Serotonin decreases the production of Th1/Th17 cytokines and elevates the frequency of regulatory CD4+ T‐cell subsets in multiple sclerosis patients

Excessive levels of proinflammatory cytokines in the CNS are associated with reduced serotonin (5‐HT) synthesis, a neurotransmitter with diverse immune effects. In this study, we evaluated the ability of exogenous 5‐HT to modulate the T‐cell behavior of patients with MS, a demyelinating autoimmune disease mediated by Th1 and Th17 cytokines. Here, 5‐HT attenuated, in vitro, T‐cell proliferation and Th1 and Th17 cytokines production in cell cultures from MS patients. Additionally, 5‐HT reduced IFN‐γ and IL‐17 release by CD8⁺ T cells. By contrast, 5‐HT increased IL‐10 production by CD4⁺ T cells from MS patients. A more accurate analysis of these IL‐10‐secreting CD4⁺ T cells revealed that 5‐HT favors the expansion of FoxP3⁺CD39⁺ regulatory T cells (Tregs) and type 1 regulatory T cells. Notably, this neurotransmitter also elevated the frequency of Treg17 cells, a novel regulatory T‐cell subset. The effect of 5‐HT in upregulating CD39⁺ Treg and Treg17 cells was inversely correlated with the number of active brain lesions. Finally, in addition to directly reducing cytokine production by purified Th1 and Th17 cells, 5‐HT enhanced in vitro Treg function. In summary, our data suggest that serotonin may play a protective role in the pathogenesis of MS.     

Reduced TCR‐dependent activation through citrullination of a T‐cell epitope enhances Th17 development by disruption of the STAT3/5 balance

Citrullination is a post‐translational modification of arginine that commonly occurs in inflammatory tissues. Because T‐cell receptor (TCR) signal quantity and quality can regulate T‐cell differentiation, citrullination within a T‐cell epitope has potential implications for T‐cell effector function. Here, we investigated how citrullination of an immunedominant T‐cell epitope affected Th17 development. Murine naïve CD4⁺ T cells with a transgenic TCR recognising p89‐103 of the G1 domain of aggrecan (agg) were co‐cultured with syngeneic bone marrow‐derived dendritic cells (BMDC) presenting the native or citrullinated peptides. In the presence of pro‐Th17 cytokines, the peptide citrullinated on residue 93 (R93Cit) significantly enhanced Th17 development whilst impairing the Th2 response, compared to the native peptide. T cells responding to R93Cit produced less IL‐2, expressed lower levels of the IL‐2 receptor subunit CD25, and showed reduced STAT5 phosphorylation, whilst STAT3 activation was unaltered. IL‐2 blockade in native p89‐103‐primed T cells enhanced the phosphorylated STAT3/STAT5 ratio, and concomitantly enhanced Th17 development. Our data illustrate how a post‐translational modification of a TCR contact point may promote Th17 development by altering the balance between STAT5 and STAT3 activation in responding T cells, and provide new insight into how protein citrullination may influence effector Th‐cell development in inflammatory disorders.     

Alloreactive regulatory T cells generated with retinoic acid prevent skin allograft rejection

CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells mediate immunological self‐tolerance and suppress immune responses. Retinoic acid (RA), a natural metabolite of vitamin A, has been reported to enhance the differentiation of Treg cells in the presence of TGF‐β. In this study, we show that the co‐culture of naive T cells from C57BL/6 mice with allogeneic antigen‐presenting cells (APCs) from BALB/c mice in the presence of TGF‐β, RA, and IL‐2 resulted in a striking enrichment of Foxp3⁺ T cells. These RA in vitro‐induced regulatory T (RA‐iTreg) cells did not secrete Th1‐, Th2‐, or Th17‐related cytokines, showed a nonbiased homing potential, and expressed several cell surface molecules related to Treg‐cell suppressive potential. Accordingly, these RA‐iTreg cells suppressed T‐cell proliferation and inhibited cytokine production by T cells in in vitro assays. Moreover, following adoptive transfer, RA‐iTreg cells maintained Foxp3 expression and their suppressive capacity. Finally, RA‐iTreg cells showed alloantigen‐specific immunosuppressive capacity in a skin allograft model in immunodeficient mice. Altogether, these data indicate that functional and stable allogeneic‐specific Treg cells may be generated using TGF‐β, RA, and IL‐2. Thus, RA‐iTreg cells may have a potential use in the development of more effective cellular therapies in clinical transplantation.     

IL4I1: Key immunoregulator at a crossroads of divergent T‐cell functions

The interleukin (IL)‐4‐induced gene1 (IL4I1), which encodes the L‐amino acid oxidase enzyme, plays an important immunoregulatory role. Indeed, this enzyme which is produced by B cells—including neoplastic B cells—dendritic cells and macrophages has been shown to inhibit proliferation, cytotoxicity and IFN‐γ production by tumor‐infiltrating CD8⁺ T cells, thus favoring tumor escape. Moreover, the same gene has been found to be constitutively expressed by CD4⁺ T helper 17 (Th17) cells, where it down‐regulates cell proliferation through a reduction of CD3 chains expression in the T‐cell receptor complex, thus impairing IL‐2 production, and by maintaining in the same cells a high expression of Tob1, which inhibits cell cycle entry, through a still unknown mechanism. Finally, IL4I1 has been shown to drive the differentiation of naive T cells into inducible regulatory T (iTreg) cells. Taken together, IL4I1 down‐regulates the effector CD8⁺ T‐cell response, promotes the development of iTreg cells and limits the expansion of Th17 cells, thus not only favoring tumor escape, but also reducing the potentially dangerous effects of adaptive immune responses in chronic inflammatory disorders.     

IL4‐induced gene 1 is secreted at the immune synapse and modulates TCR activation independently of its enzymatic activity

Amino‐acid catabolizing enzymes produced by mononuclear phagocytes play a central role in regulating the immune response. The mammalian phenylalanine‐catabolizing enzyme IL4‐induced gene 1 (IL4I1) inhibits effector T lymphocyte proliferation and facilitates regulatory T‐cell development. IL4I1 expression by macrophages of various human tumors may affect patient prognosis as it facilitates tumor escape from the T‐cell response in murine models. Its enzymatic activity appears to participate in its effects, but some actions of IL4I1 remain unclear. Here, we show that the presence of IL4I1 during T‐cell activation decreases early signaling events downstream of TCR stimulation, resulting in global T‐cell inhibition which is more pronounced when there is CD28 costimulation. Surprisingly, the enzymatic activity of IL4I1 is not involved. Focal secretion of IL4I1 into the immune synaptic cleft and its binding to CD3⁺ lymphocytes could be important in IL4I1 immunosuppressive mechanism of action.     

Enrichment of regulatory T cells in invasive breast tumor correlates with the upregulation of IL‐17A expression and invasiveness of the tumor

Breast cancer is a leading cause of neoplasia‐associated death in women worldwide. Regulatory T (Treg) and Th17 cells are enriched within some tumors, but the role these cells play in invasive ductal carcinoma (IDC) of the breast is unknown. We show that CD25⁺CD4⁺ T cells from PBMCs and tumor express high levels of Foxp3, GITR, CTLA‐4, and CD103, indicating that tumor‐infiltrating Treg cells are functional and possibly recruited by CCL22. Additionally, we observed upregulation of Th17‐related molecules (IL‐17A, RORC, and CCR6) and IL‐17A produced by tumor‐infiltrating CD4⁺ and CD8⁺ T lymphocytes. The angiogenic factors CXCL8, MMP‐2, MMP‐9, and vascular endothelial growth factor detected within the tumor are possibly induced by IL‐17 and indicative of poor disease prognosis. Treg and Th17 cells were synchronically increased in IDC patients, with positive correlation between Foxp3, IL‐17A, and RORC expression, and associated with tumor aggressiveness. Therefore, Treg and Th17 cells can affect disease progression by Treg‐cell‐mediated suppression of the effector T‐cell response, as indicated by a decrease in the proliferation of T cells isolated from PBMCs of IDC patients and induction of angiogenic factors by IL‐17‐producing Th17. The understanding of regulation of the Treg/Th17 axis may result in novel perspectives for the control of invasive tumors.     

TLR ligands of ryegrass pollen microbial contaminants enhance Th1 and Th2 responses and decrease induction of Foxp3hi regulatory T cells

Microbial contamination of grass pollens could affect sensitization, subsequent allergic response, and efficacy of allergen‐specific immunotherapy. We investigated whether bacterial immunomodulatory substances can direct PBMC responses of allergic and nonatopic subjects against ryegrass pollen (RGP) toward Th1, Th2, or regulatory T (Treg) cells. Aqueous extracts of RGP with high or low LPS were fractionated into large and small molecular weight (MW) components by diafiltration. CFSE‐labeled PBMCs from allergic and nonatopic subjects were stimulated with RGP extracts (RGPEs) and analyzed for cytokine secretion and T‐cell responses. High LPS RGPE increased IFN‐γ⁺ Th1 and IL‐4⁺ Th2 effector cell induction and consistently decreased CD4⁺Foxp3^hi Treg‐cell induction. IL‐10‐producing T‐cell frequency was unaltered, but IL‐10 secretion was increased by high LPS RGPE. RGPE‐stimulation of TLR‐transfected cell lines revealed that high LPS pollen also contained a TLR2‐ligand, and both batches a TLR9‐ligand. Beta‐1,3‐glucans were detected in large and small MW fractions and were also T‐cell stimulatory. In conclusion, coexposure to allergen and proinflammatory microbial stimuli does not convert an established Th2‐ into a Th1‐response. Instead, proinflammatory responses are exacerbated and Foxp3^hi Treg‐cell induction is decreased. These findings show that adjuvants for specific immunotherapy should enhance Treg cells rather than target immune deviation from Th2 to Th1.     

PI3Kγ kinase activity is required for optimal T‐cell activation and differentiation

Phosphatidylinositol‐3‐kinase gamma (PI3Kγ) is a leukocyte‐specific lipid kinase with signaling function downstream of G protein‐coupled receptors to regulate cell trafficking, but its role in T cells remains unclear. To investigate the requirement of PI3Kγ kinase activity in T‐cell function, we studied T cells from PI3Kγ kinase‐dead knock‐in (PI3Kγ^KD/KD) mice expressing the kinase‐inactive PI3Kγ protein. We show that CD4⁺ and CD8⁺ T cells from PI3Kγ^KD/KD mice exhibit impaired TCR/CD28‐mediated activation that could not be rescued by exogenous IL‐2. The defects in proliferation and cytokine production were also evident in naïve and memory T cells. Analysis of signaling events in activated PI3Kγ^KD/KD T cells revealed a reduction in phosphorylation of protein kinase B (AKT) and ERK1/2, a decrease in lipid raft formation, and a delay in cell cycle progression. Furthermore, PI3Kγ^KD/KD CD4⁺ T cells displayed compromised differentiation toward Th1, Th2, Th17, and induced Treg cells. PI3Kγ^KD/KD mice also exhibited an impaired response to immunization and a reduced delayed‐type hypersensitivity to Ag challenge. These findings indicate that PI3Kγ kinase activity is required for optimal T‐cell activation and differentiation, as well as for mounting an efficient T cell‐mediated immune response. The results suggest that PI3Kγ kinase inhibitors could be beneficial in reducing the undesirable immune response in autoimmune diseases.     

Boswellic acids reduce Th17 differentiation via blockade of IL‐1β‐mediated IRAK1 signaling

Interferon‐gamma producing CD4⁺ T (Th1) cells and IL‐17‐producing CD4⁺ T (Th17) cells are involved in the pathogenesis of several autoimmune diseases including multiple sclerosis. Therefore, the development of treatment strategies controlling the generation and expansion of these effector cells is of high interest. Frankincense, the resin from trees of the genus Boswellia, and particularly its prominent bioactive compound acetyl‐11‐keto‐β‐boswellic acid (AKBA), have potent anti‐inflammatory properties. Here, we demonstrate that AKBA is able to reduce the differentiation of human CD4⁺ T cells to Th17 cells, while slightly increasing Th2‐ and Treg‐cell differentiation. Furthermore, AKBA reduces the IL‐1β‐triggered IL‐17A release of memory Th17 cells. AKBA may affect IL‐1β signaling by preventing IL‐1 receptor‐associated kinase 1 phosphorylation and subsequently decreasing STAT3 phosphorylation at Ser727, which is required for Th17‐cell differentiation. The effects of AKBA on Th17 differentiation and IL‐17A release make the compound a good candidate for potential treatment of Th17‐driven diseases.     

TGF‐β induces the differentiation of human CXCL13‐producing CD4+ T cells

In the ectopic lymphoid‐like structures present in chronic inflammatory conditions such as rheumatoid arthritis, a subset of human effector memory CD4⁺ T cells that lacks features of follicular helper T (Tfh) cells produces CXCL13. Here, we report that TGF‐β induces the differentiation of human CXCL13‐producing CD4⁺ T cells from naïve CD4⁺ T cells. The TGF‐β‐induced CXCL13‐producing CD4⁺ T cells do not express CXCR5, B‐cell lymphoma 6 (BCL6), and other Tfh‐cell markers. Furthermore, expression levels of CD25 (IL‐2Rα) in CXCL13‐producing CD4⁺ T cells are significantly lower than those in FoxP3⁺ in vitro induced Treg cells. Consistent with this, neutralization of IL‐2 and knockdown of STAT5 clearly upregulate CXCL13 production by CD4⁺ T cells, while downregulating the expression of FoxP3. Furthermore, overexpression of FoxP3 in naïve CD4⁺ T cells downregulates CXCL13 production, and knockdown of FoxP3 fails to inhibit the differentiation of CXCL13‐producing CD4⁺ T cells. As reported in rheumatoid arthritis, proinflammatory cytokines enhance secondary CXCL13 production from reactivated CXCL13‐producing CD4⁺ T cells. Our findings demonstrate that CXCL13‐producing CD4⁺ T cells lacking Tfh‐cell features differentiate via TGF‐β signaling but not via FoxP3, and exert their function in IL‐2‐limited but TGF‐β‐rich and proinflammatory cytokine‐rich inflammatory conditions.     

Enhanced suppressor function of TIM‐3+FoxP3+ regulatory T cells

T‐cell immunoglobulin and mucin domain 3 (TIM‐3) is an Ig‐superfamily member expressed on IFN‐γ‐secreting Th1 and Tc1 cells and was identified as a negative regulator of immune tolerance. TIM‐3 is expressed by a subset of activated CD4⁺ T cells, and anti‐CD3/anti‐CD28 stimulation increases both the level of expression and the number of TIM‐3⁺ T cells. In mice, TIM‐3 is constitutively expressed on natural regulatory T (Treg) cells and has been identified as a regulatory molecule of alloimmunity through its ability to modulate CD4⁺ T‐cell differentiation. Here, we examined TIM‐3 expression on human Treg cells to determine its role in T‐cell suppression. In contrast to mice, TIM‐3 is not expressed on Treg cells ex vivo but is upregulated after activation. While TIM‐3⁺ Treg cells with increased gene expression of LAG3, CTLA4, and FOXP3 are highly efficient suppressors of effector T (Teff) cells, TIM‐3^? Treg cells poorly suppressed Th17 cells as compared with their suppression of Th1 cells; this decreased suppression ability was associated with decreased STAT‐3 expression and phosphorylation and reduced gene expression of IL10, EBI3, GZMB, PRF1, IL1Rα, and CCR6. Thus, our results suggest that TIM‐3 expression on Treg cells identifies a population highly effective in inhibiting pathogenic Th1‐ and Th17‐cell responses.     

Lack of Mycobacterium tuberculosis–specific interleukin‐17A–producing CD4+ T cells in active disease

Protective immunity to Mycobacterium tuberculosis (Mtb) is commonly ascribed to a Th1 profile; however, the involvement of Th17 cells remains to be clarified. Here, we characterized Mtb‐specific CD4⁺ T cells in blood and bronchoalveolar lavages (BALs) from untreated subjects with either active tuberculosis disease (TB) or latent Mtb infection (LTBI), considered as prototypic models of uncontrolled or controlled infection, respectively. The production of IL‐17A, IFN‐γ, TNF‐α, and IL‐2 by Mtb‐specific CD4⁺ T cells was assessed both directly ex vivo and following in vitro antigen‐specific T‐cell expansion. Unlike for extracellular bacteria, Mtb‐specific CD4⁺ T‐cell responses lacked immediate ex vivo IL‐17A effector function in both LTBI and TB individuals. Furthermore, Mtb‐specific Th17 cells were absent in BALs, while extracellular bacteria‐specific Th17 cells were identified in gut biopsies of healthy individuals. Interestingly, only Mtb‐specific CD4⁺ T cells from 50% of LTBI but not from TB subjects acquired the ability to produce IL‐17A following Mtb‐specific T‐cell expansion. Finally, IL‐17A acquisition by Mtb‐specific CD4⁺ T cells correlated with the coexpression of CXCR3 and CCR6, currently associated to Th1 or Th17 profiles, respectively. Our data demonstrate that Mtb‐specific Th17 cells are selectively undetectable in peripheral blood and BALs from TB patients.     

Overcoming regulatory T‐cell suppression by a lyophilized preparation of Streptococcus pyogenes

Cancer vaccines have yet to yield clinical benefit, despite the measurable induction of humoral and cellular immune responses. As immunosuppression by CD4⁺CD25⁺ regulatory T (Treg) cells has been linked to the failure of cancer immunotherapy, blocking suppression is therefore critical for successful clinical strategies. Here, we addressed whether a lyophilized preparation of Streptococcus pyogenes (OK‐432), which stimulates Toll‐like receptors, could overcome Treg‐cell suppression of CD4⁺ T‐cell responses in vitro and in vivo. OK‐432 significantly enhanced in vitro proliferation of CD4⁺ effector T cells by blocking Treg‐cell suppression and this blocking effect depended on IL‐12 derived from antigen‐presenting cells. Direct administration of OK‐432 into tumor‐associated exudate fluids resulted in a reduction of the frequency and suppressive function of CD4⁺CD25⁺Foxp3⁺ Treg cells. Furthermore, when OK‐432 was used as an adjuvant of vaccination with HER2 and NY‐ESO‐1 for esophageal cancer patients, NY‐ESO‐1–specific CD4⁺ T‐cell precursors were activated, and NY‐ESO‐1–specific CD4⁺ T cells were detected within the effector/memory T‐cell population. CD4⁺ T‐cell clones from these patients had high‐affinity TCRs and recognized naturally processed NY‐ESO‐1 protein presented by dendritic cells. OK‐432 therefore inhibits Treg‐cell function and contributes to the activation of high‐avidity tumor antigen‐specific naive T‐cell precursors.     

Leptin deficiency impairs maturation of dendritic cells and enhances induction of regulatory T and Th17 cells

Leptin is an adipose‐secreted hormone that plays an important role in both metabolism and immunity. Leptin has been shown to induce Th1‐cell polarization and inhibit Th2‐cell responses. Additionally, leptin induces Th17‐cell responses, inhibits regulatory T (Treg) cells and modulates autoimmune diseases. Here, we investigated whether leptin mediates its activity on T cells by influencing dendritic cells (DCs) to promote Th17 and Treg‐cell immune responses in mice. We observed that leptin deficiency (i) reduced the expression of DC maturation markers, (ii) decreased DC production of IL‐12, TNF‐α, and IL‐6, (iii) increased DC production of TGF‐β, and (iv) limited the capacity of DCs to induce syngeneic CD4⁺ T‐cell proliferation. As a consequence of this unique phenotype, DCs generated under leptin‐free conditions induced Treg or T_H17 cells more efficiently than DCs generated in the presence of leptin. These data indicate important roles for leptin in DC homeostasis and the initiation and maintenance of inflammatory and regulatory immune responses by DCs.