Immunoregulatory Cytokine IL-10 Research Articles

IL-21 conditions antigen-presenting human γδ T-cells to promote IL-10 expression in naïve and memory CD4+ T-cells.

Direct interaction between T-cells exerts a major influence on tissue immunity and inflammation across multiple body sites including the human gut, which is highly enriched in 'unconventional' lymphocytes such as γδ T-cells. We previously reported that microbial activation of human Vγ9/Vδ2+ γδ T-cells in the presence of the mucosal damage-associated cytokine IL-15 confers the ability to promote epithelial barrier defence, specifically via induction of IL-22 expression in conventional CD4+ T-cells. In the current report, we assessed whether other cytokines enriched in the gut milieu also functionally influence microbe-responsive Vγ9/Vδ2 T-cells. When cultured in the presence of IL-21, Vγ9/Vδ2 T-cells acquired the ability to induce expression of the immunoregulatory cytokine IL-10 in both naïve and memory CD4+ T-cells, at levels surpassing those induced by monocytes or monocyte-derived DCs. These findings identify an unexpected influence of IL-21 on Vγ9/Vδ2 T-cell modulation of CD4+ T-cell responses. Further analyses suggested a possible role for CD30L and/or CD40L reverse signalling in mediating IL-10 induction by IL-21 conditioned Vγ9/Vδ2 T-cells. Our findings indicate that the local microenvironment exerts a profound influence on Vγ9/Vδ2 T-cell responses to microbial challenge, leading to induction of distinct functional profiles among CD4+ T-cells that may influence inflammatory events at mucosal surfaces. Targeting these novel pathways may offer therapeutic benefit in disorders such as inflammatory bowel disease.

Discovery of Clinical Candidate GLPG3970: A Potent and Selective Dual SIK2/SIK3 Inhibitor for the Treatment of Autoimmune and Inflammatory Diseases.

The salt-inducible kinases (SIKs) SIK1, SIK2, and SIK3 belong to the adenosine monophosphate-activated protein kinase (AMPK) family of serine/threonine kinases. SIK inhibition represents a new therapeutic approach modulating pro-inflammatory and immunoregulatory pathways that holds potential for the treatment of inflammatory diseases. Here, we describe the identification of GLPG3970 (32), a first-in-class dual SIK2/SIK3 inhibitor with selectivity against SIK1 (IC50 of 282.8 nM on SIK1, 7.8 nM on SIK2 and 3.8 nM on SIK3). We outline efforts made to increase selectivity against SIK1 and improve CYP time-dependent inhibition properties through the structure-activity relationship. The dual activity of 32 in modulating the pro-inflammatory cytokine TNFα and the immunoregulatory cytokine IL-10 is demonstrated in vitro in human primary myeloid cells and human whole blood, and in vivo in mice stimulated with lipopolysaccharide. Compound 32 shows dose-dependent activity in disease-relevant mouse pharmacological models.

Cytokine and T cell responses in post-chikungunya viral arthritis: A cross-sectional study.

To define the relationship between chronic chikungunya post-viral arthritis disease severity, cytokine response and T cell subsets in order to identify potential targets for therapy. Participants with chikungunya arthritis were recruited from Colombia from 2019-2021. Arthritis disease severity was quantified using the Disease Activity Score-28 and an Arthritis-Flare Questionnaire adapted for chikungunya arthritis. Plasma cytokine concentrations (interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, interferon-γ and tumor necrosis factor (TNF)) were measured using a Meso Scale Diagnostics assay. Peripheral blood T cell subsets were measured using flow cytometry. Among participants with chikungunya arthritis (N = 158), IL-2 levels and frequency of regulatory T cells (Tregs) were low. Increased arthritis disease activity was associated with higher levels of inflammatory cytokines (IL-6, TNF and CRP) and immunoregulatory cytokine IL-10 (p<0.05). Increased arthritis flare activity was associated with higher Treg frequencies (p<0.05) without affecting T effector (Teff) frequencies, Treg/Teff ratios and Treg subsets. Finally, elevated levels of IL-2 were correlated with increased Treg frequency, percent Tregs out of CD4+ T cells, and Treg subsets expressing immunosuppressive markers, while also correlating with an increased percent Teff out of live lymphocytes (p<0.05). Chikungunya arthritis is characterized by increased inflammatory cytokines and deficient IL-2 and Treg responses. Greater levels of IL-2 were associated with improved Treg numbers and immunosuppressive markers. Future research may consider targeting these pathways for therapy.

Tonsillar cytotoxic CD4 T cells are involved in the control of EBV primary infection in children

CD4 T cells play a key role in Epstein Barr virus (EBV) infection, by modulating latent antigen expression, and exhibiting cytotoxic and regulatory properties. Our aim was to evaluate the presence of Granzyme B (GZMB) and Foxp3 CD4 T cells at different EBV infection status and latency profiles. We examined CD4, GZMB, Foxp3, IL10, TGF-β, CD4-GZMB and CD4-Foxp3 expression at the tonsils of pediatric patients with different infective status and EBV latency profiles. CD4+, GZMB+, Foxp3+, CD4-GZMB+ and CD4-Foxp3+ cell counts were higher at the interfollicular region. Higher expression of CD4-GZMB was found in primary infected patients compared to healthy carriers. In patients that expressed latency III antigens, we demonstrated lower CD4+, CD4-GZMB+, CD4-Foxp3+ expression; a negative correlation between the immunoregulatory cytokine IL-10+ and GZMB+ as well as a positive correlation of IL-10+ and CD4+. In patients expressing the lytic protein BMRF1, a positive correlation of TGF-β+ with CD4-GZMB+ and CD4-Foxp3+ was observed. Our findings indicate that CD4-GZMB+ cells are involved in the restriction of primary EBV infection in pediatric patients, which could partially explain the lack of symptoms, whereas both CD4-GZMB+ and CD4-Foxp3+ cells could be involved in the modulation of latency.

Blockade of CCR4 breaks immune tolerance in chronic hepatitis B patients by modulating regulatory pathways

BackgroundImmunotargets including checkpoint inhibitors and toll-like receptor 8 agonists have recently gained attention for the recovery of hepatitis B virus (HBV)-specific T cell exhaustion in chronic hepatitis B(CHB). Chemokine receptors have a similar significant role during viral infections; however, their role in CHB remains poorly understood. Therefore, in this study we evaluated the role of chemokine receptor 4 (CCR4) in deriving immunosuppression during CHB.MethodsWe characterized CCR4+CD8+ T cells in CHB and identified their involvement in immunosuppression. Further, we examined if CCR4 blockade with mogamulizumab antibody can recover the functional exhaustion in HBsAg-specific T cells.ResultsCHB patients exhibit higher frequency of CCR4+CD8+ T cells that increase with higher HBsAg levels and fibrosis scores. In vitro, HBs antigen triggers CCR4 expression. These cells express multiple inhibitory receptors and exhibit immunosuppressive functions by producing excessive immunoregulatory cytokines IL-4, IL-5, IL-10 and TGF-β1. CCR4 Blockade significantly boosted HBsAg-specific antiviral-cytokine production(IFN-γ, TNF-α and IL-21) in T cells through enhancing their proliferation capacity and polarizing these cells towards T helper 1(Th1) and T follicular helper cells(TFH) in case of CD4 cells, and cytotoxic T cell 1(TC1) and cytotoxic T follicular(TCF) cells in case of CD8. Cytotoxic potential was improved, while no induction of immunosuppressive-cytokines was seen after anti-CCR4 treatment thereby eliminating the risk of treatment-induced immunosuppression. CCR4 blockade inhibited the development and effector function of Tregs by controlling their expansion and TGF-β1 production preventing Tregs-induced immunotolearance.ConclusionsCCR4 blockade reconstitutes antiviral immune response in T cells and limits the immunosuppressive functions of Tregs, representing them as a promising immunotherapeutic target for functional cure of CHB.

Immune Microenvironment in Sporadic Early-Onset versus Average-Onset Colorectal Cancer

The incidence of left-sided colon and rectal cancer in young people are increasing worldwide, but its causes are poorly understood. It is not clear if the tumor microenvironment is dependent on age of onset, and little is known about the composition of tumor-infiltrating T cells in early-onset colorectal cancer (EOCRC). To address this, we investigated T-cell subsets and performed gene expression immune profiling in sporadic EOCRC tumors and matched average-onset colorectal cancer (AOCRC) tumors. Left-sided colon and rectal tumors from 40 cases were analyzed; 20 EOCRC (<45 years) patients were matched 1:1 to AOCRC (70-75 years) patients by gender, tumor location, and stage. Cases with germline pathogenic variants, inflammatory bowel disease or neoadjuvant-treated tumors were excluded. For T cells in tumors and stroma, a multiplex immunofluorescence assay combined with digital image analysis and machine learning algorithms was used. Immunological mediators in the tumor microenvironment were assessed by NanoString gene expression profiling of mRNA. Immunofluorescence revealed no significant difference between EOCRC and AOCRC with regard to infiltration of total T cells, conventional CD4+ and CD8+ T cells, regulatory T cells, or γδ T cells. Most T cells were located in the stroma in both EOCRC and AOCRC. Immune profiling by gene expression revealed higher expression in AOCRC of the immunoregulatory cytokine IL-10, the inhibitory NK cell receptors KIR3DL3 and KLRB1 (CD161), and IFN-a7 (IFNA7). In contrast, the interferon-induced gene IFIT2 was more highly expressed in EOCRC. However, in a global analysis of 770 tumor immunity genes, no significant differences could be detected. T-cell infiltration and expression of inflammatory mediators are similar in EOCRC and AOCRC. This may indicate that the immune response to cancer in left colon and rectum is not related to age of onset and that EOCRC is likely not driven by immune response deficiency.

DOP04 Single cell RNA sequencing reveals a potential role for IFNγ in priming circulating monocytes for intestinal inflammatory function in Crohn’s Disease.

Abstract Background Hyper-inflammatory monocyte-derived cells accumulate in the intestine and contribute to pathology in inflammatory bowel disease (IBD). Our previous work has shown that intestinal monocyte-derived cells from Crohn’s Disease patients have a reduced response to the immunoregulatory cytokine IL-10 and that this property is already established in circulating monocytes prior to their arrival in the intestine. Here, we have used scRNAseq and functional experiments to define the scope of IBD-associated changes in blood monocytes and explore the role of inflammatory cytokines in shaping these. Methods Total monocytes were isolated from the peripheral blood of 12 newly diagnosed, treatment naive IBD patients with active endoscopic disease (6 Crohn’s Disease [CD] and 6 Ulcerative Colitis [UC]) and 10 healthy controls (HC) by negative magnetic selection. scRNAseq was performed using the 10x Genomics Chromium 3’ assay at a target capture rate of 1000 cells/sample and sequencing at ~50,000 reads/cell. Data were analysed using Cell Ranger, Seurat, Metascape and Gene Set Enrichment Analysis. Blood CD14+ monocytes from healthy donors were pre-incubated in medium alone or with IFNγ (100U/ml) for 24 hours. Inhibition of LPS-induced TNFα production by IL-10 was measured by intracellular cytokine staining and flow cytometry. Results UMAP clustering identified six transcriptionally distinct monocyte clusters: clusters 0, 1 and 2 mapped to classical monocytes; cluster 3 to non-classical monocytes; cluster 4 to intermediate monocytes; and cluster 5 contained platelet signals. The proportion of cluster 4 was increased in CD patients compared to HC. Differential gene expression analysis of CD and UC monocyte clusters compared to healthy controls identified a total of 246 differentially expressed genes (DEGs) in CD and 149 in UC. Only 39 of these DEGs were significantly affected in both diseases. Both CD and UC DEGs were significantly enriched for pathways related to cytokine signalling and inflammation (Figure 1). IFNγ and TNFα -response gene sets were positively enriched in CD, but negatively enriched in UC (Figure 2). Pre-treatment of healthy monocytes with IFNγ in vitro reduced their response to IL-10 as measured by inhibition of LPS-induced TNFα (Figure 3). Conclusion Expression of genes associated with inflammation and cytokine signaling are altered in blood monocytes from IBD patients implying their modification by systemic signals prior to arrival in the intestinal mucosa. Cytokine- (IFNγ and TNFα) stimulated genes are differentially affected in CD and UC monocytes. In CD, systemic IFNγ may reduce the responsiveness of monocytes to IL-10 to prime them for inflammatory function.

Eomes is sufficient to regulate IL-10 expression and cytotoxic effector molecules in murine CD4+ T cells.

The T-box transcription factors T-bet and Eomesodermin regulate type 1 immune responses in innate and adaptive lymphocytes. T-bet is widely expressed in the immune system but was initially identified as the lineage-specifying transcription factor of Th1 CD4+ T cells, where it governs expression of the signature cytokine IFN- γ and represses alternative cell fates like Th2 and Th17. T-bet's paralog Eomes is less abundantly expressed and Eomes+ CD4+ T cells are mostly found in the context of persistent antigen exposure, like bone marrow transplantation, chronic infection or inflammation as well as malignant disorders. However, it has remained unresolved whether Eomes executes similar transcriptional activities as T-bet in CD4+ T cells. Here we use a novel genetic approach to show that Eomes expression in CD4+ T cells drives a distinct transcriptional program that shows only partial overlap with T-bet. We found that Eomes is sufficient to induce the expression of the immunoregulatory cytokine IL-10 and, together with T-bet, promotes a cytotoxic effector profile, including Prf1, Gzmb, Gzmk, Nkg7 and Ccl5, while repressing alternative cell fates. Our results demonstrate that Eomes+ CD4+ T cells, which are often found in the context of chronic antigen stimulation, are likely to be a unique CD4+ T cell subset that limits inflammation and immunopathology as well as eliminates antigen-presenting and malignant cells.

Analysis and therapeutic targeting of the EP300 and CREBBP acetyltransferases in anaplastic large cell lymphoma and Hodgkin lymphoma.

Anaplastic large cell lymphoma (ALCL) and classical Hodgkin lymphoma (HL) share a similar cytological and high surface expression of CD30, and novel therapeutic strategies are needed. The EP300 and CREBBP acetyltransferases play essential roles in the pathogenesis of non-Hodgkin B cell lymphoma, but their functions in ALCL and HL are unknown. In the current study, we investigated the physiological roles of EP300 and CREBBP in both ALCL and HL, and exploited the therapeutic potential of EP300/CREBBP small molecule inhibitors that target either the HAT or bromodomain activities. Our studies demonstrated distinct roles for EP300 and CREBBP in supporting the viability of ALCL and HL, which was bolstered by the transcriptome analyses. Specifically, EP300 but not CREBBP directly modulated the expression of oncogenic MYC/IRF4 network, surface receptor CD30, immunoregulatory cytokines IL10 and LTA, and immune checkpoint protein PD-L1. Importantly, EP300/CREBBP HAT inhibitor A-485 and bromodomain inhibitor CPI-637 exhibited strong activities against ALCL and HL in vitro and in xenograft mouse models, and inhibited PD-L1 mediated tumor immune escape. Thus, our studies revealed critical insights into the physiological roles of EP300/CREBBP in these lymphomas, and provided opportunities for developing novel strategies for both targeted and immune therapies.

Lactic acid bacteria derived-bioactive molecules condition an in vitro cell model into a tolerogenic dendritic-like cell phenotype

Abstract Retinoic acid (RA) is a vitamin A metabolite; CD103+ dendritic cells (DCs) acquire a tolerogenic phenotype through an RA-dependent mechanism, driving the differentiation of gut-homing regulatory T cells in the gut mucosa. Growing evidence shows bioactive molecules derived from lactic acid bacteria exhibit immunomodulatory activity by interacting with antigen presenting cells (APCs), stimulating their differentiation and altering their phenotype to further shape adaptive immunity. We utilized the monocytic THP-1 cell line as an in vitro model to examine the impact of the Lacticaseibacillus rhamnosus secretome (LrS) and interactions with RA on APC differentiation into a tolerogenic dendritic-like cell (DLC) phenotype. THP-1 monocytes were treated with Phorbol 12-myristate 13-acetate to drive differentiation into macrophages, then conditioned with the LrS and/or RA for 24, 48 or 72 hours. Expression of key DC markers CD11b, CD11c, DC-SIGN, CD83, CD80 and CD103 increased, while expression of proinflammatory marker CD64, co-stimulatory marker CD86 and MHC class II molecule HLA-DR was reduced. Cytokine profiling revealed LrS + RA co-treatment increased the production of immunoregulatory cytokines IL-10 and IL-1RA. Expression of NFKB1 and NFKB2, key genes involved in the activation of the NF-κB canonical and non-canonical pathways was induced by 48 hours of LrS conditioning, accompanied by increased expression of CD40 and IDO1, indicating an ability to drive DLC maturation and induce co-stimulatory capacity. Deciphering the mechanisms behind these types of microbiota-host interactions gives insight into how microbial metabolites can influence immune maturation and maintain immune homeostasis. Supported by Natural Sciences and Engineering Research Council of Canada Discovery Grant RGPIN-2017-05237

NK Cells Produce High Levels of IL-10 Early after Allogeneic Stem Cell Transplantation and Suppress Development of Acute GVHD

Natural killer (NK) cells reconstitute rapidly following allogeneic stem cell transplantation (allo-SCT) at a time when alloreactive T cell immunity is being established. We investigated very early NK cell reconstitution in 82 patients following T cell-depleted allo-SCT. NK cell number increased rapidly, exceeding T cell reconstitution such that the NK:T cell ratio was over 40 by day 14. NK cells at day 14 (NK-14) were donor-derived, intensely proliferating and expressed chemokine receptors targeted to lymphoid and peripheral tissue. Spontaneous production of the immunoregulatory cytokine IL-10 was observed in over 70% of cells and transcription of cytokines and growth factors was augmented. NK-14 cell number was inversely correlated with the incidence of grade II-IV acute graft versus host disease (GVHD). These findings reveal that robust reconstitution of immunoregulatory NK cells by day 14 after allo-SCT is an important determinant of clinical outcome and suggest NK cells may suppress development of the T cell-mediated alloreactive immune response through production of IL-10. DisclosuresNo relevant conflicts of interest to declare.

Glucocorticoids Reduce the Number and Function of CD19+CD24hiCD38hi Regulatory B Cells in Patients with Immune Thrombocytopenia

IntroductionIL-10-producing regulatory B cells (Bregs) are important modulators of immune regulation, and in humans this population has been shown to be enriched within the CD19+CD24hiCD38hi B cell population in peripheral blood. The production of IL-10 by Bregs can be induced through activation of B cells through toll like receptor (TLR) and B cell receptor (BCR) ligation, or indirectly through T cell-mediated activation via CD40:CD40L interactions. Loss of Breg-mediated suppression, either through numerical deficiency or loss of function, has been implicated in the pathogenesis of several autoimmune and inflammation-mediated diseases, including chronic Immune Thrombocytopenia (ITP; Li et al, 2012, Blood, 120:3318-3325). The purpose of this study was to extend this observation by interrogating the effect of systemic immunosuppressive treatment, in particular with glucocorticoids (GCs), on Bregs from the peripheral blood of patients with ITP.MethodsFollowing informed written consent, peripheral blood was drawn from patients with ITP registered at University Hospitals Bristol, UK, and healthy volunteers. Peripheral blood mononuclear cells were isolated by density gradient centrifugation, and multiparameter flow cytometry performed to quantify the frequency of CD19+CD24hiCD38hi B cells. For functional studies, CD19+ B cells and CD4+ T cells were isolated from the peripheral blood of healthy donors using fluorescence activated cell sorting (FACS) and subsequently co-cultured in a 1:1 ratio. Cells were activated with either anti-CD3 (indirect B cell activation via T cells) or a combination of CpG and IgM (direct B cell activation), in the presence or absence of the synthetic GC Dexamethasone (Dex). After 72 hours, intracellular IL-10 expression in B cells was quantified by flow cytometry.ResultsNo significant difference in the proportion of CD19+CD24hiCD38hi Bregs was seen between newly diagnosed ITP patients (n=7) before treatment had started, compared with healthy donors (n=7), (6.5% vs 8.2%, p=0.3176; Figure 1). However, patients receiving GC treatment (n=8) had a significant reduction in the proportion of Bregs when compared with both healthy donors (2.5% vs 8.2%, p=0.0012), and untreated ITP patients (2.5% vs 6.5%, p=0.0093). Similarly, ITP patients receiving another commonly used immunosuppressive drug, mycophenolate mofetil (MMF, n=10), had a significant reduction in the proportion of Bregs compared with both healthy donors (1.7% vs 8.2%, p=0.0004), and untreated ITP patients (1.7% vs 6.5%, p=0.0042). Patients treated with a thrombopoietin (TPO) mimetic (n=6) (not immunosuppressive) had no reduction in the proportion of Bregs compared with untreated ITP patients (5.37% vs 6.49% p=0.4452). Furthermore, in cultures activated by CpG and IgM, Dex did not significantly affect the number of IL-10-producing B cells (5.4% vs 5.4%, p=0.9232, n=10). However, in anti-CD3 activated cultures, Dex significantly reduced the proportion of IL-10 producing B cells (2.0% vs 0.8%, p=0.0094, n=8).ConclusionsThese observations suggest that a deficiency of Bregs in the peripheral blood is not a result of disease activity in ITP patients, but rather the consequence of immunosuppressive drug treatment. Consistent with this, the effect of Dex on B cell IL-10 production is likely to be an indirect phenomenon through the suppression of T cell-mediated activation of Breg function. Overall, these observations show that immunosuppressive treatments such as GCs can deplete Breg numbers in the peripheral blood and, through a T cell mediated mechanism, reduce their ability to produce the immunoregulatory cytokine IL-10. [Display omitted] DisclosuresBradbury:BMS Pfizer: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Other: Travel support; Bayer: Other: Conference attendence; Amgen: Other: Conference attendence. Lee:University of Bristol: Patents & Royalties: International Patent Application No: PCT/GB2014/053804 / US Patent application No. 61/919,404 (A biomarker and companion therapeutic for steroid refractory diseases); EMD Serono: Consultancy; Nanomerics: Consultancy; Roche: Consultancy, Research Funding.

IL-10 Receptor Deficiency Aggravates Exhaustion of CD8+ T-Cells and Impedes Their Control of Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is associated with substantial alteration in T-cell composition and function. However, the role of T-cells in CLL remains largely controversial. While T-cells can induce pro-survival signals in leukemic cells, there are several indications for existing, yet defective, anti-tumor immune responses in CLL patients. Here, we utilized the Eµ-TCL1 (TCL1) mouse model of CLL to delineate whether anti-tumoral immune responses exist in CLL, and characterize the phenotype and function of these potential anti-tumoral immune populations.First, we adoptively transferred TCL1 leukemic cells (TCL1 AT) into Rag2-/- mice, which lack B- and T-cells, and observed that these mice had significantly shorter survival compared to wild type (WT) recipients. Antibody-mediated depletion of CD8+, but not CD4+, T-cells (Figure 1A), or neutralization of IFNγ after TCL1 AT resulted in a substantial increase in CLL cell numbers in blood, bone marrow and secondary lymphoid organs, indicating that CD8+ T-cells play a crucial role in suppressing CLL progression in an IFNγ-dependent manner.Thus, we further studied the composition of CD8+ T-cells after TCL1 AT, and thereby identified a population of CD127low CD44int-hi effector cells that progressively increase along with disease. These cells presented the highest levels of activation markers (CD69, CD137 and GITR), degranulation capacity and Granzyme B (GzmB) production. Moreover, this population was composed of oligoclonal T-cells, as shown by T-cell receptor sequencing, indicating an enrichment of tumor-reactive T-cells. Of interest, expression of the inhibitory receptor, PD-1, was strictly confined to this effector CD8+ population, and we detected two distinct PD-1+ subsets, PD-1int and PD-1hi, that exhibited considerable phenotypical and functional differences. PD-1hi cells showed higher levels of activation, yet with increased expression of exhaustion markers (Lag3 and CD244). In contrast, PD-1int cells possessed superior functional capacity, as evident by higher cytokine (IFNγ, IL-2 and TNFα) release, stronger degranulation capacity and increased GzmB production. Gene expression profiling (GEP) revealed that PD-1int cells resemble effector T-cells in acute infections, while the PD-1hi subset is more similar to exhausted T-cells in chronic infections. Accordingly, PD-1hi cells expressed high levels of transcription factors mediating terminal differentiation, such as Batf and Blimp1, while PD-1int cells expressed memory transcription factors, like Tcf7.As GEP data showed that PD-1int cells expressed higher levels of Il10rb, we studied the role of the immunoregulatory cytokine IL-10 in controlling the balance between PD-1int and PD-1hi subsets. Using IL-10-GFP (tiger) mice, we observed IL-10 production by multiple cell types upon TCL1 AT. To assess the role of IL-10 in CLL, we treated CLL-bearing mice with IL-10R-blocking antibodies, and unexpectedly detected higher CLL load in all analyzed organs in anti-IL-10R-treated mice. Although effector CD8+ T-cells exhibited higher activation levels after IL-10R blockade, they were severely skewed towards PD-1hi cells with an almost complete loss of the PD-1int subset. This was accompanied by higher expression of inhibitory receptors and a substantial decrease in GzmB release and cytokine production. Moreover, GEP revealed a dramatic decrease in cell cycle and proliferation genes in PD-1hi cells following IL-10R blockade, which was further confirmed by Ki67 staining. To examine whether these effects are T-cell intrinsic, we transferred CD8+ T-cells from WT or Il10rb-/- mice into Rag2-/- mice followed by TCL1 AT. While WT T-cells substantially decreased the rate of CLL progression, Il10rb-/- cells were significantly inferior in controlling CLL in these mice (Figure 1B). Moreover, Il10rb-/- CD8+ T-cells were mostly PD-1hi cells with an increase in exhaustion markers and substantial drop in cytokine and GzmB production.Collectively, this study describes a novel role of IL-10 in maintaining immune responses in CLL by protecting anti-tumoral effector CD8+ T-cells from activation-induced exhaustion. Furthermore, these data suggest IL-10 as a therapeutic target in CLL, potentially in combination with immune checkpoint inhibitors. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

STING Agonist Mitigates Experimental Autoimmune Encephalomyelitis by Stimulating Type I IFN-Dependent and -Independent Immune-Regulatory Pathways.

The cGAS-cyclic GMP-AMP (cGAMP)-stimulator of IFN genes (STING) pathway induces a powerful type I IFN (IFN-I) response and is a prime candidate for augmenting immunity in cancer immunotherapy and vaccines. IFN-I also has immune-regulatory functions manifested in several autoimmune diseases and is a first-line therapy for relapsing-remitting multiple sclerosis. However, it is only moderately effective and can induce adverse effects and neutralizing Abs in recipients. Targeting cGAMP in autoimmunity is unexplored and represents a challenge because of the intracellular location of its receptor, STING. We used microparticle (MP)-encapsulated cGAMP to increase cellular delivery, achieve dose sparing, and reduce potential toxicity. In the C57BL/6 experimental allergic encephalomyelitis (EAE) model, cGAMP encapsulated in MPs (cGAMP MPs) administered therapeutically protected mice from EAE in a STING-dependent fashion, whereas soluble cGAMP was ineffective. Protection was also observed in a relapsing-remitting model. Importantly, cGAMP MPs protected against EAE at the peak of disease and were more effective than rIFN-β. Mechanistically, cGAMP MPs showed both IFN-I-dependent and -independent immunosuppressive effects. Furthermore, it induced the immunosuppressive cytokine IL-27 without requiring IFN-I. This augmented IL-10 expression through activated ERK and CREB. IL-27 and subsequent IL-10 were the most important cytokines to mitigate autoreactivity. Critically, cGAMP MPs promoted IFN-I as well as the immunoregulatory cytokines IL-27 and IL-10 in PBMCs from relapsing-remitting multiple sclerosis patients. Collectively, this study reveals a previously unappreciated immune-regulatory effect of cGAMP that can be harnessed to restrain T cell autoreactivity.

The chemokine CX3CL1/fractalkine regulates immunopathogenesis during fungal-associated allergic airway inflammation.

Individuals that present with difficult-to-control asthma and sensitivity to one or more fungal species are categorized as a subset of severe asthma patients belonging to a group herein referred to as severe asthma with fungal sensitization (SAFS). We have previously reported the identification of numerous cytokines and chemokines that were elevated in human asthmatics that were sensitized to fungi vs. nonfungal sensitized asthmatics. Here, we show that the unique chemokine CX3CL1 (fractalkine) is elevated in both bronchoalveolar lavage fluid and sputum from human asthmatics sensitized to fungi, implicating an association with CX3CL1 in fungal asthma severity. In an experimental model of fungal-associated allergic airway inflammation, we demonstrate that the absence of CX3CR1 signaling unexpectedly resulted in a profound impairment in lung function. Histological assessment of lung tissue revealed an unrestricted inflammatory response that was subsequently characterized by enhanced levels of neutrophils, eosinophils, and inflammatory monocytes. Neutrophilic inflammation correlated with elevated IL-17A, proinflammatory cytokines (TNF-α, IL-1α, and IL-1β), neutrophil survival factors (granulocyte colony-stimulating factor), and neutrophil-targeting chemokines (CCL3 and CCL4). Eosinophilia correlated with elevated type 2 responses (IL-5 and IL-13) whereas inflammatory monocyte levels correlated with elevated type 1 responses (IFN-γ and CXCL9) and survival factors (macrophage colony-stimulating factor). Despite enhanced inflammatory responses, the immunoregulatory cytokine IL-10 and the natural inhibitor of IL-1 signaling, IL-1RA, were significantly elevated rather than impaired. Regulatory T-cell levels were unchanged, as were levels of the anti-inflammatory cytokines IL-35 and IL-38. Taken together, the CX3CL1/CX3CR1 axis preserves lung function during fungal-associated allergic airway inflammation through a nonclassical immunoregulatory mechanism.

Escherichiacoli-Specific CD4+ T Cells Have Public T-Cell Receptors and Low Interleukin 10 Production in Crohn's Disease.

Background & AimsCrohn’s disease (CD) likely represents decreased immune tolerance to intestinal bacterial antigens. Most CD patients have high titers of antibodies to intestinal commensal proteins, including the outer membrane porin C (OmpC) of Escherichia coli.MethodsBy using major histocompatibility complex II tetramers, we identified an HLA-DRB1∗15:01-restricted peptide epitope of OmpC recognized by CD4+ T cells in peripheral blood mononuclear cells from HLA-DRB1∗15:01+ healthy control (HC) and CD patients.ResultsThe precursor frequency of these cells in CD correlated with anti-OmpC IgA titers, but did not differ from that of HCs. In both cohorts, they showed a CD161+, integrin α4β7+ phenotype ex vivo by flow cytometry, distinct from the C-X-C Motif Chemokine Receptor 3 phenotype of autologous influenza hemagglutinin (Flu) peptide-specific T cells. The T-cell receptor α and β chains of in vitro–expanded OmpC-specific T-cell clones often contained public amino acid sequences that were identical in cells from different patients. Expanded T-cell clones from CD subjects produced significantly less interleukin (IL)10 (P < .0001) than those from HCs, and a trend toward decreased production of the T helper 2 cell–associated IL4, IL5, and IL13 by CD clones also was seen.ConclusionsBoth HCs and CD patients have detectable OmpC-specific T cells in circulation, with similar immunophenotypes and often identical T-cell–receptor sequences. However, expanded clones from patients with CD produce less of the immunoregulatory cytokine IL10, showing a selective defect in the regulatory function of intestinal microbial antigen-specific T cells in patients with CD.

Differential Modulation of Human Innate Lymphoid Cell (ILC) Subsets by IL-10 and TGF-\u03b2

Using multiparameter flow cytometry human innate lymphoid cell (ILC) subsets can be detected in the circulation, in relatively low frequencies. Despite the low frequency of ILCs in circulation, ex vivo experiments have demonstrated that these ILCs release extremely large per cell quantities of signature ILC cytokines following activation. To determine how activated ILC cytokine production is regulated, ILC subsets were activated in the presence or absence of the immunoregulatory cytokines IL-10 and TGF-β. An examination of circulating ILC subsets revealed surface expression of IL-10Rα and mRNA expression of both IL-10Rα and TGF-βR1 for all ILC subsets. Stimulated ILC1 production of IFN-γ was decreased by TGF-β and not IL-10. Interestingly, ILC2s stimulated in the presence of IL-10 had a marked reduction in cytokine production of IL-5 and IL-13 while TGF-β had no effect on ILC2 cytokine production. Ex vivo activated ILC1 and ILC2 subsets were also found to be a source of the immunoregulatory cytokine IL-10, raising the potential for ILC-mediated regulation of immune cells. These findings demonstrate the differential effects of immunoregulatory cytokines IL-10 and TGF-β on activated ILC1 and ILC2 populations ex vivo.

CONTEMPORARY IDEAS ABOUT PATHOPHYSIOLOGICAL MECHANISMS OF IMMUNITY DESTRUCTION IN THE PRECLINICAL STAGE OF TYPE 1 DIABETES

The results of own studies on the role of the immune system in mechanisms for development of type 1 diabetes mellitus (T1DM) in humans that have been performed for 20 years in 612 normoglycemic children of Ukraine with a burden heredity which are positive and negative against pancreatic islet autoantibodies (OAA) are summarized in the article according to the program of the IPDM. There were confirmed data that the high titre of two (especially IA-2A and GADA) and more OAA is a reliable biomarker for diagnosing the presence of preclinical asymptomatic stage of T1DM and predicting its appearance in advance (sometimes for years) in still healthy people. The preclinical stage in OAA-positive children is characterized by moderate neutrophilopenia, monocytosis with a change in their ultrastructure, a decrease in the absolute number of CD3 + T, CD4 + T and CD8 + T lymphocytes and CD56 + cells. In the subpopulation of CD4 + T cells, a disorder of the submicroscopic organization of Holl's bodies is noted. At the same time, there is an increase in the level of proinflammatory cytokines IL-1α, IL-1β, IL-6 and TNFα, as well as chemokines IL-8 and IL-10 and simultaneous decrease of immunoregulatory cytokines IL-4 and IL-10. The obtained data indicate that already in the preclinical stage of T1DM immunity is significantly impaired in the body of normoglycemic children, that is consistent with the recently expressed opinion that the T1DM should be diagnosed not only when most of the beta cells have been destroyed, but in the stage of pre-diabetes, when immune intervention preventing the progression of the disease is still possible.

Macrophage galactose-type lectin (MGL) is induced on M2 microglia and participates in the resolution phase of autoimmune neuroinflammation

BackgroundMultiple sclerosis (MS) involves a misdirected immune attack against myelin in the brain and spinal cord, leading to profound neuroinflammation and neurodegeneration. While the mechanisms of disease pathogenesis have been widely studied, the suppression mechanisms that lead to the resolution of the autoimmune response are still poorly understood. Here, we investigated the role of the C-type lectin receptor macrophage galactose-type lectin (MGL), usually expressed on tolerogenic antigen-presenting cells (APCs), as a negative regulator of autoimmune-driven neuroinflammation.MethodsWe used in silico, immunohistochemical, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry analysis to explore the expression and functionality of MGL in human macrophages and microglia, as well as in MS post-mortem tissue. In vitro, we studied the capacity of MGL to mediate apoptosis of experimental autoimmune encephalomyelitis (EAE)-derived T cells and mouse CD4+ T cells. Finally, we evaluated in vivo and ex vivo the immunomodulatory potential of MGL in EAE.ResultsMGL plays a critical role in the resolution phase of EAE as MGL1-deficient (Clec10a−/−) mice showed a similar day of onset but experienced a higher clinical score to that of WT littermates. We demonstrate that the mouse ortholog MGL1 induces apoptosis of autoreactive T cells and diminishes the expression of pro-inflammatory cytokines and inflammatory autoantibodies. Moreover, we show that MGL1 but not MGL2 induces apoptosis of activated mouse CD4+ T cells in vitro. In human settings, we show that MGL expression is increased in active MS lesions and on alternatively activated microglia and macrophages which, in turn, induces the secretion of the immunoregulatory cytokine IL-10, underscoring the clinical relevance of this lectin.ConclusionsOur results show a new role of MGL-expressing APCs as an anti-inflammatory mechanism in autoimmune neuroinflammation by dampening pathogenic T and B cell responses, uncovering a novel clue for neuroprotective therapeutic strategies with relevance for in MS clinical applications.

Repeated clinical malaria episodes are associated with modification of the immune system in children

BackgroundThere are over 200 million reported cases of malaria each year, and most children living in endemic areas will experience multiple episodes of clinical disease before puberty. We set out to understand how frequent clinical malaria, which elicits a strong inflammatory response, affects the immune system and whether these modifications are observable in the absence of detectable parasitaemia.MethodsWe used a multi-dimensional approach comprising whole blood transcriptomic, cellular and plasma cytokine analyses on a cohort of children living with endemic malaria, but uninfected at sampling, who had been under active surveillance for malaria for 8 years. Children were categorised into two groups depending on the cumulative number of episodes experienced: high (≥ 8) or low (< 5).ResultsWe observe that multiple episodes of malaria are associated with modification of the immune system. Children who had experienced a large number of episodes demonstrated upregulation of interferon-inducible genes, a clear increase in circulating levels of the immunoregulatory cytokine IL-10 and enhanced activation of neutrophils, B cells and CD8+ T cells.ConclusionTranscriptomic analysis together with cytokine and immune cell profiling of peripheral blood can robustly detect immune differences between children with different numbers of prior malaria episodes. Multiple episodes of malaria are associated with modification of the immune system in children. Such immune modifications may have implications for the initiation of subsequent immune responses and the induction of vaccine-mediated protection.