Mesenchymal stem cell inhibition of T-helper 17 cell- differentiation is triggered by cell-cell contact and mediated by prostaglandin E2 via the EP4 receptor

Mesenchymal stem cells (MSCs) inhibit T‐cell activation and proliferation but their effects on individual T‐cell‐effector pathways and on memory versus naïve T cells remain unclear. MSC influence on the differentiation of naïve and memory CD4⁺ T cells toward the Th17 phenotype was examined. CD4⁺ T cells exposed to Th17‐skewing conditions exhibited reduced CD25 and IL‐17A expression following MSC co‐culture. Inhibition of IL‐17A production persisted upon re‐stimulation in the absence of MSCs. These effects were attenuated when cell–cell contact was prevented. Th17 cultures from highly purified naïve‐ and memory‐phenotype responders were similarly inhibited. Th17 inhibition by MSCs was reversed by indomethacin and a selective COX‐2 inhibitor. Media from MSC/Th17 co‐cultures contained increased prostaglandin E2 (PGE2) levels and potently suppressed Th17 differentiation in fresh cultures. MSC‐mediated Th17 inhibition was reversed by a selective EP4 antagonist and was mimicked by synthetic PGE2 and a selective EP4 agonist. Activation‐induced IL‐17A secretion by naturally occurring, effector‐memory Th17 cells from a urinary obstruction model was also inhibited by MSC co‐culture in a COX‐dependent manner. Overall, MSCs potently inhibit Th17 differentiation from naïve and memory T‐cell precursors and inhibit naturally‐occurring Th17 cells derived from a site of inflammation. Suppression entails cell‐contact‐dependent COX‐2 induction resulting in direct Th17 inhibition by PGE2 via EP4.     

Human TSLP and TLR3 ligands promote differentiation of Th17 cells with a central memory phenotype under Th2-polarizing conditions

Background Human thymic stromal lymphopoietin (TSLP) is expressed in the human asthmatic lung and activates dendritic cells (DCs) to strongly induce proallergic T‐helper type 2 (Th2) cell responses, suggesting that TSLP plays a critical role in the pathophysiology of human asthma. Th2 cells are predominantly involved in mild asthma, whereas a mixture of Th1 and Th2 cells with neutrophilic inflammation, probably induced by Th17, affects more severe asthmatic disease. Exacerbation of asthmatic inflammation is often triggered by airway‐targeting RNA viral infection; virus‐derived double‐stranded RNA, Toll‐like receptor (TLR)3 ligand, activates bronchial epithelial cells to produce pro‐inflammatory mediators, including TSLP. Objective Because TSLPR‐expressing DCs express TLR3, we examined how the relationship between TSLP and TLR3 ligand stimulation influences DC activation. Methods CD11c⁺DCs purified from adult peripheral blood were cultured in TLR ligands containing media with or without TSLP and then co‐cultured with allogeneic naïve CD4⁺T cells. Results CD11c⁺ DCs responded to a combination of TSLP and TLR3 ligand, poly(I : C), to up‐regulate expression of the functional TSLP receptor and TLR3. Although TSLP alone did not induce IL‐23 production by DCs, poly(I : C) alone primed DCs for the production of IL‐23, and a combination of TSLP and poly(I : C) primed DCs for further production of IL‐23. The addition of poly(I : C) did not inhibit TSLP‐activated DCs to prime naïve CD4⁺ T cells to differentiate into inflammatory Th2 cells. Furthermore, DCs activated by a combination of TSLP and poly(I : C) primed more naïve CD4⁺ T cells to differentiate into Th17‐cytokine–producing cells with a central memory T cell phenotype compared with DCs activated by poly(I : C) alone. Conclusions These results suggest that through DC activation, human TSLP and TLR3 ligands promote differentiation of Th17 cells with the central memory T cell phenotype under Th2‐polarizing conditions.     

ICOS cooperates with CD28, IL-2, and IFN-gamma and modulates activation of human naïve CD4+ T cells

Several sets of data indicate that ICOS regulates cytokine production in activated T cells, but is less effective on naïve T cells. This work evaluates ICOS function in human naïve CD4⁺ T cells through an assessment of the effect of soluble forms of the ICOS and CD28 physiological ligands on activation driven by anti‐CD3 mAb. ICOS strikingly potentiated secretion of IL‐2, IFN‐γ, IL‐10, and TNF‐α, but not IL‐4, promoted by optimal stimulation of CD3+CD28, and it was the key switching‐factor of activation when cells received suboptimal stimulation of CD3+CD28 or stimulation of CD3 alone in the presence of exogenous IL‐2. In these conditions, blockade of IL‐2 and IFN‐γ showed that ICOS builds up a positive feedback loop with IFN‐γ, which required IL‐2 and was inhibited by IL‐4. By contrast, in the absence of CD28 triggering or exogenous IL‐2, ICOS‐induced costimulation mainly supported expression of TGF‐β1 and FoxP3 and differentiation of regulatory T cells capable to inhibit proliferation of naïve CD4⁺ T cells driven by allogeneic cells. These data suggest that ICOS favors differentiation of Th effector cells when cooperates with appropriate activation stimuli such as CD3+CD28 or CD3+IL‐2, whereas it supports differentiation of regulatory T cells when costimulatory signals are insufficient.     

In CD28‐costimulated human naïve CD4+ T cells,I‐κB kinase controls the expression of cell cycle regulatory proteins via interleukin‐2‐independent mechanisms

Stimulation of naïve CD4⁺ T cells through engagement of the T‐cell receptor (TCR) and the CD28 co‐receptor initiates cell proliferation which critically depends on interleukin (IL)‐2 secretion and subsequent autocrine signalling via the IL‐2 receptor. However, several studies indicate that in CD28‐costimulated T cells additional IL‐2‐independent signals are also required for cell proliferation. In this study, using a neutralizing anti‐human IL‐2 antibody and two selective, structurally unrelated, cell‐permeable I‐κB kinase (IKK) inhibitors, BMS‐345541 and PS‐1145, we show that in human naïve CD4⁺ T cells stimulated through a short engagement of the TCR and the CD28 co‐receptor, IKK controls the expression of the cell cycle regulatory proteins cyclin D3, cyclin E and cyclin‐dependent kinase 2 (CDK2) and the stability of the F‐box protein S‐phase kinase‐associated protein 2 (SKP2) and its co‐factor CDC28 protein kinase regulatory subunit 1B (CKS1B), through IL‐2‐independent mechanisms.     

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.     

Allergen‐specific naïve and memory CD4+ T cells exhibit functional and phenotypic differences between individuals with or without allergy

Although allergen‐specific CD4⁺ T cells are detectable in the peripheral blood of both individuals with or without allergy, their frequencies and phenotypes within the memory as well as naïve repertoires are incompletely known. Here, we analyzed the DRB1*0401‐restricted responses of peripheral blood‐derived memory (CD4⁺CD45RO⁺) and naïve (CD4⁺CD45RA⁺) T cells from subjects with or without allergy against the immunodominant epitope of the major cow dander allergen Bos d 2 by HLA class II tetramers in vitro. The frequency of Bos d 2_127–142‐specific memory T cells in the peripheral blood‐derived cultures appeared to be higher in subjects with allergy than those without, whereas naïve Bos d 2_127–142‐specific T cells were detectable in the cultures of both groups at nearly the same frequency. Surprisingly, the TCR avidity of Bos d 2_127–142‐specific T cells of naïve origin, as assessed by the intensity of HLA class II tetramer staining, was found to be higher in individuals with allergy. Upon restimulation, long‐term Bos d 2_127–142‐specific T‐cell lines generated from both memory and naïve T‐cell pools from individuals with allergy proliferated more strongly, produced more IL‐4 and IL‐10, and expressed higher levels of CD25 but lower levels of CXCR3 than the T‐cell lines from individuals without allergy, demonstrating differences also at the functional level. Collectively, our current results suggest that not only the memory but also the naïve allergen‐specific T‐cell repertoires differ between individuals with or without allergy.     

Prolonged exposure of naïve CD8+ T cells to interleukin-7 or interleukin-15 stimulates proliferation without differentiation or loss of telomere length

Interleukin (IL)‐7 and IL‐15 are cytokines implicated in homeostatic control of the peripheral CD8 T‐cell pool. We compared the effects of IL‐7 and IL‐15 on survival and proliferation of purified human CD8⁺ T‐cell subsets. Low concentrations of either cytokine reduced the spontaneous apoptosis of all subsets, and enhancement of survival corresponded to the extent of Bcl‐2 up‐regulation. Surprisingly, although minimal proliferation of naïve CD8⁺ T cells was observed during the first week of culture with cytokines, a marked expansion of these cells occurred at later time points, particularly in response to IL‐15. This occurred largely without phenotypic change or acquisition of effector function, indicating a dissociation of differentiation from proliferation. Notably, progression of naïve CD8⁺ T cells through several cell divisions resulted in up‐regulation of telomerase and the maintenance of telomere length. These data show that IL‐7 and IL‐15 induce cell proliferation and rescue from apoptosis in a concentration, time and subset‐dependent manner, and have implications for the homeostatic expansion of the naïve CD8⁺ T‐cell pool.     

TLR5 functions as an endocytic receptor to enhance flagellin-specific adaptive immunity

Innate immune activation via TLR induces dendritic cell maturation and secretion of inflammatory mediators, generating favorable conditions for naïve T‐cell activation. Here, we demonstrate a previously unknown function for TLR5, namely that it enhances MHC class‐II presentation of flagellin epitopes to CD4⁺ T cells and is required for induction of robust flagellin‐specific adaptive immune responses. Flagellin‐specific CD4⁺ T cells expanded poorly in TLR5‐deficient mice immunized with flagellin, a deficiency that persisted even when additional TLR agonists were provided. Flagellin‐specific IgG responses were similarly depressed in the absence of TLR5. In marked contrast, TLR5‐deficient mice developed robust flagellin‐specific T‐cell responses when immunized with processed flagellin peptide. Surprisingly, the adaptor molecule Myd88 was not required for robust CD4⁺ T‐cell responses to flagellin, indicating that TLR5 enhances flagellin‐specific CD4⁺ T‐cell responses in the absence of conventional TLR signaling. A requirement for TLR5 in generating flagellin‐specific CD4⁺ T‐cell activation was also observed when using an in vitro dendritic cell culture system. Together, these data uncover an Myd88‐independent function for dendritic cell TLR5 in enhancing the presentation of peptides to flagellin‐specific CD4⁺ T cells.     

Dysregulation of toll‐like receptor (TLR) 2 expression on monocytes and upregulation of the frequency of T cells expressing TLR2 in patients with chronic hepatitis C virus infection

Toll‐like receptors (TLRs) initiate inflammatory responses that may play a role in disease progression in patients infected with hepatitis C virus (HCV). TLR2 and TLR4 surface expression were assessed on CD14⁺ monocytes, CD4⁺ and CD8⁺ T cells in treatment naïve patients with chronic HCV infection with fibrosis, without fibrosis, co‐infected with human immunodeficiency virus (HIV), and in healthy controls. Increased expression of TLR2 was found on monocytes in HCV‐infected patients with fibrosis (p < 0.01), co‐infected with HIV (p = 0.03), and possibly in patients without fibrosis (p = 0.07) when compared to controls. TLR2 positive CD4⁺ and CD8⁺ T cells were upregulated in patients with fibrosis only (p < 0.01). However, expression of TLR2 was not associated with T cell activation. TLR4 expression was similar in patients and healthy controls. In conclusion, TLR2 expression on monocytes and the frequency of T cells expressing TLR2 may contribute to disease progression in chronic HCV infection.     

Periodontitis‐associated pathogens P. gingivalis and A. actinomycetemcomitans activate human CD14+ monocytes leading to enhanced Th17/IL‐17 responses

The Th17/IL‐17 pathway is implicated in the pathogenesis of periodontitis (PD), however the mechanisms are not fully understood. We investigated the mechanism by which the periodontal pathogens Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa) promote a Th17/IL‐17 response in vitro, and studied IL‐17⁺ CD4⁺ T‐cell frequencies in gingival tissue and peripheral blood from patients with PD versus periodontally healthy controls. Addition of Pg or Aa to monocyte/CD4⁺ T‐cell co‐cultures promoted a Th17/IL‐17 response in vitro in a dose‐ and time‐dependent manner. Pg or Aa stimulation of monocytes resulted in increased CD40, CD54 and HLA‐DR expression, and enhanced TNF‐α, IL‐1β, IL‐6 and IL‐23 production. Mechanistically, IL‐17 production in Pg‐stimulated co‐cultures was partially dependent on IL‐1β, IL‐23 and TLR2/TLR4 signalling. Increased frequencies of IL‐17⁺ cells were observed in gingival tissue from patients with PD compared to healthy subjects. No differences were observed in IL‐17⁺ CD4⁺ T‐cell frequencies in peripheral blood. In vitro, Pg induced significantly higher IL‐17 production in anti‐CD3 mAb‐stimulated monocyte/CD4⁺ T‐cell co‐cultures from patients with PD compared to healthy controls. Our data suggest that periodontal pathogens can activate monocytes, resulting in increased IL‐17 production by human CD4⁺ T cells, a process that appears enhanced in patients with PD.     

TLR9 stimulation drives naïve B cells to proliferate and to attain enhanced antigen presenting function

Mechanisms that regulate naïve B cell proliferation and function are incompletely defined. In this study, we test the hypothesis that naïve B cell expansion, survival and ability to present antigen to T lymphocytes can be directly modulated by Toll‐like receptor (TLR) agonists. In the absence of B cell receptor stimulation, CpG oligonucleotide, a TLR9 agonist, was particularly efficient in inducing naïve B cell proliferation and survival. Although the expanded naïve B cells did not mature into CD27⁺ or IgG⁺ memory B cells, these cells did differentiate into IgM‐secreting cells with increased surface expression of HLA‐DR, CD40 and CD80. This was associated with an increased potential for these B cells to activate allogeneic T cells. We propose that the activation and expansion of naïve B cells induced by TLR9 agonists could enhance the potential of these cells to interact with cognate antigens and facilitate cell‐mediated immune responses.     

DRB1*0402 may influence arthritis by promoting naive CD4+ T‐cell differentiation in to regulatory T cells

HLA‐DRB1*0401 expression in humans has been associated with a predisposition to developing rheumatoid arthritis (RA) and collagen‐induced arthritis (CIA), while HLA‐DRB1*0402 is not associated with susceptibility. Here, we determined if mice transgenic (Tg) for human *0401 have a CD4⁺ T‐cell repertoire that predetermines proinflammatory cytokine production. The data show that both *0401 and *0402 Tg mice can produce TH1/TH17 cytokines, although the kinetics of response may be different. However, in the context of antigen‐specific responses in a CIA model, *0402 Tg mice generate a TH2 response that may explain their resistance to developing arthritis. In addition, a significant subset of naïve CD4⁺ T cells from *0402 Tg mice can be activated in polarizing conditions to differentiate into Treg cells that produce IFN‐γ. *0401 Tg mice harbor memory CD4⁺ T cells that differentiate into IL‐17⁺ cells in various polarizing conditions. Our data suggest that *0401 Tg mice generate a strong immune response to lipopolysaccharide and may be efficient in clearing infection, and may *0401 have been evolutionarily selected for this ability. Autoimmunity, such as RA, could likely be a bystander effect of the cytokine storm that, along with the presence of low Treg‐cell numbers in *0401 Tg mice, causes immune dysregulation.     

A distinct Toll-like receptor repertoire in human tonsillar B cells, directly activated by PamCSK, R-837 and CpG-2006 stimulation

Toll‐like receptors (TLRs) recognize specific pathogen‐associated molecular patterns (PAMPs), which subsequently trigger innate immunity. Recent data also suggest a role for TLRs in the direct activation of adaptive immune cells. In the present study, the expression and function of TLR1–TLR10 were characterized in purified human tonsillar B cells, focusing on differences among CD19⁺ CD38^– CD27^– (naïve B cells), CD19⁺ IgD^– CD27^–[germinal centre (GC) B cells] and CD19⁺ CD38^– CD27⁺ (memory B cells) cells. The study was also designed to compare the TLR expression in B cells obtained from infected and hyperplastic tonsils that served as controls. The results demonstrated a distinct repertoire of TLRs, in which TLR1, TLR2, TLR7, TLR9 and TLR10 predominated. No differences were found among naïve, GC and memory B cells. Tonsillar infection did not substantially alter the TLR expression profile in ex vivo‐isolated B‐cell subsets. Purified CD19⁺ B cells stimulated with Pam₃CSK₄, R‐837 and CpG oligodeoxynucleotide (ODN) 2006, via TLR1/TLR2, TLR7 and TLR9, respectively, showed an induction of interleukin‐6 secretion and an up‐regulated expression of human leucocyte antigen (HLA)‐DR. Collectively, the present study demonstrates that B cells exhibit constitutively high levels of specific TLRs, which are not developmentally regulated during the B‐cell differentiation process. Ongoing microbial infections, such as chronic tonsillitis, do not appear to affect the TLR profile in B cells. Furthermore, the distinct expression of TLRs allows B cells to respond directly to the cognate PAMPs. This further emphasizes the role of TLRs in directly activating adaptive immune cells.     

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.