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.     

B cells expressing IFN‐γ suppress Treg‐cell differentiation and promote autoimmune experimental arthritis

Clinical efficacy in the treatment of rheumatoid arthritis with anti‐CD20 (Rituximab)‐mediated B‐cell depletion has garnered interest in the mechanisms by which B cells contribute to autoimmunity. We have reported that B‐cell depletion in a murine model of proteoglycan‐induced arthritis (PGIA) leads to an increase in Treg cells that correlate with decreased autoreactivity. Here, we demonstrate that the increase in Treg cells after B‐cell depletion is due to an increase in the differentiation of naïve CD4⁺ T cells into Treg cells. Since the development of PGIA is dependent on IFN‐γ and B cells are reported to produce IFN‐γ, we hypothesized that B‐cell‐specific IFN‐γ plays a role in the development of PGIA. Accordingly, mice with B‐cell‐specific IFN‐γ deficiency were as resistant to the induction of PGIA as mice that were completely IFN‐γ deficient. Importantly, despite a normal frequency of IFN‐γ‐producing CD4⁺ T cells, B‐cell‐specific IFN‐γ‐deficient mice exhibited a higher percentage of Treg cells compared with that in WT mice. These data indicate that B‐cell IFN‐γ production inhibits Treg‐cell differentiation and exacerbates arthritis. Thus, we have established that IFN‐γ, specifically derived from B cells, uniquely contributes to the pathogenesis of autoimmunity through prevention of immunoregulatory mechanisms.     

IFN‐γ stimulates CpG‐induced IL‐10 production in B cells via p38 and JNK signalling pathways

The production of IL‐10, a potent immunosuppressive cytokine, must be strictly regulated to ensure a balanced immune response. IFN‐γ, a key cytokine in multiple immune processes and pathologies, is known as an inhibitor of IL‐10 production by monocytes and macrophages, but also has some regulatory functions. In the present study, we explored the role of IFN‐γ on Toll‐like receptor (TLR)‐induced IL‐10 production in murine peritoneal and spleen cells and in human peripheral blood mononuclear cells. IFN‐γ inhibited IL‐10 production induced by TLR2, TLR3, TLR4 and TLR7/8 agonists, but stimulated IL‐10 production when cells were triggered with CpG oligodeoxynucleotides, a specific TLR9 agonist. The stimulatory effect of IFN‐γ on TLR9‐induced IL‐10 was restricted to B cells. In line with the increased IL‐10, B cells stimulated with CpG and IFN‐γ profoundly inhibited CD4 T cell proliferation. Further research into the mechanisms involved, revealed that the mitogen‐activated protein kinases p38 and JNK are essential players in this stimulatory effect, and that the phosphatase MKP1 – an inhibitor of p38 and JNK activity – is downregulated after combined stimulation with IFN‐γ and CpG. Our data may represent a novel immunoregulatory role of IFN‐γ in B cells after triggering of TLR9, by stimulating IL‐10 production.     

Direct recognition of LPS by human but not murine CD8+ T cells via TLR4 complex

LPS comprises a major PAMP and is a key target of the immune system during bacterial infection. While LPS can be recognised by innate immune cells via the TLR4 complex, it is unknown whether T lymphocytes, especially CD8⁺ T cells are also capable of doing so. We report here that naïve human CD8⁺ T cells, after activation by TCR stimulation, express surface TLR4 and CD14. These activated CD8⁺ T cells can then secrete high concentrations of IFN‐γ, granzyme and perforin in response to LPS. These effects can be specifically inhibited using siRNA for TLR4. Furthermore, LPS can synergise with IL‐12 to polarise the CD8⁺ T cells into cytotoxic T‐cell 1 (Tc1) that produce IFN‐γ but not IL‐4, with or without TCR activation. Moreover, CD8⁺CD45RO⁺ memory T cells constitutively expressed TLR4 and markedly enhanced IFN‐γ production when stimulated with LPS. In contrast, activated murine CD8⁺ T cells lack TLR4 and CD14 expression and fail to respond to LPS for proliferation and cytokine production. Thus, human but not murine CD8⁺ T cells are able to directly recognise bacterial LPS via LPS receptor complex and TLR4 provides a novel signal for the activation of effector and memory human CD8⁺ T cells.     

Sympathetic neural signaling via the β2‐adrenergic receptor suppresses T‐cell receptor‐mediated human and mouse CD8+ T‐cell effector function

Postganglionic sympathetic neurons innervate secondary lymphoid organs and secrete norepinephrine (NE) as the primary neurotransmitter. NE binds and signals through five distinct members of the adrenergic receptor family. In this study, we show elevated expression of the β2‐adrenergic receptor (ADRB2) on primary human CD8⁺ effector memory T cells. Treatment of both human and murine CD8⁺ T cells with NE decreased IFN‐γ and TNF‐α secretion and suppressed their cytolytic capacity in response to T‐cell receptor (TCR) activation. The effects of NE were specifically reversed by β 2‐specific antagonists. Adrb2^?/? CD8⁺ T cells were completely resistant to the effects of NE. Further, the ADRB2‐specific pharmacological ligand, albuterol, significantly suppressed effector functions in both human and mouse CD8⁺ T cells. While both TCR activation and stimulation with IL‐12 + IL‐18 were able to induce inflammatory cytokine secretion, NE failed to suppress IFN‐γ secretion in response to IL‐12 + IL18. Finally, the long‐acting ADRB2‐specific agonist, salmeterol, markedly reduced the cytokine secretion capacity of CD8⁺ T cells in response to infection with vesicular stomatitis virus. This study reveals a novel intrinsic role for ADRB2 signaling in CD8⁺ T‐cell function and underscores the novel role this pathway plays in adaptive T‐cell responses to infection.     

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.     

IL‐18Rα‐deficient CD4+ T cells induce intestinal inflammation in the CD45RBhi transfer model of colitis despite impaired innate responsiveness

IL‐18 has been implicated in inflammatory bowel disease (IBD), however its role in the regulation of intestinal CD4⁺ T‐cell function remains unclear. Here we show that murine intestinal CD4⁺ T cells express high levels of IL‐18Rα and provide evidence that IL‐18Rα expression is induced on these cells subsequent to their entry into the intestinal mucosa. Using the CD45RB^hi T‐cell transfer colitis model, we show that IL‐18Rα is expressed on IFN‐γ⁺, IL‐17⁺, and IL‐17⁺IFN‐γ⁺ effector CD4⁺ T cells in the inflamed colonic lamina propria (cLP) and mesenteric lymph node (MLN) and is required for the optimal generation and/or maintenance of IFN‐γ‐producing cells in the cLP. In the steady state and during colitis, TCR‐independent cytokine‐induced IFN‐γ and IL‐17 production by intestinal CD4⁺ T cells was largely IL‐18Rα?dependent. Despite these findings however, IL‐18Rα?deficient CD4⁺ T cells induced comparable intestinal pathology to WT CD4⁺ T cells. These findings suggest that IL‐18‐dependent cytokine induced activation of CD4⁺ T cells is not critical for the development of T‐cell‐mediated colitis.     

IFN‐γ regulates survival and function of tumor‐induced CD11b+Gr‐1high myeloid derived suppressor cells by modulating the anti‐apoptotic molecule Bcl2a1

Myeloid derived suppressor cells (MDSCs) play a critical role in suppression of immune responses in cancer and inflammation. Here, we describe how regulation of Bcl2a1 by cytokines controls the suppressor function of CD11b⁺Gr‐1^high granulocytic MDSCs. Coculture of CD11b⁺Gr‐1^high granulocytic MDSCs with antigen‐stimulated T cells and simultaneous blockade of IFN‐γ by the use of anti‐IFN‐γ blocking antibody, IFN‐γ^?/? effector T cells, IFN‐γR^?/? MDSCs or STAT1^?/? MDSCs led to upregulation of Bcl2a1 in CD11b⁺Gr‐1^high cells, improved survival, and enhanced their suppressor function. Molecular studies revealed that GM‐CSF released by antigen‐stimulated CD8⁺ T cells induced Bcl2a1 upregulation, which was repressed in the presence of IFN‐γ by a direct interaction of phosphorylated STAT‐1 with the Bcl2a1 promotor. Bcl2a1 overexpressing granulocytic MDSCs demonstrated prolonged survival and enhanced suppressor function in vitro. Our data suggest that IFN‐γ/ STAT1‐dependent regulation of Bcl2a1 regulates survival and thereby suppressor function of granulocytic MDSCs.     

Siglec‐1 inhibits RSV‐induced interferon gamma production by adult T cells in contrast to newborn T cells

Interferon gamma (IFN‐γ) plays an important role in the antiviral immune response during respiratory syncytial virus (RSV) infections. Monocytes and T cells are recruited to the site of RSV infection, but it is unclear whether cell‐cell interactions between monocytes and T cells regulate IFN‐γ production. In this study, micro‐array data identified the upregulation of sialic acid‐binding immunoglobulin‐type lectin 1 (Siglec‐1) in human RSV‐infected infants. In vitro, RSV increased expression of Siglec‐1 on healthy newborn and adult monocytes. RSV‐induced Siglec‐1 on monocytes inhibited IFN‐γ production by adult CD4⁺ T cells. In contrast, IFN‐γ production by RSV in newborns was not affected by Siglec‐1. The ligand for Siglec‐1, CD43, is highly expressed on adult CD4⁺ T cells compared to newborns. Our data show that Siglec‐1 reduces IFN‐γ release by adult T cells possibly by binding to the highly expressed CD43. The Siglec‐1‐dependent inhibition of IFN‐γ in adults and the low expression of CD43 on newborn T cells provides a better understanding of the immune response against RSV in early life and adulthood.     

CD70 and IFN‐1 selectively induce eomesodermin or T‐bet and synergize to promote CD8+ T‐cell responses

CD70‐mediated stimulation of CD27 is an important cofactor of CD4⁺ T‐cell licensed dendritic cells (DCs). However, it is unclear how CD70‐mediated stimulation of T cells is integrated with signals that emanate from signal 3 pathways, such as type‐1 interferon (IFN‐1) and IL‐12. We find that while stimulation of CD27 in isolation drives weak Eomesodermin^hiT‐bet^lo CD8⁺ T‐cell responses to OVA immunization, profound synergistic expansion is achieved by cotargeting TLR. This cooperativity can substantially boost antiviral CD8⁺ T‐cell responses during acute infection. Concomitant stimulation of TLR significantly increases per cell IFN‐γ production and the proportion of the population with characteristics of short‐lived effector cells, yet also promotes the ability to form long‐lived memory. Notably, while IFN‐1 contributes to the expression of CD70 on DCs, the synergy between CD27 and TLR stimulation is dependent upon IFN‐1's effect directly on CD8⁺ T cells, and is associated with the increased expression of T‐bet in T cells. Surprisingly, we find that IL‐12 fails to synergize with CD27 stimulation to promote CD8⁺ T‐cell expansion, despite its capacity to drive effector CD8⁺ T‐cell differentiation. Together, these data identify complex interactions between signal 3 and costimulatory pathways, and identify opportunities to influence the differentiation of CD8⁺ T‐cell responses.     

Innate IFN‐γ Production by Subsets of Natural Killer Cells,Natural Killer T Cells and γδ T Cells in Response to Dying Bacterial‐Infected Macrophages

Interferon‐γ (IFN‐γ) activation of macrophages is a crucial step in the early innate defence against bacterial infection. This innate IFN‐γ is thought to be produced mainly by natural killer (NK) cells through activation with interleukin (IL)‐12p70 secreted by macrophages and dendritic cells (DCs) that have sensed bacterial products. However, a number of reports have shown that bacterial stimuli are unable to induce macrophages and/or DCs to produce sufficient amounts of IL‐12p70 unless these cells are primed by IFN‐γ. It remains, therefore, unsettled how initial IFN‐γ is produced. In a previous study, we reported a novel IFN‐γ production pathway that was associated with cell death in macrophages caused by intracellular bacteria like Listeria monocytogenes (LM) and Shigella flexneri. In this study, we showed that cell death of bone‐marrow‐derived macrophage (BMM) cells following in vitro infection with Staphylococcus aureus (SA), an extracellular bacterium, can also stimulate this IFN‐γ production pathway. We also unequivocally demonstrated by using BMM cells from IL‐12‐deficient mice that the bacterial‐infected macrophage cell death‐mediated IFN‐γ production can occur without IL‐12 although the magnitude of the response is much smaller than that in the presence of IL‐12. The enhancing effect of IL‐12 on this response proved to be attributable to the negligible amounts (0.5～1.5 pg/ml) of IL‐12p70 but not to the large amounts of IL‐12p40 that were both secreted by SA‐ and LM‐infected macrophages. Taken all together, we propose that macrophage cell death caused by bacteria may trigger the initial IFN‐γ production at an early stage of bacterial infection.     

Innate IFN‐γ promotes development of experimental autoimmune encephalomyelitis: A role for NK cells and M1 macrophages

The role of IFN‐γ in the pathogenesis of autoimmune diseases is controversial. Although Th1 cells can induce experimental autoimmune encephalomyelitis (EAE), IFN‐γ can suppress Th17 cells that are pathogenic in EAE. Here we show that NK cells provide an early source of IFN‐γ during development of EAE. Depletion of NK cells or neutralization of IFN‐γ delayed the onset of EAE and was associated with reduced infiltration of IL‐17⁺ and GM‐CSF⁺ T cells into the CNS. In the passive transfer model, immune cells from myelin oligodendrocyte glycoprotein (MOG)‐immunized IFN‐γ^?/? mice failed to induce EAE, despite producing IL‐17 and GM‐CSF. The macrophages expressed markers of M2 activation and the T cells had low very late antigen‐4 (VLA‐4) expression and failed to infiltrate the CNS. Addition of recombinant IFN‐γ to immune cells from the IFN‐γ^?/? mice activated M1 macrophages and restored VLA‐4 expression, migratory, and encephalitogenic activity of T cells. Furthermore, treatment of recipient mice with anti‐VLA‐4 neutralizing antibody abrogated EAE induced by transfer of T cells from WT mice. Our findings demonstrate IFN‐γ‐producing T cells are not required for development of EAE, but NK cell‐derived IFN‐γ has a key role in promoting M1 macrophage expansion and VLA‐4‐mediated migration of encephalitogenic T cells into the CNS.     

IL‐33 promotes innate IFN‐γ production and modulates dendritic cell response in LCMV‐induced hepatitis in mice

Recent studies have revealed IL‐33 as a key factor in promoting antiviral T‐cell responses. However, it is less clear as to how IL‐33 regulates innate immunity. In this study, we infected wild‐type (WT) and IL‐33^?/? mice with lymphocytic choriomeningitis virus and demonstrated an essential role of infection‐induced IL‐33 expression for robust innate IFN‐γ production in the liver. We first show that IL‐33 deficiency resulted in a marked reduction in the number of IFN‐γ⁺ γδ T and NK cells, but an increase in that of IL‐17⁺ γδ T cells at 16 h postinfection. Recombinant IL‐33 (rIL‐33) treatment could reverse such deficiency via increasing IFN‐γ‐producing γδ T and NK cells, and inhibiting IL‐17⁺ γδ T cells. We also found that rIL‐33‐induced type 2 innate lymphoid cells were not involved in T‐cell responses and liver injury, since the adoptive transfer of type 2 innate lymphoid cells neither affected the IFN‐γ and TNF‐α production in T cells, nor liver transferase levels in lymphocytic choriomeningitis virus infected mice. Interestingly, we found that while IL‐33 was not required for costimulatory molecule expression, it was critical for DC proliferation and cytokine production. Together, this study highlights an essential role of IL‐33 in regulating innate IFN‐γ‐production and DC function during viral hepatitis.     

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.     

Novel CD8+ Treg suppress EAE by TGF‐β‐ and IFN‐γ‐dependent mechanisms

Although CD8⁺ Treg‐mediated suppression has been described, CD8⁺ Treg remain poorly characterized. Here we identify a novel subset of CD8⁺ Treg that express latency‐associated peptide (LAP) on their cell surface (CD8⁺LAP⁺ cells) and exhibit regulatory activity in vitro and in vivo. Only a small fraction of CD8⁺LAP⁺ cells express Foxp3 or CD25, although the expression levels of Foxp3 for these cells are higher than their LAP^? counterparts. In addition to TGF‐β, CD8⁺LAP⁺ cells produce IFN‐γ, and these cells suppress EAE that is dependent on both TGF‐β and IFN‐γ. In an adoptive co‐transfer model, CD8⁺LAP⁺ cells suppress myelin oligodendrocyte glycoprotein (MOG)‐specific immune responses by inducing or expanding Foxp3⁺ cells and by inhibiting proliferation and IFN‐γ production in vivo. Furthermore, in vivo neutralization of IFN‐γ and studies with IFN‐γ‐deficient mice demonstrate an important role for IFN‐γ production in the function of CD8⁺LAP⁺ cells. Our findings identify the underlying mechanisms that account for the immunoregulatory activity of CD8⁺ T cells and suggest that induction or amplification of CD8⁺LAP⁺ cells may be a therapeutic strategy to help control autoimmune processes.     

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.