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.     

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.     

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.     

TSST-1 induces Th1 or Th2 differentiation in naïve CD4+ T cells in a dose- and APC-dependent manner

Superantigens are potent activators of the immune system, causing a variety of diseases, ranging from food poisoning to septic shock. Here, we examined the effects of different toxic shock syndrome toxin 1 (TSST‐1) concentrations on the activation, proliferation and synthesis of interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4) in purified naïve human CD4⁺ T cells in a serum‐free in vitro system. TSST‐1 given in low doses (1–10 pg/ml) generates a pronounced T helper 2 (Th2)‐like cytokine profile, characterized by elevated IL‐4‐expressing T‐cell populations and reduced IFN‐γ‐producing populations, whereas higher doses (100 pg/ml) induce a Th1‐like profile, with increased expression of IFN‐γ and reduced expression of IL‐4. These patterns were even more pronounced by adding exogenous cytokines like IL‐12 and IL‐4 and by the type of antigen‐presenting cells (APCs). Thus, B cells induced Th2 shifts, whereas monocytes favoured Th1 induction. Moreover, IL‐12 in conditions with B cells counteracted their Th2 bias. Interestingly, in purified naïve T‐cell cultures, containing a small population of HLA‐DR⁺ T cells, Th1/Th2 differentiation can be induced by TSST‐1 too. There, Th‐cell polarization is strongly dependent on TSST‐1 concentration, indicating that this is a key parameter in regulating the differentiation of T cells. In conclusion, our data show that Th1/Th2 differentiation of TSST‐1‐stimulated naïve T cells is controlled by the type of APCs, and in APC‐depleted cultures, it depends on the presence of HLA‐DR⁺ cells and TSST‐1 concentration.     

Reduced TCR‐dependent activation through citrullination of a T‐cell epitope enhances Th17 development by disruption of the STAT3/5 balance

Citrullination is a post‐translational modification of arginine that commonly occurs in inflammatory tissues. Because T‐cell receptor (TCR) signal quantity and quality can regulate T‐cell differentiation, citrullination within a T‐cell epitope has potential implications for T‐cell effector function. Here, we investigated how citrullination of an immunedominant T‐cell epitope affected Th17 development. Murine naïve CD4⁺ T cells with a transgenic TCR recognising p89‐103 of the G1 domain of aggrecan (agg) were co‐cultured with syngeneic bone marrow‐derived dendritic cells (BMDC) presenting the native or citrullinated peptides. In the presence of pro‐Th17 cytokines, the peptide citrullinated on residue 93 (R93Cit) significantly enhanced Th17 development whilst impairing the Th2 response, compared to the native peptide. T cells responding to R93Cit produced less IL‐2, expressed lower levels of the IL‐2 receptor subunit CD25, and showed reduced STAT5 phosphorylation, whilst STAT3 activation was unaltered. IL‐2 blockade in native p89‐103‐primed T cells enhanced the phosphorylated STAT3/STAT5 ratio, and concomitantly enhanced Th17 development. Our data illustrate how a post‐translational modification of a TCR contact point may promote Th17 development by altering the balance between STAT5 and STAT3 activation in responding T cells, and provide new insight into how protein citrullination may influence effector Th‐cell development in inflammatory disorders.     

The influence of bone marrow‐ and synovium‐derived mesenchymal stromal cells from osteoarthritis patients on regulatory T cells in co‐culture

There is increasing evidence that inflammation in the synovium plays a major role in the progression of osteoarthritis (OA). However, the immunogenic properties of mesenchymal stromal cells (MSCs), which are considered to regulate immunity in various diseases, remain largely unknown in OA. The purpose of this study was to determine the influence of MSCs from OA patients on regulatory T cells (T_regs) in an allogeneic co‐culture model. Bone marrow (BM) and synovial membrane (SM) were harvested from hip joints of OA patients and co‐cultured with lymphocytes enriched in CD4⁺CD25⁺CD127^– regulatory T cells (T_reg⁺LC) from healthy donors. T_reg proportions and MSC markers were assessed by flow cytometry. Cytokine levels were assessed after 2 and 5 days of co‐cultivation. Additionally, T_reg⁺LC cultures were analysed in the presence of interleukin (IL)‐6 and MSC‐supernatant complemented medium. B‐MSCs and S‐MSCs were able to retain the T_reg proportion compared to lymphocyte monocultures. T cell–MSC co‐cultures showed a significant increase of IL‐6 compared to MSC cultures. S‐MSCs produced higher amounts of IL‐6 compared to B‐MSCs, both in single and T cell co‐cultures. The effect of retaining the T_reg percentage could be reproduced partially by IL‐6 addition to the medium, but could only be observed fully when using MSC culture supernatants. Our data demonstrate that retaining the T_reg phenotype in MSC–T cell co‐cultures can be mediated by MSC derived from OA patients. IL‐6 plays an important role in mediating these processes. To our knowledge, this study is the first describing the interaction of MSCs from OA patients and T_regs in an allogeneic co‐culture model.     

High-frequency and adaptive-like dynamics of human CD1 self-reactive T cells

CD1 molecules present lipid antigens to T cells. An intriguing subset of human T cells recognize CD1‐expressing cells without deliberately added lipids. Frequency, subset distribution, clonal composition, naïve‐to‐memory dynamic transition of these CD1 self‐reactive T cells remain largely unknown. By screening libraries of T‐cell clones, generated from CD4⁺ or CD4^?CD8^? double negative (DN) T cells sorted from the same donors, and by limiting dilution analysis, we find that the frequency of CD1 self‐reactive T cells is unexpectedly high in both T‐cell subsets, in the range of 1/10–1/300 circulating T cells. These T cells predominantly recognize CD1a and CD1c and express diverse TCRs. Frequency comparisons of T‐cell clones from sorted naïve and memory compartments of umbilical cord and adult blood show that CD1 self‐reactive T cells are naïve at birth and undergo an age‐dependent increase in the memory compartment, suggesting a naïve/memory adaptive‐like population dynamics. CD1 self‐reactive clones exhibit mostly Th1 and Th0 functional activities, depending on the subset and on the CD1 isotype restriction. These findings unveil the unanticipated relevance of self‐lipid T‐cell response in humans and clarify the basic parameters of the lipid‐specific T‐cell physiology.     

Expansion of activated human naïve T-cells precedes effector function

Naïve T‐cells divide and mature, both functionally and phenotypically, upon stimulation through the T‐cell receptor. Although much is known about the overall changes that occur in naïve cells upon TCR stimulation, and the different memory/effector populations that arise following stimulation, the relationship between cell division and functional and phenotypical changes that occur after activation is poorly understood. Here, we examine the early stages of human naïve and antigen‐experienced T‐cell activation, and the relationship between cell division and acquisition of effector function during the transition from resting antigen‐experienced or naïve T‐cells into effector cells. Stimulated naïve T‐cells proliferate prior to acquisition of effector function, as measured by cytokine production and expression of effector‐associated cell surface molecules. Additionally, we show that interlukin‐7 (IL‐7) can drive proliferation of naïve T‐cells without TCR:MHC peptide interactions. IL‐7 alone does not, however, drive the proliferation of antigen‐experienced T‐cells. Memory T‐cells will divide in response to exogenous IL‐7 but only in the presence of naïve T‐cells and IL‐2. This study contributes to the current understanding of the mechanistic differences between naïve and memory T‐cell responses by defining the functional and phenotypic changes that occur to T‐cells after stimulation.     

Lck regulates IL-10 expression in memory-like Th1 cells

The Src family kinase Lck is thought to facilitate Th2 differentiation; however, its role in Th1 cells has not been well explored. Using mice that lack Lck in mature T cells, we find that lck^−/− Th1 skewed cells have normal expression of T‐bet and produce IFN‐γ at WT levels. However, there is a 3‐fold increase in IL‐10 producing cells in the mutant cultures. These cells do not have elevated levels of IL‐4, GATA3, IL‐17 or Foxp3, indicating that they are not Th2, Th17, or Foxp3⁺ T regulatory cells (Treg). Nor do these cells behave in a similar manner as the type 1 Treg. Most of the IL‐10 in the lck^−/− Th1 cultures is derived from the memory/activated subset, as the cytokine profile from Th1 cultures established from purified CD62L⁺ (naïve) cells are similar to WT cells. Furthermore, this IL‐10 expression appears to be dependent on IL‐12 and correlates with elevated c‐Maf. These data highlight a previously unappreciated role for Lck in regulating IL‐10 in Th1 cells.     

Mycobacterium tuberculosis RpfE promotes simultaneous Th1‐ and Th17‐type T‐cell immunity via TLR4‐dependent maturation of dendritic cells

Reciprocal induction of the Th1 and Th17 immune responses is essential for optimal protection against Mycobacterium tuberculosis (Mtb); however, only a few Mtb antigens are known to fulfill this task. A functional role for resuscitation‐promoting factor (Rpf) E, a latency‐associated member of the Rpf family, in promoting naïve CD4⁺ T‐cell differentiation toward both Th1 and Th17 cell fates through interaction with dendritic cells (DCs) was identified in this study. RpfE induces DC maturation by increasing expression of surface molecules and the production of IL‐6, IL‐1β, IL‐23p19, IL‐12p70, and TNF‐α but not IL‐10. This induction is mediated through TLR4 binding and subsequent activation of ERK, p38 MAPKs, and NF‐κB signaling. RpfE‐treated DCs effectively caused naïve CD4⁺ T cells to secrete IFN‐γ, IL‐2, and IL‐17A, which resulted in reciprocal expansions of the Th1 and Th17 cell response along with activation of T‐bet and RORγt but not GATA‐3. Furthermore, lung and spleen cells from Mtb‐infected WT mice but not from TLR4^?/? mice exhibited Th1 and Th17 polarization upon RpfE stimulation. Taken together, our data suggest that RpfE has the potential to be an effective Mtb vaccine because of its ability to activate DCs that simultaneously induce both Th1‐ and Th17‐polarized T‐cell expansion.     

Dendritic cells erase bad memory

DC not only activate CD4 T (Th) cell and cytotoxic CD8 T cell (CTL) responses against pathogens, but they also tolerize autoreactive T cells in order to avoid autoimmunity. Previous studies have demonstrated that steady‐state DC can tolerize naïve CTL, naïve Th cells and memory CTL. A study in this issue of the European Journal of Immunology demonstrates that DC also tolerize memory Th cells. This is arguably most critical for developing therapies against autoimmune disease; first, because Th cells are the central regulators of all adaptive immune responses, and second because memory, rather than naïve T cells are the clinically relevant cells in established autoimmune diseases. This study fosters hope that DC‐based specific immunotherapies for common autoimmune diseases are possible.     

The immunosuppressive enzyme IL4I1 promotes FoxP3+ regulatory T lymphocyte differentiation

IL4I1 (interleukin‐4‐induced gene 1) is a phenylalanine oxidase produced mainly by APCs of myeloid origin, and converts phenylalanine (Phe) to phenylpyruvate, hydrogen peroxide, and ammonia. We have previously shown that IL4I1 is highly expressed by tumor‐associated macrophages from various human cancers and facilitates immune evasion from the cytotoxic response in a murine tumor model. Indeed, IL4I1 inhibits T‐cell proliferation via hydrogen peroxide toxicity on effector/memory T cells. Here, we explored the effect of IL4I1 on naïve CD4⁺ T‐cell differentiation. We show that IL4I1 stimulates the generation of Foxp3⁺ regulatory T (Treg) cells in vitro from human and mouse T cells. This effect was observed with IL4I1 from different sources, including the naturally produced enzyme. Conversely, IL4I1 limits Th1 and Th2 polarization while modifying the Th17 phenotype, in particular, by inducing its own production. Analysis of Treg‐cell induction under conditions of Phe deprivation and hydrogen peroxide addition suggests that Phe consumption by the enzyme participates in Treg‐cell enrichment. In line with this hypothesis, IL4I1 inhibits mTORC1 signaling shortly after T‐cell activation. Thus, the IL4I1 enzyme may act on T cells both by direct inhibition of effector cell proliferation and by indirect immunoregulation mediated by Treg‐cell induction.     

T‐bet is essential for Th1‐mediated,but not Th17‐mediated,CNS autoimmune disease

T cells that produce both IL‐17 and IFN‐γ, and co‐express ROR‐γt and T‐bet, are often found at sites of autoimmune inflammation. However, it is unknown whether this co‐expression of T‐bet with ROR‐γt is a prerequisite for immunopathology. We show here that T‐bet is not required for the development of Th17‐driven experimental autoimmune encephalomyelitis (EAE). The disease was not impaired in T‐bet^?/? mice and was associated with low IFN‐γ production and elevated IL‐17 production among central nervous system (CNS) infiltrating CD4⁺ T cells. T‐bet^?/? Th17 cells generated in the presence of IL‐6/TGF‐β/IL‐1 and IL‐23 produced GM‐CSF and high levels of IL‐17 and induced disease upon transfer to naïve mice. Unlike their WT counterparts, these T‐bet^?/– Th17 cells did not exhibit an IL‐17→IFN‐γ switch upon reencounter with antigen in the CNS, indicating that this functional change is not critical to disease development. In contrast, T‐bet was absolutely required for the pathogenicity of myelin‐responsive Th1 cells. T‐bet‐deficient Th1 cells failed to accumulate in the CNS upon transfer, despite being able to produce GM‐CSF. Therefore, T‐bet is essential for establishing Th1‐mediated inflammation but is not required to drive IL‐23‐induced GM‐CSF production, or Th17‐mediated autoimmune inflammation.     

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.     

The Wnt inhibitor secreted Frizzled‐Related Protein 1 (sFRP1) promotes human Th17 differentiation

Wnt/β‐catenin signaling plays a crucial role during embryogenesis and tumorigenesis, and in T cells, promotes the differentiation of Th2 cells. However, the role of Wnt signals in the differentiation and maintenance of human Th17 cells remains poorly understood. We found that the higher levels of IL‐17 in the synovial fluid of rheumatoid arthritis (RA) patients compared with that of osteoarthritis (OA) patients were associated with a higher concentration of sFRP1 (secreted Frizzled‐Related Protein 1), an inhibitor of the Wnt/β‐catenin pathway. The addition of sFRP1 during TCR‐mediated stimulation induced a significant increase in IL‐17 production by both naïve and memory CD4⁺ T cells. Moreover, under Th17‐differentiation conditions, the addition of sFRP1 significantly reduced the requirement for TGF‐β. Mechanistically, we observed that sFRP1 significantly enhanced the phosphorylation of Smad2/3 in CD4⁺ T cells upon TGF‐β stimulation and that blocking TGF‐β signaling abolished the Th17‐promoting activity of sFRP1. Our findings reveal a novel function for sFRP1 as a potent inducer of human Th17‐cell differentiation. Consequently, sFRP1 may represent a promising target for the treatment of Th17‐mediated disease in humans.