CD39+ Regulatory T cells suppress generation and differentiation of Th17 cells in human malignant pleural effusion via a LAP-dependent mechanism

Background

Both regulatory T cells (Tregs) and T helper IL-17-producing cells (Th17 cells) have been found to be involved in human malignancies, however, the possible implication of Tregs in regulating generation and differentiation of Th17 cells in malignant pleural effusion remains to be elucidated.

Methods

The numbers of both CD39⁺Tregs and Th17 cells in malignant pleural effusion and peripheral blood from patients with lung cancer were determined by flow cytometry. The regulation and mechanism of Tregs on generation and differentiation of Th17 cells were explored.

Results

Both CD39⁺Tregs and Th17 cells were increased in malignant pleural effusion when compared with blood, and the numbers of CD39⁺Tregs were correlated negatively with those of Th17 cells. It was also noted that high levels of IL-1β, IL-6, and TGF-β1 could be observed in malignant pleural effusion when compared the corresponding serum, and that pleural CD39⁺Tregs could express latency-associated peptide on their surface. When naïve CD4⁺ T cells were cocultured with CD39⁺Tregs, Th17 cell numbers decreased as CD39⁺Treg numbers increased, addition of the anti-latency-associated peptide mAb to the coculture reverted the inhibitory effect exerted by CD39⁺Tregs.

Conclusions

Therefore, the above results indicate that CD39⁺Tregs inhibit generation and differentiation of Th17 cells via a latency-associated peptide-dependent mechanism.     

Expression and Function of TNF and IL-1 Receptors on Human Regulatory T Cells

Regulatory T cells (Tregs) suppress immune activation and are critical in preventing autoimmune diseases. While the ability of Tregs to inhibit proliferation of other T cells is well established, it is not yet clear whether Tregs also modulate inflammatory cytokines during an immune response. Here, we show that the expression of inflammatory cytokine receptors IL-1R1 and TNFR2 were higher on resting mature Tregs compared to naïve or memory T cells. While upon activation through the T cell receptor (TCR), expression of IL-1R1 and TNFR2 were upregulated on all T cell subsets, IL-1R1 maintained significantly higher expression on activated Tregs as compared to other T cell subsets. The decoy receptor for IL-1 (IL-1R2) was not expressed by any of the resting T cells but was rapidly upregulated and preferentially expressed upon TCR-stimulation on Tregs. In addition, we found that Tregs also expressed high levels of mRNA for IL-1 antagonist, IL-1RA. TCR-stimulation of naïve T cells in the presence of TGFβ, which induces FOXP3 expression, however did not result in upregulation of IL-1R1 or IL-1R2. In addition, ectopic expression of FOXP3 in non-Tregs, while causing significant upregulation of IL-1R1 and IL-1R2, did not achieve the levels seen in bona fide Tregs. We also determined that resting human Tregs expressing IL-1R1 did not have higher suppressive capacity compared to IL-1R1- Tregs, suggesting that IL-1R1 does not discriminate suppressive resting Tregs in healthy individuals. Functionally, activated human Tregs displayed a capacity to neutralize IL-1β, which suggests a physiological significance for the expression of IL-1 decoy receptor on Tregs. In conclusion, our findings that human Tregs preferentially express receptors for TNF and IL-1 suggest a potential function in sensing and dampening local inflammation.     

Decreased interleukin 27 expression is associated with active uveitis in Beh?et’s disease

Instruction

Interleukin 27 (IL-27) is an important regulator of the proinflammatory T-cell response. In this study, we investigated its role in the pathogenesis of Behçet’s disease (BD).

Methods

IL-27 mRNA in peripheral blood mononuclear cells (PBMCs) was examined by performing RT-PCRs. Cytokine levels in sera or supernatants of PBMCs, naïve CD4⁺ T cells, dendritic cells (DCs) and DC/T cells were determined by enzyme-linked immunosorbent assay. We used RNA interference in naïve CD4⁺ T cells to study the role of interferon regulatory factor 8 (IRF8) in the inhibitory effect of IL-27 on Th17 cell differentiation. Flow cytometry was used to evaluate the frequency of IL-17- and interferon γ–producing T cells.

Results

The expression of IL-27p28 mRNA by PBMCs and IL-27 in the sera and supernatants of cultured PBMCs were markedly decreased in patients with active BD. A higher frequency of IL-17-producing CD4⁺ T (Th17) cells and increased IL-17 production under Th17 polarizing conditions were observed in patients with active BD. IL-27 significantly inhibited Th17 cell differentiation. Downregulation of IRF8 by RNA interference abrogated the suppressive effect of IL-27 on Th17 differentiation. IL-27 inhibited the production of IL-1β, IL-6 and IL-23, but promoted IL-10 production, by DCs. IL-27-treated DCs inhibited both the Th1 and Th17 cell responses.

Conclusions

The results of the present study suggest that a decreased IL-27 expression is associated with disease activity in BD patients. Low IL-27 expression may result in a higher Th1 and Th17 cell response and thereby promote the autoinflammatory reaction observed in BD. Manipulation of IL-27 may offer a new treatment modality for this disease.     

Superior Cervical Ganglia Neurons Induce Foxp3+ Regulatory T Cells via Calcitonin Gene-Related Peptide

The nervous and immune systems communicate bidirectionally, utilizing diverse molecular signals including cytokines and neurotransmitters to provide an integrated response to changes in the body’s internal and external environment. Although, neuro-immune interactions are becoming better understood under inflammatory circumstances and it has been evidenced that interaction between neurons and T cells results in the conversion of encephalitogenic T cells to T regulatory cells, relatively little is known about the communication between neurons and naïve T cells. Here, we demonstrate that following co-culture of naïve CD4⁺ T cells with superior cervical ganglion neurons, the percentage of Foxp3 expressing CD4⁺CD25⁺ cells significantly increased. This was mediated in part by immune-regulatory cytokines TGF-β and IL-10, as well as the neuropeptide calcitonin gene-related peptide while vasoactive intestinal peptide was shown to play no role in generation of T regulatory cells. Additionally, T cells co-cultured with neurons showed a decrease in the levels of pro-inflammatory cytokine IFN-γ released upon in vitro stimulation. These findings suggest that the generation of Tregs may be promoted by naïve CD4⁺ T cell: neuron interaction through the release of neuropeptide CGRP.     

p38α Protein Negatively Regulates T Helper Type 2 Responses by Orchestrating Multiple T Cell Receptor-associated Signals

Mitogen-activated protein kinase p38α is a critical regulator of certain inflammatory diseases. However, its role in T helper type 2 (Th2) responses and allergic inflammation remains unknown. Here we show an increase in the production of interleukin-4 (IL-4) in p38α^−/− CD4⁺ T cells in response to antigen stimulation. p38α-deficient naïve CD4⁺ T cells preferentially differentiate into Th2 cells through increased endogenous production of IL-4. Consistent with those results, we also observed decreased expression of p38α during T helper cell differentiation. Furthermore, deficiency of p38α alters the balance in the expression of NFATc1 and NFATc2 under steady-state conditions and enhances the expression and nuclear translocation of NFATc1 in CD4⁺ T cells upon antigen stimulation. Knockdown of NFATc1 significantly inhibits Th2 differentiation in p38α^−/− T cells but not in p38α^+/− T cells. p38α deficiency also inhibits the activation of Akt but enhances the activation of ERK in response to T cell receptor engagement without impacting IL-2/Stat5 signaling. In a model of ovalbumin-induced acute allergic airway inflammation, mice with induced deletion of p38α show elevated serum ovalbumin-specific IgE level, increased infiltration of eosinophils, and higher concentrations of Th2 cytokines including IL-4 and IL-5 in the bronchoalveolar lavage fluid relative to control mice. Taken together, p38α regulates multiple T cell receptor-associated signals and negatively influences Th2 differentiation and allergic inflammation.     

Dendritic Cells Induce a Subpopulation of IL-12Rβ2-Expressing Treg that Specifically Consumes IL-12 to Control Th1 Responses

Cytokines secreted from dendritic cells (DCs) play an important role in the regulation of T helper (Th) cell differentiation and activation into effector cells. Therefore, controlling cytokine secretion from DCs may potentially regulate Th differentiation/activation. DCs also induce de-novo generation of regulatory T cells (Treg) that modulate the immune response. In the current study we used the mixed leukocyte reaction (MLR) to investigate the effect of allospecific Treg on IL-12, TNFα and IL-6 secretion by DCs. Treg cells were found to markedly down-regulate IL-12 secretion from DCs following stimulation with TLR7/8 agonist. This down-regulation of IL-12 was neither due to a direct suppression of its production by the DCs nor a result of marked DC death. We found that IL-12 was rather actively consumed by Treg cells. IL-12 consumption was mediated by a subpopulation of IL-12Rβ2-expressing Treg cells and was dependent on MHC class-II expressed on dendritic cells. Furthermore, IL-12 consumption by Tregs increased their suppressive effect on T cell proliferation and Th1 activation. These results provide a new pathway of Th1 response regulation where IL-12 secreted by DCs is consumed by a sub-population of IL-12Rβ2-expressing Treg cells. Consumption of IL-12 by Tregs not only reduces the availability of IL-12 to Th effector cells but also enhances the Treg immunosuppressive effect. This DC-induced IL-12Rβ2-expressing Treg subpopulation may have a therapeutic advantage in suppressing Th1 mediated autoimmunity.     

Exposure to Tobacco Smoking Induces a subset of Activated Tumor-resident Tregs in Non-Small Cell Lung Cancer

Tobacco smoking is the major cause of non-small-cell-lung cancer (NSCLC). However, it is barely known how smoking impact the tumor immune environment (TIME) of lung cancer.We integrated single-cell RNA-seq and bulk RNA-seq data from several studies to systematically study the impact of smoking on T cells in treatment naïve NSCLC patients. We defined a set of smoking-induced differentially expressed genes (SIDEGs) in different cells in TIME.. Specifically, we defined a smoking-related tumor-specific Treg subset, ADAM12⁺ CTLA4⁺ Tregs according to the trajectory analysis and highly express genes in cell adhesion pathways and lipid metabolism. Using independent datasets from treatment naïve patients, we found that the fraction of ADAM12⁺ CTLA4⁺ Tregs are significantly increased in patients with smoking history. Moreover, the fraction of ADAM12⁺ CTLA4⁺ Tregs are positively correlated with the fraction of exhausted T cells. Additionally, we reconstructed the spatial organization of the tumor immune microenvironment and found that ADAM12⁺ CTLA4⁺ Tregs more actively communicate with LAYN⁺CD8⁺ exhausted T cells compared with ADAM12⁻CTLA4⁺ Tregs.Our data demonstrate that smoking induced a unique subset of tumor-specific activated Tregs which interact with exhausted T cells in the TIME. Our findings not only explained how smoking impact the TIME but also provide new targets and biomarkers for precision immunotherapy of lung cancer.     

The Role of SnoN in Transforming Growth Factor β1-induced Expression of Metalloprotease-Disintegrin ADAM12

Increased expression of metalloprotease-disintegrin ADAM12 is a hallmark of several pathological conditions, including cancer, cardiovascular disease, and certain inflammatory diseases of the central nervous system or the muscoskeletal system. We show that transforming growth factor β1 (TGFβ1) is a potent inducer of ADAM12 mRNA and protein in mouse fibroblasts and in mouse and human mammary epithelial cells. Induction of ADAM12 is detected within 2 h of treatment with TGFβ1, is Smad2/Smad3-dependent, and is a result of derepression of the Adam12 gene. SnoN, a negative regulator of the TGFβ signaling pathway, is a master regulator of ADAM12 expression in response to TGFβ1 stimulation. Overexpression of SnoN in NIH3T3 cells reduces the magnitude of ADAM12 induction by TGFβ1 treatment. Down-regulation of SnoN expression by short hairpin RNA enhances TGFβ1-induced expression of ADAM12. In a panel of TGFβ1-responsive cancer cell lines with high expression of SnoN, induction of ADAM12 by TGFβ1 is significantly impaired, suggesting that the endogenous SnoN plays a role in regulating ADAM12 expression in response to TGFβ1. Identification of SnoN as a repressor of the ADAM12 gene should contribute to advances in the studies on the role of ADAM12 in tumor progression and in the development of other pathologies.     

IFN-β Inhibits the Increased Expression of IL-9 during Experimental Autoimmune Uveoretinitis

Purpose

It has been shown that IL-9 plays a proinflammatory role in the pathogenesis of certain autoimmune diseases. This study was designed to investigate the possible role of IL-9 in the development of experimental autoimmune uveoretinitis (EAU) and the effect of IFN-β on its expression.

Methods

EAU was induced in B10RIII mice by immunization with interphotoreceptor retinoid-binding protein peptide 161–180 (IRBP_161–180). IFN-β was administered subcutaneously to IRBP_161–180 immunized mice every other day from day one before immunization to the end of the study. Splenocytes and draining lymph node (DLN) cells from EAU mice or control mice or EAU mice treated with IFN-β or PBS were stimulated with anti-CD3/CD28 or IRBP_161–180 for 3 days. Naïve T cells cultured under Th1 or Th17 polarizing conditions were incubated in the presence or absence of IFN-β for 4 days. Effector/memory T cells were activated by anti-CD3/CD28 in the presence or absence of IFN-β for 3 days. IFN-β-treated monocytes were cocultured with naïve T cells or effector/memory T cells for 3 days. Culture supernatants were collected and IL-9 was detected by ELISA.

Results

IL-9 expression in splenocytes and DLN cells was increased in EAU mice during the inflammatory phase and returned back to lower levels during the recovery phase. IFN-β in vivo treatment significantly inhibited EAU activity in association with a down-regulated expression of IL-9. In vitro polarized Th1 and Th17 cells both secreted IL-9 and the addition of IFN-β suppressed production of IL-9 by both Th subsets. Beside its effect on polarized Th cells, IFN-β also suppressed the secretion of IL-9 by effector/memory T cells. However, IFN-β-treated monocytes had no effect on the production of IL-9 when cocultured with naïve or effector/memory T cells.

Conclusion

IL-9 expression is increased during EAU which could be suppressed by IFN-β.     

Novel IL27p28/IL12p40 Cytokine Suppressed Experimental Autoimmune Uveitis by Inhibiting Autoreactive Th1/Th17 Cells and Promoting Expansion of Regulatory T Cells

IL-12 family cytokines are important in host immunity. Whereas some members (IL-12, IL-23) play crucial roles in pathogenesis of organ-specific autoimmune diseases by inducing the differentiation of Th1 and Th17 lymphocytes, others (IL-27 and IL-35) suppress inflammatory responses and limit tissue injury induced by these T cell subsets. In this study, we have genetically engineered a novel IL27p28/IL12p40 heterodimeric cytokine (p28/p40) that antagonizes signaling downstream of the gp130 receptor. We investigated whether p28/p40 can be used to ameliorate uveitis, a CNS inflammatory disease. Experimental autoimmune uveitis (EAU) is the mouse model of human uveitis and is mediated by Th1 and Th17 cells. We show here that p28/p40 suppressed EAU by inhibiting the differentiation and inflammatory responses of Th1 and Th17 cells while promoting expansion of IL-10⁺- and Foxp3⁺-expressing regulatory T cells. Lymph node cells from mice treated with p28/p40 blocked adoptive transfer of EAU to naïve syngeneic mice by immunopathogenic T cells and suppressive effects of p28/p40 derived in part from antagonizing STAT1 and STAT3 pathways induced by IL-27 and IL-6. Interestingly, IL27p28 also suppressed EAU, but to a lesser extent than p28/p40. The inhibition of uveitogenic lymphocyte proliferation and suppression of EAU by p28/p40 and IL27p28 establish efficacy of single chain and heterodimeric IL-12 family cytokines in treatment of a CNS autoimmune disease. Creation of the biologically active p28/p40 heterodimeric cytokine represents an important proof-of-concept experiment, suggesting that cytokines comprising unique IL-12 α- and β-subunit pairing may exist in nature and may constitute a new class of therapeutic cytokines.     

Histone Deacetylase Inhibitors Modulate Interleukin 6-dependent CD4+ T Cell Polarization in Vitro and in Vivo

Histone deacetylase (HDAC) inhibitors have been associated primarily with an anti-proliferative effect in vitro and in vivo. Recent data provide evidence for an anti-inflammatory potency of HDAC inhibitors in models of experimental colitis. Because the balance of T cell subpopulations is critical for the balance of the mucosal immune system, this study explores the regulatory potency of HDAC inhibitors on T cell polarization as a mechanistic explanation for the observed anti-inflammatory effects. Although HDAC inhibition suppressed the polarization toward the pro-inflammatory T helper 17 (Th17) cells, it enhanced forkhead box P3 (FoxP3)⁺ regulatory T cell polarization in vitro and in vivo at the site of inflammation in the lamina propria. This was paralleled by a down-regulation of the interleukin 6 receptor (IL-6R) on naïve CD4⁺ T cells on the mRNA as well as on the protein level and changes in the chromatin acetylation at the IL6R gene and its promoter. Downstream of the IL-6R, HDAC inhibition was followed by a decrease in STAT3 phosphorylation as well as retinoic acid receptor-related orphan receptor γT (RORγT) expression, thus identifying the IL-6/STAT3/IL-17 pathway as an important target of HDAC inhibitors. These results directly translated to experimental colitis, where IL-6R expression was suppressed in naïve T cells, paralleled by a significant reduction of Th17 cells in the lamina propria of ITF2357-treated animals, resulting in the amelioration of disease. This study indicates that, in experimental colitis, inhibition of HDAC exerts an anti-inflammatory potency by directing T helper cell polarization via targeting the IL-6 pathway.     

Ursolic acid inhibits Th17 cell differentiation via STAT3/RORγt pathway and suppresses Schwann cell-mediated Th17 cell migration by reducing CXCL9/10 expression

Th17 cells represent important immune cells. Ursolic acid (UA) can regulate immune cell activities. This study was aimed to explore the effects of UA on Th17 cell differentiation and Schwann cell(SCs)-mediated migration and the potential mechanism. Naïve CD4⁺ T cells were isolated from rat peripheral blood, induced for Th17 cell differentiation, and treated with UA. The proportion of Th17 cells was detected by flow cytometry assay. SCs were co-cultured with Th17 cells. Th17 cell migration was detected by Transwell assay. The molecule expression was determined by Western blot and qRT-PCR. UA inhibited the Th17 cell differentiation and suppressed the STAT3/RORγt pathway. STAT3 overexpression up-regulated p-STAT3 and RORγt expression and promoted Th17 cell differentiation under the UA treatment. In LPS- and IFN-γ-stimulated-SCs, UA suppressed the expression of chemokines CXCL9/10, but had no significant effect of CCL20 expression. The expression CXCL9/10 receptor CXCR3 was higher in Th17 cells than that in Treg cells, while the expression CCL20 receptor CCR6 was lower in Th17 cells than that in Treg cells. UA, anti-CXCR3, and anti-CCR6 treatment inhibited SCs-mediated Th17 cell migration, and anti-CXCR3 exerted stronger inhibitory effect than Anti-CCR6. UA inhibited Th17 cell differentiation through STAT3/RORγt pathway and suppressed Th17 cell migration through down-regulating CXCL9/10 expression in SCs.     

Interferon-α-conditioned human monocytes combine a Th1-orienting attitude with the induction of autologous Th17 responses: role of IL-23 and IL-12

IFN-α exerts multiple effects leading to immune protection against pathogens and cancer as well to autoimmune reactions by acting on monocytes and dendritic cells. We analyzed the versatility of human monocytes conditioned by IFN-α towards dendritic cell differentiation (IFN-DC) in shaping the autologous T-helper response. Priming of naïve CD4 T cells with autologous IFN-DC in the presence of either SEA or anti-CD3, resulted, in addition to a prominent expansion of CXCR3+ IFN-γ-producing CD4 Th1 cells, in the emergence of two distinct subsets of IL-17-producing CD4 T cells: i) a predominant Th17 population selectively producing IL-17 and expressing CCR6; ii) a minor Th1/Th17 population, producing both IL-17 and IFN-γ. After phagocytosis of apoptotic cells, IFN-DC induced Th17 cell expansion and IL-17 release. Notably, the use of neutralizing antibodies revealed that IL-23 was an essential cytokine in mediating Th17 cell development by IFN-DC. The demonstration of the IFN-DC-induced expansion of both Th1 and Th17 cell populations reveals the intrinsic plasticity of these DC in orienting the immune response and provides a mechanistic link between IFN-α and the onset of autoimmune phenomena, which have been correlated with both IL-17 production and exposure to IFN-α.     

Ustekinumab Improves Psoriasis without Altering T Cell Cytokine Production,Differentiation, and T Cell Receptor Repertoire Diversity

Ustekinumab is a fully human IgG1κ monoclonal antibody targeting interleukin (IL)-12/23 p40 subunit. The role of IL-12/23-mediated pathway in the mechanism of various inflammatory disorders especially psoriasis has been well recognized. Recently the long-term efficacy and safety of ustekinumab in patients with moderate-to-severe psoriasis has been evaluated in phase 2/3 clinical trials, and the results showed no significant risk for serious adverse effects, infections, or malignancies. Ustekinumab inhibits the function of the IL-12/23 p40 subunit, and therefore it is believed that inhibition of IL-12 p40 pathway decreases IFN-γ production. The major concern for the use of ustekinumab is the possibility of increased immunosuppression due to low IFN-γ production. However, the effects of ustekinumab on CD4⁺ T cell function have not been fully investigated so far. In this study, we explored changes in cytokine production by memory CD4⁺ T cells as well as in the differentiation of naïve T cells to helper T cell (Th) 1, Th2, or Th17 cells in psoriasis patients treated with ustekinumab. The effect of the treatment on T cell receptor repertoire diversity was also evaluated. The results showed that ustekinumab improves clinical manifestation in patients with psoriasis without affecting cytokine production in memory T cells, T cell maturation, or T cell receptor repertoire diversity. Although the number of patients is limited, the present study suggests that T cell immune response remains unaffected in psoriasis patients treated with ustekinumab.     

Heat shock protein X purified from Mycobacterium tuberculosis enhances the efficacy of dendritic cells-based immunotherapy for the treatment of allergic asthma

Dendritic cells play an important role in determining whether naïve T cells mature into either Th1 or Th2 cells. We determined whether heat-shock protein X (HspX) purified from Mycobacterium tuberculosis regulates the Th1/Th2 immune response in an ovalbumin (OVA)-induced murine model of asthma. HspX increased interferon-gamma, IL-17A, -12 and transforming growth factor (TGF)-β production and T-bet gene expression but reduced IL-13 production and GATA-3 gene expression. HspX also inhibited asthmatic reactions as demonstrated by an increase in the number of eosinophils in bronchoalveolar lavage fluid, inflammatory cell infiltration in lung tissues, airway luminal narrowing, and airway hyper-responsiveness. Furthermore, HspX enhanced OVA-induced decrease of regulatory T cells in the mediastinal lymph nodes. This study provides evidence that HspX plays critical roles in the amelioration of asthmatic inflammation in mice. These findings provide new insights into the immunotherapeutic role of HspX with respect to its effects on a murine model of asthma. BMB Reports 2015; 48(3): 178-183]     

Naïve and Memory CD4 T Cells Differ in Their Susceptibilities to Human Immunodeficiency Virus Type 1 Infection following CD28 Costimulation: Implications for Transmission and Pathogenesis

In vitro evidence suggests that memory CD4⁺ cells are preferentially infected by human immunodeficiency virus type 1 (HIV-1), yet studies of HIV-1-infected individuals have failed to detect preferential memory cell depletion. To explore this paradox, we stimulated CD45RA⁺ CD4⁺ (naïve) and CD45RO⁺ CD4⁺ (memory) cells with antibodies to CD3 and CD28 and infected them with either CCR5-dependent (R5) or CXCR4-dependent (X4) HIV-1 isolates. Naïve CD4⁺ cells supported less X4 HIV replication than their memory counterparts. However, naïve cells were susceptible to R5 viral infection, while memory cells remained resistant to infection and viral replication. As with the unseparated cells, mixing the naïve and memory cells prior to infection resulted in cells resistant to R5 infection and highly susceptible to X4 infection. While both naïve and memory CD4⁺ subsets downregulated CCR5 expression in response to CD28 costimulation, only the memory cells produced high levels of the β-chemokines RANTES, MIP-1α, and MIP-1β upon stimulation. Neutralization of these β-chemokines rendered memory CD4⁺ cells highly sensitive to infection with R5 HIV-1 isolates, indicating that downregulation of CCR5 is not sufficient to mediate complete protection from CCR5 strains of HIV-1. These results indicate that susceptibility to R5 HIV-1 isolates is determined not only by the level of CCR5 expression but also by the balance of CCR5 expression and β-chemokine production. Furthermore, our results suggest a model of HIV-1 transmission and pathogenesis in which naïve rather than memory CD4⁺ T cells serve as the targets for early rounds of HIV-1 replication.