell therapies. This approach utilizes an optimal panel of diverse human cell-lines to capture the large majority of protein-coding gene expression in adult human tissues. We apply this cell-line set to test Jurkat and primary T cells engineered with a dual-signal integrator, called TmodTM, that contains an activating receptor (activator) and a separate inhibitory receptor (blocker). In proof-of-concept experiments, we use CD19 as the activating antigen and HLA-A*02 as the blocker antigen. This specific Tmod system, which employs a blocker targeting a ubiquitously expressed HLA class I antigen to inhibit CAR activation, has an inherent mechanism for selectivity/safety, designed to activate only when a specific HLA class I antigen is lost. Nonetheless, it is important to test off-target reactivity in functional assays, especially given the disconnect between ligand-binding and function among T cell receptors (TCRs) and chimeric antigen receptors (CARs). We show these cell-based assays yield consistent results with high sensitivity and specificity. The general strategy is likely applicable to more traditional single-receptor CAR- and TCR-T therapeutics.

Genetic variant within CDK6 regulates immune response to palbociclib treatment.

Everyone carries a set of genetic variants that contribute to regulation of the levels of blood cells, with unknown clinical impact. One of them, rs445 within the cell-cycle checkpoint gene CDK6, reduces the levels of myeloid cell types including granulocytes. We treated CD3+ T cells and whole blood with palbociclib in 41 individuals, who were stratified by genotype for analyses. In T cells we assessed cell cycle and apoptosis, whereas in whole blood, apoptosis in activated (CD11b+), unactivated (CD11b-) granulocytes, cytotoxic (CD8 + CD4-), and helper (CD8-CD4+) T cells. We find that rs445 modulates the immune response of CD8+ T cells. It also increases the level of apoptotic CD11b + activated granulocytes after palbociclib treatment, which, in synergy with neutropenia, may affect drug related adverse events. These results suggest that the effect of palbociclib treatment may depend on underlying genetically encoded individual immune response as well as the direct response to the drug.

Impaired antibacterial response of liver sinusoidal Vγ9+Vδ2+ T cells in patients with chronic liver disease.

OBJECTIVE: The liver acts as a frontline barrier against diverse gut-derived pathogens, and the sinusoid is the primary site of liver immune surveillance. However, little is known about liver sinusoidal immune cells in the context of chronic liver disease (CLD). Here, we investigated the antibacterial capacity of liver sinusoidal γδ T cells in patients with various CLDs. DESIGN: We analysed the frequency, phenotype and functions of human liver sinusoidal γδ T cells from healthy donors and recipients with CLD, including HBV-related CLD (liver cirrhosis (LC) and/or hepatocellular carcinoma (HCC)), alcoholic LC and LC or HCC of other aetiologies, by flow cytometry and RNA-sequencing using liver perfusates obtained during living donor liver transplantation. We also measured the plasma levels of D-lactate and bacterial endotoxin to evaluate bacterial translocation. RESULTS: The frequency of liver sinusoidal Vγ9+Vδ2+ T cells was reduced in patients with CLD. Immunophenotypic and transcriptomic analyses revealed that liver sinusoidal Vγ9+Vδ2+ T cells from patients with CLD were persistently activated and pro-apoptotic. In addition, liver sinusoidal Vγ9+Vδ2+ T cells from patients with CLD showed significantly decreased interferon (IFN)-γ production following stimulation with bacterial metabolites and Escherichia coli. The antibacterial IFN-γ response of liver sinusoidal Vγ9+Vδ2+ T cells significantly correlated with liver function, and inversely correlated with the plasma level of D-lactate in patients with CLD. Repetitive in vitro stimulation with E. coli induced activation, apoptosis and functional impairment of liver sinusoidal Vγ9+Vδ2+ T cells. CONCLUSION: Liver sinusoidal Vγ9+Vδ2+ T cells are functionally impaired in patients with CLD. Bacterial translocation and decreasing liver functions are associated with functional impairment of liver sinusoidal Vγ9+Vδ2+ T cells.

Synovial single-cell heterogeneity, zonation and interactions: a patchwork of effectors in arthritis.

Despite extensive research, there is still no treatment that would lead to remission in all patients with rheumatoid arthritis as our understanding of the affected site, the synovium, is still incomplete. Recently, single-cell technologies helped to decipher the cellular heterogeneity of the synovium; however, certain synovial cell populations, such as endothelial cells or peripheral neurons, remain to be profiled on a single-cell level. Furthermore, associations between certain cellular states and inflammation were found; whether these cells cause the inflammation remains to be answered. Similarly, cellular zonation and interactions between individual effectors in the synovium are yet to be fully determined. A deeper understanding of cell signalling and interactions in the synovium is crucial for a better design of therapeutics with the goal of complete remission in all patients.

Generation of functional human thymic cells from induced pluripotent stem cells.

BACKGROUND: The thymus is a glandular organ that is essential for the formation of the adaptive immune system by educating developing T cells. The thymus is most active during childhood and involutes around the time of adolescence, resulting in a severe reduction or absence of naive T-cell output. The ability to generate a patient-derived human thymus would provide an attractive research platform and enable the development of novel cell therapies. OBJECTIVES: This study sought to systematically evaluate signaling pathways to develop a refined direct differentiation protocol that generates patient-derived thymic epithelial progenitor cells from multiple induced pluripotent stem cells (iPSCs) that can further differentiate into functional patient-derived thymic epithelial cells on transplantation into athymic nude mice. METHODS: Directed differentiation of iPSC generated TEPs that were transplanted into nude mice. Between 14 and 19 weeks posttransplantation, grafts were removed and analyzed by flow cytometry, quantitative PCR, bulk RNA sequencing, and single-cell RNA sequencing for markers of thymic-cell and T-cell development. RESULTS: A direct differentiation protocol that allows the generation of patient-derived thymic epithelial progenitor cells from multiple iPSC lines is described. On transplantation into athymic nude mice, patient-derived thymic epithelial progenitor cells further differentiate into functional patient-derived thymic epithelial cells that can facilitate the development of T cells. Single-cell RNA sequencing analysis of iPSC-derived grafts shows characteristic thymic subpopulations and patient-derived thymic epithelial cell populations that are indistinguishable from TECs present in primary neonatal thymus tissue. CONCLUSIONS: These findings provide important insights and resources for researchers focusing on human thymus biology.

Infiltrating T lymphocytes and programmed cell death protein-1/programmed death-ligand 1 expression in endometriosis-associated ovarian cancer.

OBJECTIVE: To characterize T lymphocyte infiltration and programmed cell death protein-1 (PD-1)/programmed death-ligand 1 (PD-L1) expression in early-stage endometriosis-associated ovarian cancer (EAOC), ovarian endometriosis (OE), atypical endometriosis (AE), and deep endometriosis (DE). DESIGN: Case-control, retrospective study. SETTING: Research University Hospital. PATIENT(S): A total of 362 patients with a histologic diagnosis of EAOC, OE, AE, or DE were identified between 2000 and 2019 from Fondazione Policlinico Universitario Agostino Gemelli IRCCS and Gemelli Molise SpA tissue data banks. A 1:1 propensity score-matched method yielded matched pairs of 55 subjects with EAOC, 55 patients with OE, 12 patients with AE, and 42 patients with DE, resulting in no differences in family history of cancer, parity, and use of oral contraceptives. INTERVENTION(S): Immunohistochemistry assays using the following primary antibodies: CD3+; CD4+; CD8+; PD-1; and PD-L1. MAIN OUTCOME MEASURE(S): To characterize T lymphocyte infiltration and PD-1/PD-L1 expression in 4 different endometriosis-related diseases. RESULT(S): Endometriosis-associated ovarian cancer cases displayed significantly higher levels of PD-1/PD-L1 expression compared with all other endometriosis-related diseases (vs. OE vs. AE vs. DE). Moreover, a significantly lower count of infiltrating T lymphocytes was observed in EAOC cases compared with OE ones. Finally, one-third of OE cases showed a cancer-like PD-1/PD-L1 expression profile. CONCLUSION(S): Endometriosis-associated ovarian cancer is characterized by higher levels of PD-1/PD-L1 expression compared with benign endometriosis-related diseases. This profile was found in one-third of clinically benign cases, suggesting that it develops early in the carcinogenesis process.

Mesenchymal Stem Cells Enhance Chemotaxis of Activated T Cells through the CCL2-CCR2 Axis In Vitro.

Activation and migration of donor T cells to the host target organs are critical mechanisms in the pathogenesis of graft-versus-host disease (GVHD). The role of monocyte chemoattractant protein-1 (MCP-1/CCL2) and its receptor CCR2 in the recruitment of T cells during immune or inflammatory response is also well known. For elucidation of the mechanism of the therapeutic effect of human bone marrow derived-mesenchymal stem cells (MSC) in GVHD, we studied the effect of these cells on migration of activated donor T cells through the CCL2-CCR2 axis in vitro. MSC were expanded from donors' bone marrow mononuclear cells. After co-culturing of IL-2-activated T cells with allogeneic MSC at different ratios, the levels of CCL2 in supernatants were measured by ELISA, and CCR2 expression in CD4+/CD8+ T cells subsets were detected by flow cytometry. The effect of MSC on the migration of activated T cells in the Transwell system was studied in the absence or presence of CCL2. Our results show that CCL2 levels in supernatants of co-cultures were significantly higher than in MSC monoculture and this increase depended on the number of MSC. MSC inhibited proliferation of T cells, but did not change the percentages of CD4+ and CD8+ T cells subsets. MSC can up-regulate the CCR2 expression in CD8+ subsets rather than in CD4+ subsets; MSC enhanced migration of IL-2-activated T cells to CCL2 by increasing the expression of CCR2. The data demonstrate that MSC can enhance chemotaxis of cytokine-activated T cells through the CCL2-CCR2 axis in vitro.

Genetic re-direction of canine primary T cells for clinical trial use in pet dogs with spontaneous cancer.

Immunocompetent pet dogs develop spontaneous, human-like cancers, representing a parallel patient population for the investigation of chimeric antigen receptor (CAR) therapies. We have optimized a retrovirus-based protocol to efficiently CAR transduce primary T cells from healthy and tumor-bearing dogs. While transduction efficiencies and CAR-T expansion vary among dogs, CAR expression is typically higher and more stable compared with previous protocols, thus enabling human and comparative oncology researchers to use the dog as a pre-clinical model for human CAR-T cell research. For complete details on the use and execution of this protocol, please refer to Panjwani et al. (2020).

Easy or Not-The Advances of EZH2 in Regulating T Cell Development, Differentiation, and Activation in Antitumor Immunity.

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of polycomb repressive complex 2 (PRC2), which regulates downstream gene expression by trimethylation of lysine 27 in histone H3 (H3K27me3). EZH2 mutations or overexpressions are associated with many types of cancer. As inhibition of EZH2 activity could upregulate the expression of tumor suppressor genes, EZH2 has recently become an interesting therapeutic target for cancer therapy. Moreover, accumulating evidence has shown that EZH2 may contribute to the regulation of immune cells, especially T cells. EZH2 regulates T cell development, differentiation, and function, suggesting that EZH2 also regulates immune homeostasis in addition to tumor suppressor genes. Moreover, EZH2 can regulate T cell fate by targeting non-T cell factors such as metabolism, cytokines, and myeloid-derived suppressor cells. The role of EZH2 in this process has not been fully addressed. This review discusses up-to-date research on EZH2-mediated regulation of immunological function and the progress of immunological therapeutic strategies based on this regulation.

A regulatory network of microRNAs confers lineage commitment during early developmental trajectories of B and T lymphocytes.

The commitment of hematopoietic multipotent progenitors (MPPs) toward a particular lineage involves activation of cell type-specific genes and silencing of genes that promote alternate cell fates. Although the gene expression programs of early-B and early-T lymphocyte development are mutually exclusive, we show that these cell types exhibit significantly correlated microRNA (miRNA) profiles. However, their corresponding miRNA targetomes are distinct and predominated by transcripts associated with natural killer, dendritic cell, and myeloid lineages, suggesting that miRNAs function in a cell-autonomous manner. The combinatorial expression of miRNAs miR-186-5p, miR-128-3p, and miR-330-5p in MPPs significantly attenuates their myeloid differentiation potential due to repression of myeloid-associated transcripts. Depletion of these miRNAs caused a pronounced de-repression of myeloid lineage targets in differentiating early-B and early-T cells, resulting in a mixed-lineage gene expression pattern. De novo motif analysis combined with an assay of promoter activities indicates that B as well as T lineage determinants drive the expression of these miRNAs in lymphoid lineages. Collectively, we present a paradigm that miRNAs are conserved between developing B and T lymphocytes, yet they target distinct sets of promiscuously expressed lineage-inappropriate genes to suppress the alternate cell-fate options. Thus, our studies provide a comprehensive compendium of miRNAs with functional implications for B and T lymphocyte development.

The Host Response to Viral Infections Reveals Common and Virus-Specific Signatures in the Peripheral Blood.

Viruses cause a wide spectrum of clinical disease, the majority being acute respiratory infections (ARI). In most cases, ARI symptoms are similar for different viruses although severity can be variable. The objective of this study was to understand the shared and unique elements of the host transcriptional response to different viral pathogens. We identified 162 subjects in the US and Sri Lanka with infections due to influenza, enterovirus/rhinovirus, human metapneumovirus, dengue virus, cytomegalovirus, Epstein Barr Virus, or adenovirus. Our dataset allowed us to identify common pathways at the molecular level as well as virus-specific differences in the host immune response. Conserved elements of the host response to these viral infections highlighted the importance of interferon pathway activation. However, the magnitude of the responses varied between pathogens. We also identified virus-specific responses to influenza, enterovirus/rhinovirus, and dengue infections. Influenza-specific differentially expressed genes (DEG) revealed up-regulation of pathways related to viral defense and down-regulation of pathways related to T cell and neutrophil responses. Functional analysis of entero/rhinovirus-specific DEGs revealed up-regulation of pathways for neutrophil activation, negative regulation of immune response, and p38MAPK cascade and down-regulation of virus defenses and complement activation. Functional analysis of dengue-specific up-regulated DEGs showed enrichment of pathways for DNA replication and cell division whereas down-regulated DEGs were mainly associated with erythrocyte and myeloid cell homeostasis, reactive oxygen and peroxide metabolic processes. In conclusion, our study will contribute to a better understanding of molecular mechanisms to viral infections in humans and the identification of biomarkers to distinguish different types of viral infections.

T cell landscape and dynamics in immunoglobulin light chain amyloidosis before and after daratumumab-based therapy.

Amyloid light-chain (AL) is characterized by the presence of small, poorly proliferating plasma cell clones with the production and deposition of light chains into tissues. T cell changes within the tumour microenvironment in AL are poorly understood. By sequencing at a single-cell level of CD3+ T cells purified from bone marrow (BM) and blood of newly diagnosed AL patients before and after a combination of daratumumab with cyclophosphamide, bortezomib, and dexamethasone (Dara-BCD), we analysed the transcriptomic features of T cells and found an expansion, activation and type I cytokine upregulation in BM and circulating T cells after the treatment. More prominent changes were shown in CD8+ T cells. In particular, we found the presence of CD8+ BM resident memory T cells (TRM ) with high expression of inhibitory molecules in AL patients at diagnosis. After Dara-BCD, these TRM cells were quickly activated with downregulation of suppressive molecules and upregulation of IFNG expression. These data collectively demonstrate that Dara-based therapy in patients with AL amyloidosis promotes anti-tumour T cell responses. The similar transcriptomic features of BM and circulating T cells before and after therapy further provide a less invasive approach for molecular monitoring of T cell response in AL amyloidosis.

The histone demethylase Lsd1 regulates multiple repressive gene programs during T cell development.

Analysis of the transcriptional profiles of developing thymocytes has shown that T lineage commitment is associated with loss of stem cell and early progenitor gene signatures and the acquisition of T cell gene signatures. Less well understood are the epigenetic alterations that accompany or enable these transcriptional changes. Here, we show that the histone demethylase Lsd1 (Kdm1a) performs a key role in extinguishing stem/progenitor transcriptional programs in addition to key repressive gene programs during thymocyte maturation. Deletion of Lsd1 caused a block in late T cell development and resulted in overexpression of interferon response genes as well as genes regulated by the Gfi1, Bcl6, and, most prominently, Bcl11b transcriptional repressors in CD4+CD8+ thymocytes. Transcriptional overexpression in Lsd1-deficient thymocytes was not always associated with increased H3K4 trimethylation at gene promoters, indicating that Lsd1 indirectly affects the expression of many genes. Together, these results identify a critical function for Lsd1 in the epigenetic regulation of multiple repressive gene signatures during T cell development.