PolyI:C plus IL‐2 or IL‐12 induce IFN‐γ production by human NK cells via autocrine IFN‐β

NK lymphocytes and type I IFN (IFN‐α/β) are major actors of the innate anti‐viral response that also influence adaptive immune responses. We evaluated type I IFN production by human NK cells in response to polyI:C, a potent type I IFN‐inducing TLR3 agonist. PolyI:C plus IL‐2/IL‐12 induced IFN‐β (but not IFN‐α) mRNA expression and protein production by highly pure human NK cells and by the human NK cell line NK92. Neutralizing anti‐IFNAR1 or anti‐IFN‐β Ab prevented the production of IFN‐γ induced by polyI:C plus IL‐2/IL‐12. Similarly, IFN‐γ production induced by polyI:C plus IL‐12 was reduced in NK cells isolated from IFNAR1^?/? compared with WT mice. The ability of polyI:C plus IL‐12 to induce IFN‐γ production was related to an increase of TLR3, Mda5 and IFNAR expression and by an increase of STAT1 and STAT4 phosphorylation. Collectively, these data demonstrate that NK cells, in response to polyI:C plus IL‐2/IL‐12, produce IFN‐β that induce, in an autocrine manner, the production of IFN‐γ and thereby highlight that NK cells may control the outcome of protective or injurious immune responses through type I IFN secretion.     

IFN‐λ‐mediated IL‐12 production in macrophages induces IFN‐γ production in human NK cells

With increasing interest in alternative options to interferon‐alpha‐based treatments, IFN‐λ has shown therapeutic promise in a variety of diseases. Although the antiviral activity of IFN‐λ has been extensively studied, there is limited knowledge regarding the immunological functions of IFN‐λ and how these differ from those of other classes of IFNs. In this study, we investigated the effects of IFN‐λ on primary human NK cells, both in a direct and indirect capacity. We demonstrate that in contrast to interferon‐alpha, IFN‐λ is unable to directly stimulate NK cells, due to the absence of IFN‐λ receptor chain 1 (IFN‐λR1) on NK cells. However, IFN‐λ, in combination with TLR4 challenge, is able to induce the production of select members of the IL‐12 family of cytokines in monocyte‐derived macrophages. We further show that through macrophage‐mediated IL‐12 production, IFN‐λ is able to indirectly affect NK cells and ultimately induce IFN‐γ production.     

Micro‐RNA‐155 inhibits IFN‐γ signaling in CD4+ T cells

Micro‐RNA (miR) are increasingly recognized as critical regulators of tissue‐specific patterns of gene expression. CD4⁺ T cells lacking miR‐155, for example, exhibit bias towards Th2 differentiation, indicating that the absence of individual miR could alter CD4⁺ T‐cell differentiation. We now show that miR‐155 is induced upon T‐cell activation and that it promotes Th1 differentiation when over‐expressed in activated CD4⁺ T cells. Antagonism of miR‐155 leads to induction of IFN‐γ receptor α‐chain (IFN‐γRα, and a functional miR‐155 target site is identified within the 3′ untranslated region of IFN‐γRα. These results identify IFN‐γRα as a second miR‐155 target in T cells and suggest that miR‐155 contributes to Th1 differentiation in CD4⁺ T cells by inhibiting IFN‐γ signaling.     

DX5+CD4+ T cells modulate CD4+ T‐cell response via inhibition of IL‐12 production by DCs

DX5⁺CD4⁺ T cells have been shown to dampen collagen‐induced arthritis and delayed‐type hypersensitivity reactions in mice. These cells are also potent modulators of T‐helper cell responses through direct effects on CD4⁺ T cells in an IL‐4 dependent manner. To further characterize this T‐cell population, we studied their effect on DCs and the potential consequences on T‐cell activation. Here, we show that mouse DX5⁺CD4⁺ T cells modulate DCs by robustly inhibiting IL‐12 production. This modulation is IL‐10 dependent and does not require cell contact. Furthermore, DX5⁺CD4⁺ T cells modulate the surface phenotype of LPS‐matured DCs. DCs modulated by DX5⁺CD4⁺ T‐cell supernatant express high levels of the co‐inhibitor molecules PDL‐1 and PDL‐2. OVA‐specific CD4⁺ T cells primed with DCs exposed to DX5⁺CD4⁺ T‐cell supernatant produce less IFN‐γ than CD4⁺ T cells primed by DCs exposed to either medium or DX5^?CD4⁺ T‐cell supernatant. The addition of IL‐12 to the co‐culture with DX5⁺ DCs restores IFN‐γ production. When IL‐10 present in the DX5⁺CD4⁺ T‐cell supernatant is blocked, DCs re‐establish their ability to produce IL‐12 and to efficiently prime CD4⁺ T cells. These data show that DX5⁺CD4⁺ T cells can indirectly affect the outcome of the T‐cell response by inducing DCs that have poor Th1 stimulatory function.     

IL‐10 produced by activated human B cells regulates CD4+ T‐cell activation in vitro

IL‐10‐producing regulatory B cells have been identified in mice and shown to downregulate inflammation, making them potentially important for maintenance of tolerance. In this study, we isolated B cells from human blood and spleen, and showed that after a short period of ex vivo stimulation a number of these cells produced IL‐10. The IL‐10‐producing B cells did not fall within a single clearly defined subpopulation, but were enriched in both the memory (CD27⁺) and the transitional (CD38^high) B‐cell compartments. Combined CpG‐B+anti‐Ig stimulation was the most potent IL‐10 stimulus tested. B cells stimulated in this way inhibited CD4⁺CD25^? T‐cell proliferation in vitro by a partially IL‐10‐dependent mechanism. These findings imply that manipulating IL‐10 production by human B cells could be a useful therapeutic strategy for modulating immune responses in humans.     

IL‐17‐producing γδ T cells

IL‐17 is produced not only by CD4⁺ αβ T cells, but also CD8⁺ αβ T cells, NKT cells, and γδ T cells, plus some non‐T cells, including macrophages and neutrophils. The ability of IL‐17 to deploy neutrophils to sites of inflammation imparts this cytokine with a key role in diseases of several types. Surprisingly, γδ T cells are responsible for much of the IL‐17 produced in several disease models, particularly early on.     

γδ T cells restrain extrathymic development of Foxp3+‐inducible regulatory T cells via IFN‐γ

Inducible Treg (iTreg) cells generated from Ag‐stimulated naïve CD4⁺ T cells in the periphery play an important role in regulating immune responses. TGF‐β is a key cytokine that promotes this conversion process; however, how this process is regulated in vivo remains unclear. Here, we report that γδ T cells play a crucial role in controlling iTreg generation and suppressor function. Ag‐induced iTreg generation was significantly enhanced in C57BL/6 mice in the absence of γδ T cells. Inhibition of iTreg conversion was mediated by IFN‐γ produced by activated γδ T cells but not by activated CD4⁺ T cells. BM chimera experiments further confirmed γδ‐derived IFN‐γ‐dependent mechanism in regulating iTreg generation in vivo. Lastly, human peripheral blood γδ T cells also interfere with iTreg conversion via IFN‐γ. Our results suggest a novel function of γδ T cells in limiting the generation of iTreg cells, potentially balancing immunity and tolerance.     

Mycobacterium tuberculosis PstS1 amplifies IFN‐γ and induces IL‐17/IL‐22 responses by unrelated memory CD4+ T cells via dendritic cell activation

The immunological mechanisms that modulate protection during Mycobacterium tuberculosis (Mtb) infection or vaccination are not fully understood. Secretion of IFN‐γ and, to a lesser extent, of IL‐17 by CD4⁺ T cells plays a major role both in protection and immunopathology. Few Mtb Ags interacting with DCs affect priming, activation, and regulation of Ag‐unrelated CD4⁺ T‐cell responses. Here we demonstrate that PstS1, a 38 kDa‐lipoprotein of Mtb, promotes Ag‐independent activation of memory T lymphocytes specific for Ag85B or Ag85A, two immunodominant protective Ags of Mtb. PstS1 expands CD4⁺ and CD8⁺ memory T cells, amplifies secretion of IFN‐γ and IL‐22 and induces IL‐17 production by effector memory cells in an Ag‐unrelated manner in vitro and in vivo. These effects were mediated through the stimulation of DCs, particularly of the CD8α^? subtype, which respond to PstS1 by undergoing phenotypic maturation and by secreting IL‐6, IL‐1β and, to a lower extent, IL‐23. IL‐6 secretion by PstS1‐stimulated DCs was required for IFN‐γ, and to a lesser extent for IL‐22 responses by Ag85B‐specific memory T cells. These results may open new perspectives for immunotherapeutic strategies to control Th1/Th17 immune responses in Mtb infections and in vaccinations against tuberculosis.     

Regulatory effects of IFN‐β on production of osteopontin and IL‐17 by CD4+ T Cells in MS

IFN‐β currently serves as one of the major treatments for MS. Its anti‐inflammatory mechanism has been reported as involving a shift in cytokine balance from Th1 to Th2 in the T‐cell response against elements of the myelin sheath. In addition to the Th1 and Th2 groups, two other important pro‐inflammatory cytokines, IL‐17 and osteopontin (OPN), are believed to play important roles in CNS inflammation in the pathogenesis of MS. In this study, we examined the potential effects of IFN‐β on the regulation of OPN and IL‐17 in MS patients. We found that IFN‐β used in vitro at 0.5–3 ng/mL significantly inhibited the production of OPN in primary T cells derived from PBMC. The inhibition of OPN was determined to occur at the CD4⁺ T‐cell level. In addition, IFN‐β inhibited the production of IL‐17 and IL‐21 in CD4⁺ T cells. It has been described that IFN‐β suppresses IL‐17 production through the inhibition of a monocytic cytokine, the intracellular translational isoform of OPN. Our further investigation demonstrated that IFN‐β also acted directly on the CD4⁺ T cells to regulate OPN and IL‐17 expression through the type I IFN receptor‐mediated activation of STAT1 and suppression of STAT3 activity. Administration of IFN‐β to EAE mice ameliorated the disease severity. Furthermore, spinal cord infiltration of OPN⁺ and IL‐17⁺ cells decreased in IFN‐β‐treated EAE mice along with decreases in serum levels of OPN and IL‐21. Importantly, decreased OPN production by IFN‐β treatment contributes to the reduced migratory activity of T cells. Taken together, the results from both in vitro and in vivo experiments indicate that IFN‐β treatment can down‐regulate the OPN and IL‐17 production in MS. This study provides new insights into the mechanism of action of IFN‐β in the treatment of MS.     

TGF‐β interactions with IL‐1 family members trigger IL‐4‐independent IL‐9 production by mouse CD4+ T cells

TGF‐β and IL‐4 were recently shown to selectively upregulate IL‐9 production by naïve CD4⁺ T cells. We report here that TGF‐β interactions with IL‐1α, IL‐1β, IL‐18, and IL‐33 have equivalent IL‐9‐stimulating activities that function even in IL‐4‐deficient animals. This was observed after in vitro antigenic stimulation of immunized or unprimed mice and after polyclonal T‐cell activation. Based on intracellular IL‐9 staining, all IL‐9‐producing cells were CD4⁺ and 80–90% had proliferated, as indicated by reduced CFSE staining. In contrast to IL‐9, IL‐13 and IL‐17 were strongly stimulated by IL‐1 and either inhibited (IL‐13) or were unaffected (IL‐17) by addition of TGF‐β. IL‐9 and IL‐17 production also differed in their dependence on IL‐2 and regulation by IL‐1/IL‐23. As IL‐9 levels were much lower in Th2 and Th17 cultures, our results identify TGF‐β/IL‐1 and TGF‐β/IL‐4 as the main control points of IL‐9 synthesis.     

IL‐12 and IL‐4 activate a CD39‐dependent intrinsic peripheral tolerance mechanism in CD8+ T cells

Immune responses to protein antigens involve CD4⁺ and CD8⁺ T cells, which follow distinct programs of differentiation. Naïve CD8 T cells rapidly develop cytotoxic T‐cell (CTL) activity after T‐cell receptor stimulation, and we have previously shown that this is accompanied by suppressive activity in the presence of specific cytokines, i.e. IL‐12 and IL‐4. Cytokine‐induced CD8⁺ regulatory T (Treg) cells are one of several Treg‐cell phenotypes and are Foxp3^? IL‐10⁺ with contact‐dependent suppressive capacity. Here, we show they also express high level CD39, an ecto‐nucleotidase that degrades extracellular ATP, and this contributes to their suppressive activity. CD39 expression was found to be upregulated on CD8⁺ T cells during peripheral tolerance induction in vivo, accompanied by release of IL‐12 and IL‐10. CD39 was also upregulated during respiratory tolerance induction to inhaled allergen and on tumor‐infiltrating CD8⁺ T cells. Production of IL‐10 and expression of CD39 by CD8⁺ T cells was independently regulated, being respectively blocked by extracellular ATP and enhanced by an A2A adenosine receptor agonist. Our results suggest that any CTL can develop suppressive activity when exposed to specific cytokines in the absence of alarmins. Thus negative feedback controls CTL expansion under regulation from both nucleotide and cytokine environment within tissues.     

CCR6 and NK1.1 distinguish between IL‐17A and IFN‐γ‐producing γδ effector T cells

γδ T cells are a potent source of innate IL‐17A and IFN‐γ, and they acquire the capacity to produce these cytokines within the thymus. However, the precise stages and required signals that guide this differentiation are unclear. Here we show that the CD24^low CD44^high effector γδ T cells of the adult thymus are segregated into two lineages by the mutually exclusive expression of CCR6 and NK1.1. Only CCR6⁺ γδ T cells produced IL‐17A, while NK1.1⁺ γδ T cells were efficient producers of IFN‐γ but not of IL‐17A. Their effector phenotype correlated with loss of CCR9 expression, particularly among the NK1.1⁺ γδ T cells. Accordingly, both γδ T‐cell subsets were rare in gut‐associated lymphoid tissues, but abundant in peripheral lymphoid tissues. There, they provided IL‐17A and IFN‐γ in response to TCR‐specific and TCR‐independent stimuli. IL‐12 and IL‐18 induced IFN‐γ and IL‐23 induced IL‐17A production by NK1.1⁺ or CCR6⁺ γδ T cells, respectively. Importantly, we show that CCR6⁺ γδ T cells are more responsive to TCR stimulation than their NK1.1⁺ counterparts. In conclusion, our findings support the hypothesis that CCR6⁺ IL‐17A‐producing γδ T cells derive from less TCR‐dependent selection events than IFN‐γ‐producing NK1.1⁺ γδ T cells.     

Allogeneic induced human FOXP3+IFN‐γ+ T cells exhibit selective suppressive capacity

Human induced CD4⁺CD25⁺ T cells have been shown to express FOXP3, similar to naturally occurring Treg cells (nTreg). However, the suppressive capacity of these cells is still under debate. The current study was designed to investigate functional characteristics of CD25⁺FOXP3⁺ derived from CD25^? T cells. Stimulation of CD25^? PBMC with allogeneic PBMC resulted in production of CD4⁺CD25^high T cells. This process was more rapid and prominent when highly mature DC were used for stimulation. The resultant CD4⁺CD25^high population concurrently exhibited regulatory markers FOXP3, CTLA‐4, GITR, and inflammatory cytokines IL‐2 and IFN‐γ. These human‐induced FOXP3⁺IFN‐γ⁺ T cells were shown, for the first time, to markedly inhibit alloreactive T‐cell expansion, similar to nTreg. However, in contrast to nTreg, the induced CD4⁺CD25⁺FOXP3⁺ cells did not suppress proliferation against a third party donor stimulus or CMV. This suggested that the cell population possessed a more selective suppressive capacity targeted against the original stimulus only. The induced human CD4⁺CD25⁺FOXP3⁺ subset derived from CD25^? T cells, while expressing inflammatory cytokines, exhibits a suppressive cell contact‐dependent effect, restricted against T cells responding to the original stimulus. Such unique properties suggest that these cells are potentially ideal for the use as post‐transplant GVH disease prophylaxis.     

IL‐15‐dependent CD8+CD122+ T cells ameliorate experimental autoimmune encephalomyelitis by modulating IL‐17 production by CD4+ T cells

Interleukin‐15 (IL‐15) is an inflammatory cytokine whose role in autoimmune diseases has not been fully elucidated. Th17 cells have been shown to play critical roles in experimental autoimmune encephalomyelitis (EAE) models. In this study, we demonstrate that blockade of IL‐15 signaling by TMβ‐1 mAb treatment aggravated EAE severity. The key mechanism was not NK‐cell depletion but depletion of CD8⁺CD122⁺ T cells. Adoptive transfer of exogenous CD8⁺CD122⁺ T cells to TMβ‐1‐treated mice rescued animals from severe disease. Moreover, transfer of preactivated CD8⁺CD122⁺ T cells prevented EAE development and significantly reduced IL‐17 secretion. Naïve effector CD4⁺CD25^? T cells cultured with either CD8⁺CD122⁺ T cells from wild‐type mice or IL‐15 transgenic mice displayed lower frequencies of IL‐17A production with lower amounts of IL‐17 in the supernatants when compared with production by effector CD4⁺CD25^? T cells cultured alone. Addition of a neutralizing antibody to IL‐10 led to recovery of IL‐17A production in Th17 cultures. Furthermore, coculture of CD8⁺CD122⁺ T cells with effector CD4⁺ T cells inhibited their proliferation significantly, suggesting a regulatory function for IL‐15 dependent CD8⁺CD122⁺ T cells. Taken together, these observations suggest that IL‐15, acting through CD8⁺CD122⁺ T cells, has a negative regulatory role in reducing IL‐17 production and Th17‐mediated EAE inflammation.     

Enrichment of IL‐17A+IFN‐γ+ and IL‐22+IFN‐γ+ T cell subsets is associated with reduction of NKp44+ILC3s in the terminal ileum of Crohn's disease patients

Crohn's disease (CD) is a chronic inflammatory condition of the human gastrointestinal tract whose aetiology remains largely unknown. Dysregulated adaptive immune responses and defective innate immunity both contribute to this process. In this study, we demonstrated that the interleukin (IL)‐17A⁺interferon (IFN)‐γ⁺ and IL‐22⁺IFN‐γ⁺ T cell subsets accumulated specifically in the inflamed terminal ileum of CD patients. These cells had higher expression of Ki‐67 and were active cytokine producers. In addition, their proportions within both the IL‐17A‐producer and IL‐22‐producer populations were increased significantly. These data suggest that IL‐17A⁺IFN‐γ⁺ and IL‐22⁺IFN‐γ⁺ T cell subsets might represent the pathogenic T helper type 17 (Th17) population in the context of intestinal inflammation for CD patients. In the innate immunity compartment we detected a dramatic alteration of both phenotype and function of the intestinal innate lymphoid cells (ILCs), that play an important role in the maintenance of mucosal homeostasis. In the inflamed gut the frequency of the NKp44^–CD117^–ILC1s subset was increased significantly, while the frequency of NKp44⁺ILC3s was reduced. Furthermore, the frequency of human leucocyte antigen D‐related (HLA‐DR)‐expressing‐NKp44⁺ILC3s was also reduced significantly. Interestingly, the decrease in the NKp44⁺ILC3s population was associated with an increase of pathogenic IL‐17A⁺IFN‐γ⁺ and IL‐22⁺IFN‐γ⁺ T cell subsets in the adaptive compartment. This might suggest a potential link between NKp44⁺ILC3s and the IL‐17A⁺IFN‐γ⁺ and IL‐22⁺IFN‐γ⁺ T cell subsets in the terminal ileum of CD patients.