Then, the cells were cultured in 30 wells of 96-well round-bottomed plates (Corning Inc., Corning, NY) at a density of 2 × 105 cells per well in 150 μl of complete RPMI-1640 medium supplemented with 2 mm l-glutamine, 20 μm 2-mercaptoethanol,

sodium pyruvate, non-essential amino acids, 100 IU/ml penicillin and 100 μg/ml streptomycin, 10 mm HEPES (all from Lonza, Verviers, Belgium), and 5% inactivated human AB serum (Sigma-Aldrich, St Louis, MO) together with p143–160 of Equ c 1 (10 μg/ml) at + 37°C. On day 5, 50 μl of fresh medium was added together with recombinant human IL-2 (rIL-2, final concentration 10 IU/ml; Miltenyi Biotec, Bergisch Gladbach, www.selleckchem.com/products/Everolimus(RAD001).html Germany). On day 10, the cells were restimulated with p143–160 along with 1·5 × 105 γ-irradiated (3000 rads) autologous PBMCs as MK-1775 solubility dmso antigen-presenting

cells (APCs) and rIL-2 (10 IU/ml) in a total volume of 150 μl. On day 15, 50 μl of fresh medium was added together with rIL-2 (final concentration 10 IU/ml). Finally, on day 20, the wells were split to create two replicate plates by transferring 50 μl of cell suspension per well to new 96-well daughter plates. Cultures in one of the daughter plates were stimulated with Equ c 1143–160 (10 μg/ml) and the other served as a control plate. Proliferation was measured, as described below. Positive cultures (stimulation index > 2) were transferred onto a 48-well plate and restimulated with Equ c 1143–160 (10 μg/ml) and rIL-2 (25 IU/ml) in the presence of 106 γ-irradiated autologous PBMCs as APCs. The cell lines were incubated for 14 days and supplemented with fresh medium and rIL-2 (25 IU/ml) every

2–3 days before analyses. The T-cell proliferation assays were set up in triplicates on 96-well round-bottomed plates with 2·5 × 104 T cells and 5 × 104 autologous PBMCs together with the Equ c 1 peptide p143–160 (10 μg/ml) and rEqu c 1 (100 μg/ml). The plates were then incubated for 3 days at Oxalosuccinic acid +37°C, after which the cells were pulsed for 16 hr with 1·0 μCi of [3H]thymidine (GE Healthcare, Little Chalfont, UK) per well and harvested onto glass-fibre filters (Wallac, Turku, Finland). Thymidine incorporation was then measured by scintillation counting (MicroBeta Trilux 1450, Wallac), and the results were displayed as mean counts per minute (CPM) or as stimulation indices (SI; CPM of a stimulated culture divided by CPM of an unstimulated culture). The HLA restriction of the p143–160-specific TCLs was studied by inhibiting the proliferative response to p143–160 with monoclonal antibodies (1 μg/ml) to HLA-DR (clone L243) and HLA-DQ (clone SPVL3), as described previously.[14] A response with at least a 50% inhibition to p143–160 was considered significant. In addition, allogeneic, partially HLA-matched PBMCs from a person expressing only one shared allele with the subject from whom the TCL was derived were used in proliferation assays.

It has been shown that the addition of erythrocytes to cultured slanDC blocks their capacity

to produce IL-12 and TNF-α via the interaction of CD47 on erythrocytes and the corresponding ligand signal regulatory protein α on slanDC.4 After slanDC leave the bloodstream and infiltrate the tissue, as it was shown in inflamed skin of AD lesions, the control by erythrocytes is lost. Our study suggests that histamine might take over this control function in the tissue because we could show that histamine reduces the highly pro-inflammatory capacity of slanDC, particularly via activation of the H4R. To be sure that histamine stimulation does not reduce cytokine production in general, we investigated IL-10 as a see more cytokine OSI-906 nmr that is associated with anti-inflammatory actions. Interleukin-10 reduces the production of IL-2, TNF-α, IFN-γ and co-stimulatory molecules and was shown to counteract the inflammatory response in allergic contact dermatitis.24 In our study we could not observe a significant effect of histamine receptor activation on the release of IL-10. As a result, it can be assumed that H4R and H2R stimulation on slanDC specifically down-regulate the production of the pro-inflammatory mediators TNF-α and IL-12, whereas the level of the anti-inflammatory mediator IL-10 is not affected. The differential regulation of cytokine release by histamine might

be explained by varying signalling processes involved. For example, it was shown that the activation of mitogen-activated protein kinase signalling mediates histamine-induced down-regulation of IL-12p70 in monocytes,15 but on the other hand induces IL-10 production in monocytes.25 Our observations fit the current understanding

of the role of the H4R on antigen-presenting cells. Several studies have shown that the H4R on DC has an anti-inflammatory role: on MoDC, monocytes and inflammatory dendritic epidermal cells the production of IL-12 and CCL2 was down-regulated after H4R activation.15–17 In response to the reduced presence of these mediators, Etofibrate Th1 polarization is impaired and a lower number of macrophages and T cells is attracted to the site of the immune response, respectively. We can draw the conclusion that the stimulation of the H4R on DC, and as shown here in particular on slanDC, could greatly reduce the inflammatory responses taking place in the course of inflammatory skin diseases like AD and H4R agonists therefore might represent potential therapeutic tools in these kinds of diseases. This study was supported by grants from the Deutsche Forschungsgemeinschaft DFG: Gu434/5-1 and GRK1441/1 and the European Community (COST action BM0806). Maria Gschwandtner was supported by a grant from the Hannover Biomedical Research School. The authors declare no conflict of interest.

Secretory IgA is the predominant class of Ig found in human breast milk. This class of non-inflammatory Ig inhibits microbial colonization through decreased adherence of bacteria and viruses to mucosal surfaces and thereby protects against gut and respiratory infections in breastfed children [7]. IgA can also trap food antigens, leading to immune exclusion of dietary antigens by favouring degradation

by pancreatic enzymes [47]. In addition to immune exclusion, IgA can exert immunoregulatory effects [17–20]. The epidemiological evidence of food allergy prevention by IgA [48–51] might be explained by AZD6244 these two mechanisms. As the majority of inhaled antigens reach the gut [52], the presence of milk-borne Der p-specific IgA may then protect the newborn

from respiratory allergens as proposed for food allergens. Notably, we found anti-Der p IgA in all colostrum samples tested. The range of values was broad, and we did not observe significant differences in antibody concentrations between atopic and non-atopic mothers. One previous study assessed the presence of IgA to cat allergen in human breast milk from atopic and non-atopic mothers. This study also found a similar concentration of IgA in both groups [26]. The absence of an effect of atopy on IgA levels could be explained by the fact that IgA class switching depends mainly on the presence of TGF-β [53]. In fact, we found similar levels of TGF-β in colostrum of atopic and non-atopic mothers, and we observed that both total IgA and Der p-specific Selleckchem LY2109761 IgA levels correlated with TGF-β levels in colostrum (Figure S1). In addition to IgA specific for respiratory allergen, our study demonstrated, for the first time, the presence of Der p-specific IgG in colostrum. Der p-specific IgG concentrations were higher in colostrum from atopic mothers compared to non-atopic mothers, and colostrum levels correlated with maternal IgE serum levels. It is worth noting Paclitaxel molecular weight that colostrum Der p-specific IgG concentration correlated with maternal serum IgG levels in the non-atopic

but not in the atopic group. IgG in colostrum could come from maternal serum, as supported by the observation that intravenous administration of Ig to immunodeficient mothers results in the presence of Ig in breast milk [54]. In addition, IgG maybe synthesized locally in the mammary gland. The latter mechanism may operate in the atopic group because there was no correlation between maternal serum and colostrum Der p-specific IgG levels in that group. Studies in rodents suggest that, as in the placenta, FcRn can be involved in IgG transfer across mammary gland epithelium [55]. Notably, in contrast to IgA that stays in the gut lumen, anti-Der p IgG can then be transferred to the neonate by FcRn expressed in the human proximal intestine [39, 56].

Vascular endothelial growth factor and angiopoietin-2 genes and protein expression, endothelial

proliferation as well as free radical levels and antioxidants were assessed in the germinal matrix, white matter and cortex of pups exposed to 100% oxygen and to 21% oxygen. Results: Exposure with 100% oxygen for 1 h did not adversely exacerbate the incidence of glycerol-induced IVH in premature rabbit pups. Compared with room air, 100% oxygen enhanced mRNA expression of both vascular endothelial growth factor and angiopoietin-2 as well as reactive oxygen species levels in the germinal matrix. Hyperoxia did not affect endothelial proliferation, PI3K inhibitor apoptosis or neuronal degeneration in the forebrain. Conclusion: Our data suggest that 100% oxygen exposure for 1 h does not increase the risk of IVH or cerebral injury in premature rabbit pups. Although extrapolating rabbit neural developmental data into humans has obvious limitations, we speculate that hyperoxia of short duration at birth in premature infants may not result in major neurological adverse effects. “
“The aim of this study was to evaluate whether transplantation of human bone marrow stromal cell-derived Schwann cells (hBMSC-SC) promotes functional recovery Depsipeptide concentration after contusive spinal cord injury of adult rats. Human bone marrow stromal cells (hBMSC) were cultured from

bone marrow of adult human patients and induced into Schwann cells (hBMSC-SC) in vitro.

Schwann cell phenotype was confirmed by immunocytochemistry. Growth factors secreted from hBMSC-SC were detected using cytokine antibody array. Immunosppressed rats were laminectomized and their spinal cords were contused using NYU impactor (10 g, 25 mm). Nine days after injury, a mixture of Matrigel and hBMSC-SC (hBMSC-SC group) was injected into the lesioned site. Five weeks after transplantation, cresyl-violet staining revealed that the area of cystic cavity was smaller in the hBMSC-SC group than that in the control group. Immunohistochemstry revealed that the number of anti-growth-associated protein-43-positive Non-specific serine/threonine protein kinase nerve fibers was significantly larger in the hBMSC-SC group than that in the control group. At the same time, the number of tyrosine hydroxylase- or serotonin-positive fibers was significantly larger at the lesion epicenter and caudal level in the hBMSC-SC group than that in the control group. In electron microscopy, formation of peripheral-type myelin was recognized near the lesion epicenter in the hBMSC-SC group. Hind limb function recovered significantly in the hBMSC-SC group compared with the control group. In conclusion, the functions of hBMSC-SC are comparable to original Schwann cells in rat spinal cord injury models, and are thus potentially useful treatments for patients with spinal cord injury.

When logistic regression was used to carry out association analysis after modelling the SNP effects as additive, dominant or recessive, SNP6 (rs7749390, located on the splice site of the exon/intron of ifngr1 gene) showed a significant difference in co-dominant (OR: 1.86, 95% CI: 1.04–3.31) and log-additive (OR: 1.35, Galunisertib cell line 95% CI: 1.02–1.80) models, and P-values were <0.05 after adjustment for sex (Table 4). The log-additive model was accepted as the best inheritance model because of the smaller Akaike information criterion (AIC) value

(565.6). The other SNP showed no association with tuberculosis in any of the five inheritance models (all P > 0.05, data not shown). Pairwise LD between the three SNP of the ifng gene and the other four SNP of ifngr1 was calculated for the cases and controls in the Chinese Han population. The three SNP of the ifng gene had no association with tuberculosis (data not shown). D′ and r for all possible pairs of the SNP in the ifngr1 gene are shown in Table 5. We found strong LD (D′ > 0.75) between the following pairs of the markers in the ifngr1 gene: SNP4/SNP5 (D′ = 0.941), SNP4/SNP6 (D′ = 0.830) and SNP5/SNP6 HER2 inhibitor (D′ = 0.998). Therefore, we constructed SNP4/SNP5/SNP6/SNP7 as haplotype blocks in the ifngr1 gene (because the distance was about 141 bp between SNP6 and

SNP7, SNP7 was list to the haplotype analysis). The frequencies of the estimated haplotypes are presented in Table 6. The association analysis of the haplotypes with tuberculosis is shown in Table 6. The haplotype of SNP4/SNP5/SNP6/SNP7 showed significant association with the disease (P = 0.00079). The HSP90 C-A-A-TT haplotype was observed more frequently in the cases than in the controls (OR: 3.96, 95% CI: 1.90–8.21). The association analysis of haplotypes was adjusted also by sex. In China, tuberculosis is still prevalent with about 5 million cases every year. As only about 10% of the population that

is infected by M. tuberculosis will develop clinical tuberculosis, differences in host immunity and genetic factors may account for the development of tuberculosis after infection [3]. In this study, we tested the hypothesis that the ifng and ifngr1 genes play a role in the pathogenesis of tuberculosis. Seven SNP in these two genes were selected as the gene markers for association analysis. It is accepted generally that IFN-γ plays a pivotal role in the pathogenesis of tuberculosis [7]. Abnormality of the ifng gene is considered as one of the causative factors [8]. An initial study of the association of the ifng gene with tuberculosis indicated that allele A of the +874 A/T in the first intron region was a susceptibility factor for M. tuberculosis infection, both by population- and family-based analysis.

In addition, knock-down of pro-IL-16 expression using #1 siRNA was further confirmed in Western blot analysis using fractionated samples; pro-IL-16 expression

in both nuclear and cytoplasmic extracts prepared from either non-treated or LPS-treated resting B cells was efficiently inhibited (Fig. 4B). selleck chemicals llc Collectively, we successfully impaired pro-IL-16 expression in 38B9 resting B cells using siRNA. Cyclin-dependent kinase (CDK) inhibitor p27kip plays an important role in controlling cell proliferation; degradation of p27kip stimulates cell-cycle transition from the G0 to the S phase, and this process is promoted by the G1 cyclin-CDK complex [25]. In addition, p27 kip downregulates tumour metabolism by changing the cell cycle [26], and its stability is affected by the SCFSkp2 ubiquitin E3 ligase complex [27]. Skp2 is a key component required for ubiquitination and subsequent degradation of p27kip and these two molecules, Skp2 and p27kip, are inversely involved in cell-cycle

regulation. Because pro-IL-16 is known to be critically involved in cell-cycle progression in T cells and overexpression of pro-IL-16 inhibited proliferation of resting B cells, we investigated whether the inhibitory Selleck GSK1120212 role of pro-IL-16 in resting B cell proliferation is associated with the levels of Skp2 and p27kip (Fig. 5). As shown in Fig. 5, knock-down of pro-IL-16 using siRNA resulted in the reduction of p27kip expression as evidenced by Western blot analysis. We detected increased Wilson disease protein expression of Skp2 by knocking-down pro-IL-16 using siRNA, as expected. Although the difference between control and pro-IL-16

siRNA-treated cells was somewhat lower than that observed in LPS non-treated cells, pro-IL-16 siRNA treatment of 38B9 resting B cells reduced p27kip expression and increased Skp2 expression. Collectively, these data suggest that pro-IL-16 exerts its inhibitory function on resting B cell proliferation by reducing the level of Skp2, which degrades p27kip, thereby elevating levels of p27kip. We previously demonstrated that ERK/p38 MAP kinases are involved in mitogen-activated resting B cells proliferation and differentiation and that these kinases are also involved in MHC class II-mediated negative signalling [16, 17, 28]. Consequently, we examined the influence of knock-down of pro-IL-16 using siRNA on the level of MAP kinases (Fig. 6). As shown in Fig. 6, knock-down of pro-IL-16 increased the levels of activated ERK1/2 and p38 MAP kinases, but the level of activated JNK1/2 decreased. A similar pattern of ERK1/2, p38 MAP kinase and JNK1/2 expression was previously observed in LPS-treated resting B cells. Taken together, our results demonstrate that pro-IL-16 transduces inhibitory signalling through MHC class II molecules by inhibiting MAP kinase activation.

IL-12 and type-I IFN were shown to support programming of memory CD8+ T cells in response to Selleckchem Vadimezan L. monocytogenes and VV infection 10. Moreover, it was recently shown that prolonged IL-2 signaling on CD8+ T cells during the priming with LCMV promotes SLEC differentiation 15, 16. Thus, depending on the nature of the infection, the associated cytokine milieu critically regulates effector and memory CD8+ T-cell development. Although there are several in vivo studies focusing on the role of IL-12 in this fate decision process 3–5, a direct role of type-I IFN in the instruction of SLEC versus MPEC differentiation has so far not been studied in vivo. Here, we have

addressed the requirement of type-I IFN signaling on the early fate decision of CD8+ T cells in a type-I IFN biased cytokine milieu as found in LCMV

infection. We provide evidence that direct type-I IFN signaling in CD8+ T cells augments the level of the transcription factor T-bet and thereby instructs the transition of CD8+ T cells toward an SLEC phenotype. CD8+ T cells lacking the type-I IFN receptor fail to form SLECs but instead preferentially give rise to MPECs. Although the primary expansion of these cells is strongly reduced, they have the capacity to develop into functional PDGFR inhibitor memory cells. In summary, the data presented here demonstrate that during infections associated with abundant levels of type-I IFN, the early lineage choice toward the differentiation of SLECs is mediated by direct type-I IFN signaling on CD8+ T cells, identifying type-I IFN as an old important factor instructing the early division between the effector and memory CD8+ T-cell pool. To investigate the role of direct type-I IFN signaling on the SLEC versus MPEC fate decision of CD8+ T cells, we used an established LCMV8.7 and vaccinia virus expressing the LCMV glycoprotein (VVG2) co-infection model 17 combined with adoptive transfer of LCMV gp33-specific

TCR-transgenic CD8+ T cells (P14) which are either sufficient or deficient for type-I IFN signaling (hereafter: WT P14 and interferon-alpha receptor (IFNAR)−/− P14 respectively). Using this system we were able to generate a type-I IFN-dominated inflammatory environment induced by LCMV8.7 infection in face of antigen presentation exclusively derived from a recombinant VVG2, as P14 cells only recognize their cognate epitope expressed by VVG2 but do not recognize the mutant gp33 (V35L) epitope expressed by LCMV8.7. We chose this co-infection system as it avoids the LCMV-inherent abundant antigen presentation and hence puts more emphasis on the role of the cytokine milieu in CD8+ T-cell priming and differentiation. Consistent with previous findings upon single infection with WT LCMV 17–19, WT and IFNAR−/− P14 cells underwent substantial expansion during the first three days after LCMV8.7 and VVG2 co-infection.

26 The identity and purity of the isolated molecules were tested using sodium dodecyl sulphate–polyacrylamide click here gel electrophoresis (SDS-PAGE) and Coomassie Blue or silver staining (not shown). CatG from human sputum or from neutrophils was purchased from Sigma-Aldrich (St Louis, MO); CatL and CatB were

purchased from Caltag (Burlingame, CA) or R & D Systems (Minneapolis, MN). Full-length or soluble MHC II or DM molecules (100 μg/ml) were incubated with different isolated cathepsins (50–100 ng protein) in reaction buffer [phosphate-buffered saline (PBS), pH 7·2, 2·5 mm dithiothreitol (DTT) or 0·1 m citrate, pH 5·0–6·0, and 2·5 mm DTT] at 37° for various times (routinely 2 hr). Digestion products were resolved by SDS-PAGE and analysed by silver staining. Soluble HLA-DR1 expressed in Schneider cells and purified26 was used for digestion with CatG. The digested products were separated MAPK Inhibitor Library cell line by SDS-PAGE followed by transfer to an Immulon-PSQ membrane (Millipore, Billerica, MA). The membrane was stained with Coomassie Blue and air-dried. The bands were cut out and submitted for N-terminal sequencing to the Protein and Nucleic Acid Facility (Stanford University School of Medicine). Soluble HLA-DR1 expressed in Escherichia coli (a kind gift

from L. Stern, Biochemistry and Molecular Pharmacology, University of Massachusetts, Worcester, MA) was used for digestion with CatG and stained with

Gelcode Blue (Pierce, Rockford, IL). Prominent CatG cleavage products were excised, reduced with DTT and alkylated with iodoacetamide. Duplicate gel pieces for each band were digested with either Arg-C or Glu-C (Sigma-Aldrich) and peptides were extracted using established protocols.30 Protease digests were subjected Progesterone to reverse-phase high-performance liquid chromatography (HPLC) separation and the HPLC eluant was spotted to MALDI target plates for MALDI-TOF/TOF mass spectrometry (MS) (Applied Biosystems 4700, Foster City, CA) analysis. Peptides were identified by tandem mass spectrometry (MS/MS) analysis utilizing the Mascot search engine. Recombinant soluble HLA-DR1 molecules were loaded with 100-fold excess of a 7-amino-4-methylcoumarin-3-acetic acid (AMCA)-labelled variant of the influenza A hemagglutinin (HA) 307-319 peptide (AMCA-HA) (a kind gift from L. Stern) in PBS overnight at 37°. Free AMCA-HA was removed by centrifuging the binding reactions through spin columns (Sephadex G50 Superfine; BioRad, Hercules, CA) according to the manufacturer’s instructions. Binding stoichiometry was determined by absorption spectrophotometry at 280 and 350 nm, as described previously.31 HLA-DR molecules were 70–90% loaded with AMCA-HA. HLA-DR1/AMCA-HA complexes were incubated with 50 ng of CatG in CatG digestion buffer (PBS, pH 7·4, and 0·05% Tween-20).

1d). Some of the lineage markers used for rhesus macaque cells differed from those used for human cells. CD20 replaced CD19 for staining of rhesus B cells as mentioned above. CD56 was excluded from the rhesus staining

panel because it is expressed both on rhesus natural killer cells and on subpopulations of monocytes and mDCs.14,15,38,39 The total DC population was further subdivided into mDCs and pDCs based on their expression of CD11c and CD123, respectively (Fig. 1d). For these stainings, the same clones of antibodies worked well for both human and rhesus DCs. We found no significant difference in the percentage of rhesus pDCs (0·07 ± 0·06%) and human pDCs (0·16 ± 0·28%) see more of total PBMCs (P = 0·145) (Fig. 1e). In contrast, the percentage of rhesus mDCs (0·31 ± 0·19%) was lower than of human mDCs (0·83 ± 0·63%) (P = 0·0003). These levels are comparable to values reported in previous studies.14,15,26,40,41

We next compared the proliferative response of rhesus and human B cells to selected TLR Birinapant datasheet ligands (TLR3, 7/8, 9 ligands) in vitro. We first analysed the proliferation of B cells in total PBMC cultures induced by the three distinct classes of CpG ODN (A, B and C), the imidazoquinoline compound 3M-0012 referred to as TLR7/8-L binding both TLR7 and TLR8, and polyI:C binding TLR3. Proliferation was measured using thymidine incorporation at day 5 of culture. Both human and rhesus B cells express TLR8 and TLR9 but not TLR3 and TLR7.26,42 According to this expression pattern, we observed that all the CpG classes and TLR7/8-L induced significant proliferation compared with unstimulated cultures in both the rhesus and human culture systems (Fig. 2a,b). In contrast, poly I:C did not induce proliferation. CpG class B and C as well as TLR7/8-L induced the strongest proliferation both in rhesus and human cultures. However, while CpG C was significantly more potent in its ability to induce

proliferation in rhesus cultures than the other ligands, CpG B was superior in the human cultures, consistent with previous reports.2,43 The proliferative response was also examined using CFSE dilution allowing us to determine the identity of the proliferating cells (Fig. 2a,b, histograms). The vast majority of cells that divided within the nearly PBMCs were found to be CD20+ and CD19+ B cells in the rhesus and human cultures, respectively (data not shown), indicating that mainly B cells proliferated in response to these TLR ligands. In general, rhesus B cells showed lower proliferative capacity compared with human B cells, as found by both detection methods. Human B cells also exhibited somewhat higher spontaneous proliferation in the unstimulated cultures. Taken together, we concluded that rhesus macaque and human B cells proliferated in response to the same TLR ligands, with only CpG B and CpG C displaying a difference in rank order.

H10/0.05% DMSO was used as a negative control and PHA was used as a positive control. The following day, the cells were discarded and the plate was incubated with

biotinylated anti-IFN-γ antibody (Mabtech) for 3 h at 37°C, followed by streptavidin-conjugated alkaline phosphatase (Mabtech) for 1 h at 37°C. The plate was developed with alkaline phosphatase conjugate substrate (Bio-Rad). Spots were counted using an automated ELISpot plate reader selleck compound (AID Systems, Germany) and the frequencies of IFN-γ-producing cells were expressed as IFN-γ SFU per 106 PBMCs. The Kruskal–Wallis test followed by Dunn’s multiple Enzalutamide research buy comparisons post-test (multiple group comparisons), Wilcoxon matched-pairs

test, Spearman’s rank test and paired t-test were performed using GraphPad Prism version 5. p values of <0.05 were considered statistically significant. This work was supported by the Oxford NIHR Biomedical Research Centre, UK. A.M., P.B. and L.D. are Jenner Investigators. The authors declare no financial or commercial conflict of interest. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Supplementary Figure 1 Progressive gating strategy used to identify CD4+ T cells, CD8+ T cells and CD19+ B cells

within the lymphocyte population and CD3- CD14+ cells within the monocyte population. Supplementary Figure 2 Frequencies of CD4+ T cells, CD8+ T cells, CD19+ B cells and CD14+ monocytes that constitutively express IL-10 among ART-naive patients (n=25), ART-treated patients (n=20) and uninfected controls (n=5). Supplementary Figure 3 Effect of depletion of HIV-1 gag-specific IL-10+ CD8+ T cells on HIV-1 gag-induced expression of HLA-DR and CD38 on CD4+ T cells. Supplementary Figure MG-132 mw 4 (A) CD38 expression on CD14+ monocytes from infected (p24 Ag+) and mock-infected PBMC (n = 4) after 3 and 5 days’ culture. CD8+ T cell-depleted PBMC from four HIV-negative subjects were activated for 3 days with phytohaemagglutinin and then infected with HIV-1BaL in the presence of IL-2, using a MOI to achieve infection of 5-10% CD4+ T cells, as indicated by expression of p24 antigen (p24 Ag). (B) Representative plots showing p24 Ag expression in monocytes from mock-infected PBMC cultures (left) and HIV-1BaL-infected PBMC cultures (right) after 3 days.