Human Th17 Research Articles

Response Gene to Complement-32 Expression Is Upregulated in Lupus T cells and Promotes IL-17A Expression

Abstract RGC (Response Gene to Complement)-32 is a cell cycle regulator expressed in normal tissues, tumors and a variety of cell lines. RGC-32 is preferentially upregulated in murine Th17 cells and promotes their differentiation. Patients with Systemic Lupus Erythematosus (SLE) display increased serum levels, expanded frequency of IL-17 producing cells. Whether RGC-32 plays a role in human Th17 differentiation pathway and in Th17 abnormalities in lupus patients has not yet been investigated. RGC-32 mRNA expression was determined in CD4+ T cells stimulated under Th0, Th1, Th2, Th17 and Treg conditions and the effect of RGC-32 overexpression and silencing under Th17 conditions was determined by nucleofection. RGC-32 expression was evaluated by RT-PCR, flow cytometry and cDNA Array in PBMC from lupus patients vs controls. RGC-32 mRNA expression was upregulated by TCR stimulation and TGFβ, and was more robust under Th17 (3.2 ± fold) and Treg (2.6 ± 0.8 fold) vs. Th1 (1.3 ± 0.4 fold) and Th2 (1.8 ± 0.1 fold) conditions. Overexpression or silencing of RGC-32 in CD4+ T cells upregulated, respectively downregulated IL-17A transcript levels and protein secretion. RGC-32 mRNA and protein level was significantly increased in CD19+ B cells and CD3+ T from lupus patients compared to controls. These results suggest that RGC-32 promotes the differentiation of human Th17 cells. Furthermore, T cells from patients with SLE exhibit increased expression of RGC-32 compared to controls. These data support the idea that RGC-32 signaling may enhance disease expression in SLE by promoting abnormalities in the Th17 pathway and provide a compelling rationale for further investigating the therapeutic potential of blocking RGC-32 in SLE.

CCR2 mediates transendothelial migration of human pathogenic Th17 cells

Abstract Leukocyte extravasation is a multistep process characterized by rolling and arrest on the endothelium followed by transendothelial migration (TEM). Arrest and TEM by T cells typically requires the activities of chemokines and their receptors. Previously, we showed that CCR2 identifies highly differentiated, readily activated CD4+ memory T cells in human blood with diverse effector capabilities, and we showed that CCR6 is the signature chemokine receptor for Th17 cells. Here we describe studies of the migratory capabilities and effector profiles of CD4+ CCR6+ CCR2+ as compared with CD4+ CCR6+ CCR2− and CD4+ CCR6− CCR2− T cells from human blood. Using flow chamber assays with TNFa-activated human umbilical vein endothelial cells, we found that among the CCR6+ cells, only the CCR2+ subset was able to cross the endothelial monolayer efficiently. Using antibodies against the CCR6 ligand, CCL20, and a specific inhibitor of CCR2, we found that CCR6 was important for arrest and that CCR2 was critical for subsequent TEM. Using intracellular staining of cells activated ex vivo, we found that although IL-17-expressing cells were found within all CCR6+ subsets, the CCR6+CCR2+ cells were distinguished by their production of IFNg, GM-CSF, and TNFa, thereby demonstrating the cytokine profile of pathogenic Th17 cells as identified in mouse models of autoimmune disease. Taken together, our studies suggest that chemokine receptors on memory Th cells play non-redundant roles in the egress from blood, that providing T cells with CCR6 and CCR2 could enhance their trafficking into tissue, and that, on the other hand, blocking CCR2 alone could prevent the extravasation of pathogenic Th17 cells and ameliorate tissue damage in Th17-cell driven disease.

Identification of a conserved multi-lineage Il9 enhancer

Abstract The immune system provides resistance to the myriad of pathogens in the environment, but can also respond inappropriately causing allergic inflammation and autoimmune disease. CD4+ T cells, which play a crucial role in adaptive immune system, can be divided into subsets based on their effector functions. T helper 9 (Th9) cells, derived by the IL-4/STAT6 and TGFβ signaling pathways, produce IL-9 as a hallmark cytokine. Through IL-9 production, Th9 cells protect against parasite infection but are also involved in allergic inflammation and autoimmune diseases. In this study, we demonstrate that a conserved noncoding sequence (CNS) located 25kb upstream of the Il9 transcription start site, termed Il9 CNS-25, is critical for regulating Il9 expression in Th cell subsets and IL-9 producing CD8+ T cells (Tc9). Th9 cells derived from Il9 CNS-25 mutant (Il9ΔCNS-25) mice produce significantly less IL-9. Il9 CNS-25 was bound by factors that promote Il9 gene expression including STATs, IRF4 and BATF, promoted chromatin modifications and transcription factor binding at the promoter, and mediated accessibility of the locus. Il9ΔCNS-25 mice showed attenuated airway inflammation compared to control mice. The Il9 CNS-25 region in mice is conserved with an IL9 CNS-18 region in the human genome. We deleted CNS-18 in primary human Th9 cells and observed diminished IL-9 production. Thus, we have demonstrated that the Il9 CNS-25/IL9 CNS-18 elements are respectively critical for Il9/IL9 gene expression in Th9 cells and other IL-9 producing T cell subsets.

CD28 suppresses IL-23- and IL-1β-driven human Th17 development

Abstract Th17 cells drive autoimmune disease but also control commensal microbes. A common link between antigens from self-proteins or commensal microbiota is relatively low activation of T cell receptor and costimulation signaling. Indeed, strong TCR/CD28 stimulation suppressed Th17 cell differentiation from human naïve T cells, but not effector/memory cells. CD28 suppressed the classical Th17 transcriptional program, while inducing known Th17-regulators, and acted through an Akt-dependent mechanism. Th17 cells differentiated without CD28 were not anergic: they showed robust proliferation and maintained Th17 cytokine production following restimulation. IL-23 and IL-1β promoted glucose uptake and increased glycolysis. Although modestly increased compared to CD28 costimulation, glycolysis was necessary to support Th17 differentiation, indicating that cytokine-mediated metabolic shifts were sufficient to obviate the classical requirement for CD28 in Th17 differentiation. Together these data propose that in humans, strength of TCR/CD28/Akt activation serves as a rheostat tuning the magnitude of Th17 development driven by IL-23 and IL-1β.

Arginine methylation drives inflammatory T cell responses and autoimmunity

Abstract Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system that is the leading cause of disability in 2 million young adults aged 20–40 worldwide. Heightened pro-inflammatory T helper (Th) 1 and Th17 cell responses and deficient Th2 and regulatory T (Treg) cell responses drive myelin sheath damage and disability in MS. Thus, therapeutic strategies that modulate inflammatory T cell responses would be beneficial to the field. Epigenetic modifications are well known to provide control over cellular phenotype, and pan-methyltransferase inhibitors have been shown to suppress Experimental Autoimmune Encephalomyelitis (EAE), the mouse model of MS. However, the methylation enzyme responsible for this therapeutic effect remains unknown. Protein arginine methyltransferase 5 (PRMT5) is the only enzyme known to catalyze symmetric dimethylation (SDM) on arginine residues of histones, mediating transcriptional repression. Although PRMT5 is a known cancer driver, until recently, there was no known role for PRMT5 in T cells. We found that PRMT5 protein was up-regulated during re-activation of memory human Th1 cells, and that PRMT5-selective inhibitors suppressed inflammatory T cell proliferation. Further, we developed a conditional PRMT5 knockout mouse model in which PRMT5 knockout can be induced in CD4+ T cells. With this model, we genetically validated our findings that PRMT5 drives CD4+ T cell proliferation. Importantly, we translated our findings in vivo by showing that PRMT5 inhibitors suppressed established EAE disease severity. Overall, these data support a role for PRMT5 in pathogenic memory T cell responses and suggest its promise as a potential therapeutic target for autoimmune diseases such as MS.

Inhibition of Inositol-Requiring Enzyme 1 α in Established TH2 Cells Decreases the Secretion of Cytokines IL-5 and IL-13, but Not IL-4

Abstract TH2 cells are crucial in immune responses against extracellular pathogens; however, they can play a predominant role in a number of diseases due to inappropriate IL-4, IL-5, and IL-13 cytokine expression. Therefore, understanding how these cells develop and regulate their effector functions is of great importance. In response to an excess of unfolded proteins, serine-threonine kinase Inositol-Requiring Enzyme 1 α or IRE1α activates the unfolded protein response, UPR, which promotes increased protein folding capacity. T cell activation induces ER stress, and inhibition of IRE1α RNase activity in activated primary cells, via treatment of the cells with the commercially available drug 4μ8c, results in the reduction of IL-5, IL-4, and IL-13. Prior to this work, it was unknown if 4μ8c could inhibit TH2 cytokines in established TH2 cells. Treatment of a mouse TH2 cell line and differentiated human TH2 cells with 4μ8c results in inhibition of IL-5 and IL-13, but not IL-4 as measured by ELISA. The reduced cytokine expression is not due to differences in mRNA stability or mRNA levels; it appears to be due to a defect in secretion, as the cells appear to produce cytokines IL-5 and IL-13 as measured by flow cytometry. This suggests that the inhibition of these cytokines is due to post-translational processes. IL-5 and IL-13 produced by TH2 cells are critical for promoting chronic, inflammatory asthma, and 4μ8c blocks their expression in T cells in vitro. Future studies will determine if 4μ8c treatment can ameliorate the effects of these cytokines in a disease model. This work is supported by the NSU Faculty Research Council grant and OK-INBRE NIH P20GM103447.

066 Glucocorticoids promote intrinsic human Th17 differentiation

Although Th17 cells are critical to host protection at epithelial barriers, they can play a pathogenic role in inflammatory and autoimmune diseases. Glucocorticoids (GC) are therapeutically used to suppress undesired inflammation, but the common persistence or even increase of Th17 responses in GC-treated patients led us to investigate the effect of GC on Th17 development. We found that GC trigger a Th17 differentiation program in naïve human CD4 T cells in the absence of antigen-presenting cells or exogenous cytokines. GC-induced Th17 cells expressed IL-10 and their development was associated with an inhibition of Th2, and particularly Th1 differentiation. GC induction of Th17 differentiation was not linked to increased IL-1β, IL-6 and TGF-β, yet GC downregulated IL-2. Consistently, the addition of exogenous IL-2 prevented the GC-induced increase in Th17/Th1 ratios. Also, IL-2/IL-2R blocking enhanced Th17/Th1 ratios, altogether suggesting a mechanistic role for IL-2 regulation in GC-induced Th17 polarization. Functionally, supernatants of GC-differentiated T cells were superior to those of non-GC-differentiated T cells at inducing antimicrobial peptides and pro-IL-1β in keratinocytes, despite decreased IFN-γ and increased IL-10. Finally, GC also favored Th17 over Th1 among memory T cells from blood and skin. Altogether, our data define GC as human Th17 polarizing factors.

1031 Regulation of IL-13 production by human Th2 cell and Tc2 T cells fromperipheral blood and atopic lesional skin by S.aureus and their cell-wall products

1031 Regulation of IL-13 production by human Th2 cell and Tc2 T cells fromperipheral blood and atopic lesional skin by S.aureus and their cell-wall products

006 TGF-β- and IL-4-induced PPAR-γ promotes IL-9 expression in human TH2 cells

006 TGF-β- and IL-4-induced PPAR-γ promotes IL-9 expression in human TH2 cells

T helper 17.1 cells associate with multiple sclerosis disease activity: perspectives for early intervention.

Interleukin-17-expressing CD4+ T helper 17 (Th17) cells are considered as critical regulators of multiple sclerosis disease activity. However, depending on the species and pro-inflammatory milieu, Th17 cells are functionally heterogeneous, consisting of subpopulations that differentially produce interleukin-17, interferon-gamma and granulocyte macrophage colony-stimulating factor. In the current study, we studied distinct effector phenotypes of human Th17 cells and their correlation with disease activity in multiple sclerosis patients. T helper memory populations single- and double-positive for C-C chemokine receptor 6 (CCR6) and CXC chemokine receptor 3 (CXCR3) were functionally assessed in blood and/or cerebrospinal fluid from a total of 59 patients with clinically isolated syndrome, 35 untreated patients and 24 natalizumab-treated patients with relapsing-remitting multiple sclerosis, and nine patients with end-stage multiple sclerosis. Within the clinically isolated syndrome group, 23 patients had a second attack within 1 year and 26 patients did not experience subsequent attacks during a follow-up of >5 years. Low frequencies of T helper 1 (Th1)-like Th17 (CCR6+CXCR3+), and not Th17 (CCR6+CXCR3-) effector memory populations in blood strongly associated with a rapid diagnosis of clinically definite multiple sclerosis. In cerebrospinal fluid of clinically isolated syndrome and relapsing-remitting multiple sclerosis patients, Th1-like Th17 effector memory cells were abundant and showed increased production of interferon-gamma and granulocyte macrophage colony-stimulating factor compared to paired CCR6+ and CCR6-CD8+ T cell populations and their blood equivalents after short-term culturing. Their local enrichment was confirmed ex vivo using cerebrospinal fluid and brain single-cell suspensions. Across all pro-inflammatory T helper cells analysed in relapsing-remitting multiple sclerosis blood, Th1-like Th17 subpopulation T helper 17.1 (Th17.1; CCR6+CXCR3+CCR4-) expressed the highest very late antigen-4 levels and selectively accumulated in natalizumab-treated patients who remained free of clinical relapses. This was not found in patients who experienced relapses during natalizumab treatment. The enhanced potential of Th17.1 cells to infiltrate the central nervous system was supported by their predominance in cerebrospinal fluid of early multiple sclerosis patients and their preferential transmigration across human brain endothelial layers. These findings reveal a dominant contribution of Th1-like Th17 subpopulations, in particular Th17.1 cells, to clinical disease activity and provide a strong rationale for more specific and earlier use of T cell-targeted therapy in multiple sclerosis.

IL-23 and IL-1β Drive Human Th17 Cell Differentiation and Metabolic Reprogramming in Absence of CD28 Costimulation

Th17 cells drive autoimmune disease but also control commensal microbes. A common link among antigens from self-proteins or commensal microbiota is relatively low activation of Tcell receptor (TCR) and costimulation signaling. Indeed, strong TCR/CD28 stimulation suppressed Th17 cell differentiation from human naive Tcells, but not effector/memory cells. CD28 suppressed the classical Th17 transcriptional program, while inducing known Th17 regulators, and acted through an Akt-dependent mechanism. Th17 cells differentiated without CD28 were not anergic: they showed robust proliferation and maintained Th17 cytokine production following restimulation. Interleukin (IL)-23 and IL-1β promoted glucose uptake and increased glycolysis. Although modestly increased compared to CD28 costimulation, glycolysis was necessary to support Th17 differentiation, indicating that cytokine-mediated metabolic shifts were sufficient to obviate the classical requirement for CD28 in Th17 differentiation. Together, these data propose that, in humans, strength of TCR/CD28/Akt activation serves as a rheostat tuning the magnitude of Th17 development driven by IL-23 and IL-1β.

What We're Reading

Getting stabilized (from cegoh via Pixabay)A link has been found between oncogenic RAS signaling and increased immuno-suppressive expression of PD-L1. The mechanism relies on stabilization of the PD-L1 transcript, with RAS signaling culminating in phosphorylation and inactivation of TTP, a factor involved in degrading PD-L1 transcripts. As TTP inactivation causes accumulation of PD-L1 mRNA, interfering with the RAS pathway increases TTP binding to AU-rich elements of the transcripts, decreases PD-L1 protein production, and provides a means to enhance antitumor immunity.Coelho MA, …, Downward J. Immunity 2017 Dec 19;47:1083–99.e6.Resolvin D2 (from Roadnottaken via Wikimedia Commons)The goal of radio-, chemo-, and targeted therapy of tumors is to destroy tumor cells. However, the debris left from cell destruction can promote tumor growth through induction of local inflammation and cytokine release. Resolvins, as their name implies, quietly clean up and tune down inflammation. Addition of RvD1, RvD2, or RvE1 successfully cleared debris in a process dependent on phagocytosis by macrophages and reduction of inflammatory cytokine production.Sulciner ML, …, Panigrahy D. J Exp Med 2018 Jan 2;215:115–140.Shielded from removal (from LavaSnake via Wikimedia Commons)The proportion of patients that respond to PD-1 blockade remains low. PD-1 inhibits through recruitment of SHP2 and subsequent dephosphorylation of tyrosines involved in propagating TCR signals. SAP was found bound to intracellular PD-1 complexes and shielded phosphates from dephosphorylation by SHP2. Increased expression of SAP correlated with decreased inhibitory signaling through PD-1. Thus, therapeutically targeting SHP2 or SAP to increase T-cell responses could provide an approach to enhance responses to PD-1 blockade.Peled M, …, Mor, A. Proc Natl Acad Sci USA 2018 Jan 16;115:E468–77.Targeted IFN delivery plus TNF (from Cauwels et al.)Interferons can support antitumor activity, but have pleiotropic effects. Injection of exogenous IFN is associated with adverse side effects. Removing IFN's ability to bind to its widely expressed natural receptor, and providing it with an alternative segment that binds to Clec9, a DC-specific protein, allowed targeting of minute quantities of IFN to tumoral DCs without toxicity, and, with other therapies, completely eradicated tumors considered to be poorly immunogenic.Cauwels A, …, Tavernier J. Cancer Res 2018 Jan 15;78:463–74.Neutrophil enablers (from BruceBlaus via Wikimedia Commons)A hallmark of progression in many solid tumors is a preponderance of myeloid-derived cells and an increase in circulating myeloid-lineage cells. Nonmetastatic lung adenocarcinomas were found to disrupt bone marrow homeostasis in humans and mice. Osteocalcin+ osteoblasts increase in the bone marrow, and are responsible for the expansion of tumor-promoting SiglecFhigh neutrophils that accumulate in the tumor. The results highlight that cancer is a systemic disease, with cross-talk between lung tumors and bone enhancing tumor progression.Engblom C, …, Pittet MJ. Science 2017 Dec 1;358:eaal5081.Gastric tumor (from MF Press et al, Mol Cancer Therapeutics 2017)Although CTLA-4 acts as an inhibitor of antitumor responses, paradoxically, people with genetically-reduced expression of CTLA-4 have a greater risk for gastric cancer (GC). Insufficiency of CTLA-4 expression in mice instigates an inflammatory state that becomes premalignant and progresses to GC. The progression is not caused by dysregulation of microbiota, and can be transferred with spleen cells to Rag1-deficient mice. Th2 cytokines IL4 and IL13 were essential for tumorigenesis, inducing epigenetic disruptions in human GC, and Th1 or Th17 cells were not involved.Miska J, …, Chen Z. J Exp Med 2018 Jan 26. DOI: 10.1084/jem.20171971.

The transcription factors Runx3 and ThPOK cross-regulate acquisition of cytotoxic function by human Th1 lymphocytes.

Cytotoxic CD4 (CD4CTX) T cells are emerging as an important component of antiviral and antitumor immunity, but the molecular basis of their development remains poorly understood. In the context of human cytomegalovirus infection, a significant proportion of CD4 T cells displays cytotoxic functions. We observed that the transcriptional program of these cells was enriched in CD8 T cell lineage genes despite the absence of ThPOK downregulation. We further show that establishment of CD4CTX-specific transcriptional and epigenetic programs occurred in a stepwise fashion along the Th1-differentiation pathway. In vitro, prolonged activation of naive CD4 T cells in presence of Th1 polarizing cytokines led to the acquisition of perforin-dependent cytotoxic activity. This process was dependent on the Th1 transcription factor Runx3 and was limited by the sustained expression of ThPOK. This work elucidates the molecular program of human CD4CTX T cells and identifies potential targets for immunotherapy against viral infections and cancer.

PKCεpromotes human Th17 differentiation: Implications in the pathophysiology of psoriasis.

PKCε is implicated in Tcell activation and proliferation and is overexpressed in CD4+ -Tcells from patients with autoimmune Hashimoto's thyroiditis. Although this might induce the suspicion that PKCε takes part in autoimmunity, its role in the molecular pathophysiology of immune-mediated disorders is still largely unknown. We studied PKCε expression in circulating CD4+ -Tcells from patients with psoriasis, a skin disorder characterized by an increased amount of Th17 cells, a CD4+ subset that is critical in the development of autoimmunity. Although the mechanisms that underlie Th17 differentiation in humans are still unclear, we here show that: (i) PKCε is overexpressed in CD4+ -Tcells from psoriatic patients, and its expression positively correlates with the severity of the disease, being reduced by effective phototherapy; (ii) PKCε interacts with Stat3 during Th17 differentiation and its overexpression results in an enhanced expression of Stat3 and pStat3(Ser727); iii) conversely, when PKCε is forcibly downregulated, CD4+ -Tcells show lower levels of pStat3(Ser727) expression and defective in vitro expansion into the Th17-lineage. These data provide a novel insight into the molecular mechanisms of Th17 cell polarization that is known to play a crucial role in autoimmunity, pinpointing PKCε as a potential target in Th17-mediated diseases.

Human TH17 cell development requires processing of dendritic cell–derived CXCL8 by neutrophil elastase

Human TH17 cell development requires processing of dendritic cell–derived CXCL8 by neutrophil elastase

CXCL4 is a novel inducer of human Th17 cells and correlates with IL-17 and IL-22 in psoriatic arthritis.

CXCL4 regulates multiple facets of the immune response and is highly upregulated in various Th17‐associated rheumatic diseases. However, whether CXCL4 plays a direct role in the induction of IL‐17 production by human CD4+ T cells is currently unclear. Here, we demonstrated that CXCL4 induced human CD4+ T cells to secrete IL‐17 that co‐expressed IFN‐γ and IL‐22, and differentiated naïve CD4+ T cells to become Th17‐cytokine producing cells. In a co‐culture system of human CD4+ T cells with monocytes or myeloid dendritic cells, CXCL4 induced IL‐17 production upon triggering by superantigen. Moreover, when monocyte‐derived dendritic cells were differentiated in the presence of CXCL4, they orchestrated increased levels of IL‐17, IFN‐γ, and proliferation by CD4+ T cells. Furthermore, the CXCL4 levels in synovial fluid from psoriatic arthritis patients strongly correlated with IL‐17 and IL‐22 levels. A similar response to CXCL4 of enhanced IL‐17 production by CD4+ T cells was also observed in patients with psoriatic arthritis. Altogether, we demonstrate that CXCL4 boosts pro‐inflammatory cytokine production especially IL‐17 by human CD4+ T cells, either by acting directly or indirectly via myeloid antigen presenting cells, implicating a role for CXCL4 in PsA pathology.

Identity and Diversity of Human Peripheral Th and T Regulatory Cells Defined by Single-Cell Mass Cytometry.

Human CD3+CD4+ Th cells, FOXP3+ T regulatory (Treg) cells, and T regulatory type 1 (Tr1) cells are essential for ensuring peripheral immune response and tolerance, but the diversity of Th, Treg, and Tr1 cell subsets has not been fully characterized. Independent functional characterization of human Th1, Th2, Th17, T follicular helper (Tfh), Treg, and Tr1 cells has helped to define unique surface molecules, transcription factors, and signaling profiles for each subset. However, the adequacy of these markers to recapitulate the whole CD3+CD4+ T cell compartment remains questionable. In this study, we examined CD3+CD4+ T cell populations by single-cell mass cytometry. We characterize the CD3+CD4+ Th, Treg, and Tr1 cell populations simultaneously across 23 memory T cell-associated surface and intracellular molecules. High-dimensional analysis identified several new subsets, in addition to the already defined CD3+CD4+ Th, Treg, and Tr1 cell populations, for a total of 11 Th cell, 4 Treg, and 1 Tr1 cell subsets. Some of these subsets share markers previously thought to be selective for Treg, Th1, Th2, Th17, and Tfh cells, including CD194 (CCR4)+FOXP3+ Treg and CD183 (CXCR3)+T-bet+ Th17 cell subsets. Unsupervised clustering displayed a phenotypic organization of CD3+CD4+ T cells that confirmed their diversity but showed interrelation between the different subsets, including similarity between Th1-Th2-Tfh cell populations and Th17 cells, as well as similarity of Th2 cells with Treg cells. In conclusion, the use of single-cell mass cytometry provides a systems-level characterization of CD3+CD4+ T cells in healthy human blood, which represents an important baseline reference to investigate abnormalities of different subsets in immune-mediated pathologies.

R-loops cause genomic instability in T helper lymphocytes from patients with Wiskott-Aldrich syndrome

Wiskott-Aldrich syndrome (WAS), X-linked thrombocytopenia (XLT), and X-linked neutropenia, which are caused by WAS mutations affecting Wiskott-Aldrich syndrome protein (WASp) expression or activity, manifest in immunodeficiency, autoimmunity, genomic instability, and lymphoid and other cancers. WASp supports filamentous actin formation in the cytoplasm and gene transcription in the nucleus. Although the genetic basis for XLT/WAS has been clarified, the relationships between mutant forms of WASp and the diverse features of these disorders remain ill-defined. We sought to define how dysfunctional gene transcription is causally linked to the degree of TH cell deficiency and genomic instability in the XLT/WAS clinical spectrum. In human TH1- or TH2-skewing cell culture systems, cotranscriptional R-loops (RNA/DNA duplex and displaced single-stranded DNA) and DNA double-strand breaks (DSBs) were monitored in multiple samples from patients with XLT and WAS and in normal T cells depleted of WASp. WASp deficiency provokes increased R-loops and R-loop-mediated DSBs in TH1 cells relative to TH2 cells. Mechanistically, chromatin occupancy of serine 2-unphosphorylated RNA polymerase II is increased, and that of topoisomerase 1, an R-loop preventing factor, is decreased at R-loop-enriched regions of IFNG and TBX21 (TH1 genes) in TH1 cells. These aberrations accompany increased unspliced (intron-retained) and decreased spliced mRNA of IFNG and TBX21 but not IL13 (TH2 gene). Significantly, increased cellular load of R-loops and DSBs, which are normalized on RNaseH1-mediated suppression of ectopic R-loops, inversely correlates with disease severity scores. Transcriptional R-loop imbalance is a novel molecular defect causative in TH1 immunodeficiency and genomic instability in patients with WAS. The study proposes that cellular R-loop load could be used as a potential biomarker for monitoring symptom severity and prognostic outcome in the XLT-WAS clinical spectrum and could be targeted therapeutically.

Effector and Regulatory T Cells Roll at High Shear Stress by Inducible Tether and Sling Formation

The adaptive immune response involves Tcell differentiation and migration to sites of inflammation. Tcell trafficking is initiated by rolling on inflamed endothelium. Tethers and slings, discovered in neutrophils, facilitate cell rolling at high shear stress. Here, we demonstrate that the ability to form tethers and slings during rolling is highly inducible in T helper 1 (Th1), Th17, and regulatory T (Treg) cells but less in Th2 cells. Invivo, endogenous Treg cells rolled stably in cremaster venules at physiological shear stress. Quantitative dynamic footprinting nanoscopy of Th1, Th17, and Treg cells uncovered the formation of multiple tethers per cell. Human Th1 cells also showed tethers and slings. RNA sequencing (RNA-seq) revealed the induction of cell migration and cytoskeletal genes in sling-forming cells. We conclude that differentiated CD4 Tcells stabilize rolling by inducible tether and sling formation. These phenotypic changes approximate the adhesion phenotype of neutrophils and support CD4 Tcell access to sites of inflammation.

Transcriptional signature of human pro-inflammatory TH17 cells identifies reduced IL10 gene expression in multiple sclerosis

We have previously reported the molecular signature of murine pathogenic TH17 cells that induce experimental autoimmune encephalomyelitis (EAE) in animals. Here we show that human peripheral blood IFN-γ+IL-17+ (TH1/17) and IFN-γ−IL-17+ (TH17) CD4+ T cells display distinct transcriptional profiles in high-throughput transcription analyses. Compared to TH17 cells, TH1/17 cells have gene signatures with marked similarity to mouse pathogenic TH17 cells. Assessing 15 representative signature genes in patients with multiple sclerosis, we find that TH1/17 cells have elevated expression of CXCR3 and reduced expression of IFNG, CCL3, CLL4, GZMB, and IL10 compared to healthy controls. Moreover, higher expression of IL10 in TH17 cells is found in clinically stable vs. active patients. Our results define the molecular signature of human pro-inflammatory TH17 cells, which can be used to both identify pathogenic TH17 cells and to measure the effect of treatment on TH17 cells in human autoimmune diseases.