Increased Frequency of T Follicular Helper Cells and Elevated Interleukin-27 Plasma Levels in Patients with Pemphigus

Pemphigus is an autoimmune disease in which IgG auto-antibodies (auto-ab) against the desmosomal cadherins desmoglein (Dsg) 3 and Dsg1 cause loss of epidermal keratinocyte adhesion. Aim of this study was to investigate cytokines derived from antigen-presenting cells (APC) and their relation to CD4⁺ T cell subpopulations and to the auto-ab response in pemphigus. In this regard, patients with pemphigus were compared to patients with myasthenia gravis (MG), an unrelated auto-ab–mediated autoimmune disease, and healthy controls. In pemphigus and MG, the plasma concentrations of the APC-derived immunomodulatory cytokine IL-27 were highly increased. Strikingly, IL-27 strongly correlated with Dsg-specific IgG auto-ab titers. T helper (Th) 17 cells were augmented in both pemphigus and MG patients while T follicular helper (Tfh) cells, which are essential in providing B cell help, were increased only in pemphigus along with increasing plasma concentrations of IL-21, a cytokine produced by Th17 and Tfh cells. Moreover, we could detect Dsg3-specific autoreactive T cells producing IL-21 upon ex vivo stimulation with Dsg3. These findings suggest that IL-27 and IL-21-producing T cells, are involved in the pathogenesis of pemphigus. The further characterization of IL-21-producing T cells and of the role of IL-27 will lead to a more defined understanding of the auto-ab response in pemphigus.     

Unique T Cells with Unconventional Cytokine Profiles Induced in the Livers of Mice during Schistosoma mansoni Infection

During infection with Schistosoma, serious hepatic disorders are induced in the host. The liver possesses unique immune systems composed of specialized cells that differ from those of other immune competent organs or tissues. Host immune responses change dramatically during Schistosoma mansoni infection; in the early phase, Th1-related responses are induced, whereas during the late phase Th2 reactions dominate. Here, we describe unique T cell populations induced in the liver of mice during the period between Th1- and Th2-phases, which we term the transition phase. During this phase, varieties of immune cells including T lymphocytes increase in the liver. Subsets of CD4⁺ T cells exhibit unique cytokine production profiles, simultaneously producing both IFN-γ and IL-13 or both IFN-γ and IL-4. Furthermore, cells triply positive for IFN-γ, IL-13 and IL-4 also expand in the S. mansoni-infected liver. The induction of these unique cell populations does not occur in the spleen, indicating it is a phenomenon specific to the liver. In single hepatic CD4⁺ T cells showing the unique cytokine profiles, both T-bet and GATA-3 are expressed. Thus, our studies show that S. mansoni infection triggers the induction of hepatic T cell subsets with unique cytokine profiles.     

Loss of Circulating CD4 T Cells with B Cell Helper Function during Chronic HIV Infection

The interaction between follicular T helper cells (T_FH) and B cells in the lymph nodes and spleen has a major impact on the development of antigen-specific B cell responses during infection or vaccination. Recent studies described a functional equivalent of these cells among circulating CD4 T cells, referred to as peripheral T_FH cells. Here, we characterize the phenotype and in vitro B cell helper activity of peripheral T_FH populations, as well as the effect of HIV infection on these populations. In co-culture experiments we confirmed CXCR5+ cells from HIV-uninfected donors provide help to B cells and more specifically, we identified a CCR7^highCXCR5^highCCR6^highPD-1^high CD4 T cell population that secretes IL-21 and enhances isotype-switched immunoglobulin production. This population is significantly decreased in treatment-naïve, HIV-infected individuals and can be recovered after anti-retroviral therapy. We found impaired immunoglobulin production in co-cultures from HIV-infected individuals and found no correlation between the frequency of peripheral T_FH cells and memory B cells, or with neutralization activity in untreated HIV infection in our cohort. Furthermore, we found that within the peripheral T_FH population, the expression level of T_FH-associated genes more closely resembles a memory, non-T_FH population, as opposed to a T_FH population. Overall, our data identify a heterogeneous population of circulating CD4 T cells that provides in vitro help to B cells, and challenges the origin of these cells as memory T_FH cells.     

IL-33 Induces IL-9 Production in Human CD4+ T Cells and Basophils

IL-33, an IL-1 family member and ligand for the IL-1 receptor-related protein ST2, has been associated with induction of Th2 cytokines such as IL-4, IL-5, and IL-13. Here, we report that IL-33 can initiate IL-9 protein secretion in vitro in human CD4+ T cells and basophils isolated from peripheral blood. TGF-β has been described as a critical factor for IL-9 induction in Th2 cells; however, we found that TGF-β also induces co-production of IL-9 in purified, naïve (>99%) CD4⁺CD45RA⁺CD45RO⁻CD25⁻ T cells differentiated towards a Th1 profile. Subsequently, it was demonstrated that TGF-β is important, although not an absolute requirement, for IL-9 production in CD4+ T cells. IL-9 production by purified (>95%) human basophils, cultured for 24 h with IL-3 or IL-33, was found, with a strong synergy between the two, likely to be explained by the IL-3 upregulated ST2 expression. Collectively, these data indicate that barrier functioning cells are important for the regulation of IL-9 production by immune cells in inflamed tissue.     

Regulation of Autoimmune Germinal Center Reactions in Lupus-Prone BXD2 Mice by Follicular Helper T Cells

BXD2 mice spontaneously develop autoantibodies and subsequent glomerulonephritis, offering a useful animal model to study autoimmune lupus. Although initial studies showed a critical contribution of IL-17 and Th17 cells in mediating autoimmune B cell responses in BXD2 mice, the role of follicular helper T (Tfh) cells remains incompletely understood. We found that both the frequency of Th17 cells and the levels of IL-17 in circulation in BXD2 mice were comparable to those of wild-type. By contrast, the frequency of PD-1⁺CXCR5⁺ Tfh cells was significantly increased in BXD2 mice compared with wild-type mice, while the frequency of PD-1⁺CXCR5⁺Foxp3⁺ follicular regulatory T (Tfr) cells was reduced in the former group. The frequency of Tfh cells rather than that of Th17 cells was positively correlated with the frequency of germinal center B cells as well as the levels of autoantibodies to dsDNA. More importantly, CXCR5⁺ CD4⁺ T cells isolated from BXD2 mice induced the production of IgG from naïve B cells in an IL-21-dependent manner, while CCR6⁺ CD4⁺ T cells failed to do so. These results together demonstrate that Tfh cells rather than Th17 cells contribute to the autoimmune germinal center reactions in BXD2 mice.     

Involvement of IL-18 in the Expansion of Unique Hepatic T Cells with Unconventional Cytokine Profiles during Schistosoma mansoni Infection

Infection with schistosomes invokes severe fibrotic granulomatous responses in the liver of the host. Schistosoma mansoni infection induces dramatic fluctuations in Th1 or Th2 cytokine responses systemically; Th1 reactions are provoked in the early phase, whilst Th2 responses become dominant after oviposition begins. In the liver, various unique immune cells distinct from those of conventional immune competent organs or tissues exist, resulting in a unique immunological environment. Recently, we demonstrated that S. mansoni infection induces unique CD4⁺ T cell populations exhibiting unconventional cytokine profiles in the liver of mice during the period between Th1- and Th2-phases, which we term the transition phase. They produce both IFN-γ and IL-4 or both IFN-γ and IL-13 simultaneously. Moreover, T cells secreting triple cytokines IFN-γ, IL-13 and IL-4 were also induced. We term these cells Multiple Cytokine Producing Hepatic T cells (MCPHT cells). During the transition phase, when MCPHT cells increase, IL-18 secretion was up-regulated in the liver and sera. In S. mansoni-infected IL-18-deficient mice, expansion of MCPHT cells was curtailed. Thus our data suggest that IL-18 produced during S. mansoni infection play a role in the expansion of MCPHT cells.     

IL-4-based helper activity of CD4 T cells is radiation sensitive

The capacity of CD4⁺ T cells to induce IgG synthesis in B cells has been known to be radioresistant for more than 20 years. However, the radiation sensitivity of helper T cells with regard to their ability to induce the synthesis of isotypes other than IgG has not been studied. We therefore irradiated KLH-primed lymph node T cells and examined their capacity to induce IgG, IgM, and IgE synthesis in hapten-primed B cells. We demonstrated that while the capacity of KLH-primed lymph node cells to induce IgG synthesis was not affected by irradiation, the capacity of such T cells to induce IgE synthesis was greatly reduced by γ-irradiation. This was consistent with our observations that IL-4 and IL-5 synthesis in such cells was greatly diminished by irradiation, whereas IL-2 synthesis was only minimally affected. A similar differential sensitivity to irradiation of the helper activity of Th1 and Th2 clones was observed with regard to their ability to induce IgE and IgG synthesis under cognate conditions. Irradiation greatly inhibited the capacity of Th2 clones to induce IgE synthesis, but only minimally affected the capacity of Th1 clones to induce IgG synthesis in primed B cells. The capacity of irradiated Th2 clones to induce IgE synthesis was restored by the addition of IL-4 and IL-5. These results taken together indicated that the sensitivity to irradiation of T helper cells with regard to the induction of IgE but not IgG synthesis was due to the sensitivity to irradiation of the production of IL-4 but not of IL-2. Thus, although some functions of CD4⁺ T cells are resistant to radiation, other functions, particularly those that depend on the production of IL-4 and IL-5, are greatly diminished by ionizing radiation.     

Effects of Sustained Sleep Restriction on Mitogen-Stimulated Cytokines,Chemokines and T Helper 1/ T Helper 2 Balance in Humans

Background

Recent studies suggest that acute sleep deprivation disrupts cellular immune responses by shifting T helper (Th) cell activity towards a Th2 cytokine profile. Since little is known about more long-term effects, we investigated how five days of sleep restriction would affect pro-inflammatory, chemotactic, Th1- and Th2 cytokine secretion.

Methods

Nine healthy males participated in an experimental sleep protocol with two baseline sleep-wake cycles (sleep 23.00 – 07.00 h) followed by 5 days with restricted sleep (03.00 – 07.00 h). On the second baseline day and on the fifth day with restricted sleep, samples were drawn every third hour for determination of cytokines/chemokines (tumor necrosis factor alpha (TNF-α), interleukin (IL) -1β, IL-2, IL-4 and monocyte chemoattractant protein-1 (MCP-1)) after in vitro stimulation of whole blood samples with the mitogen phytohemagglutinin (PHA). Also leukocyte numbers, mononuclear cells and cortisol were analysed.

Results

5-days of sleep restriction affected PHA-induced immune responses in several ways. There was a general decrease of IL-2 production (p<.05). A shift in Th1/Th2 cytokine balance was also evident, as determined by a decrease in IL2/IL4 ratio. No other main effects of restricted sleep were shown. Two significant interactions showed that restricted sleep resulted in increased TNF-α and MCP-1 in the late evening and early night hours (p’s<.05). In addition, all variables varied across the 24 h day.

Conclusions

5-days of sleep restriction is characterized by a shift towards Th2 activity (i.e. lower 1L-2/IL-4 ratio) which is similar to the effects of acute sleep deprivation and psychological stress. This may have implications for people suffering from conditions characterized by excessive Th2 activity like in allergic disease, such as asthma, for whom restricted sleep could have negative consequences.     

Genetic Deletion of Mst1 Alters T Cell Function and Protects against Autoimmunity

Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4⁺ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1^−/− B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1^−/− CD4⁺ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4⁺ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.     

CD103+CD11b+ Dendritic Cells Induce Th17 T Cells in Muc2-Deficient Mice with Extensively Spread Colitis

Mucus alterations are a feature of ulcerative colitis (UC) and can drive inflammation by compromising the mucosal barrier to luminal bacteria. The exact pathogenesis of UC remains unclear, but CD4⁺ T cells reacting to commensal antigens appear to contribute to pathology. Given the unique capacity of dendritic cells (DCs) to activate naive T cells, colon DCs may activate pathogenic T cells and contribute to disease. Using Muc2^-/- mice, which lack a functional mucus barrier and develop spontaneous colitis, we show that colitic animals have reduced colon CD103⁺CD11b^- DCs and increased CD103^-CD11b⁺ phagocytes. Moreover, changes in colonic DC subsets and distinct cytokine patterns distinguish mice with distally localized colitis from mice with colitis spread proximally. Specifically, mice with proximally spread, but not distally contained, colitis have increased IL-1β, IL-6, IL-17, TNFα, and IFNγ combined with decreased IL-10 in the distal colon. These individuals also have increased numbers of CD103⁺CD11b⁺ DCs in the distal colon. CD103⁺CD11b⁺ DCs isolated from colitic but not noncolitic mice induced robust differentiation of Th17 cells but not Th1 cells ex vivo. In contrast, CD103^-CD11b⁺ DCs from colitic Muc2^-/- mice induced Th17 as well as Th1 differentiation. Thus, the local environment influences the capacity of intestinal DC subsets to induce T cell proliferation and differentiation, with CD103⁺CD11b⁺ DCs inducing IL-17-producing T cells being a key feature of extensively spread colitis.     

CD8+ T Cells Mediate Female-Dominant IL-4 Production and Airway Inflammation in Allergic Asthma

The prevalence and severity of bronchial asthma are higher in females than in males after puberty. Although antigen-specific CD8⁺ T cells play an important role in the development of asthma through their suppressive effect on cytokine production, the contribution of CD8⁺ T cells to sex differences in asthmatic responses remains unclear. In the present study, we investigated the sex-specific effect of CD8⁺ T cells in the suppression of asthma using an ovalbumin mouse model of asthma. The number of inflammatory cells in bronchoalveolar lavage (BAL) fluid, lung type 2 T-helper cytokine levels, and interleukin-4 (IL-4) production by bronchial lymph node cells were significantly higher in female wild-type (WT) mice compared with male mice, whereas no such sex differences were observed between male and female cd8α-disrupted mice. The adaptive transfer of male, but not female, CD8⁺ T cells reduced the number of inflammatory cells in the recovered BAL fluid of male recipient mice, while no such sex difference in the suppressive activity of CD8⁺ T cells was observed in female recipient mice. Male CD8⁺ T cells produced higher levels of IFN-γ than female CD8⁺ T cells did, and this trend was associated with reduced IL-4 production by male, but not female, CD4⁺ T cells. Interestingly, IFN-γ receptor expression on CD4⁺ T cells was significantly lower in female mice than in male mice. These results suggest that female-dominant asthmatic responses are orchestrated by the reduced production of IFN-γ by CD8⁺ T cells and the lower expression of IFN-γ receptor on CD4⁺ T cells in females compared with males.     

IL-17 Induction by ArtinM is Due to Stimulation of IL-23 and IL-1 Release and/or Interaction with CD3 in CD4+ T Cells

ArtinM is a D-mannose-binding lectin extracted from the seeds of Artocarpus heterophyllus that interacts with TLR2 N-glycans and activates antigen-presenting cells (APCs), as manifested by IL-12 production. In vivo ArtinM administration induces Th1 immunity and confers protection against infection with several intracellular pathogens. In the murine model of Candida albicans infection, it was verified that, in addition to Th1, ArtinM induces Th17 immunity manifested by high IL-17 levels in the treated animals. Herein, we investigated the mechanisms accounting for the ArtinM-induced IL-17 production. We found that ArtinM stimulates the IL-17 production by spleen cells in BALB/c or C57BL/6 mice, a response that was significantly reduced in the absence of IL-23, MyD88, or IL-1R. Furthermore, we showed that ArtinM directly induced the IL-23 mRNA expression and the IL-1 production by macrophages. Consistently, in cell suspensions depleted of macrophages, the IL-17 production stimulated by ArtinM was reduced by 53% and the exogenous IL-23 acted synergistically with ArtinM in promoting IL-17 production by spleen cell suspensions. We verified that the absence of IL-23, IL-1R, or MyD88 inhibited, but did not block, the IL-17 production by ArtinM-stimulated spleen cells. Therefore, we investigated whether ArtinM exerts a direct effect on CD4⁺ T cells in promoting IL-17 production. Indeed, spleen cell suspensions depleted of CD4⁺ T cells responded to ArtinM with very low levels of IL-17 release. Likewise, isolated CD4⁺ T cells under ArtinM stimulus augmented the expression of TGF-β mRNA and released high levels of IL-17. Considering the observed synergism between IL-23 and ArtinM, we used cells from IL-23 KO mice to assess the direct effect of lectin on CD4⁺ T cells. We verified that ArtinM increased the IL-17 production significantly, a response that was inhibited when the CD4⁺ T cells were pre-incubated with anti-CD3 antibody. In conclusion, ArtinM stimulates the production of IL-17 by CD4⁺ T cells in two major ways: (I) through the induction of IL-23 and IL-1 by APCs and (II) through the direct interaction with CD3 on the CD4⁺ T cells. This study contributes to elucidation of mechanisms accounting for the property of ArtinM in inducing Th17 immunity and opens new perspectives in designing strategies for modulating immunity by using carbohydrate recognition agents.     

Cutting Edge: Suppression of GM-CSF Expression in Murine and Human T Cells by IL-27

GM-CSF is a potent proinflammatory cytokine that plays a pathogenic role in the CNS inflammatory disease experimental autoimmune encephalomyelitis. As IL-27 alleviates experimental autoimmune encephalomyelitis, we hypothesized that IL-27 suppresses GM-CSF expression by T cells. We found that IL-27 suppressed GM-CSF expression in CD4(+) and CD8(+) T cells in splenocyte and purified T cell cultures. IL-27 suppressed GM-CSF in Th1, but not Th17, cells. IL-27 also suppressed GM-CSF expression by human T cells in nonpolarized and Th1- but not Th17-polarized PBMC cultures. In vivo, IL-27p28 deficiency resulted in increased GM-CSF expression by CNS-infiltrating T cells during Toxoplasma gondii infection. Although in vitro suppression of GM-CSF by IL-27 was independent of IL-2 suppression, IL-10 upregulation, or SOCS3 signaling, we observed that IL-27-driven suppression of GM-CSF was STAT1 dependent. Our findings demonstrate that IL-27 is a robust negative regulator of GM-CSF expression in T cells, which likely inhibits T cell pathogenicity in CNS inflammation.     

Nitric oxide mediates interleukin-1 induced inhibition of glycosaminoglycan synthesis in rat articular cartilage

Interleek-1beta (IL-1) is a key mediator of cartilage matrix degradation in osteoarthritis and rheumatoid arthritis. It was found that the IL-1-induced suppression of glycosaminoglycan (GAG) synthesis in rat articular cartilage occurred simultaneously with the accumulation of nitrite (a metabolite of nitric oxide (NO) in aqueous milieu) in the culture medium. NO-synthase inhibitors, L-NMMA and L-NIO, inhibited both these IL-1 effects. Dexamethasone suppressed GAG synthesis additively to IL-1, but did not alter nitrite accumulation. Three NO-donors (GEA 3175, SNAP and SIN-1) also had an inhibitory effect on cartilage GAG synthesis. Therefore, it is concluded that IL-1 induced suppression of GAG synthesis in rat articular cartilage is mediated by the production of NO.     

CD4+ T cells kill HLA-class-II-antigen-positive melanoma cells presenting peptide in vitro

Purpose: Most melanoma cell lines express HLA class II antigens constitutively or can be induced to do so with interferon γ (IFNγ). We have previously demonstrated that peptide-specific CD4⁺ T cells proliferate in response to HLA-class-II-antigen-mediated peptide presentation by melanoma cells in vitro and produce interleukin-10 (IL-10) and (IFNγ). We asked whether the responding T cells kill the tumor cells and, if so, whether direct cell contact was required. Methods: Two HLA class II⁺ melanoma cell lines derived from metastases were co-cultured with a human CD4⁺ T cell clone specific for influenza hemagglutinin peptide (HA). T cells, melanoma, and HA were co-cultured for 48 h. Melanoma cells with and without HA and/or T cells served as controls. After 36 h, the medium was removed for cytokine analysis by enzyme-linked immunosorbent assay (ELISA). Twelve hours later non-adherent cells were washed away and the adherent melanoma cells were trypsinized and counted. Dual-chamber culture plates were used to determine whether cell contact and/or exposure to cytokine were required for tumor cell death. Results: Melanoma cell counts were over 80% lower in wells containing T cells than in wells with melanoma and peptide alone (P < 0.05). ELISA of supernatants revealed production of IFNγ and IL-10 by the responding T cells. Direct T cell contact with tumor cells was not required for tumor cell death, as melanoma cells were killed when they shared medium but had no contact with T cells responding to peptide presentation by HLA-class-II-antigen-positive melanoma cells in a separate chamber. Blocking antibody to IFNγ but not IL-10 prevented melanoma cell death at levels of cytokine similar to that present in co-culture assays. Conclusions: Peptide-specific CD4⁺ T cells kill melanoma cells in vitro when they recognize peptide presented by the tumor cell in the context of HLA class II antigen. Direct cell contact is not required, suggesting that it is a cytokine-mediated event. Immunotherapy, using primed CD4⁺ T cells and peptide, may be beneficial in patients whose tumors express HLA class II antigens or can be induced to do so with IFNγ. Received: 1 July 1999 / Accepted: 17 September 1999