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.     

Modified vaccinia Ankara‐expressing Ag85A,a novel tuberculosis vaccine,is safe in adolescents and children,and induces polyfunctional CD4+ T cells

Modified vaccinia Ankara‐expressing Ag85A (MVA85A) is a new tuberculosis (TB) vaccine aimed at enhancing immunity induced by BCG. We investigated the safety and immunogenicity of MVA85A in healthy adolescents and children from a TB endemic region, who received BCG at birth. Twelve adolescents and 24 children were vaccinated and followed up for 12 or 6 months, respectively. Adverse events were documented and vaccine‐induced immune responses assessed by IFN‐γ ELISpot and intracellular cytokine staining. The vaccine was well tolerated and there were no vaccine‐related serious adverse events. MVA85A induced potent and durable T‐cell responses. Multiple CD4⁺ T‐cell subsets, based on expression of IFN‐γ, TNF‐α, IL‐2, IL‐17 and GM‐CSF, were induced. Polyfunctional CD4⁺ T cells co‐expressing IFN‐γ, TNF‐α and IL‐2 dominated the response in both age groups. A novel CD4⁺ cell subset co‐expressing these three Th1 cytokines and IL‐17 was induced in adolescents, while a novel CD4⁺ T‐cell subset co‐expressing Th1 cytokines and GM‐CSF was induced in children. Ag‐specific CD8⁺ T cells were not detected. We conclude that in adolescents and children MVA85A safely induces the type of immunity thought to be important in protection against TB. This includes induction of novel Th1‐cell populations that have not been previously described in humans.     

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.     

Emergence of a distinct HIV‐specific IL‐10‐producing CD8+ T‐cell subset with immunomodulatory functions during chronic HIV‐1 infection

Interleukin‐10 (IL‐10) plays a key role in regulating proinflammatory immune responses to infection but can interfere with pathogen clearance. Although IL‐10 is upregulated throughout HIV‐1 infection in multiple cell subsets, whether this is a viral immune evasion strategy or an appropriate response to immune activation is unresolved. Analysis of IL‐10 production at the single cell level in 51 chronically infected subjects (31 antiretroviral (ART) naïve and 20 ART treated) showed that a subset of CD8⁺ T cells with a CD25^neg FoxP3^neg phenotype contributes substantially to IL‐10 production in response to HIV‐1 gag stimulation. The frequencies of gag‐specific IL‐10‐ and IFN‐γ‐producing T cells in ART‐naïve subjects were strongly correlated and the majority of these IL‐10⁺ CD8⁺ T cells co‐produced IFN‐γ; however, patients with a predominant IL‐10⁺/IFN‐γ^neg profile showed better control of viraemia. Depletion of HIV‐specific CD8⁺ IL‐10⁺ cells from PBMCs led to upregulation of CD38 on CD14⁺ monocytes together with increased IL‐6 production, in response to gag stimulation. Increased CD38 expression was positively correlated with the frequency of the IL‐10⁺ population and was also induced by exposure of monocytes to HIV‐1 in vitro. Production of IL‐10 by HIV‐specific CD8⁺ T cells may represent an adaptive regulatory response to monocyte activation during chronic infection.     

CD161++CD8+ T cells,including the MAIT cell subset,are specifically activated by IL‐12+IL‐18 in a TCR‐independent manner

CD161⁺⁺CD8⁺ T cells represent a novel subset that is dominated in adult peripheral blood by mucosal‐associated invariant T (MAIT) cells, as defined by the expression of a variable‐α chain 7.2 (Vα7.2)‐Jα33 TCR, and IL‐18Rα. Stimulation with IL‐18+IL‐12 is known to induce IFN‐γ by both NK cells and, to a more limited extent, T cells. Here, we show the CD161⁺⁺ CD8⁺ T‐cell population is the primary T‐cell population triggered by this mechanism. Both CD161⁺⁺Vα7.2⁺ and CD161⁺⁺Vα7.2^? T‐cell subsets responded to IL‐12+IL‐18 stimulation, demonstrating this response was not restricted to the MAIT cells, but to the CD161⁺⁺ phenotype. Bacteria and TLR agonists also indirectly triggered IFN‐γ expression via IL‐12 and IL‐18. These data show that CD161⁺⁺ T cells are the predominant T‐cell population that responds directly to IL‐12+IL‐18 stimulation. Furthermore, our findings broaden the potential role of MAIT cells beyond bacterial responsiveness to potentially include viral infections and other inflammatory stimuli.     

Interleukin‐4 downregulates CD127 expression and activity on human thymocytes and mature CD8+ T cells

Signaling via the IL‐7 receptor complex (IL‐7Rα/CD127 and IL‐2Rγ/CD132) is required for T‐cell development and survival. Decreased CD127 expression has been associated with persistent viral infections (e.g. HIV, HCV) and cancer. Many IL‐2Rγ‐sharing (γ_C) cytokines decrease CD127 expression on CD4⁺ and CD8⁺ T cells in mice (IL‐2, IL‐4, IL‐7, IL‐15) and in humans (IL‐2, IL‐7), suggesting a common function. IL‐4 is of particular interest as it is upregulated in HIV infection and in thyroid and colon cancers. The role of IL‐4 in regulating CD127 expression and IL‐7 activity in human thymocytes and mature CD8⁺ T cells is unknown and was therefore investigated. IL‐4 decreased CD127 expression on all thymocyte subsets tested and only on naïve (CD45RA⁺) CD8⁺ T cells, without altering membrane‐bound CD127 mRNA expression. Pre‐treatment of thymocytes or CD8⁺ T cells with IL‐4 inhibited IL‐7‐mediated phosphorylation of STAT5 and decreased proliferation of CD8⁺ T cells. By downregulating CD127 expression and signaling on developing thymocytes and CD8⁺ T cells, IL‐4 is a potential contributor to impaired CD8⁺ T‐cell function in some anti‐viral and anti‐tumor responses. These findings are of particular consequence to diseases such as HIV, HCV, RSV, measles and cancer, in which CD127 expression is decreased, IL‐7 activity is impaired and IL‐4 concentrations are elevated.     

Different interleukin‐17‐secreting Toll‐like receptor+ T‐cell subsets are associated with disease activity in multiple sclerosis

Signalling through Toll‐like receptors (TLRs) may play a role in the pathogenesis of autoimmune diseases, such as multiple sclerosis (MS). In the present study, the expression of TLR‐2, ‐4 and ‐9 was significantly higher on CD4⁺ and CD8⁺ T‐cells from MS patients compared to healthy individuals. Following in‐vitro activation, the proportion of interleukin (IL)‐17⁺ and IL‐6⁺ CD4⁺ and CD8⁺ T‐cells was higher in the patients. In addition, the proportion of IFN‐γ‐secreting TLR⁺ CD8⁺ T‐cells was increased in MS patients. Among different IL‐17⁺ T‐cell phenotypes, the proportion of IL‐17⁺ TLR⁺ CD4⁺ and CD8⁺ T‐cells producing IFN‐γ or IL‐6 were positively associated with the number of active brain lesions and neurological disabilities. Interestingly, activation of purified CD4⁺ and CD8⁺ T‐cells with ligands for TLR‐2 (Pam3Csk4), TLR‐4 [lipopolysaccharide (LPS)] and TLR‐9 [oligodeoxynucleotide (ODN)] directly induced cytokine production in MS patients. Among the pathogen‐associated molecular patterns (PAMPs), Pam3Csk4 was more potent than other TLR ligands in inducing the production of all proinflammatory cytokines. Furthermore, IL‐6, IFN‐γ, IL‐17 and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) levels produced by Pam3Csk4‐activated CD4⁺ cells were directly associated with disease activity. A similar correlation was observed with regard to IL‐17 levels released by Pam3Csk4‐stimulated CD8⁺ T‐cells and clinical parameters. In conclusion, our data suggest that the expansion of different T helper type 17 (Th17) phenotypes expressing TLR‐2, ‐4 and ‐9 is associated with MS disease activity, and reveals a preferential ability of TLR‐2 ligand in directly inducing the production of cytokines related to brains lesions and neurological disabilities.     

CD8+ T cells producing IL‐3 and IL‐5 in non‐IgE‐mediated eosinophilic diseases

The cytokines IL‐5, IL‐3, and GM‐CSF are crucial for eosinophil development, survival, and function. To better understand their role in non‐IgE‐mediated eosinophilic diseases, we investigated plasma levels of these cytokines as well as cytokine expression in peripheral blood T cells. While we did not find any evidence for an involvement of T‐cell‐derived GM‐CSF, some of these patients did show an increased proportion of IL‐5‐ or IL‐3‐producing CD4⁺ T cells. However, in a significant proportion of patients, IL‐5‐producing CD8⁺ T cells, so‐called Tc2 cells, which in healthy donors can only be detected at very low levels, were prominent. Furthermore, increased IL‐3 production by CD8⁺ T cells was also observed, strongly supporting the notion that CD8⁺ T cells, not just CD4⁺ T cells, must also be considered as a potential source of the cytokines promoting eosinophilia.     

Type I interferon potentiates T‐cell receptor mediated induction of IL‐10‐producing CD4+ T cells

Type I interferons (IFNs) have the dual ability to promote the development of the immune response and exert an anti‐inflammatory activity. We analyzed the integrated effect of IFN‐α, TCR signal strength, and CD28 costimulation on human CD4⁺ T‐cell differentiation into cell subsets producing the anti‐ and proinflammatory cytokines IL‐10 and IFN‐γ. We show that IFN‐α boosted TCR‐induced IL‐10 expression in activated peripheral CD45RA⁺CD4⁺ T cells and in whole blood cultures. The functional cooperation between TCR and IFN‐α efficiently occurred at low engagement of receptors. Moreover, IFN‐α rapidly cooperated with anti‐CD3 stimulation alone. IFN‐α, but not IL‐10, drove the early development of type I regulatory T cells that were mostly IL‐10⁺ Foxp3^? IFN‐γ^? and favored IL‐10 expression in a fraction of Foxp3⁺ T cells. Our data support a model in which IFN‐α costimulates TCR toward the production of IL‐10 whose level can be amplified via an autocrine feedback loop.     

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.     

Both IFN‐γ and IL‐17 are required for the development of severe autoimmune gastritis

IL‐17, produced by a distinct lineage of CD4⁺ helper T (Th) cells termed Th17 cells, induces the production of pro‐inflammatory cytokines from resident cells and it has been demonstrated that over‐expression of IL‐17 plays a crucial role in the onset of several auto‐immune diseases. Here we examined the role of IL‐17 in the pathogenesis of autoimmune gastritis, a disease that was previously believed to be mediated by IFN‐γ. Significantly higher levels of IL‐17 and IFN‐γ were found in the stomachs and stomach‐draining lymph nodes of mice with severe autoimmune gastritis. Unlike IL‐17, which was produced solely by CD4⁺ T cells in gastritic mice, the majority of IFN‐γ‐producing cells were CD8⁺ T cells. However, CD8⁺ T cells alone were not able to induce autoimmune gastritis. T cells that were deficient in IL‐17 or IFN‐γ production were able to induce autoimmune gastritis but to a much lower extent compared with the disease induced by wild‐type T cells. These data demonstrate that production of neither IL‐17 nor IFN‐γ by effector T cells is essential for the initiation of autoimmune gastritis, but suggest that both are required for the disease to progress to the late pathogenic stage that includes significant tissue disruption.     

Signal 3 requirement for memory CD8+ T-cell activation is determined by the infectious pathogen

The relevance of direct inflammatory signals (signal 3) for the activation of memory CD8⁺ T cells during recall responses is so far unknown. We therefore investigated the direct impact of IL‐12 and type I IFN on the formation, recall potential and protective capacity of memory T cells. Using CD8⁺ T cells deficient for IL‐12 or type I IFN receptors in an adoptive transfer system, we generated memory populations after infection with vaccinia virus, lymphocytic choriomeningitis virus or Listeria monocytogenes. The results demonstrate that in the absence of signal 3 cytokines during primary infection, functional memory T cells were formed. After retransfer into naïve mice, signal 3‐deficient memory T cells were able to specifically lyse target cells in vivo under non‐infectious conditions. However, after reinfection, secondary effector CD8⁺ T cells lacking signal 3 were impaired in expansion and protective capacity dependent on the nature of the pathogen. We conclude that memory CD8⁺ T cells depend on a signal 3 for expansion, independent of signals obtained during priming, thereby being influenced by the pathogen‐induced inflammatory milieu during secondary infection. In summary, our results reveal an essential role for direct inflammatory cytokine signaling in secondary T‐cell responses.     

CTLA‐4 (CD152) enhances the Tc17 differentiation program

Although CD8⁺ T cells that produce IL‐17 (Tc17 cells) have been linked to host defense, Tc17 cells show reduced cytotoxic activity, which is the characteristic function of CD8⁺ T cells. Here, we show that CTLA‐4 enhances the frequency of IL‐17 in CD8⁺ T cells, indicating that CTLA‐4 (CD152) specifically promotes Tc17 differentiation. Simultaneous stimulation of CTLA‐4^+/+ and CTLA‐4^?/? T cells in cocultures and agonistic CTLA‐4 stimulation unambiguously revealed a cell‐intrinsic mechanism for IL‐17 control by CTLA‐4. The quality of CTLA‐4‐induced Tc17 cells was tested in vivo, utilizing infection with the facultative intracellular bacterium Listeria monocytogenes (LM). Unlike CTLA‐4^+/+ Tc17 cells, CTLA‐4^?/? were nearly as efficient as Tc1 CTLA‐4^+/+ cells in LM clearance. Additionally, adoptively transferred CTLA‐4^?/? Tc17 cells expressed granzyme B after rechallenge, and produced Tc1 cytokines such as IFN‐γ and TNF‐α, which strongly correlate with bacterial clearance. CTLA‐4^+/+ Tc17 cells demonstrated a high‐quality Tc17 differentiation program ex vivo, which was also evident in isolated IL‐17‐secreting Tc17 cells, with CTLA‐4‐mediated enhanced upregulation of Tc17‐related molecules such as IL‐17A, RORγt, and IRF‐4. Our results show that CTLA‐4 promotes Tc17 differentiation that results in robust Tc17 responses. Its inactivation might therefore represent a central therapeutic target to enhance clearance of infection.     

CD8+ MAIT cells infiltrate into the CNS and alterations in their blood frequencies correlate with IL‐18 serum levels in multiple sclerosis

Recent findings indicate a pathogenic involvement of IL‐17‐producing CD8⁺ T cells in multiple sclerosis (MS). IL‐17 production has been attributed to a subset of CD8⁺ T cells that belong to the mucosal‐associated invariant T (MAIT) cell population. Here, we report a reduction of CD8⁺ MAIT cells in the blood of MS patients compared with healthy individuals, which significantly correlated with IL‐18 serum levels in MS patients. In vitro stimulation of peripheral blood mononuclear cells from healthy individuals and MS patients with IL‐18 specifically activated CD8⁺ MAIT cells. Moreover, IL‐18 together with T‐cell receptor stimulation induced, specifically on CD8⁺ MAIT cells, an upregulation of the integrin very late antigen‐4 that is essential for the infiltration of CD8⁺ T cells into the CNS. Notably, we were able to identify CD8⁺ MAIT cells in MS brain lesions by immunohistochemistry while they were almost absent in the cerebrospinal fluid (CSF). In summary, our findings indicate that an IL‐18–driven activation of CD8⁺ MAIT cells contributes to their CNS infiltration in MS, in turn leading to reduced CD8⁺ MAIT‐cell frequencies in the blood. Therefore, CD8⁺ MAIT cells seem to play a role in the innate arm of immunopathology in MS.     

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.     

CXCL4 is a novel inducer of human Th17 cells and correlates with IL‐17 and IL‐22 in psoriatic arthritis

CXCL4 regulates multiple facets of the immune response and is highly upregulated in various Th17‐associated rheumatic diseases. However, whether CXCL4 plays a direct role in the induction of IL‐17 production by human CD4⁺ T cells is currently unclear. Here, we demonstrated that CXCL4 induced human CD4⁺ T cells to secrete IL‐17 that co‐expressed IFN‐γ and IL‐22, and differentiated naïve CD4⁺ T cells to become Th17‐cytokine producing cells. In a co‐culture system of human CD4⁺ T cells with monocytes or myeloid dendritic cells, CXCL4 induced IL‐17 production upon triggering by superantigen. Moreover, when monocyte‐derived dendritic cells were differentiated in the presence of CXCL4, they orchestrated increased levels of IL‐17, IFN‐γ, and proliferation by CD4⁺ T cells. Furthermore, the CXCL4 levels in synovial fluid from psoriatic arthritis patients strongly correlated with IL‐17 and IL‐22 levels. A similar response to CXCL4 of enhanced IL‐17 production by CD4⁺ T cells was also observed in patients with psoriatic arthritis. Altogether, we demonstrate that CXCL4 boosts pro‐inflammatory cytokine production especially IL‐17 by human CD4⁺ T cells, either by acting directly or indirectly via myeloid antigen presenting cells, implicating a role for CXCL4 in PsA pathology.     

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.