Although the mechanism by which H1R and H2R regulates T-cell effector function is poorly understood, possible mechanisms

include multiple signaling through HRs, receptor density on a particular cell type, the use of different second messenger molecule/pathways or direct/indirect effect on T cells, APCs, or both. Therefore, while H1R and H2R signaling clearly influences CD4+ T-cell differentiation and effector functions, HR signaling may also contribute to EAE pathogenesis by acting in other cells types associated with disease and remains the subject of future studies. Pathophysiology associated with MS is thought to be initiated by peripheral autoreactive T ABT-263 cell line cells that cross the BBB and elicit neuroinflammation or autoimmune responses that are secondary to the events initiated by the CNS tissue [[43]]. Unlike other HRs, H3R is expressed primarily on nonhematopoietic cells. It is predominantly expressed presynaptically and regulates the release of HA and other neurotransmitters. H3RKO mice develop significantly more severe acute early phase disease, neuropathology, and increased

BBB barrier permeability compared with B6 mice. T cells from H3RKO mice restimulated ex vivo with MOG35–55 had greater expression of MIP-2, IP-10, and CXCR3 with no significant difference in the Th1, Th2, or Th17 cytokine production [[18]]. H4R expression is confined mainly on hematopoietic cells and its activation can result in actin polymerization, upregulation LDK378 supplier of adhesion molecules, and chemotaxis of many immune cells [[44-46]]. However, recently H4R has been Protein kinase N1 shown to be functionally expressed in

the CNS [[17]]. H4RKO mice develop more severe MOG35–55 induced EAE, augmented neuroinflammation, and increased BBB permeability compared with B6 mice. Similar to H3RKO mice, H4RKO mice had no effect on the production of Th1, Th2, or Th17 cytokines in ex vivo recall assays [[34]]. Based on the phenotypes observed in the single HRKO animals, it was surprising for us to find no difference in the production of IFN-γ by H1H2RKO CD4 T cells in ex vivo recall assays, nor a difference in BBB permeability in H3H4RKO mice. Importantly, however, H1H2RKO mice had a significant decrease in BBB permeability while H3H4RKO mice had significantly increased production of IFN-γ and IL-17 compared with B6 mice. The observed phenotypes in H1RKO and H2RKO mice parallels the phenotypes seen in H3H4RKO mice while the H3RKO and H4RKO phenotypes mimic those of H1H2RKO mice. The basis of this yin-yang effect is unknown but may be due to differential cross-regulation of HR expression. Here, we show that in the absence of a single HR, the expression of the remaining HRs is increased above B6 levels in CD4+ T cells.

In addition, mRNA expression of IFN-stimulated genes such as MxA, a key effector molecule of the innate antiviral response [24], and RIG-I was also increased 4 days p.i. (Fig. 5C). Collectively, these data demonstrate that HTNV-infected human lung epithelial cells produce IFN-β and type III IFN. At this point, we investigated whether IFN is absolutely required for HTNV-triggered modulation of MHC-I expression. For this purpose, we used Vero E6 cells. Although they lack type I IFN genes [25],

Vero E6 cells increased MHC-I expression in response to HTNV (Fig. 6A). This could have been due to type III IFN induced by HTNV. We tested this possibility by adding IFN-λ1 to uninfected Vero E6 cells and subsequent FACS analysis. As shown in Fig. 6B (left graph), MHC-I surface expression on uninfected Vero E6 cells was significantly upregulated by exogenously added IFN-α but not by IFN-λ1.

However, Ibrutinib a comparatively weak induction of MxA expression was observed in IFN-λ1-treated Vero E6 cells (Fig. 6B, right graph) in accordance with previous results obtained by other Deforolimus investigators [26]. Increased MHC-I surface expression was also detected on uninfected Vero E6 cells after transfection of HTNV RNA (Fig. 6C). Vesicular stomatitis virus (VSV) RNA was used as a positive control as it is a known strong stimulator of innate responses. Importantly, type III IFN was found in the supernatant of Vero E6 cells after transfection with VSV RNA but not HTNV RNA (Fig. 6D). Collectively, these data suggest that an IFN-independent mechanism contributes to HTNV-induced MHC-I upregulation. Similar to epithelial cells, HTNV-infected DCs upregulated expression of HLA-I and other immunologically important molecules such as ICAM-1 (Fig. 7A). Thus, we next asked whether HTNV induces cross-presentation that is crucial for induction of antiviral CD8+ T-cell responses. We used pp65, a human cytomegalovirus (HCMV) encoded matrix protein, as a model antigen to test cross-presentation by HTNV-infected DCs. Immature DCs expressing the human HLA-I molecule HLA-A2 were either left uninfected or infected with BCKDHB HTNV. These cells were then

fed with lysates derived from uninfected (control) or HCMV-infected (pp65 containing) fibroblasts. After co-culture with pp65-specific HLA-A2-restricted T lymphocytes at different ratios, IFN-γ production was analyzed (Fig. 7B). Large amounts of IFN-γ were found in co-cultures of pp65-specific T cells with DCs in the presence of phorbol 12-myristate 13-acetate , a strong polyclonal T-cell stimulus. Intriguingly, substantial IFN-γ secretion occurred also in co-cultures of pp65-specific T cells with HTNV-infected DCs that had been fed with pp65-containing lysates. Importantly, uninfected DCs incubated with pp65-containing lysates did not activate T cells. As expected, co-culture of pp65-specific T cells with either uninfected or HTNV-infected human DCs fed with control lysates did not result in IFN-γ secretion.

Moreover, both studies, Jang et al. [24] and our, showed that the total frequency of the AA haplotype was highest (90.3% and 85.3%, respectively) and the GG haplotype was lowest (4.5% and 0.6%, respectively) in diseased patients and controls. Some authors have reported that gender differences in the disease phenotype among patients

with RA; however, no statistically Crizotinib manufacturer gender differences were noted at diagnosis (Table 1). Our findings have shown that both analysed IL-17F gene polymorphisms were not associated with gender. We also have shown that the impact of the His161Arg IL-17F gene polymorphism was more significant than that of the Glu126Gly. Our detailed genotype–phenotype analysis indicated that IL-17F 161Arg variant was Pexidartinib ic50 associated with higher number of tender joints (P = 0.03), higher mean value of DAS-28-CRP and higher HAQ score, suggesting that this polymorphism might be associated with an increased disease activity (Table 4). Moreover, our findings have shown that patients with RA with rare allele of the IL-17F Glu126Gly variant had a tendency to have longer

disease duration than a carrier of two wild-type alleles (P = 0.07, Table 5). Perhaps the IL-17F His161Arg and/or Glu126Gly substitution may directly regulate the IL-17F expression. IL-17A, IL-17F and IL-23 may play an important role in T-cell-triggered inflammation by upregulating some of gene products involved in cell activation, proliferation and growth and it is an important inductor of various cytokines and chemokines that are crucial in regulating inflammatory response [37]. Our hypothesis suggests

that polymorphisms in the IL-17 gene may cause redundant production of some proinflammatory Tyrosine-protein kinase BLK cytokines, such as IL-1β and TNF-α, which can mediate inflammatory pathology in many autoimmune diseases, including RA. In addition, in autoimmune diseases, TNF-α is responsible for the inflammatory and protective aspects, and IL-1β is responsible for the destructive processes [37]. Moreover, IL-1β polymorphism was also associated with the parameters of disease activity [data not shown]. And maybe the relationship between IL-17F and severity of RA is connected with expression of IL-1β or other proinflammatory cytokines. Only two other genetic studies have shown relationship between IL-17 family cytokine and RA, however, they analysed IL-17A but not IL-17F [38, 39]. Nordang GB et al. [39] analysed the IL-17 gene by tagging the main genetic variation and they found a weak but significant correlation with the IL-17A promoter polymorphism, rs2275913, in Norwegian patients with RA. However, Furuya et al. [38] examined the association between SE, age at RA onset, radiographic progression in Japanese patients with early RA and three SNPs in the IL-17A gene, rs3804513, rs3748067, rs1974226. They suggested that rs3804513 IL-17A gene polymorphism may be associated with radiographic progression in patients with RA.

[33-36]. Palinauskas et al. [33] infected 5 passerine species with the same generalist Plasmodium relictum (lineage

SGS1) and investigated the parasitaemia OTX015 molecular weight and the associated costs for the hosts. While starlings (Sturnus vulgaris) were fully resistant to the infection, the other four species showed a variable pattern of resistance/tolerance. House sparrows (Passer domesticus) were partially resistant because 50% of inoculated birds established a successful infection, whereas 100% of chaffinches (Fringilla coelebs), crossbills (Loxia curvirostra) and siskins (Carduelis spinus) were susceptible to the infection. Within the susceptible species, infection intensity showed huge variation with siskins and crossbills having the highest peak of parasitaemia. However, when looking at the reduction in haematocrit (the proportion of red blood cells, a good proxy of infection-induced fitness cost), only the two species

with experimental highest parasitaemia seemed to suffer from the infection. This study therefore strongly suggests that avian Apoptosis Compound Library price hosts exhibit interspecific variation in their propensity to be resistant/tolerant to Plasmodium parasites. The co-infection with two Plasmodium species (Plasmodium relictum and Plasmodium ashfordi) led to a very different outcome depending on the host species [34]. Whereas starlings were again fully resistant to the infection by the two parasites, siskins and crossbills were highly susceptible, with parasitaemia in double-infected birds being higher than in single infected hosts. The two susceptible species appear to differ in terms of tolerance to the infection.

Indeed, even though siskins and crossbills have similar peak parasitaemia, siskins paid a much smaller cost of infection (a smaller reduction in haematocrit values and no infection-induced mortality). This experimental work therefore shows that generalist malaria parasites infecting a large number of host species nevertheless achieve quite different infection dynamics and incur quite different costs for their hosts possibly due to a combination of resistance and tolerance processes. A pending question is what accounts for this interspecific pattern of resistance/tolerance even for closely related host Obeticholic Acid clinical trial species. Variation in life history traits among species has been suggested to explain specific propensity to invest in costly inflammatory response [20]. However, the species used by Palinauskas et al. [33, 34] have similar paces of life. Immunologically naïve hosts, in particular those that have not coevolved with avian malaria, are predicted to suffer more from infection. The accidental introduction of avian malaria in the Hawaiian archipelago provides a textbook illustration of a rapid evolutionary change in a novel host–parasite association.

In the absence of commensal microbiota, mice have been shown to be unable to efficiently resist infections at different anatomical sites, such as influenza A in the lung and oral Listeria infection [21, 25, 26, 53, 58]. Mice lacking commensal microbiota have also been shown not to develop pathology in experimental models of autoimmunity, such as those for multiple CP-868596 chemical structure sclerosis and arthritis [59-61]. These mice have also been shown to respond poorly to different types of cancer immune- and chemotherapy [22, 62]. Although the precise mechanisms behind these observations still need to be clarified, GF or antibiotics-treated animals have been shown to have a reduced number of different subsets of T cells,

such as Th1 and Th17 cells constitutively producing IFN-γ and IL-17, respectively. They also present an expansion of Treg cells, and fail to activate innate resistance and adaptive immunity responses to systemic infections [20, 21, 53, 26]. Thus, in the absence of the commensal microbiota, a decreased Alectinib solubility dmso inflammatory and immune setting is established, which is lower than that required for optimal responses to stimuli. While the microbiota at all barrier surfaces is likely able to contribute

to local immunity [57], the systemic immune homeostatic effect of the microbiota has been largely ascribed to the gut microbiota [21, 25]. Colonization of the skin of GF mice with bacterial species that efficiently reconstitute skin immunity has been shown to have no systemic effect, for example, skin bacterial

colonization does not enhance the activation of Th1 cells and Th17 cells in the intestinal lamina propria [53]. The possible predominant effect of the gut microbiota at the systemic level may be due to its higher diversity and higher total number of microorganisms (up to a trillion) than that in other organ [63], as well as to the large surface area that the gut mucosa and the associated Cobimetinib immune organs comprise. However, because most experimental evidence is based on the use of GF mice or use of oral antibiotics that may deplete the microbiota at sites other than the gut, for example, in the oral cavity, it is possible that the microbiota at all barrier sites in combination may contribute to the observed systemic effects. Moreover, despite the great variation among microbiota at different body sites, the community types present at the different anatomical barriers have been found to be predictive of each other [64]: thus, it is possible that the observation of a correlation between a particular immune phenotype and the microbiota of a given organ, for example, the gut, may reflect the contribution of other organs, for example, the oral cavity. The epithelial barrier is maintained not only by the presence of tight junctions among epithelial cells and physicochemical barriers, such as keratin and mucous layers, but also by active mechanisms mediated by soluble products (e.g.

The ability of the DNA vaccine constructs to elicit cellular immune responses makes them an attractive weapon as a safer vaccine candidate for preventive and therapeutic applications against tuberculosis. Tuberculosis (TB) is a major local, regional and global infectious disease problem with about 9 million new cases and

2 million deaths every year [1]. Mycobacterium tuberculosis kills more adults each year than any other single pathogen. The vaccination with Mycobacterium bovis bacille Calmette Guerin (BCG) is considered to be the most important tool to protect against TB [2]. In spite of its widespread use and many advantages like being inexpensive, safe at birth, given as a single shot and provision of some protection against leprosy, BCG vaccination remains controversial [2–4]. Daporinad mouse The protection afforded by BCG vaccination has shown wide variations in different parts of the world, and its impact on the global problem of TB remains unclear [5]. Estimates of protection given by BCG against pulmonary TB vary greatly [4]. For example, a trial in British school children, in 1952, showed about 80% efficacy, whereas the Chingleput trial in India showed zero efficacy

of protection against adult pulmonary selleck chemicals TB, after BCG vaccination [4, 6]. This variability has been attributed to various factors including strain variation in BCG preparations, environmental influences such as sunlight exposure, poor cold-chain maintenance, genetic or nutritional differences between populations and exposure Selleck Temsirolimus to environmental mycobacterial infections etc. [5]. In addition, because of sharing most of the antigens, BCG vaccination induces a delayed-type hypersensitivity skin response to the purified protein derivative of M. tuberculosis (the stimulus used to test the individuals for tuberculous infection), which cannot be distinguished from exposure to M. tuberculosis [7]. This makes the use

of tuberculin skin test difficult for diagnostic or epidemiological purposes. Furthermore, BCG vaccination cannot be used in all groups of people, e.g. WHO has recommended that children with symptoms of HIV or AIDS should receive all the vaccines except BCG. This is because BCG is a live attenuated vaccine that might cause disease in immuno-compromised people rather than giving immunity [8]. Thus, there is an urgent need to develop M. tuberculosis-specific and safer vaccines against TB [6, 9]. The development of a better BCG vaccine or alternative vaccines needs the identification and evaluation of antigens recognized by protective immune responses [9]. In previous studies, we have identified RD1 PE35 (Rv3872), PPE68 (Rv3873), EsxA (Rv3874), EsxB (Rv3875) and RD9 EsxV (Rv3619c) as M. tuberculosis-specific antigens [10–13]. Furthermore, in vitro studies in patients with TB and healthy subjects infected with M. tuberculosis have shown that these antigens induced cellular immune responses that correlate with protection [9].

In contrast to naturally occurring CD4+CD25+ Tregs, DN T cells have to be activated by antigen-presenting cells (APCs) to induce their regulatory

potential. The suppressive activity of DN T cells is neither mediated indirectly by modulation of APCs nor by competition for T-cell growth factors. Furthermore, DN T-cell-mediated suppression toward responder T cells is TCR dependent and requires novel protein synthesis. In contrast to murine CHIR99021 DN T cells, which eliminate effector T cells via Fas/FasL or perforin/granzyme, human DN T cells suppress proliferation of responder T cells by cell contact-dependent mechanisms. Taken together, our data indicate that human DN T cells exert strong immunosuppressive effects on both CD4+ and CD8+ T cells and may serve as a new therapeutic approach to treat autoimmunity and transplant rejection. Suppression of immune responses by Tregs is critical

for the induction and maintenance of self-tolerance. Tregs have been shown to be involved in downregulating immune responses Torin 1 nmr in autoimmunity, transplant rejection, graft-versus-host disease (GvHD), and tumor immunity 1–3. Numerous studies demonstrated that a variety of T-cell subsets possess immunoregulatory properties: the population of thymus-derived naturally occurring CD4+CD25+ forkhead box P3 (Foxp3)+ T cells is currently the most extensively investigated subset of Tregs and their role has been studied in a wide range else of animal models and in humans 4–7. However, inducible Tregs such as T-regulatory type 1 (Tr1) cells,

T-helper 3 (Th3) cells, CD8+CD28− T cells, and TCR-αβ+ CD4−CD8− double-negative (DN) T cells are generated in the periphery and also show the ability to inhibit immune responses 8–11. In both mice and humans, about 1–5% of all peripheral T cells are of TCR-αβ+ DN phenotype 11, 12. These cells express a specific set of cell surface molecules and show a characteristic cytokine profile 11, 13. The group of L. Zhang was the first to identify and characterize the immunoregulatory function of DN T cells. They have demonstrated that murine DN T cells specifically eliminate activated anti-donor CD4+, CD8+ T cells and B cells 11, 13–15. Moreover, adoptive transfer of DN T cells prolongs skin and heart allograft survival in murine models 11, 13, 16–19. Others have shown that mouse DN T cells are highly potent in suppressing T-cell responses both in vitro and in vivo in an antigen-specific manner and therefore induce skin and islet allograft survival 20. Even now, the function and ontogeny of human DN T cells still remains elusive. Of interest, in a recent clinical report, an inverse linear relationship between the severity of GvHD and the frequency of DN T cells could be demonstrated in patients after allogeneic stem cell transplantation 21.

The hierarchy of resistance to suppression described in this AIG model has implications for the design

of Treg-based therapies in terms of which responses can be targeted effectively by Tregs, and which type of Tregs are most appropriate for the job. This was highlighted by a further study in this experimental system, which illustrated once again the additive effects of activation status and antigen specificity in determining the capacity of Tregs to modulate autoaggressive responses. Only antigen-specific (not polyclonal) iTreg can suppress the development of Th17-induced pathology in the gastritis model [96]. A similar pattern of responsiveness to Treg-induced suppression ZD1839 price has been observed in several other model systems. The ameliorative effect of all trans-retinoic acid treatment on the development of type 1 diabetes is dependent upon an expansion of FoxP3+

Tregs which suppress the generation of IFN-γ but not IL-17 responses [97]. We have found that Tregs isolated from the central nervous system (CNS) of mice with EAE suppress IFN-γ production efficiently by CNS-derived effector T cells in co-culture, but are unable to suppress their production of IL-17 [76]. Our own unpublished studies also suggest that polarized myelin-responsive Th17 populations are relatively resistant to Treg-mediated suppression of their proliferation in vitro, compared to their Th1 counterparts. selleckchem Consistent data from human studies show that Th17 cells are resistant to Treg-mediated suppression at the level of proliferation [98], as well as cytokine production [99]. Extrapolation of these in vitro studies would suggest that Th17

cells might preferentially resist Treg-mediated control of their clonal expansion in vivo. As yet, this has not been Protein tyrosine phosphatase tested formally. It therefore appears that Th1 responses are perhaps the most acutely sensitive to Treg-mediated suppression, while Th17 responses appear most resistant. The basis for differential sensitivity to regulation remains unclear. However, factors associated with Th17 responses (IL-6, IL-21, TNF-α and potentially IL-17 itself) impair the suppressive capacity of Tregs and may thus prevent suppression of Th17 responses selectively. Several studies have presented persuasive arguments that the suppressive function of Tregs must, at times, be subverted to allow inflammatory immune responses to effectively eliminate pathogens. Central to this hypothesis is the ability of the innate immune system to sense the presence of a pathogen via Toll-like receptor (TLR) signalling and respond by producing proinflammatory cytokines such as IL-6, which overcome Treg-mediated suppression [100]. IL-6 blockade has been shown to restrain the development of both Th1 and Th17 responses following immunization [101]. IL-6 influences the development and expansion of effector and Treg cell responses as well as Treg function, and this has been demonstrated most elegantly in the EAE model.

The chronic phase of Chagas disease is either asymptomatic or may lead to cardiac and digestive system pathology.

Chagas heart GSI-IX mw disease is a potentially fatal dilated cardiomyopathy that develops in 30% of T. cruzi-infected individuals [2] and is responsible for the largest number of deaths among chagasic patients. Clinical treatment of chagasic cardiomyopathy-associated hypertension in chagasic patients includes sodium restriction and additional treatment with digitalis, diuretics or angiotensin-converting enzyme (ACE) inhibitors, such as captopril [3,4]. As true for other ACE inhibitors, captopril has also been reported to reduce heart inflammation and fibrosis [5]. ACE has a dual role in vascular homeostasis. Acting primarily in the renin–angiotensin system, ACE processes the inactive intermediate angiotensin I (Ang I), generating the vasopressor octapeptide angiotensin II (Ang II). Although Ang II may bind to different subtypes of G protein coupled

receptors, excessive formation of this agonist may increase intracellular volume, peripheral vascular resistance and blood pressure [5]. ACE inhibitors such as captopril exert their anti-hypertensive effects by inhibiting ACE-dependent formation of the vasopressor Ang II and by attenuating ACE (kininase II)-dependent degradation of bradykinin (BK) or find more lysyl-bradykinin (LBK) [6]. Termed collectively as ‘kinins’, BK/LBK are short-lived peptides liberated from an internal moiety of high or low molecular weight kininogens by the action of specialized proteases of host [7] or microbial origin [8,9].

Once released, BK/LBK exert their vasodilating function by triggering endothelium BK2R, a constitutively expressed G-protein coupled receptor (GPCR) [10]. Alternatively, the released kinins old undergo processing by kininase I, generating arginine-truncated metabolites (des-Arg-kinin) that activate BK1R, an inducible subtype of kinin receptor up-regulated in inflamed tissues [11], while losing affinity for BK2R. Studies on cruzipain, a lysosomal cysteine protease characterized previously as a kinin-releasing enzyme of T. cruzi[12], provided the first evidence that pathogen uptake is driven by the activation of kinin receptors (BK2R and BK1R) [13,14]. Whether involving human endothelial cells or murine cardiomyocytes, these in vitro studies revealed that addition of captopril to the interaction medium potentiated parasite invasion via the kinin signalling pathway [13,14]. More recently, it was reported that BK/LBK induces the maturation of dendritic cells (DCs) through the signalling of BK2R [15,16]. Further underscoring the importance of kinins and ACE to pathogenic outcome, Monteiro and co-workers [17] demonstrated that ACE inhibitors (single-dose administration) potentiated paw oedema evoked by trypomastigotes through mechanisms involving co-operation between Toll-like receptor (TLR)-2 and BK2R.

Carbonyl iron was added to PBMC at 37°C for 60 min to remove phagocytic cells (Invitrogen). The B and CD4+ T cells were removed by positive selection with immunomagnetic beads: CD19 pan B cell and CD4 beads (Dynal; Invitrogen) at 4°C for 30 min. The remaining cells were incubated with 0·4 μg/ml purified anti-CD28 antibody (BD Biosciences, Oxford, UK) (4°C for 20 min) followed by anti-mouse IgG beads (Dynal; Invitrogen) at 4°C for 30 min. The purity of the negatively isolated CD8+CD28− Treg expressing CD3 was >95%, as determined by flow cytometric analysis. For T cell

and monocyte isolation the T cell-negative (Dynal; Invitrogen) and CD14-positive isolation kits (Dynal; Invitrogen) were used, respectively, according to

the manufacturer’s instructions. Selleck Rucaparib Heparinized PB (100 μl) was incubated with antibodies for 20 min at 4°C, then with 2 ml fluorescence activated cell sorter (FACS) lysing solution (BD Biosciences) for 10 min at room temperature and washed twice in immunofluorescence buffer (IFB) (phosphate-buffered saline with 0·05% sodium azide and 0·1% bovine serum albumin) for 5 min and fixed in 1% paraformaldehyde in IFB (Sigma, Poole, UK). PBMC in IFB were surface-stained with required antibodies for 20 min on ice, Talazoparib ic50 washed twice in IFB and fixed for analysis. Analysis for all samples was carried out with a FACSCalibur flow cytometer (BD Biosciences) using CellQuest software (BD Biosciences). CD8+CD28− Treg were placed in co-culture with autologous responder PBMC Etofibrate at ratios of

1:1, 0·2:1 and 0·1:1 (PBMC 105 cells/well). Cultures were stimulated with anti-CD3 antibody (1/1000 dilution) [muromonab-CD3 (OKT3)] [American Type Cell Collection (ATCC), Rockville, MD, USA] in 96-well flat-bottomed plates (Corning Costar, Sunderland, UK) and incubated in a 5% CO2 humidified atmosphere at 37°C for 72 h. CD8+CD28− Treg were co-cultured with either allogeneic responder T cells from HC or RA(MTX). Each HC or RA(MTX) CD8+CD28− Treg sample was co-cultured with autologous T cells or allogeneic T cells isolated from two HC and two RA(MTX). Cultures were stimulated with CD3/CD28 beads (Dynal, Invitrogen) and incubated for 72 h at 37°C. TNF inhibitor [infliximab (IFX), 10 ng/ml; Remicade®, Centocor, the Netherlands], anti-TGF-β1 antibody (5 μg/ml, clone 1D11, mIgG1; R&D Systems, Abingdon, UK) and LEAF™ purified mouse IgG1, k isotype control (clone MG1-45; Biolegend, Cambridge, UK), were added at the start of culture in the functional assays. All reagents were added to either the 1:1 co-culture or PBMC alone. CD8+CD28− Treg were co-cultured with autologous responder PBMC and CD14+ cells at a ratio of 1:1:1 in the presence or absence of a semi-permeable membrane held in a TW (0·4 μm pore size) (Corning Costar).