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.     

Serotonin decreases the production of Th1/Th17 cytokines and elevates the frequency of regulatory CD4+ T‐cell subsets in multiple sclerosis patients

Excessive levels of proinflammatory cytokines in the CNS are associated with reduced serotonin (5‐HT) synthesis, a neurotransmitter with diverse immune effects. In this study, we evaluated the ability of exogenous 5‐HT to modulate the T‐cell behavior of patients with MS, a demyelinating autoimmune disease mediated by Th1 and Th17 cytokines. Here, 5‐HT attenuated, in vitro, T‐cell proliferation and Th1 and Th17 cytokines production in cell cultures from MS patients. Additionally, 5‐HT reduced IFN‐γ and IL‐17 release by CD8⁺ T cells. By contrast, 5‐HT increased IL‐10 production by CD4⁺ T cells from MS patients. A more accurate analysis of these IL‐10‐secreting CD4⁺ T cells revealed that 5‐HT favors the expansion of FoxP3⁺CD39⁺ regulatory T cells (Tregs) and type 1 regulatory T cells. Notably, this neurotransmitter also elevated the frequency of Treg17 cells, a novel regulatory T‐cell subset. The effect of 5‐HT in upregulating CD39⁺ Treg and Treg17 cells was inversely correlated with the number of active brain lesions. Finally, in addition to directly reducing cytokine production by purified Th1 and Th17 cells, 5‐HT enhanced in vitro Treg function. In summary, our data suggest that serotonin may play a protective role in the pathogenesis of MS.     

The intestinal expansion of TCRγδ+ and disappearance of IL4+ T cells suggest their involvement in the evolution from potential to overt celiac disease

Celiac disease (CD) is characterized by a spectrum of intestinal inflammatory lesions. Most patients have villous atrophy (overt‐CD), while others have a morphologically normal mucosa, despite the presence of CD‐specific autoantibodies (potential‐CD). As the mechanism responsible for villous atrophy is not completely elucidated, we investigated biomarkers specific for the different celiac lesions. Phenotype and cytokine production of intestinal mucosa cells were analyzed by flow cytometry in gut biopsies of children with overt‐ or potential‐CD and in healthy controls. Density of TCRγδ⁺ T cells was found markedly enhanced in intestinal mucosa of children with overt‐CD compared to potential‐CD or controls. By contrast, very few IL4⁺ T cells infiltrated the mucosa with villous atrophy compared to morphologically normal mucosa. IL4⁺ T cells were classical CD4⁺ T‐helper cells (CD161^?), producing or not IFN‐γ, and negative for IL17A. Our study demonstrated that the transition to villous atrophy in CD patients is characterized by increased density of TCRγδ⁺ T cells, and concomitant disappearance of IL4⁺ cells. These findings suggest that immunomodulatory mechanisms are active in potential‐CD to counteract the inflammatory cascade responsible of villous atrophy. Further studies are required to validate the use of IL4⁺ and TCRγδ⁺ T cells as biomarkers of the different CD forms.     

Early effector maturation of naïve human CD8+ T cells requires mitochondrial biogenesis

The role of mitochondrial biogenesis during naïve to effector differentiation of CD8⁺ T cells remains ill explored. In this study, we describe a critical role for early mitochondrial biogenesis in supporting cytokine production of nascent activated human naïve CD8⁺ T cells. Specifically, we found that prior to the first round of cell division activated naïve CD8⁺ T cells rapidly increase mitochondrial mass, mitochondrial respiration, and mitochondrial reactive oxygen species (mROS) generation, which were all inter‐linked and important for CD8⁺ T cell effector maturation. Inhibition of early mitochondrial biogenesis diminished mROS dependent IL‐2 production – as well as subsequent IL‐2 dependent TNF, IFN‐γ, perforin, and granzyme B production. Together, these findings point to the importance of mitochondrial biogenesis during early effector maturation of CD8⁺ T cells.     

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.     

IL‐4 and IL‐15 promotion of virtual memory CD8+ T cells is determined by genetic background

Virtual memory (VM) CD8⁺ T cells are present in unimmunized mice, yet possess T‐cell receptors specific for foreign antigens. To date, VM cells have only been characterized in C57BL/6 mice. Here, we assessed the cytokine requirements for VM cells in C57BL/6 and BALB/c mice. As reported previously, VM cells in C57BL/6 mice rely mostly on IL‐15 and marginally on IL‐4. In stark contrast, VM cells in BALB/c mice rely substantially on IL‐4 and marginally on IL‐15. Further, NKT cells are the likely source of IL‐4, because CD1d‐deficient mice on a BALB/c background have significantly fewer VM cells. Notably, this NKT/IL‐4 axis contributes to appropriate effector and memory T‐cell responses to infection in BALB/c mice, but not in C57BL/6 mice. However, the effects of IL‐4 are manifest prior to, rather than during, infection. Thus, cytokine‐mediated control of the precursor population affects the development of virus‐specific CD8⁺ T‐cell memory. Depending upon the genetic background, different cytokines encountered before infection may influence the subsequent ability to mount primary and memory anti‐viral CD8⁺ T‐cell responses.     

Phenotypic differences of CD4+ T cells in response to red blood cell immunization in transfused sickle cell disease patients

Alloimmunization against red blood cells (RBCs) is the main immunological risk associated with transfusion in patients with sickle cell disease (SCD). However, about 50–70% of SCD patients never get immunized despite frequent transfusion. In murine models, CD4⁺ T cells play a key role in RBC alloimmunization. We therefore explored and compared the CD4⁺ T‐cell phenotypes and functions between a group of SCD patients (n = 11) who never became immunized despite a high transfusion regimen and a group of SCD patients (n = 10) who had become immunized (at least against Kidd antigen b) after a low transfusion regimen. We studied markers of CD4⁺ T‐cell function, including TLR, that directly control lymphocyte function, and their spontaneous cytokine production. We also tested responders for the cytokine profile in response to Kidd antigen b peptides. Low TLR2/TLR3 expression and, unexpectedly, strong expression of CD40 on CD4⁺ T cells were associated with the nonresponder status, whereas spontaneous expression of IL‐10 by CD4⁺ T cells and weak Tbet expression were associated with the responder status. A Th17 profile was predominant in responders when stimulated by Jb^k. These findings implicate CD4⁺ T cells in alloimmunization in humans and suggest that they may be exploited to differentiate responders from nonresponders.     

Reduced maternal regulatory T cell numbers and increased T helper type 2 cytokine production are associated with elevated levels of immunoglobulin E in cord blood

Background There is evidence that the basis of an atopic‐skewed immune response is acquired early in life, perhaps at the fetal stage. Thus, we hypothesized that the development of the fetal immune system might be influenced by maternal regulatory T cells (Treg) and maternal T cell cytokine production during pregnancy. The aim of the present study was to assess the influence of maternal Treg and cytokine production during pregnancy on Treg and atopy at birth. Methods Within the mother–child study LINA (Lifestyle and Environmental factors and their Influence on Newborns Allergy risk), we determined the frequency and function of Treg and the total IgE concentration in pregnant women in the 34th week of gestation and in corresponding cord bloods at birth (n=24). Furthermore, we assessed how maternal mitogen‐induced T‐helper type 1/T‐helper type 2 and inflammatory cytokines influence the level of cord blood Treg and IgE. Results Frequencies of CD4⁺CD25^high T cells were higher (P=0.001), whereas percentages of FOXP3⁺ T cells were lower (P<0.001) in cord blood cells compared with maternal blood. Reduced maternal CD4⁺CD25^high Treg frequencies correlated with increased total IgE concentrations at the 34th week of gestation (r=?0.32, P=0.028) and with increased IgE concentrations in cord blood (r=?0.50, P<0.001). Elevated maternal mitogen‐induced Th2 cytokine production was related to increased total IgE levels in the serum of corresponding cord bloods (IL‐4, r=0.53; IL‐5, r=0.43; IL‐13, r=0.52). Conclusions Because cord blood IgE has been shown to be predictive for allergic diseases in early childhood, our results indicate that reduced maternal Treg numbers and increased Th2 cytokine production during pregnancy might influence the allergy risk of the child. Cite this as: D. Hinz, J. C. Simon, C. Maier‐Simon, L. Milkova, S. Röder, U. Sack, M. Borte, I. Lehmann and G. Herberth, Clinical & Experimental Allergy, 2010 (40) 419–426.     

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.     

Fasciola hepatica tegumental antigens induce anergic‐like T cells via dendritic cells in a mannose receptor‐dependent manner

FoxP3⁺ Treg cells and anergic T cells are the two regulatory phenotypes of T‐cell responses associated with helminth infection. Here, we examine the T‐cell responses in mice during Fasciola hepatica infection, and to its tegumental coat antigens (FhTeg) that are shed from the fluke every 2–3 h. FhTeg comprises a rich source of glycoproteins, mainly oligomannose N‐glycans that bind to mannose receptor. This study demonstrated a novel mechanism for the T‐cell unresponsiveness observed during F. hepatica infection and after injection with FhTeg. Markers of T‐cell anergy, such as GRAIL, EGR2, ICOS, and ITCH, are enhanced amongst CD4⁺ T‐cell populations during infection and following FhTeg injection. This is characterized by a lack of cytokine responses and reduced proliferative activity, which can be reversed with the addition of IL‐2. FhTeg‐activated dendritic cells (DCs) suppress T cells in vitro as measured by enhanced GRAIL and CTLA4 by RNA and suppressed cytokine expression in anti‐CD3 stimulated CD4⁺ T cells. FhTeg‐treated DCs have enhanced MR expression, which is critical for DC‐CD4⁺ T‐cell communication. Taken together, this study presents markers of anergy in a mouse model of F. hepatica infection, and improves our understanding of host–pathogen interactions and how helminths modulate host immunity.     

IL‐15‐dependent CD8+CD122+ T cells ameliorate experimental autoimmune encephalomyelitis by modulating IL‐17 production by CD4+ T cells

Interleukin‐15 (IL‐15) is an inflammatory cytokine whose role in autoimmune diseases has not been fully elucidated. Th17 cells have been shown to play critical roles in experimental autoimmune encephalomyelitis (EAE) models. In this study, we demonstrate that blockade of IL‐15 signaling by TMβ‐1 mAb treatment aggravated EAE severity. The key mechanism was not NK‐cell depletion but depletion of CD8⁺CD122⁺ T cells. Adoptive transfer of exogenous CD8⁺CD122⁺ T cells to TMβ‐1‐treated mice rescued animals from severe disease. Moreover, transfer of preactivated CD8⁺CD122⁺ T cells prevented EAE development and significantly reduced IL‐17 secretion. Naïve effector CD4⁺CD25^? T cells cultured with either CD8⁺CD122⁺ T cells from wild‐type mice or IL‐15 transgenic mice displayed lower frequencies of IL‐17A production with lower amounts of IL‐17 in the supernatants when compared with production by effector CD4⁺CD25^? T cells cultured alone. Addition of a neutralizing antibody to IL‐10 led to recovery of IL‐17A production in Th17 cultures. Furthermore, coculture of CD8⁺CD122⁺ T cells with effector CD4⁺ T cells inhibited their proliferation significantly, suggesting a regulatory function for IL‐15 dependent CD8⁺CD122⁺ T cells. Taken together, these observations suggest that IL‐15, acting through CD8⁺CD122⁺ T cells, has a negative regulatory role in reducing IL‐17 production and Th17‐mediated EAE inflammation.     

Lack of Mycobacterium tuberculosis–specific interleukin‐17A–producing CD4+ T cells in active disease

Protective immunity to Mycobacterium tuberculosis (Mtb) is commonly ascribed to a Th1 profile; however, the involvement of Th17 cells remains to be clarified. Here, we characterized Mtb‐specific CD4⁺ T cells in blood and bronchoalveolar lavages (BALs) from untreated subjects with either active tuberculosis disease (TB) or latent Mtb infection (LTBI), considered as prototypic models of uncontrolled or controlled infection, respectively. The production of IL‐17A, IFN‐γ, TNF‐α, and IL‐2 by Mtb‐specific CD4⁺ T cells was assessed both directly ex vivo and following in vitro antigen‐specific T‐cell expansion. Unlike for extracellular bacteria, Mtb‐specific CD4⁺ T‐cell responses lacked immediate ex vivo IL‐17A effector function in both LTBI and TB individuals. Furthermore, Mtb‐specific Th17 cells were absent in BALs, while extracellular bacteria‐specific Th17 cells were identified in gut biopsies of healthy individuals. Interestingly, only Mtb‐specific CD4⁺ T cells from 50% of LTBI but not from TB subjects acquired the ability to produce IL‐17A following Mtb‐specific T‐cell expansion. Finally, IL‐17A acquisition by Mtb‐specific CD4⁺ T cells correlated with the coexpression of CXCR3 and CCR6, currently associated to Th1 or Th17 profiles, respectively. Our data demonstrate that Mtb‐specific Th17 cells are selectively undetectable in peripheral blood and BALs from TB patients.     

Peritoneal cavity B‐1a cells promote peripheral CD4+ T‐cell activation

Innate‐like murine B‐1a cells are well known for their ability to secrete natural IgM. Their non‐Ab mediated functions, including Ag presentation to CD4⁺ T cells, are less well explored. Using combined adoptive transfer experiments with peptide‐pulsed peritoneal cavity (PerC)‐derived B‐1a cells and CFSE‐labeled T cells, we show that B‐1a cells present Ag to CD4⁺ T cells from the periphery in vivo. In vitro characterization, using co‐cultures in which B‐1a or splenic B cells presented whole OVA protein to OVA‐specific Tg T cells, shows that B‐1a cells differentially promote intracellular cytokine‐expressing T cells. PerC‐derived B‐1a cells increase the percentage of IL‐10‐producing T cells along with IL‐4‐ and IFN‐γ‐producing CD4⁺ T cells. These data suggest that B cells in the PerC have the potential to influence peripheral immune responses without the necessity to migrate out of this location. This, to our knowledge previously undescribed, immuno‐logical pathway potentially plays a role in the presentation of gut microbiota‐derived Ags to peripheral T cells.