TLR7-mediated inhibition of

Treg-dependent immune control may contribute to the loss of peripheral tolerance in SLE and to the generation of protective immune responses against viral infections. To investigate the impact of TLR7 as well as TLR9 activation on Treg cell induction in vitro in the context of DCs, naïve CD4+CD25− T cells were cocultured with splenic CD11c+ DCs in the presence or absence of optimal doses of synthetic ligands for TLR7 (Imiquimod analogue S-27609), TLR9 (CpG 1668), and TLR4 (LPS) for comparison. To induce Foxp3 expression, naïve T cells were stimulated by plate-bound anti-CD3 antibody and were cultured with TGF-β and IL-2 for 4 days. Since comparable results were selleck chemicals obtained using soluble or immobilized

anti-CD3 for stimulation, we used plate-bound anti-CD3 throughout the study. The percentage of CD4+ T cells expressing Foxp3 and CD25 was reduced by approximately 40% in the presence of S-27609 and CpG, but not in the presence of LPS (Fig. 1A). A similar reduction in the percentage of induced Tregs was observed with two other synthetic TLR7 agonists CL-076 and R848 but also in the presence of the endogenous TLR7 ligand U1snRNP which is contained in autoimmune complexes from SLE patients (Supporting Information Fig. S1). Not only the percentage, but also the absolute numbers of Foxp3+ T cells generated in the coculture were reduced (0.96±0.59×105/well Montelukast Sodium FK506 solubility dmso with TLR7 ligand versus 1.91±0.80×105/well w/o), whereas total cell numbers remained unchanged (3.50±0.32×105/well with TLR7 ligand versus 3.32±0.45×105/well w/o). Thus, lower percentages of Foxp3+ Tregs reflect lower Treg numbers and not expansion of Foxp3− effector T cells. TLR7 and TLR9 ligands similarly reduced TGF-β-mediated conversion of truly naïve OVA-specific TCR-transgenic

CD4+ T cells into Tregs in coculture with splenic DCs which directly present OVA-derived peptides on MHC class II (Fig. 1B). Inhibition of Treg generation by TLR7 ligand could be mediated by direct effects on the developing Tregs, which have been shown to express TLR7 21. However, addition of TLR ligands had no effect on TGF-β-induced Treg generation from CD4+CD25− T cells stimulated with plate-bound anti-CD3 antibody and soluble anti-CD28 antibody in the absence of DCs (Fig. 1C, left panel). On the contrary, a significantly reduced percentage of Foxp3-expressing cells was reproducibly observed in T cells cocultured with DCs in the presence of TLR7 and TLR9 ligands for 4 days (Fig. 1C, right panel). Inhibition of Foxp3 expression by TLR7 and TLR9 ligands was dependent on the number of DCs in the coculture (Fig. 1D).

Another potential mechanism that may describe the differential effect

of auto and allospecific Treg cells on donor engraftment in this model, is related to the ability of Treg cells to regulate natural killer (NK) cell activity [34]. NK cells are known to be important contributors of rejection of parental bone marrow transplants in semi-allogeneic transplant settings [35], which is attributed to NK-cell activation upon recognition of cells “missing self” MHC Class I expression. Treg cells may therefore affect NK-cell targeting of transferred donor cells and also act to inhibit the contribution of activated NK Selleck Nutlin 3 cells toward driving the alloimmune response. Engrafted donor T cells from allospecific Treg-cell-treated animals retained the capacity to react against 3rd party alloantigens, but were unresponsive to either autologous or recipient alloantigens, confirming that allospecific Treg cells mediated donor-specific

regulation in vivo, which was sufficient to simultaneously prevent donor T-cell alloreactivity and recipient autoimmunity. Allospecific Treg cells may therefore be more beneficial for long-term clinical use than autospecific or polyclonal Treg cells, as they provide the additional benefit of permitting engraftment of donor T cells with the capacity to respond to foreign antigens, MG132 which therefore have the potential to mediate graft-versus-leukemic activity [36]. Of particular interest was the observation that although donor T cells were hyporesponsive to autologous-MHC antigen and recipient alloantigen, no CD4+CD25+FoxP3 Treg cells were detected within engrafted donor cells (not shown), implying that allospecific Treg-cell application may have mediated the deletion of autoreactive and alloreactive

donor T-cell clones, or have induced infectious tolerance [37]. The pathophysiology of cGVHD is multifaceted, involving components of both alloreactivity many and autoimmunity, whereby alloreactive donor T cells initiate the immune processes leading to cGVHD, which stimulate a cascade of autoimmune-directed responses by the recipient [12, 38]. In the cGVHD model used in this study, the resulting B-cell hyperactivity and autoantibody generation, which is characteristic of lupus [39], would have occurred through the inappropriate provision of T-cell help by alloreactive donor T cells [40]. Our findings confirm that a combination of alloimmunity and dysregulated autoimmune reactivity both play a critical role in the progression of cGVHD, and more importantly highlight that control of alloreactivity may present an optimised strategy for preventing cGVHD autoimmunity.

1A). After 5 and 8 days culture the CFSE signal of ER-MP58+ cells from the NOD fetal pancreas was dramatically decreased in line with a high proliferative activity (Fig. 4B and Supporting Information Fig. 1A). No such a decrease was detected in C57BL/6. Although a decrease of the CFSE signal was detected in the BALB/c fetal pancreas, the decrease was less compared with NOD. In the fetal liver as well as in the adult BM the majority of ER-MP58+ cells showed a low CFSE signal,

with no differences between NOD and controls. The number of CFSElow cells in the culture of ER-MP58+ cells from the NOD fetal pancreas was significantly higher compared with controls. Cells with at least 5 divisions were counted as CFSElow cells (Fig. 4C). As monocytes in the peripheral blood also express ER-MP58 these cells were analyzed for their proliferative capacity too. The CFSE signal of day 8 cultures of ER-MP58+ cells www.selleckchem.com/screening/apoptosis-library.html from the blood was not decreased, showing that ER-MP58+ peripheral

blood monocytes were not able to proliferate after GM-CSF stimulation (Supporting Information Fig. 1B). In conclusion, myeloid precursors in the NOD fetal pancreas have a specific proliferation abnormality. DCs are the first cells that start to accumulate around the islets in the pancreas at 5 weeks of age in the pre-diabetic NOD mice. selleckchem To investigate if this DC accumulation is preceded by an increased proliferation of local pancreatic precursors the pre-diabetic pancreas was studied for ER-MP58+Ki-67+ cells by immunofluorescence and FACS analysis. To assess if the proliferation abnormality in the NOD pancreas is a general phenomenon of the genetic background of these mice, the non-obese Verteporfin cell line resistant mouse (NOR) was included as an extra control. In the NOD pancreas of 5 weeks of age the number of ER-MP58+Ki-67+ cells was significantly higher compared to C57BL/6 and NOR (Fig. 5A and B). This was confirmed by FACS analysis of the pancreas of 5–week-old NOD, NOR and C57BL/6 mice (Supporting Information Fig. 2 and 5C). No significant difference in the total number of ER-MP58+ cells between NOD, NOR and C57BL/6 was detected (data not shown). Thus, proliferating

myeloid precursors are present before the DC accumulation in the NOD pre-diabetic pancreas and this is not due to the genetic background of this mouse. We here show that ER-MP58+Ly6G−CD11bhiLy6Chi and ER-MP58+Ly6G−CD11bhiLy6Clow precursors for myeloid DCs are present in the pancreas of C57BL/6 and NOD mice from embryonic (E15.5) age onwards. After sorting and culture in GM-CSF, these precursors have the potential to develop into CD11c+MHCII+CD86+ DCs capable of processing antigens. Although the number of precursors is not increased in the NOD mouse pancreas, the cells have a higher proliferative capacity in the embryonic as well as in the pre-diabetic NOD pancreas. This abnormality was specific for the pancreas and did not occur in blood, liver and BM.

Previous immunity to DENV is a major risk factor for developing severe dengue disease in humans.23 A small reliable animal model that supports functional human innate and adaptive immune responses that will further our knowledge of protective and pathological immune responses to dengue virus is therefore clearly important. Researchers have detected measurable signs of dengue disease after infection of cord-blood-engrafted NSG mice with virulent low-passage clinical strains of DENV-2.13,16 However, robust human anti-DENV adaptive immune responses were not thoroughly assessed in those studies.

this website We have shown DENV-specific HLA-A2-restricted T-cell function and modest antibody responses in cord blood HSC-engrafted NSG mice.14 The main objective of the current study was to determine whether we can detect improved adaptive immune responses to primary DENV infection in BLT-NSG mice. Here we show HLA-A2-restricted T-cell responses to multiple non-structural proteins in BLT-NSG mice at frequencies similar to those detected

in humans. We show heightened antibody responses in BLT-NSG mice compared with cord blood HSC-engrafted mice. Furthermore, B cells maintained long-term in immunized BLT-NSG mice were able to secrete DENV-specific neutralizing antibodies. We have not assessed germinal centre formation or somatic hypermutation LY2157299 molecular weight of immunoglobulin genes in B cells from BLT-NSG mice; therefore it is unclear whether these B cells can be considered bona fide memory B cells. We and others have noted that levels of haematolymphoid engraftment in BLT-NSG mice are Montelukast Sodium increased compared with levels in cord blood HSC-engrafted NSG mice.24–26 Humanized mice have demonstrated some evidence of human adaptive immune responses to Epstein–Barr virus infection, toxic shock syndrome toxin-1 and HIV infection.17,18,27,28 Human T cells are educated on autologous human thymic tissue in the BLT-NSG mice, so we speculated that DENV-specific T cells restricted by multiple

HLA alleles expressed by the donor should develop in the mice following infection. We therefore used overlapping peptide pools that encompass the entire genome to assess the breadth, magnitude and quality of DENV-specific T-cell responses. Our results demonstrate that non-structural proteins are the predominant targets of CD8 T cells. These findings are similar to findings in humans,29–31 further validating BLT-NSG mice as an animal model that can be used to measure human T-cell responses to DENV during acute infection and in memory. We detected elevated serum IgM responses, which persist for several weeks in DENV-infected BLT-NSG mice during acute infection. Furthermore, B cells obtained from splenocytes of BLT-NSG mice immunized several weeks earlier were able to secrete DENV-specific antibodies capable of neutralizing DENV infectivity in vitro.

The mature biofilm was prepared according to the protocol of Li et al. (2003) with minor modifications. Briefly, the polystyrene Petri dishes (3 cm diameter, Sarstedt) were inoculated

with 1 × 107 cells mL−1 in 3 mL of yeast–nitrogen base (YNB) medium with amino acids (Sigma-Aldrich) supplemented with 0.9%d-glucose (AppliChem, Darmstadt, Germany) at 37 °C for 90 min (adhesion phase). Biofilm was also formed in polystyrene 96-well plates (flat bottom, GSK-3 activity Sarstedt) in the same medium with the cell concentration of 107 mL−1. A 100-μL aliquot of this suspension was then applied to each well. Nonadherent cells were then removed and adherent cells were washed three times with 1 × phosphate-buffered saline (PBS). Finally, 3 mL or 100 μL of YNB medium was added and cultivation continued at 37 °C for 48 h to obtain a mature biofilm. The

mature biofilm was washed with 3 mL of 1 × PBS three times and then blocked with 1% gelatin (w/v, Oxoid, Ogdensburg, NY) dissolved in 1 × PBS at 37 °C. After 1 h, the plates were washed once with PBS–0.05% v/v Tween 20 (Sigma-Aldrich), followed by incubation with 100 μL of polyclonal anti-CR3-RP antibody diluted 1 : 100 and OKM1 mAb or TIB111 mAb (used as the control), both diluted 1 : 10 in 1 × PBS for 1 h on ice. Then samples were washed three selleck times in PBS–0.05% v/v Tween 20 followed by centrifugation to remove unbound antibody. Specific immunocomplexes were developed with goat anti-rabbit or goat anti-mouse immunoglobulin G (IgG)-(H+L) fluorescein isothiocyanate (FITC)-conjugated antibody (Bethyl Laboratories Inc., Montgomery, TX) for 1 h in the dark at room temperature. After three washing steps, the immunofluorescence signal was directly observed by microscopy (Axio Imager A.1, Carl Zeiss, Oberkochen, Germany). Etofibrate Parallel plates with the biofilm preincubated with all antibodies

were scraped and submitted for immunocytometric assay, using an indirect staining (FITC-secondary anti-rabbit IgG and anti-mouse IgG antibodies) and evaluated by flow cytometry using a Beckman Coulter FC 500 flow cytometer (Beckman Coulter Inc., Fullerton, CA) equipped with a 488-nm argon laser and a 637-nm HeNe collinear laser, and controlled by cxp software. Candida biofilm cells were gated on the basis of forward light scatter (FSC) and side light scatter (SSC) using a logarithmic scale. Gates were set to exclude debris and intact cells on a forward scatter vs. side scatter dot plot. Additionally, gates were previously optimalized on properly prepared cultures of the yeasts, budding yeasts and hyphae from Candida strains CCY (29-3-163) according to the protocol of Bujdákováet al. (1999).

While both DC populations effectively primed CD8+ T cell responses to cell-associated antigens, only mcDC were capable to prime CD4+ T cells to cell-associated antigens. Consequentially, the transfer of tumour vaccine-pulsed mcDC, but not of CD8 DCs, protected mice from subsequent tumour challenge in a vaccination model and resulted in eradication

of established tumours in a therapeutic selleck chemicals llc approach. These results show that the beneficial effect of FLT3L is associated with the induction of mcDC and suggests that selective targeting to mcDC or instilling mcDC ‘characteristics’ into conventional DC populations could significantly enhance the efficacy of tumour vaccines. Autologous tumour cell vaccines are intended to drive specific activation of the adaptive immune system for therapy of existing malignancies. The resulting in vivo destruction of tumour cells leads to an additional release of tumour antigens that further amplifies tumour-specific T cell responses [1–3]. This secondary antigenic boost has been suggested to help to enhance and sustain anti-tumour T cell responses and prevent recurrences and metastases. Dendritic cells (DC) are the only antigen-presenting cells that can adequately prime naive T cells.

The (cross)-presentation of tumour antigens by DC upon uptake of dying tumour cells/tumour cell debris has also been shown to be critical for the induction of endogenous anti-tumour T cell responses [4,5]. DCs are phenotypically and functionally heterogeneous. Selleck MK1775 At least six DC subsets have been described in mice and Oxalosuccinic acid humans: plasmacytoid DCs (pDCs), three blood-derived subsets (CD4+ DCs, CD8α+ DCs and CD4-CD8- DCs [6,7]) and two tissue-derived subsets (Langerhans’ cells and dermal/interstitial DCs)

– all of which appear to be distinct sublineages and not precursor-product-related [8–10]. However, this classification has been proved to be a simplified subdivision, as we and others have recently identified novel DC subsets that are either present in common lymphoid tissues or associated with specific organs [11–15]. Even though most DC subsets can capture proteins and cell-associated antigens and can activate CD4+ and CD8+ T cells when pulsed with cognate peptides, only few DC populations have the actual capacity to process and present tumour-derived antigens to T cells [16,17]. Cross-presentation of cell-associated antigens to CD8+ T cells in particular is believed to be limited to just one or two DC populations [17,18]. Moreover, besides the fact that only few DC subsets can present both CD4+ and CD8+ T cell epitopes from cell-associated antigens, both human and mouse studies have shown that detection and subsequent clearance of apoptotic cells leads to a tolerogenic state in DC [19–22].

After a 3-week washout period, the same animals were treated with 1 mg/kg chimeric A9H12 and were challenged the next day with a second IDR. They showed no cutaneous erythema with the 40 UI PPD dose and a milder reaction (diameter of the erythema and reaction time) with the 2000 UI PPD dose (Fig. 3a,b). One of these animals was challenged

again for a third IDR 6 weeks later (a period INCB018424 clinical trial of time sufficient to completely eliminate chimeric A9H12 from the blood representing up to 10 half-lives; data not shown) and showed a restored DTH reaction with an erythema similar to the first IDR (Fig. 3b). In a second round of experiments, three other immunized animals were treated with 0·1 mg/kg on day 1 of

the second IDR and showed a more pronounced inhibition of DTH reaction, as two of these animals did not develop any cutaneous erythema even with the 2000 UI PPD injection dose (Fig. 3d,e). The third animal developed no erythema with the 40 UI PPD injection and a decreased erythema (diameter and reaction time) with the 2000 UI PPD injection dose (Fig. 3c). The inhibitory action of chimeric A9H12 injected at 0·1 mg/kg was long-lasting, because subsequent IDRs performed 3-6 weeks after injection were similar to the second IDR performed during treatment. A 3-month washout period was actually necessary to recover a positive reaction in two of these animals (Fig. 3d,e). Skin biopsies Dehydratase were selleck inhibitor performed on

day 3 after 40 UI PPD challenges on one duplicate IDR and processed for analysis by immunofluorescence. In accordance with the clinical DTH observations, these data revealed a reduction in T cell and macrophage infiltration after administration of chimeric A9H12 at 1 and 0·1 mg/kg (Table 2 and Fig. 4), an effect that persisted partially at the third IDR (in the absence of further administration of chimeric A9H12). Both CD4+ and CD8+ T cells were found reduced in the infiltrates after treatment. In agreement with our observations in lymph nodes (Fig. 2b), LAG-3+ cells in skin biopsies represented a minority of infiltrating T cells which, none the less, was also reduced after administration of chimeric A9H12. In this study, we evaluated the biological effect of the depletion of LAG-3+ cells in a non-human primate model of delayed-type hypersensitivity. First, we demonstrated that the chimeric A9H12 anti-LAG-3 monoclonal antibody could deplete in vitro by ADCC and in vivo in lymph nodes CD4+ and CD8+ target cells expressing LAG-3+. In vivo chimeric A9H12 showed efficacy at reducing skin inflammation in a tuberculin-induced DTH model in the baboon, an effect that persisted after elimination of the antibody. Using antibodies that specifically deplete activated T cells represents a promising therapeutic strategy to prevent and/or treat autoimmune diseases and transplant rejection.

Unpulsed T2-cells, pulsed with the two other UTY-peptides or the non-T2-binging-I540S-peptide served as controls (W248-CTLs: 0–43/100,000 T cells, median: 10; T368-CTLs: 13–27/100,000 T cells, median: 18; K1234-CTLs: 3–86/100,000 T cells, median: 17; P < 0.046 to P < 0.023, Wilcoxon-test, exceptions: T2-cells versus T2-cells + W248 and K1234 + : P < 0.113 and P < 0.335, respectively). Generated female-canine-W248-specific

CTLs (Fig. 3A) recognized DLA-identical-male cell types in all three cases tested with Sunitinib up to 98/100,000 specific-spots (median: 28/100,000; E:T = 80:1; n = 3) in an MHC-I-restricted manner (: 2-30/100,000, median: 19/100,000), T368-specific cCTLs (Fig. 3B) specifically reacted against DLA-identical male-cells only in one dog (#6) (<38/100,000 T cells; : 0–6/100,000; n = 1) and K1234-specific cCTLs (Fig. 3C) induced MHC-I-restricted

IFN-γ-secretion in 2/3 samples (#4 + #6) towards male-cells (up to 338/100,000 K1234-specific T cells, median: 39/100,000; : 0–113/100,000, median: 15/100,000; P < 0.041 to P < 0.001, Palbociclib molecular weight Wilcoxon-test; n = 2). In all cases, controls, i.e. the corresponding female-DLA-identical and autologous-female cell-types (without presentation of male-restricted Y-chromosomal-peptides like UTY) were not recognized or only to low extent (W248: <29/100,000 T cells; T368: <20/100,000 T cells; K1234: <59/100,000 T cells; P < 0.046 to P < 0.002, Mann–Whitney-U-test). Supplementary exogenous peptide-addition to male-DCs revealed an increased cCTL-reactivity for all three peptides compared to the naïve male-DCs (W248: 54 ± 26 versus 35 ± 25 spots/100,000 T cells; T368: 20 ± 4 versus 11 ± 3/100,000; K1234: 117 ± 102 versus 107 ± 104/100,000;

P < 0.025 to P < 0.024, Wilcoxon-test). In contrast, male-DCs loaded with an unspecific peptide revealed low CTL-reactivity, showing the CTLs′ peptide restriction and specificity (W248 (K1234): 17 ± 11/100,000 T cells; T368 (W248): 5 ± 3; K1234 (W248): 39 ± 12; P < 0.043 to P < 0.010, Wilcoxon-test). Female-autologous and DLA-identical-female DCs were not targeted (W248: 1 ± 2/100,000 T cells; T368: 6 ± 2/100,000; K1234: 20 ± 25/100,000; all P < 0.025, MRIP Mann–Whitney-U-test), but when pulsed with hUTY-peptides, cCTL-reactivity increased (W248: 29 ± 20 spots/100,000 T cells; T368: 20 ± 4/100,000; K1234: 59 ± 40/100,000; P < 0.026 to P < 0.024, Wilcoxon-test). Besides, male-BM was the cell-type being mostly recognized by the in vitro-generated female-canine CTLs (38–338 spots/100,000 T cells), followed by male-DCs (11–181/100,000), male-PBMCs (5–109/100,000), male-monocytes (<79/100,000) and male-B cells (<33/100,000). This pattern was detected for each of the three UTY-peptides. Additionally, UTY-mRNA-expression levels (total-dog-RNA; RT-PCR) of the different hematopoietic cell-types from all animals investigated were determined semi-quantitatively (Fig.

RNA was prepared from purified B cells of Foxo1f/fCd19Cre and Foxo1f/f mice using Trizol reagent (Invitrogen) as described previously 1, 2. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA,

USA). Primers for genes of interest Roscovitine datasheet (Klf2, Klf4, Ccng2, Rbl2, Sell and Ltb) and housekeeping gene (β-actin) were optimized to amplify products between 75 and 200 nucleotides. Primer sequences are available on request. Quantitative PCR was performed with SyBr green as described previously 1, 2. A Student’s t-test was used for all comparisons. The specifics of each test (one versus two-tailed) are indicated in the figure legends. This study was supported in part by a Research Scholar Grant from the American Cancer Society (to D. A. F.) and by NIH grants AI057471 and AI061478 (to S. L. P.). The authors thank Craig Walsh and Aimee Edinger for helpful discussions, Lomon So for technical assistance and Christine McLaren for statistical analysis. Conflict

of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting www.selleckchem.com/products/lee011.html Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
“The expression of Langerhans cell (LC) and dermal dendritic cell (dDC) as well as T CD4+ and CD8+ immune responses was evaluated in the skin of BALB/c mice experimentally infected by L. (L.) amazonensis (La) and L. (V.) braziliensis (Lb). At 4th and 8th weeks post infection (PI), skin biopsies were collected to determine the parasite load and CD207+, CD11c+, CD4+, CD8+, iNOS+ cellular densities. buy Ponatinib Cytokine (IFN-γ, IL-4

and IL-10) profiles were also analysed in draining lymph node. At 4th week, the densities of CD207+ and CD11c+ were higher in the La infection, while in the Lb infection, these markers revealed a significant increase at 8th week. At 4th week, CD4+ and CD8+ were higher in the La infection, but at 8th week, there was a substantial increase in both markers in the Lb infection. iNOS+ was higher in the Lb infection at 4th and 8th weeks. In contrast, the parasite load was higher in the La infection at 4th and 8th weeks. The concentration of IFN-γ was higher in the Lb infection, but IL-4 and IL-10 were higher in the La infection at 4th and 8th weeks. These results confirm the role of the Leishmania species in the BALB/c mice disease characterized by differences in the expression of dendritic cells and cellular immune response. American cutaneous leishmaniasis (ACL) is a zoonotic protozoal disease caused by different species of Leishmania (1). In Brazil, L. (V.) braziliensis and L. (L.) amazonensis are considered the main pathogenic species causing human ACL (2). The human L. (L.

5D), the number of MR+ cells was significantly lower in the mice lacking CD73 (Fig. 5E). The decrease in the numbers of these cells was not merely a consequence of smaller tumor volumes, since

tumors of overlapping sizes (from different experiments) still showed a selective reduction of MR+ cells in the CD73-deficient host (Fig. 5E). Staining for Clever-1/stabilin-1, which is also highly enriched in DMXAA ic50 type 2 macrophages 22, confirmed this observation of CD73-dependent macrophage differentiation defect (Fig. 5F). Additional staining of intratumoral cells for FIZZ/RELM-α did not reveal differences between the genotypes (132±11 and 145±13 cells/mm2 in WT and CD73-deficient mice respectively). In this context it should be noted that although FIZZ/RELM-α is considered to be a type 2 macrophage marker, it is also expressed on other hematopoietic and non-hematopoietic cells such as adipocytes, epithelial cells and eosinophils 22–24, 28. We found fewer intratumoral macrophages expressing CD169 (sialoadhesin), which has been proposed to be central in cross-presentation GDC-0068 in vitro of tumor antigens to T cells 29, in the tumors growing in CD73-deficient mice (28±1 cells/mm2) than in WT mice (53±2 cells/mm2, p<0.01). Together, these data show that the numbers of macrophages expressing MR and Clever-1, markers compatible with the type 2 phenotype

22–24, are decreased within the tumors, if the host lacks CD73. We used the tumor-infiltrating leukocytes for quantitative PCR analyses of immune-related genes. The results showed that intratumoral CD45+ cells isolated from CD73-deficient mice had twofold more IFN-γ mRNA and also the expression of several INF-γ-inducible genes such as Smad 3, Smad 7 and Socs 2 was induced (Fig. 5G, and Supporting Information Table 1). Notably, intratumoral leukocytes from CD73-deficient mice had more than eight times higher expression of Nos2 when compared with those from WT controls. The level of IL-10 Abiraterone mRNA was not different between the genotypes, and IL-4 was not detectable in any sample. IFN-γ and Nos2 are well-established markers of

type 1 macrophage polarization 22. Therefore, these results are in line with our immunohistological data that in the absence of CD73 activity fewer tumor macrophages show a type 2-like phenotype (and consequently, since there is no difference in the total numbers of all macrophages (F4/80+ cells), more macrophages exhibit the type 1-like phenotype). Since we found that many tumor vessels were CD73+, we studied the role of this molecule in recruitment of leukocytes into the tumor. Tumor-infiltrating leukocytes were isolated from WT melanomas, and their adherence to melanoma vessels in tumors grown either in the WT or CD73-deficient mice were analyzed. When compared to the WT vasculature (100%), the binding of tumor-infiltrating leukocytes to CD73-deficient vasculature was only 45±8% (mean±SEM, p<0.02).