We thank the NIH/NCRR Resource this website for Nonhuman Primate Immune Reagents (Emory University, Atlanta, GA) for the macaque recombinant proteins; the NIH Division of Veterinary Resources (Bethesda, MD) for providing macaque blood samples; Dr Bernard A.P. Lafont (Laboratory of Molecular Microbiology, NIAID/NIH) for providing 721.221 cells; Drs Alison E. Hogg and L. Jean Patterson (Vaccine Branch, NCI/NIH) for helpful discussions; and Katherine M. McKinnon (Vaccine Branch Flow Cytometry Core, NCI/NIH) for expert advice. This research was supported by the Intramural Research Program of

the NIH, National Cancer Institute. Figure  S1. CD8α- macaque NK cells represent 35 percent of CD3-CD8α+ lymphocytes and express both CD56 and CD16. “
“Experimental autoimmune thyroiditis (EAT) is commonly induced by thyroglobulin (Tg) or Tg peptides in mice genetically susceptible to thyroiditis. In the present study, we investigated the immunogenic and pathogenic potential of a novel 20mer human Tg peptide, p2208 (amino acids 2208–2227), in mouse strains classified as low (LR) or high (HR) responders in EAT. The peptide was selected for its content in overlapping binding motifs for MHC class II products, associated with either resistance (Ab), or susceptibility

(As, Ek) to EAT. We therefore immunized LR BALB/c (H-2d) and C57BL/6 (H-2b) strains, as well as HR CBA/J (H-2k) (-)-p-Bromotetramisole Oxalate and SJL/J (H-2s) mice with 100 nmol of p2208 in adjuvant Olaparib and collected their sera, lymph nodes and thyroid glands for further analysis. The p2208 peptide was found to contain B-cell and cryptic T-cell epitope(s) in two of the

four strains examined, one LR and one HR. Specifically, it elicited direct EAT in C57BL/6 mice (two of seven mice, infiltration index 1–3), as well as in SJL/J mice (two of six mice, infiltration index 1–2). Such an EAT model could provide insights into the immunoregulatory cascades taking place in resistant hosts. “
“An oral delivery system based on ApxIIA#5-expressed on Saccharomyces cerevisiae was studied for its potential to induce immune responses in mice. Murine bone marrow-derived dendritic cells (DCs) stimulated in vitro with ApxIIA#5-expressed on S. cerevisiae upregulated the expression of maturation and activation markers, leading to production of tumor necrosis factor-α, interleukin (IL)-1β, IL-12p70 and IL-10. Presentation of these activated DCs to cluster of differentiation CD4+ T cells collected from mice that had been orally immunized with the ApxIIA#5-expressed on S. cerevisiae elicited specific T-cell proliferation.

Because infusion of haploidentical male mouse splenocytes

was found previously to prevent diabetes in NOD mice we looked for, but found no evidence of, persistent chimeric lymphocytes from haploidentical paternal origin within the dams’ splenocytes. Gestation per se appears to have no aggravating or ameliorating effects on pre-existent autoimmune beta cell destruction, but pregnancy from MHC partially Dinaciclib chemical structure mismatched males delays diabetes onset in female NOD mice. In type 1 diabetes, autoimmune mechanisms are involved in the destruction of the insulin-producing beta cells in the pancreatic islets of Langerhans leading to the eventual need for insulin replacement therapy in patients [1,2]. Pregnancy has the capacity to alter both immune CB-839 mw response and beta cell function, but its effects on the development of autoimmune diabetes are largely unknown. Pregnancy is reported to ameliorate autoimmune diseases [3–5] through establishing a privileged state of tolerance potentially by shifting immune responses towards a less inflammatory state (reviewed in Piccinni [6]). However, this may not be the case for type 1 diabetes. Pregnancy also increases insulin demand with an expansion of beta cell mass [7–9], and a

number of islet autoantibody-positive women develop diabetes during gestation [10]. Evidence in humans indicates that increasing insulin demand aggravates autoimmune diabetes, and that increasing insulin demand Adenosine triphosphate at a late stage of preclinical disease will anticipate the onset of clinical diabetes [11–13]. We reasoned that examining the effect of gestation in the non-obese diabetic (NOD) mouse may be informative with respect to accelerating or delaying the onset of autoimmune diabetes. Because it is reported that the relative matching of the fetus may be important in the maternal tolerance state, we further reasoned that partially or

fully mismatched fetuses may provide advantage in controlling maternal autoimmunity. We therefore mated NOD female mice with male NOD mice, major histocompatibility complex (MHC) haploidentical mice and fully MHC mismatched mice and followed the female mice for diabetes development during and after pregnancy. The findings of our study are inconsistent with the notion that pregnancy accelerates the development of autoimmune diabetes, but support amelioration when mating is with haploidentical males. NOD mice were obtained originally from Taconics (Germantown, NY, USA) and C57BL/6J mice from The Jackson Laboratory (Bar Harbor, ME, USA), and the colonies established in the animal facilities at the Diabetes Research Institute Munich. The frequency of diabetes within untreated female NOD mice at the time of the study was 89% at age 36 weeks. Four male CByB6F1/J mice, F1 hybrids of female BALB/cByJ and male C57BL/6J mice were purchased at age 8 weeks from The Jackson Laboratory.

In addition, I found many eosinophilic inclusion bodies in small neurons of the deeper layers of the cerebral cortex. Then, I wondered what these bodies were. Prior selleck chemicals llc to that time, it was thought that Lewy bodies only rarely occurred in the cerebral cortex. Thereafter, I also found many similar cortical eosinophilic bodies in the brain of another patient2 with similar clinical symptoms. I became interested in these cortical eosinophilic bodies. Morphologically these bodies were similar to, but somewhat different from, brain stem Lewy

bodies. Therefore, I could not identify these cortical eosinophilic bodies as Lewy bodies. Based on histochemical and electron microscopic examinations, I demonstrated that these cortical eosinophilic bodies were cortical Lewy bodies. In 1978, we reported a second paper2“Lewy bodies in cerebral cortex” in Acta Neuropathologica, based on our three cases including the first case. In that report, the close relationship between cortical Lewy bodies and neuronal cell death was for the first time

indicated by showing six developmental stages of cortical Lewy bodies. In addition, we pointed out for the first time that SCH727965 cell line the amygdala and claustrum are also predilection sites for Lewy bodies. Following these papers, some similar cases were reported in Japan. In the USA, a Japanese neuropathologist, Okazaki, and his colleagues5 reported two similar American autopsied cases without Alzheimer pathology Tenofovir purchase in 1961, but these cases had not received attention until our citation of their paper in our first paper.1 In addition, Forno et al.6 also reported a similar American case in 1978. In 1979, we reported3 two similar German cases when I was at the Max-Planck Institute of Psychiatry in Munich. These were the first DLBD cases reported in Europe. In 1984, we proposed4 the term “diffuse Lewy body disease (DLBD)” based on our 11 autopsies

including Japanese, German and Austrian cases. As we proposed it in 1980, 11 DLBD was thought to be a type of “Lewy body disease”. We classified Lewy body disease into three types: brainstem type, traditional type and diffuse type. The diffuse type is now considered DLBD, while the brain stem type is considered PD. After our proposal of DLBD in 1984, this disease received more attention among European and American researchers. In 1990, I reviewed 37 autopsied DLBD cases reported in Japan.9 Then I classified these cases into two forms: a common form with a more or less Alzheimer pathology, and a pure form without such findings. In addition, I pointed out that the clinical features differed between the two forms. In the common form, all cases showed presenile or senile onset, and the chief clinical feature was progressive dementia, followed by parkinsonism in 70% of cases, while in the pure form most cases showed early onset and the chief clinical symptom was parkinsonism, followed by dementia.

This study aimed to clarify the effect of sodium restriction on prolonging the duration between the time when eGFR is 15 mL/min/1.73 m2 p38 MAP Kinase pathway to hemodialysis (HD) induction (G5 spans). Methods: Seventy-seven type 2 DKD patients (61 men and 16 women, mean age 58.6 ± 11.2 years) were recruited. All patients underwent frequent nutritional therapy and 24-h urine collection. Sodium intake was calculated using the 24-h urine collection. Patients

were divided into the following 2 groups: adequate group (AG: n = 39) defined as patients with sodium intake < 8.0 g/day, and over-intake group (OG: n = 32) defined as sodium intake ≧ 8.0 g/day. We retrospectively evaluated the G5 span between the 2 groups. Results: The Alisertib glycated hemoglobin value was 6.4 ± 1.8% when eGFR was firstly 15 mL/min/1.73 m2. In all patients, the G5 span was 556 ± 372 days, and the sodium intake was 7.9 ± 3.2 g/day. The G5 was significantly

longer in AG than in OG (660 ± 403 days vs. 487 ± 314 days, p < 0.05). Conclusion: Sodium restriction ameliorates the progression of renal dysfunction in type 2 advanced DKD patients (CKD stage G5). RAVI RAMA1,2, RAVI RAJALAKSHMI1,2, KURIEN ABRAHAM1,2, NAIR SANJEEV1,2, YUVARAJ ANAND1,2, ABRAHAM GEORGI1,2, RAVICHANDRAN SANGEETHA2, PANDIAN DEVI1,2 1Madras Medical Mission; 2Tamilnad Kidney Research Foundation Introduction: The current scenario of global burden of diseases comprise of a triple burden of diseases of which non communicable diseases form a huge proportion. Among the non communicable diseases, chronic kidney disease has emerged a major threat in terms of complications, accessibility and availability of treatment, especially in developing countries like India. There are a few studies done on prevalence of kidney disease and our programme targets early detection of kidney disease in the form of awareness and screening programmes directed at different segments of the society. Methods: The awareness programme

comprises of powerpoint presentation on basics of kidney functions and symptoms for early detection of kidney disease. The screening programme consists of brief history of medical illness, followed by measurement Janus kinase (JAK) of body mass index and blood pressure and urine examination to look for proteinuria. Results: We have so far conducted a total of 447 programmes of which 93.5% of the programmes were targeted to urban areas and we covered 79.2% of students through our awareness programmes. Our programme identified prehypertension in 38.7% of the population screened and 24.% were identified with proteinuria. Individuals who were above 45 years of age, and those with proteinuria were found to be significantly associated with abnormal serum creatinine and eGFR.

A key feature of several of these agents is the potential to induce tolerogenic effects that outlast generalized suppression of the immune system and are therefore of particular interest for future interventions in T1D. Fc receptor non-binding anti-CD3 monoclonal antibodies (mAbs) show much promise in preliminary trials, as a short course of treatment can delay the post-diagnosis High Content Screening decline in stimulated C-peptide for up to 5 years, with depletion of T cells evident for a limited period of time (< 1 months) [13]. These agents demonstrate clearly that modulation of β cell autoimmunity in humans can be achieved

without the need for continuous immunosuppression. A recent trial using anti-CD20 (Rituxan) to target B lymphocytes in patients with recent-onset T1D [12] found that the window between generalized immunosuppression and tolerance towards β cells appears to be smaller than that of anti-CD3. This trial was nevertheless noteworthy because of the well-documented safety profile of B lymphocyte depletion. It is also known that B lymphocyte infiltration is a significant late-stage event

in T1D [14]. Thus, as no single agent demonstrates the ability to induce durable disease remission, anti-CD20 therapy could serve as a rapid, anti-inflammatory component of a rational combinational intervention [14,15]. Indeed, a further lesson from the past 20 years is that the immunological defects Protease Inhibitor Library manufacturer responsible for T1D are multiple and complex, and are not likely to be addressed with a single agent. It is more probable that multiple pathways will need to be modulated in order to achieve a lasting remission. For example, down-regulation of the inflammatory response, elimination of autoreactive effector

and memory T cells, and the induction and long-term maintenance of T and B regulatory cell populations may all be required in varied degrees to induce robust disease remission. Furthermore, given the level of β cell destruction observed at the onset of overt disease, the ideal intervention would be one that not only halts the autoimmune response, but also enhances Amisulpride β cell function or stimulates regeneration. Drugs that have shown promise either in preclinical or early clinical trials fall into a few general classes: T cell modulators [anti-CD3, anti-thymocyte globulin (ATG)], B cell-depleting agents (anti-CD20), anti-inflammatory molecules [anti-interleukin (IL)-1, anti-tumour necrosis factor (TNF)-α], antigen-specific therapies [insulin, glutamic acid decarboxylase-65 (GAD65), islet autoantigenic peptides [16]] and incretin mimetics (insulinotropic agents, such as exenatide) (see Fig. 1 and also an earlier comprehensive review by Staeva-Vieira [17]).

Representatives of five ixodid tick genera were compared, both metastriate species (D. reticulatus, R. appendiculatus, H. excavatum and A. variegatum) and a prostriate species (I. ricinus). The D. reticulatus and I. ricinus ticks were collected by flagging the vegetation in selected locations of western Slovakia previously used for tick collecting; R. appendiculatus, H. excavatum and A. variegatum were obtained from colonies maintained at the Institute of Zoology (Bratislava). Hyalomma excavatum was the kind gift of Dr Michael Samish, Kimron Veterinary Institute,

Bait Dagan, Israel. It is both a two-host ditropic tick, with larvae and nymphs feeding on the same ABT-888 individual host animal while adults feed on entirely different host species, and a three-host tick with larvae, nymphs and adults each feeding on a different animal. To maintain our H. excavatum colony, the ticks were fed on rabbits: 70–80% followed a two-host strategy while the remainder were three-host. Larvae fed for 6–9 days, nymphs for 7–10 days and adult females fed for 8–12 days to complete engorgement; larvae + nymphs (two-host strategy) completed engorgement as nymphs in 11–28 days. ZIETDFMK SGE was prepared by modifying the method of [13]. Briefly, at given times, ticks were gently removed from the laboratory animals and their

salivary glands dissected out in ice-cold sterile 0.15 m NaCl (0.9%) and washed three times in the same

solution. Salivary gland tissues were then homogenized and centrifuged at 10 000 g for 30 min at 4°C. Supernatant fluids were dried using a Speed-Vac, stored at 4°C and reconstituted in PBS before use. Pooled SGE was prepared from ticks feeding on laboratory rabbits for two time periods: 3 days representing the early (slow) period and 7 days representing Tenoxicam the late (rapid) phase of engorgement (Table 1). Before testing, the pooled dried SGE was diluted such that 10 μL contained SGE from a single tick. The hypostome of ticks is sclerotized and does not change size or shape once the tick has moulted [14]. Live ticks were immobilized on double-sided tape, and the tube-shaped hypostome from the apex to the base of the cheliceral shaft (dorsal aspect) was measured by means of an eyepiece and lens micrometre using a binocular microscope (Nikon SMZ 645; Optoteam S.R.O., Bratislava, Slovak Republic) at magnification, ×50. Antigrowth factor activities were measured using commercial ELISA kits and recombinant growth factors obtained from R&D Systems (Abingdon, UK): human fibroblast growth factor, FGF-2 (basic; DFB50); human hepatocyte growth factor, HGF (DHG00); human IL-6 (D6050); human keratinocyte growth factor, KGF (DKG00); human/mouse platelet-derived growth factor, PDGF-AA (DAA00); human stromal cell-derived factor, SDF-1α (DSA00); and human-transforming growth factor, TGF-β1 (DY240).

Supporting Information Fig. 1C and D show that in these animals, Egr2 mRNA levels were reduced in all of the T-cell populations isolated. Some Egr2 mRNA remained in the immature

DP population, but Egr2 expression was lost in more mature populations in the thymus and in peripheral T cells. Egr2 genomic deletion, mediated by CD4Cre, was effective in all subsets, with only a residual percentage of the sorted cells retaining an intact Egr2 locus. Staining for CD4 and CD8 in both Egr2-Tg and knockout Egr2f/fCD4Cre thymocytes, relative to their respective littermate controls, GSI-IX ic50 showed that, although thymocyte numbers remained similar (mean total thymocyte numbers (×106) for each genotype were: Egr2-Tg, 220.2±44.7, https://www.selleckchem.com/products/pexidartinib-plx3397.html compared with NT littermates, 222.2±57.0, and Egr2f/fCD4Cre, 153.8±47.2, compared with Egr2f/f littermates, 138.4±19.8), there were broadly reciprocal effects upon the proportion of CD4SP and CD8SP cells. Egr2-Tg mice had a 1.75-fold gain in absolute numbers of mature (TCRhi; data not shown) CD8SP (p=<0.01; Fig. 2A), leading to a skewing of the CD4:CD8 ratio from an average of 6.4 in WT littermate controls to 2.8 in their Tg counterparts. Egr2f/fCD4Cre mice had fewer CD4SP and CD8SP cells, leading to a small but significant overall reduction in the numbers and percentage of mature SP thymocytes

(p=0.05; Fig. 2B). Taken together, the phenotypes of the overexpressing and knockout lines suggest that gain of Egr2 enhances generation or survival of CD8 lineage T cells, whereas loss of Egr2 negatively affects generation or survival of both CD4 and CD8 SP thymocytes following this website positive selection. To determine whether Egr2 could misdirect lineage commitment following positive selection, we crossed Egr2-Tg mice and littermate controls with β2m−/− mice, which produce exclusively CD4SP thymocytes; MHC class II−/− mice, which only produce CD8SP thymocytes, and mice lacking both β2m and MHC class II (MHC° mice), whose thymocytes cannot progress beyond the naïve

DP stage. Expression of the Egr2 Tg further enhanced the development of CD8SP thymocytes on an MHC class II−/− background (Fig. 3, centre panel), but had no effect on generation of CD4SP thymocytes on the β2m−/− background (Fig. 3, left panel). On a β2m−/− background, there was also a small increase in the number of CD8 SP cells generated, as previously seen with Egr1 overexpression 24, but in the absence of selecting MHC, there was no change from littermate controls, in which only background levels of SP cells could be seen (Fig. 3, right panel). We also bred Egr2f/fCD4Cre mice with Tg mice expressing either the OTII TCR, in which thymocytes are selected into the CD4 lineage, or the F5 TCR, in which thymocytes are selected into the CD8 lineage, both on RAG-deficient backgrounds to preclude rearrangement and expression of endogenous TCR.

USUI JOICHI1, GLEZERMAN ILYA G3, CHANDRAN LDE225 supplier CHANDRA B4, SALVATORE STEVEN P2, FLOMBAUM CARLOS D3, SESHAN SURYA V2 1University of Tsukuba; 2Weill Cornell Medical College, Cornell University; 3Memorial Sloan-Kettering Cancer Center; 4St. Joseph’s Regional Medical Center Introduction: Cancer therapies have been supplemented by vascular endothelial growth factor(VEGF) inhibitors as anti-angiogenic agents in the recent years. The present work discloses the spectrum of pathological features in VEGF inhibitor-associated kidney disease. Methods: Pathological findings of kidney disease were retrospectively studied in 4 cancer patients treated

with VEGF inhibitors, bevacizumab (anti-VEGF-A), PS-341 nmr with chemotherapeutic agents. Results: All patients

presented with acute kidney injury. All kidney biopsies showed endothelial injury of varying severity, including one with typical active features of thrombotic microangiopathy(TMA). Evidence of chronic endothelial injury and vascular sclerosis were also observed. Furthermore, acute tubular injury with focal necrosis was seen in all cases. Conclusion: A range of renal pathologic lesions secondary to endothelial injury are noted often accompanied by acute tubular damage following anti-VEGF therapy, the most severe being TMA. The role of other nephrotoxic chemotherapeutic agents in enhancing renal injury and other host factors with possible pathological variety should be considered. RAPUR RAM1, ADIRAJU KRISHNA PRASAD2, GUDITI SWARNALATHA2, GAURISHANKAR SWARNALATHA3, KALIGOTLA VENKATA DAKSHINAMURTY3 1Sri Venkateswara Insitute of Medical Sciences, Tirupati; 2Nizam’s Institute of Medical Sciences, Hyderabad; 3Apollo Hospitals, Hyderabad Introduction: Introduction: Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired chronic disorder characterized by a triad of clinical features- haemolytic anaemia, pancytopenia, and thrombosis. Not many

reports of renal involvement in PNH are available in literature. We present a case series of PNH with renal involvement. Methods: Materials and methods: We present the data of PNH patients PRKD3 attended to departments of General Medicine and Nephrology at a government run tertiary care institute in South India. The patients’ data was maintained on an out- patient case record. The diagnosis of PNH in these patients during initially phase, between 1998 and 2004 was based on sucrose lysis and Ham’s test. After 2004, the diagnosis was based on flow cytometry to detect CD59 (MIRL), a glycoprotein, and CD55 (DAF) in regulation of complement action. Results: The patient data was collected from 1998 to 2012. There were 26 patients of paroxysmal nocturnal haemoglobinuria in this period. The mean age was 37 years and the range was 16 to 68 years. There were 14 females. ARF was noted in ten patients.

This is important as the concentration of complement proteins in serum is very high. Therefore, to inhibit complement activity in totality, either a set of inhibitory proteins or a multicomplement-binding protein could fulfil such a requirement. The complement proteins usually act on the surface of target pathogens. However, blocking of complement activation in blood-sucking H. contortus is all the more important as antibodies formed against the internal proteins of the parasite during infection

[42] in combination with complement proteins (acquired during blood meal) would damage the internal tissues with serious consequences for the parasite. Identification of H.c-C3BP should facilitate development of new therapeutics considering a key role of this protein in immune modulation. We thank Director, IVRI, Lumacaftor in vitro for providing the necessary facilities, Prof. Anil K Jaiswal, University of Maryland, USA, for mass spectrometry. This work was supported by a grant from the Department of Biotechnology, Government of India, to PJ. “
“Plasmacytoid dendritic cells (PDC) are involved in innate immunity by interferon (IFN)-α production,

and in adaptive immunity by stimulating T cells and inducing generation of regulatory T cells (Treg). In this study we studied the effects of mammalian target of rapamycin (mTOR) inhibition by rapamycin, a commonly used immunosuppressive and anti-cancer drug, on innate and adaptive immune functions of human PDC. A clinically relevant concentration Decitabine concentration of rapamycin inhibited Toll-like receptor (TLR)-7-induced IFN-α secretion potently (−64%) but TLR-9-induced IFN-α secretion only slightly (−20%), while the same concentration suppressed proinflammatory cytokine production by TLR-7-activated and TLR-9-activated PDC with similar

efficacy. Rapamycin inhibited the ability of both TLR-7-activated and TLR-9-activated PDC to stimulate production of IFN-γ and interleukin (IL)-10 by allogeneic T cells. Surprisingly, mTOR-inhibition enhanced the capacity of TLR-7-activated PDC to stimulate naive and memory T helper cell proliferation, which was caused by rapamycin-induced PAK6 up-regulation of CD80 expression on PDC. Finally, rapamycin treatment of TLR-7-activated PDC enhanced their capacity to induce CD4+forkhead box protein 3 (FoxP3)+ regulatory T cells, but did not affect the generation of suppressive CD8+CD38+lymphocyte activation gene (LAG)-3+ Treg. In general, rapamycin inhibits innate and adaptive immune functions of TLR-stimulated human PDC, but enhances the ability of TLR-7-stimulated PDC to stimulate CD4+ T cell proliferation and induce CD4+FoxP3+ regulatory T cell generation. Plasmacytoid dendritic cells (PDC) have important functions in innate and adaptive immunity. They are unique in rapidly producing massive amounts of type I interferon upon recognition of viral nucleotides or self-DNA-protein complexes by their Toll-like receptors (TLR).

While blockade of IL-6 by adding neutralizing antibodies against IL-6 and the IL-6 receptor α-chain at the beginning of the coculture greatly reduced the inhibitory effect of TLR7 Epacadostat solubility dmso ligand on TGF-β-induced Foxp3 expression (13.9±0.3 versus 52.1±3.3% inhibition, p<0.01), neutralizing antibodies against IL-4 (31.3±8.8 versus 52.1±3.3% inhibition, p=0.04) and against IFN-γ (32.5±13.9 versus 52.1±3.3% inhibition, p=0.13) had only minor effects. However, simultaneous blockade of IL-6, IFN-γ,

and IL-4 from the beginning of the coculture entirely abrogated the inhibitory effect of TLR7 ligand on Foxp3 expression after 4 days (2.4±2.3 versus 52.1±3.3% inhibition, p<0.01, Fig. 3D). Neutralization of IL-6 in the supernatant of DCs stimulated with TLR7 ligand also reduced its inhibitory effect on TGF-β-induced Foxp3 expression in TLR7−/− T cells stimulated with anti-CD3/anti-CD28 (Supporting Information Fig. S1C). Accordingly, addition of recombinant IL-6 at the beginning of the DC–T-cell coculture significantly reduced Foxp3 expression

on day 4 (Fig. 3E). Thus, mostly IL-6 and to a minor extent IFN-γ and IL-4 produced in the DC–T-cell coculture are responsible for the observed reduction in Foxp3 expression in the presence of TLR7 ligand. The reduced percentage of Foxp3-expressing T cells measured after 4 days of DC–T-cell APO866 cost coculture in the presence of TLR7 ligand could be due to reduced initial induction of Foxp3 expression by TGF-β in naïve T cells or to downregulation of Foxp3 expression during the coculture. Alternatively, reduced proliferation or survival of induced Foxp3+ as compared with Foxp3− T cells in the coculture could be responsible for the observed effect of TLR7 ligand. We therefore analyzed Foxp3 expression by flow cytometry at different time points during the DC–T-cell coculture in the presence or absence of TLR7 ligand. We observed that the initial induction of Foxp3 expression by TGF-β within the first 2–3 days was not affected by addition of TLR7 ligand (Fig. 4A, left panel). Foxp3 is functional at this

early time point, since Foxp3+ T cells isolated from TLR7 ligand containing PLEK2 cocultures at day 2 suppress responder T-cell proliferation as efficiently as Foxp3+ T cells generated in the absence of TLR7 ligand (Fig. 5A, left panel). However, the percentage of Foxp3+ cells decreased progressively after 3 days in cocultures treated with TLR7 ligand, whereas it remained relatively stable in the absence of TLR7 ligand. In addition, the mean fluorescence intensity (MFI) of Foxp3 staining in Foxp3+ cells was significantly reduced in Tregs generated in the presence of TLR7 ligand in comparison to Tregs generated in the absence of TLR7 ligand at days 4 and 5 but not at earlier time points (Fig. 4A, right panel). IL-6 is already produced early after the beginning of the coculture stimulated with TLR7 ligand (day 1) and then further increases with incubation time (Supporting Information Fig. S2A).