Naive CD4 Research Articles

CBAF complex components and MYC cooperate early in CD8+ T cell fate.

The identification of mechanisms to promote memory T (Tmem) cells has important implications for vaccination and anti-cancer immunotherapy1-4. Using a CRISPR-based screen for negative regulators of Tmem cell generation in vivo5, here we identify multiple components of the mammalian canonical BRG1/BRM-associated factor (cBAF)6,7. Several components of the cBAF complex are essential for the differentiation of activated CD8+ T cells into T effector (Teff) cells, and their loss promotes Tmem cell formation in vivo. During the first division of activated CD8+ T cells, cBAF and MYC8 frequently co-assort asymmetrically to the two daughter cells. Daughter cells with high MYC and high cBAF display a cell fate trajectory towards Teff cells, whereas those with low MYC and low cBAF preferentially differentiate towards Tmem cells. The cBAF complex and MYC physically interact to establish the chromatin landscape in activated CD8+ T cells. Treatment of naive CD8+ T cells with a putative cBAF inhibitor during the first 48 h of activation, before the generation of chimeric antigen receptor T (CAR-T) cells, markedly improves efficacy in a mousesolid tumour model. Our results establish cBAF as a negative determinant of Tmem cell fate and suggest that manipulation of cBAF early in T cell differentiation can improve cancer immunotherapy.

Impact of secondary TCR engagement on the heterogeneity of pathogen-specific CD8+ T cell response during acute and chronic toxoplasmosis.

Initial TCR engagement (priming) of naive CD8+ T cells results in T cell expansion, and these early events influence the generation of diverse effector and memory populations. During infection, activated T cells can re-encounter cognate antigen, but how these events influence local effector responses or formation of memory populations is unclear. To address this issue, OT-I T cells which express the Nur77-GFP reporter of TCR activation were paired with the parasite Toxoplasma gondii that expresses OVA to assess how secondary encounter with antigen influences CD8+ T cell responses. During acute infection, TCR stimulation in affected tissues correlated with parasite burden and was associated with markers of effector cells while Nur77-GFP- OT-I showed signs of effector memory potential. However, both Nur77-GFP- and Nur77-GFP+ OT-I from acutely infected mice formed similar memory populations when transferred into naive mice. During the chronic stage of infection in the CNS, TCR activation was associated with large scale transcriptional changes and the acquisition of an effector T cell phenotype as well as the generation of a population of CD103+ CD69+ Trm like cells. While inhibition of parasite replication resulted in reduced effector responses it did not alter the Trm population. These data sets highlight that recent TCR activation contributes to the phenotypic heterogeneity of the CD8+ T cell response but suggest that this process has a limited impact on memory populations at acute and chronic stages of infection.

Role of interferons (IFNs) in the differentiation of T peripheral helper (Tph) cells.

T follicular helper (Tfh) cells and T peripheral helper (Tph) cells produce interleukin (IL)-21 and are thought to contribute to follicular and extra-follicular B-cell activation, respectively, in autoimmune diseases. It is known that programmed cell death-1 (PD-1)-positive CXCR5+ Tfh-like cells are differentiated from human naive CD4+ T cells by IL-12 plus transforming growth factor (TGF)-β. However, it remains unclear what cytokines are required for Tph differentiation. In this study, we found that interferon (IFN)-α and IFN-β reduce the frequency of Tfh-like cells under the IL-12 plus TGF-β condition, whereas they promote generation of PD-1+CXCR5-CD4+ T cells and secretion of IL-21, IFN-γ and CXCL13. Intracellular cytokine staining and T-cell-B-cell co-culture studies indicated that IFN-α promotes generation of IL-21+IFN-γ +CXCR5-CD4+ T cells thereby enhancing B-cell helper function. By IFN-α treatment, the mRNA levels of IL21, IFNG, CXCL13, CD244, SLAMF7, GZMB and PRDM1 were significantly up-regulated but BCL6 mRNA expression was down-regulated, suggesting a Tph-related gene expression pattern. On the other hand, IL-2-neutralization increased mRNA levels of IL21, CXCL13 and CXCR5, retained BCL6, but showed no clear effect on IFNG or PRDM1. RNA sequencing analyses revealed that PD-1hiCXCR5-CD4+ T cells prepared from in vitro culture show a Tph-related gene expression pattern similar with that of PD-1hiCXCR5- Tph cells obtained from the blood of patients with systemic lupus erythematosus. From our findings, it is highly probable that type I IFNs play a key role in differentiation of Tph cells and trigger Tph cell expansion in autoimmune diseases.

Livin promotes Th2-type immune response in airway allergic diseases

ObjectivesTo investigate the effects of livin on the Th2 immune response in airway allergic diseases (AAD) and explore the interaction among livin, GATA3, IL-4 in peripheral blood CD4+ T cells of AAD patients.MethodsWT mice and livin KO mice were developed for model of AAD. Th2 cell levels in the lung tissues and spleen were assessed by flow cytometry. Also, it was assessed in the culture after exposing to livin inhibitor (Lp-15); the protein and mRNA levels of livin, GATA3 and IL-4 in peripheral blood CD4+ T cells isolated from patients with or without AAD were measured by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting, respectively. Finally, Co-immunoprecipitation (Co-IP) was employed to identify the interaction between livin and GATA3.ResultsCompared with WT mouse, Th2 cell frequency in lung tissues and spleen was significantly decreased in livin KO mouse; after adding Lp-15, the differentiation from Naive CD4+T cells in spleen to Th2 cells was blocked; the protein and mRNA levels of livin, GATA3 and IL-4 in AAD group were higher than that in control group. The levels of livin were positively correlated with IL-4, and GATA3 was also positively correlated with IL-4 and livin. GATA3 was detected in the protein complex co-precipitated with livin antibody, and livin was also detected in the protein complex co-precipitated by GATA3 antibody.ConclusionLivin increases the expression of IL-4 and facilitates naive CD4+ T cells to differentiate into Th2 cells, which triggers airway allergy.

TOX promotes follicular helper T cell differentiation in patients with primary Sjögren's syndrome.

Whether naive CD4+ T cells are dysregulated and associated with the overactivation of CD4+ T cells in primary SS (pSS) remains unclear. We aimed to explore the role and underlying mechanism of naive CD4+ T cells in pSS. We examined the activation, proliferation and differentiation of naive CD4+ T cells from pSS patients and healthy controls. Differentially expressed genes were identified using RNA sequencing, and were overexpressed or silenced to determine the gene regulating follicular helper T (Tfh) cells. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) with chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) was performed to explore the epigenetic mechanism. Naive CD4+ T cells were treated with pSS-related cytokines to explore the upstream signalling pathway. pSS naive CD4+ T cells had higher potentials of activation, proliferation and differentiation towards Tfh cells. Thymocyte selection-associated high mobility group box protein (TOX) was upregulated in pSS naive CD4+ T cells and promoted T cell activation and Tfh cell polarization. TOX silencing in pSS naive CD4+ T cells downregulated B cell lymphoma 6 (BCL6) expression and altered levels of multiple Tfh-associated genes. ChIP-seq analysis implied that TOX bound to the BCL6 locus, where there were accessible regions found by ATAC-seq. IFN-α induced TOX overexpression, which was attenuated by Janus kinase (JAK) and signal transducer and activator of transcription 1 (STAT1) inhibitors. Our data suggest that TOX in pSS naive CD4+ T cells is upregulated, which facilitates Tfh cell differentiation. Mechanistically, IFN-α induces TOX overexpression in naive CD4+ T cells through JAK-STAT1 signalling and TOX regulates BCL6 expression. Therefore, IFN-α-JAK-STAT1 signalling and TOX might be potential therapeutic targets in pSS.

Steroid nuclear receptor coactivator 2 controls immune tolerance by promoting induced Treg differentiation via up-regulating Nr4a2.

Steroid nuclear receptor coactivator 2 (SRC2) is a member of a family of transcription coactivators. While SRC1 inhibits the differentiation of regulatory T cells (Tregs) critical for establishing immune tolerance, we show here that SRC2 stimulates Treg differentiation. SRC2 is dispensable for the development of thymic Tregs, whereas naive CD4+ T cells from mice deficient of SRC2 specific in Tregs (SRC2fl/fl/Foxp3YFP-Cre) display defective Treg differentiation. Furthermore, the aged SRC2fl/fl/Foxp3YFP-Cre mice spontaneously develop autoimmune phenotypes including enlarged spleen and lung inflammation infiltrated with IFNγ-producing CD4+ T cells. SRC2fl/fl/Foxp3YFP-Cre mice also develop severer experimental autoimmune encephalomyelitis (EAE) due to reduced Tregs. Mechanically, SRC2 recruited by NFAT1 binds to the promoter and activates the expression of Nr4a2, which then stimulates Foxp3 expression to promote Treg differentiation. Members of SRC family coactivators thus play distinct roles in Treg differentiation and are potential drug targets for controlling immune tolerance.

Abstract 5222: Biomarkers associated with complete metabolic response (CMR) to nivolumab and brentuximab vedotin (nivo + BV) for the treatment of children, adolescents, and young adults (CAYA) with relapsed or refractory (R/R) classical Hodgkin lymphoma (cHL) in the CheckMate 744 study

Abstract Introduction: CAYA with R/R cHL without CMR have suboptimal outcomes. Biomarkers associated with poor outcomes in cHL include PD-L1 overexpression and presence of tumor-associated macrophages (TAMs). In the phase 2 CheckMate 744 study (NCT02927769), CAYA with R/R cHL treated with a risk-adapted approach using nivo + BV induction showed a CMR rate per BICR of 59% (26/44) in the standard risk cohort; 41% of patients (pts) did not achieve CMR (non-CMR). Here we compared biomarker data from pts achieving CMR vs non-CMR. Methods: Pts aged 5-30 y with R/R cHL received 4 cycles of nivo + BV induction. Tumor biopsy and peripheral blood samples were collected at baseline (BL) and 4 prespecified timepoints during induction. CD163+ CD68+ TAMs were quantified using multiplex immunohistochemistry. Cytokine expression in pts with CMR vs non-CMR was assessed by the limma workflow and a linear model based on changes from BL. Immune cells were analyzed by flow cytometry. Statistical testing was adjusted for repeated measures across visits; values are reported from 39/39 cytokines and 13/16 cell subsets tested. Cytokine ratios to BL were compared across all timepoints and between response groups by ANOVA. Results: Pts with CMR (n = 24/26) after nivo + BV induction had more CD163+ CD68+ TAMs than pts with non-CMR (n = 17/18) in the BL tumor microenvironment (P = 0.018). Hierarchical clustering of cytokine fold changes from BL to the indicated timepoint, between pts with CMR vs non-CMR, showed reduced levels of haptoglobin, RANTES, CRP, PARC, EN-RAGE, and TARC/CCL17, and increased levels of MCP-2, IFN-γ, IP-10, and MIG. In all pts, median concentrations of TARC, IL-2Rα, CRP, and ICAM-1 decreased significantly (all P < 0.05); a greater reduction in TARC and CRP was observed in early cycles in pts with CMR vs non-CMR. In contrast, IP-10 levels increased from BL in all pts (P = 0.0012). Four cytokines showed higher concentrations in pts with CMR vs non-CMR: TNFR2, MCP-2, vWF, and TIMP-1 (all P < 0.05). Increased numbers of lymphocytes and monocytes and decreased numbers of granulocytes were observed after induction in all pts (all P < 0.01). Pts with non-CMR had more naive (CCR7+ CD45RA+) CD4, and naive CD8 T cells than pts with CMR at BL, and more overall CD4 (CD3+ CD4+) T cells at the end of cycle 4 (all P < 0.05). Conclusions: This biomarker analysis from CheckMate 744 suggests that different immunological parameters are associated with CMR vs non-CMR to nivo + BV induction in CAYA with R/R cHL. These include increased TAMs at BL in pts with CMR, changes in cytokine levels associated with macrophage biology, and greater numbers of naive lymphocytes and CD4 T cells in pts with non-CMR. TAMs have been reported to express high PD-L1 levels; thus, increased TAM numbers may sensitize pts to nivo + BV. Citation Format: Charlie Garnett-Benson, Ron Ammar, Russell Crowe, Stephen Francis, Sahar Ansari, Alev Akyol. Biomarkers associated with complete metabolic response (CMR) to nivolumab and brentuximab vedotin (nivo + BV) for the treatment of children, adolescents, and young adults (CAYA) with relapsed or refractory (R/R) classical Hodgkin lymphoma (cHL) in the CheckMate 744 study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5222.

Vasculitogenic T Cells in Large Vessel Vasculitis

Vasculitis is an autoimmune disease of unknown etiology that causes inflammation of the blood vessels. Large vessel vasculitis is classified as either giant cell arteritis (GCA), which occurs exclusively in the elderly, or Takayasu arteritis (TAK), which mainly affects young women. Various cell types are involved in the pathogenesis of large vessel vasculitis. Among these, dendritic cells located between the adventitia and the media initiate the inflammatory cascade as antigen-presenting cells, followed by activation of macrophages and T cells contributing to vessel wall destruction. In both diseases, naive CD4+ T cells are polarized to differentiate into Th1 or Th17 cells, whereas differentiation into regulatory T cells, which suppress vascular inflammation, is inhibited. Skewed T cell differentiation is the result of aberrant intracellular signaling, such as the mechanistic target of rapamycin (mTOR) or the Janus kinase signal transducer and activator of transcription (JAK-STAT) pathways. It has also become clear that tissue niches in the vasculature fuel activated T cells and maintain tissue-resident memory T cells. In this review, we outline the most recent understanding of the pathophysiology of large vessel vasculitis. Then, we provide a summary of skewed T cell differentiation in the vasculature and peripheral blood. Finally, new therapeutic strategies for correcting skewed T cell differentiation as well as aberrant intracellular signaling are discussed.

Abstract 2061: Deep immune profiling by mass cytometry revealed an association between the state of immune system before treatment and response to checkpoint inhibitor therapy in clear cell renal cell carcinoma

Abstract Checkpoint immunotherapy (CPI) and its combination with tyrosine kinase inhibitor (TKI) therapy are widely used in the treatment of many cancers, including clear cell renal cell carcinoma (ccRCC). Although recent advances in these therapies demonstrate effectiveness, a subset of patients are unresponsive. In this study, using deep immune profiling by mass cytometry (CyTOF), we analyzed peripheral blood mononuclear cell (PBMC) samples from 86 ccRCC patients pre- and post-treatment. The cohort included patients treated with first-line CPI (n = 43), TKI (n = 15) and combinatorial CPI+TKI (n = 28), with PBMC samples collected pre- (n = 41) and post- (n = 69) therapy (n = 24 patients total with paired samples). Samples were characterized by CyTOF analysis of 30 different cell surface markers. Using t-distributed stochastic neighbor embedding (tSNE) and Phenograph clustering, the presence of immune populations and subpopulations were characterized and associated with therapeutic response as well as longitudinally tracked. While most cell types did not differ between pre- and post-treatment states, a decrease in CD8 and CD4 PD1+ T cell subsets was observed in patients treated with CPI and CPI+TKI (p < 0.001). Also, increased CD8 effector memory T cells were observed in post-treatment samples in those treated with CPI or CPI+TKI (p < 0.001).Subsequent analysis of pre-treatment samples in the context of clinical outcome revealed specific features associated with favorable response to immunotherapy. Responders (R) in comparison with non-responders (NR) were characterized by an increased number of naive CD4 T cells (p = 0.03), CD8 T cells (p = 0.038), immature NK cells (p = 0.005) and lower percentage of granulocytes (p = 0.012). Using unsupervised hierarchical clustering, 8 patient groups were identified with a unique immune profile enriched by a certain PBMC population. Three groups (G1-G3), which included a high ratio of R, were enriched by naive T cells, immature NK cells and non-classical monocytes, respectively. The G4 group, enriched with B cells, consisted of R and NR in equal numbers. The G5-G8 groups, enriched with granulocytes, classical monocytes, mature NK-cells and effector CD8 T cells, respectively, were primarily comprised of NR. Patients with favorable pre-treatment immune profiles also belonged to favorable groups post-treatment, with a similar trend observed with unfavorable profiles. In conclusion, this analysis revealed a remarkable association between the initial immune status or “portrait” of the patient before therapy and subsequent therapeutic outcome as well as the development of therapeutic resistance. Citation Format: Svetlana Shishkova, Dmitry Tabakov, Evgeny Egorov, Akshaya Ramachandran, Yang Lyu, Krystle Nomie, Jessica Brown, Vladimir Zyrin, Alexander Zaytcev, Natalia Miheecheva, Ekaterina Postovalova, Alexander Bagaev, Ravshan Ataullakhanov, James Hsieh. Deep immune profiling by mass cytometry revealed an association between the state of immune system before treatment and response to checkpoint inhibitor therapy in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2061.

Peripheral Blood T Cells of Patients with IPAH Have a Reduced Cytokine-Producing Capacity.

Pulmonary arterial hypertension (PAH) is rare disease that is categorized as idiopathic (IPAH) when no underlying cause can be identified. Lungs of most patients with IPAH contain increased numbers of T cells and dendritic cells (DCs), suggesting involvement of the immune system in its pathophysiology. However, our knowledge on circulating immune cells in IPAH is rather limited. We used flow cytometry to characterize peripheral blood DCs and T cells in treatment-naive IPAH patients, compared with connective-tissue disease-PAH (CTD-PAH) patients and healthy controls (HCs). At diagnosis, T-helper (Th) cells of IPAH patients were less capable of producing TNFα, IFNγ, IL-4 and IL-17 compared to HCs. IPAH patients showed a decreased frequency of Th2 cells and significantly enhanced expression of the CTLA4 checkpoint molecule in naive CD4+ T cells and both naive and memory CD8+ T cells. Frequencies and surface marker expression of circulating DCs and monocytes were essentially comparable between IPAH patients and HCs. Principal component analysis (PCA) separated IPAH patients—but not CTD-PAH patients—from HCs, based on T-cell cytokine profiles. At 1-year follow-up, the frequencies of IL-17+ production by memory CD4+ T cells were increased in IPAH patients and accompanied by increased proportions of Th17 and Tc17 cells, as well as decreased CTLA4 expression. Treatment-naive IPAH patients displayed a unique T-cell phenotype that was different from CTD-PAH patients and was characterized by reduced cytokine-producing capacity. These findings point to involvement of adaptive immune responses in IPAH, which may have an implication for the development of therapeutic interventions.

Towards a unified model of naive T cell dynamics across the lifespan.

Naive CD4 and CD8 T cells are cornerstones of adaptive immunity, but the dynamics of their establishment early in life and how their kinetics change as they mature following release from the thymus are poorly understood. Further, due to the diverse signals implicated in naive T cell survival, it has been a long-held and conceptually attractive view that they are sustained by active homeostatic control as thymic activity wanes. Here we use multiple modelling and experimental approaches to identify a unified model of naive CD4 and CD8 T cell population dynamics in mice, across their lifespan. We infer that both subsets divide rarely, and progressively increase their survival capacity with cell age. Strikingly, this simple model is able to describe naive CD4 T cell dynamics throughout life. In contrast, we find that newly generated naive CD8 T cells are lost more rapidly during the first 3-4 weeks of life, likely due to increased recruitment into memory. We find no evidence for elevated division rates in neonates, or for feedback regulation of naive T cell numbers at any age. We show how confronting mathematical models with diverse datasets can reveal a quantitative and remarkably simple picture of naive T cell dynamics in mice from birth into old age.

Mesothelial cell-derived antigen-presenting cancer-associated fibroblasts induce expansion of regulatory Tcells in pancreatic cancer.

Recent studies have identified a unique cancer-associated fibroblast (CAF) population termed antigen-presenting CAFs (apCAFs), characterized by the expression of major histocompatibility complex class II molecules, suggesting a function in regulating tumor immunity. Here, by integrating multiple single-cell RNA-sequencing studies and performing robust lineage-tracing assays, we find that apCAFs are derived from mesothelial cells. During pancreatic cancer progression, mesothelial cells form apCAFs by downregulating mesothelial features and gaining fibroblastic features, a process induced by interleukin-1 and transforming growth factor β. apCAFs directly ligate and induce naive CD4+ Tcells into regulatory Tcells (Tregs) in an antigen-specific manner. Moreover, treatment with an antibody targeting the mesothelial cell marker mesothelin can effectively inhibit mesothelial cell to apCAF transition and Treg formation induced by apCAFs. Taken together, our study elucidates how mesothelial cells may contribute to immune evasion in pancreatic cancer and provides insight on strategies to enhance cancer immune therapy.

Revealing Potential Spinal Cord Injury Biomarkers and Immune Cell Infiltration Characteristics in Mice.

Spinal cord injury (SCI) is a disabling condition with significant morbidity and mortality. Currently, no effective SCI treatment exists. This study aimed to identify potential biomarkers and characterize the properties of immune cell infiltration during this pathological event. To eliminate batch effects, we concurrently analyzed two mouse SCI datasets (GSE5296, GSE47681) from the GEO database. First, we identified differentially expressed genes (DEGs) using linear models for microarray data (LIMMA) and performed functional enrichment studies on those DEGs. Next, we employed bioinformatics and machine-learning methods to identify and define the characteristic genes of SCI. Finally, we validated them using immunofluorescence and qRT-PCR. Additionally, this study assessed the inflammatory status of SCI by identifying cell types using CIBERSORT. Furthermore, we investigated the link between key markers and infiltrating immune cells. In total, we identified 561 robust DEGs. We identified Rab20 and Klf6 as SCI-specific biomarkers and demonstrated their significance using qRT-PCR in the mouse model. According to the examination of immune cell infiltration, M0, M1, and M2 macrophages, along with naive CD8, dendritic cell-activated, and CD4 Follicular T cells may have a role in the progression of SCI. Therefore, Rab20 and Klf6 could be accessible targets for diagnosing and treating SCI. Moreover, as previously stated, immune cell infiltration may significantly impact the development and progression of SCI.

Single-cell RNA sequencing reveals immunological rewiring at the maternal-fetal interface following asymptomatic/mild SARS-CoV-2 infection.

While severe coronavirus 2019 (COVID-19) is associated with immune activation at the maternal-fetal interface, responses to asymptomatic/mild severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during pregnancy remain unknown. Here, we assess immunological adaptations in blood and term decidua in response to asymptomatic/mild disease in pregnant women. We report attenuated antigen presentation and type I interferon (IFN) signaling pathways, loss of tissue-resident decidual macrophages, and upregulated cytokine/chemokine signaling in monocyte-derived decidual macrophages. Furthermore, we describe increased frequencies of activated tissue-resident T cells and decreased abundance of regulatory T cells with infection while frequencies of cytotoxic CD4/CD8 T cells are increased in the blood. In contrast to decidual macrophages, type I IFN signaling is higher in decidual T cells. Finally, infection leads to a narrowing of T cell receptor diversity in both blood and decidua. Collectively, these observations indicate that asymptomatic/mild COVID-19 during pregnancy results in remodeling of the immunological landscape of the maternal-fetal interface, with a potential for long-term adverse outcomes for the offspring.

Loss of voltage-gated hydrogen channel 1 expression reveals heterogeneous metabolic adaptation to intracellular acidification by T cells

Voltage-gated hydrogen channel 1 (Hvcn1) is a voltage-gated proton channel, which reduces cytosol acidification and facilitates the production of ROS. The increased expression of this channel in some cancers has led to proposing Hvcn1 antagonists as potential therapeutics. While its role in most leukocytes has been studied in depth, the function of Hvcn1 in T cells remains poorly defined. We show that Hvcn1 plays a nonredundant role in protecting naive T cells from intracellular acidification during priming. Despite sharing overall functional impairment in vivo and in vitro, Hvcn1-deficient CD4+ and CD8+ T cells display profound differences during the transition from naive to primed T cells, including in the preservation of T cell receptor (TCR) signaling, cellular division, and death. These selective features result, at least in part, from a substantially different metabolic response to intracellular acidification associated with priming. While Hvcn1-deficient naive CD4+ T cells reprogram to rescue the glycolytic pathway, naive CD8+ T cells, which express high levels of this channel in the mitochondria, respond by metabolically compensating mitochondrial dysfunction, at least in part via AMPK activation. These observations imply heterogeneity between adaptation of naive CD4+ and CD8+ T cells to intracellular acidification during activation.

Altered Cellular Immunity and Differentially Expressed Immune-Related Genes in Patients With Systemic Sclerosis-Associated Pulmonary Arterial Hypertension.

Systemic sclerosis (SSc) is the most common connective tissue disease causing pulmonary hypertension (PAH). However, the cause and potential immune molecular events associated with PAH are still unclear. Therefore, it is particularly essential to analyze the changes in SSc-PAH–related immune cells and their immune-related genes. Three microarray datasets (GSE22356, GSE33463, and GSE19617) were obtained by the Gene Expression Omnibus (GEO). Compared with SSc, we found neutrophils have a statistically higher abundance, while T-cell CD4 naive and T-cell CD4 memory resting have a statistically lower abundance in peripheral blood mononuclear cells (PBMCs). Moreover, the results of Gene Set Enrichment Analysis (GSEA) showed there is a differential enrichment of multiple pathways between SSc and SSc-PAH. By combining differentiated expressed genes (DEGs) and immune-related genes (IRGs), fifteen IRGs were selected. In addition, we also analyzed the first five rich Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and the most abundant Gene Ontology (GO)-molecular functional terms. Furthermore, interleukin-7 receptor (IL-7R), tyrosine–protein kinase (LCK), histone deacetylase 1 (HDAC1), and epidermal growth factor receptor (EGFR) genes were identified as hub genes via protein–protein interaction (PPI) network analysis. The Comparative Toxic Genomics Database (CTD) analysis result showed that LCK, HDAC1, and EGFR have a higher score with SSc. Coexpression network analysis confirmed that IL-7R, LCK, and HDAC1 are key genes related to immune regulation in SSc without PAH and are involved in T-cell immune regulation. Subsequently, using GSE22356 and GSE33463 as the test sets and GSE19617 as the verification set, it was verified that the mRNA expression levels of the three central genes of SSc-PAH were significantly lower than those of the SSc without PAH samples. Consistent with previous predictions, the expressions of IL-7R, LCK, and HDAC1 are positively correlated with the numbers of T-cell CD4 naive and T-cell CD4 memory, while the expressions of IL-7R and LCK are negatively correlated with the numbers of neutrophils in the peripheral blood. Therefore, this evidence may suggest that these three immune-related genes: IL-7R, LCK, and HDAC1, may be highly related to the immunological changes in SSc-PAH. These three molecules can reduce T cells in SSc-PAH PBMCs through the regulation of T-cell activation, which suggests that these three molecules may be involved in the development of SSc-PAH. Meanwhile, the low expression of IL-7R, LCK, and HDAC1 detected in the peripheral blood of SSc may indicate the possibility of PAH and hopefully become a biomarker for the early detection of SSc-PAH. Finally, 49 target miRNAs of 3 specifically expressed hub genes were obtained, and 49 mRNA–miRNA pairs were identified, which provided directions for our further research.

Minocycline Alleviates White Matter Injury following Intracerebral Hemorrhage by Regulating CD4+ T Cell Differentiation via Notch1 Signaling Pathway.

Neuroinflammation is a major reason for white matter injury (WMI) after intracerebral hemorrhage (ICH). Apart from microglia/macrophage activation, T cells also play an important role in regulating immune responses after ICH. In a previous study, we have revealed the role of minocycline in modulating microglia/macrophage activation after ICH. However, the exact mechanisms of minocycline in regulating T cells differentiation after ICH are still not well understood. Hence, this study explored the relationship between minocycline and CD4+ T cell differentiation after ICH. Piglet ICH model was used to investigate naive CD4+ T cell differentiation and T cells signal gene activation after ICH with immunofluorescence and whole transcriptome sequencing. Naive CD4+ T cells and primary oligodendrocyte coculture model were established to explore the effect and mechanism of minocycline in modulating CD4+ T cell differentiation after ICH. Flow cytometry was used to indicate CD4+ T cell differentiation after ICH. The mechanism of minocycline in modulating CD4+ T cell differentiation was demonstrated with immunofluorescence and western blot. Double immunostaining of representative CD4+ T cell marker CD3 and different subtype CD4+ T cell assisted proteins (IL17, IL4, Foxp3, and IFNγ) demonstrated naive CD4+ T cell differentiation in piglet after ICH. Whole transcriptome sequencing for perihematomal white matter sorted from piglet brains indicated T cell signal gene activation after ICH. The results of luxol fast blue staining, immunofluorescent staining, and electron microscopy showed that minocycline alleviated white matter injury after ICH in piglets. For our in vitro model, minocycline reduced oligodendrocyte injury and neuroinflammation by regulating CD4+ T cell differentiation after ICH. Moreover, minocycline increased the expression of NOTCH1, ACT1, RBP-J, and NICD1 in cultured CD4+ T cell when stimulated with hemoglobin. Hence, minocycline treatment could modulate naive CD4+ T cell differentiation and attenuate white matter injury via regulating Notch1 signaling pathway after ICH.

Strong tonic TCR signaling is associated with negative regulation of naive CD4+ T cells

Abstract Naive T cells constantly experience tonic or basal TCR signals in response to self-peptides presented by MHC (pMHC) in vivo. We aimed to define how tonic TCR signaling impacts naive CD4+ T cells at the functional, transcriptional, and epigenetic levels. The transgenic Nur77-GFP reporter gene is responsive to tonic TCR signals. Naive CD4+ T cells can express a broad range of Nur77-GFP transgene expression at a single cell level, even if they express identical transgenic TCRs. Immature T cells that express high levels of Nur77-GFP following selection in the thymus tend to sustain high Nur77-GFP expression in the periphery. Naive CD4+ T cells with a Nur77-GFPHI Ly6C− phenotype experience the strongest tonic TCR signaling and in the OT-II TCR transgenic system, exhibit less sensitivity to stimulation with low doses of cognate pMHC or to weaker affinity pMHC. Analyses of gene expression in Nur77-GFPHI Ly6C− cells suggest a transcriptional program consistent with T cell activation, but also of negative regulation. Analyses of chromatin accessibility by ATAC-seq revealed over 3000 differentially accessible chromatin regions in Nur77-GFPHI Ly6C− cells, including several loci within or near genes encoding negative regulators such as Ctla-4 and Tim3. Nur77-GFPHI Ly6C− cells exhibited increased accessibility at multiple loci in the gene encoding Tox. A subset of these loci was previously reported to be more highly accessible in exhausted T cells. These findings suggest that persistent strong tonic TCR signaling may induce changes similar to those that occur during exhaustion. We propose that naive CD4+ T cells adapt to strong tonic TCR signaling and as a result, exhibit decreased sensitivity to subsequent TCR stimulations. Supported by a grant from NIH (K01AR065481)

Arthroplasty surgery alters the local immune composition of the knee joint by causing increases in specific lymphocyte populations.

Abstract Objective We aimed to determine how the innate and adaptive immune cell composition and cytokine profiles of the knee joint microenvironment are altered in replaced knees compared to native knees. Methods Upon IRB approval, we obtained peripheral blood (PBMC) and synovial fluid (SF) from 14 native knees with osteoarthritis (OA) and 12 replaced knees at least 6 months after arthroplasty surgery, comprising 26 total patients. All PBMC and SF samples were analyzed by Flow Cytometry, and utilizing multidimensional reduction analysis via TriMap and clustering analysis by FlowSOM, cell clusters were phenotyped for identification of unique populations of cells. Additionally all samples were analyzed for cytokine/chemokine profiles using 65-Plex ProcartaPlex assays. Results Overall, there was no significant different in any of the innate cell populations studied, including neutrophils, monocytes (including G-MDSCs), macrophages (M1 of M2 phenotypes), and dendritic cells. B cells, CD8 effector T cells, CD4 T cells, Naive CD4 T cells, Th17 cells, and CD4 T cells with Th1-phenotypes s were all significantly increased in replaced knees compared to native knees (p < 0.02 in each case). The replaced knee group showed elevated levels of CXCL5, HGF, IFN-gamma, IL-6, IL-16, CXCL10, CCL2 (MCP-1), CCL8, MDC, TNF-alpha, and VEGF-A compared to the native knee group (p < 0.01 for all) and a decrease in CX3CL1, CXCL11, IL-3, IL-5, CXCL9, TRAIL (p < 0.02 in all cases). Conclusions Arthroplasty surgery results in a lymphocytic shift in immune cell populations and a generally pro-inflammatory cytokine/chemokine profile. Overall, this lymphocytic shift may have implications for development of periprosthetic infections and warrants further study.

IL-17 cytokines preferentially act on naive CD4+ T cells with IL-17AF inducing the greatest transcriptomic changes.

Abstract CD4+ T-helper 17 (Th17) T cells are important in the regulation of protective immunity during infection and in self-tolerance/autoimmunity. Th17 cells, through the secretion of IL-17, act in promotion of inflammation and are thus a major potential therapeutic target in autoimmune disorders. Recent reports show that the IL-17 family cytokines, IL-17A, IL-17F and IL-17AF, can directly act on CD4+ T-cells, inducing functional changes in these cells. Here we show that functional and transcriptomic changes were preferentially induced in naïve CD4+ T-cells, but not in memory CD4+ T-cells. As early as 48 hours post-IL-17 exposure, naïve cells showed transcriptome changes, whereas memory cells remained unaffected as late as 7 days. The functional differences occurred despite similar IL-17RC and IL-17RA receptor expression on these subsets. Also, the differential IL-17-mediated changes in naïve, but not memory, cells occurred even in transwell/co-culture systems, with each subset maintaining its respective functional phenotype. Notably, there were differences in downstream transcriptional signaling by the three IL-17 cytokines. The IL-17AF heterodimer conferred both the greatest transcriptional change and most altered functional consequences. Detailed analysis of the transcriptome provides important insights into the genes and pathways that are altered as a result of IL-17-induced signaling and may serve as targets of future therapies. This work was supported, in part, by grant awards from the NIH to N.J.K. (R01 AI121567) and N.B. (F30 CA29655).