The resence of these cytokines and chemokines

at lower levels in the urine of asymptomatic control patients confirm the cell culture studies on detrusor cells. Preclinical studies have previously shown that increased urine levels of MCP-1 and CXCL1 are evidence of bladder inflammation.61 Increased production of inflammatory selleck cytokines may contribute to altered sensory processing in bladder. The higher urine cytokine levels in OAB wet relative to OAB dry might suggest a relationship between OAB symptom severity and bladder inflammation. Midstream urine specimens were collected from a prospective study of eight asymptomatic control subjects and 17 idiopathic OAB patients. The urine was analyzed by a multiplex panel screen for 12 chemokines, cytokines, growth factors and soluble receptors using Lumina xMAP technology (Austin, Texas, USA). Protein concentration values were normalized to the levels of creatinine.This analysis revealed a significant elevation of seven key proteins in the urine of OAB patients relative to controls (*P < 0.05). A greater than 10-fold elevation was measured in OAB, relative to controls, in the levels of monocyte chemotactic JQ1 datasheet protein-1 (MCP-1), soluble fraction of the

CD40 ligand (sCD40L) in urine was obtained from OAB patients relative to controls. At least fivefold elevations were detected in the levels of macrophage inflammatory protein (MIP-1β), IL-12p70/p40, IL-5, epidermal growth factor (EGF), and growth-related oncogene GRO-α compared to controls. Significant threefold elevation

was also noticed in the urine levels of sIL-2Rα, and IL-10 in the OAB group.55 The presence of elevated levels in urine of inflammatory biomarkers involved in inflammation and tissue repair suggests a role for inflammation in OAB, and may help in diagnosis and treatment of this disease. NGF is involved in the development and maintenance of specific peripheral and central populations of neuronal cells. NGF may operate through multiple pathways to ultimately regulate physiological homeostasis and behavioral coping.62 Serum NGF has been found to play an important role in the pathogenesis of autoimmune disorders and degenerative diseases. Increased serum NGF levels have been found in several medical and psychiatric disorders, such as asthma, allergy, Alzheimer disease, Palmatine CVA and physical stress.62–66 One recent study revealed that serum NGF is also increased in part of OAB patients.67 NGF is implicated mainly in inflammatory response, autoimmunity and neuronal repair. The significant correlation between serum NGF and urinary NGF levels in OAB patients indicates that a systemic inflammation might exist in part of the OAB patients. NGF might reduce the excitatory threshold of bladder to dorsal root ganglia and resulting in increased mechanosensitivity of the bladder wall.26 It is possible that circulating serum NGF elevates in changes of systemic conditions.

Using SCID-Hu mouse models, Dick and colleagues showed that only 1/250 000 AML CD34+CD38– cells were capable of establishing leukaemic haematopoiesis in the recipient [21,22]. These cells could be targeted by alloreactive T cells recognizing minor antigens on the leukaemia stem Idasanutlin mw cells [7,8]. These models should be interpreted with caution, as the

xenogeneic milieu of the recipient mouse underestimates the number of cells capable of self-renewal and do not provide clear evidence that long-lived AML progenitors are subject to the same degree of immune attack. Furthermore, they do not identify whether all subtypes of AML have comparable hierarchies of long-lived progenitors. Indeed, an alternative model of leukaemia cure is that a sustained T cell response to the progeny of the AML stem cell but not the small stem cell pool itself could contain the leukaemia at a minimal disease level, resulting in a functional cure [3]. Although the concept of immune surveillance is well accepted, evidence for IS specifically in AML is largely indirect, revealed through relationships between treatment outcome and this website immune parameters and adaptive changes made by the leukaemia favouring immune evasion, unlike viral-induced malignancies. Perhaps the most compelling evidence for a significant role of immune control of AML comes from several observations indicating that

lymphocyte recovery following induction chemotherapy is strongly predictive for outcome. T cells are reduced after chemotherapy PRKD3 but have a rapid clonogenic potential which allows a swift T cell recovery [23]. Patients achieving the highest lymphocyte counts within 6 weeks of chemotherapy have the lowest relapse rates [24–26]. Long-term survival in AML is also favoured by normalized lymphocyte counts [27]. These data all suggest that an intact immune system can protect against relapse of disease, but do not define whether the effect is mediated through T cells or NK cells. There are diverse abnormalities in AML at presentation and relapse that suggest how the leukaemia may develop despite immunosurveillance and how an established leukaemia may acquire new characteristics to defeat immune control. Figure 1 depicts the interactions between AML cells and the immune environment. Genetic features are emerging that may favour the development of AML in the presence of an intact immune system. There is an increased frequency in AML of a particular genotype of the co-stimulatory molecule cytotoxic lymphocyte antigen -4 (CTLA-4) [28]. The inhibitory KIR molecule KIR 2DL2 is expressed more frequently in AML, again suggesting a predisposition for AML through some form of immune escape [29]. There is also strong evidence that an established AML can mutate to escape immune control.

Louis, MO, USA). Sections were counterstained with Hematoxylin. Splenocytes

from naive BALB/c mice were enriched AZD0530 for CD4 or for CD8 by means of magnetic cell sorting and labeled with CFSE (Molecular Probes Invitrogen) as previously described 27. Subsequently, cells were incubated with plate-bound anti-CD3 and anti-CD28 antibodies and PI concentrations of 0, 12.5, 50 or 200 μg/mL. Th polarizing experiments were designed based on a previous publication 10. In short, CD62Lhi purified naïve CD4 T cells (CD4+CD62L+ T-cell isolation kit Miltenyi Biotec, Bergisch Gladbach) were cultured at 1×106 cells/mL with 10 μg/mL anti-CD3 and 10 μg/mL anti-CD28. For Th1 polarization cells were stimulated in the presence of IL-12 (10 ng/mL) and anti-IL-4 (10 μg/mL; purified from 11B11 hybridoma). Th2 polarizing conditions included IL-4 (10 ng/mL, R&D Systems), anti-IFN-γ (5 μg/mL; purified from

XMG1.2 hybridoma) and anti-IL-12/23 p40 (5 μg/mL; purified AZD2281 mouse from C17.8 hybridoma). Treg induction was performed with TGF-β (20 ng/mL; Preprotech, Rocky Hill, NJ), 10 nM retinoic acid (Sigma), anti-IL-4 (10 μg/mL) and anti-IFN-γ (5 μg/mL). For Th0 conditions no cytokines or antibodies were added. Th17 conditions included TGF-β (20 ng/mL), anti-IL-4 (10 μg/mL), anti-IFN-γ and IL-6 (20 ng/mL). At 72 h cytokine levels were measured in the supernatant using ELISA (murine IL-2 from BD Pharmingen; murine IL-17 coat with clone TC11-18H10.1 and detection clone TC11-8H4, Biolegend). To detect IL-4 secretion, cells were washed and restimulated with 5 ng/mL phorbol ester 4-phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich) and 100 ng/mL CAI (A23187, Sigma-Aldrich). At 24 h after restimulation murine IL-4 was detected by ELISA

with coat clone 11B11 and detection (BVD6-24G2). For analysis of division the CFSE-labeled cells were stained with fluorescently labeled anti-CD4 or anti-CD8 antibodies and CFSE peaks were analyzed by flow cytometry. For analysis of signal transduction pathways, cells of a T-cell line DN32.D3 (hereafter referred to as DN32), kindly provided by Prof. Clomifene Dr. Richard Blumberg (Harvard University, Boston, USA), were used. DN32 cells were stimulated with 5 ng/mL phorbol ester 4-phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich) and 100 ng/mL CAI (A23187, Sigma-Aldrich) in the presence of 0, 12.5, 25, 50 or 100 μg/mL PI. At 24 h IL-2 concentrations were measured in the supernatant by means of ELISA (BD Biosciences Pharmingen). After 1, 3 and 5 h of incubation with PI 50 μg/mL IL-2 mRNA levels were measured by means of quantitative PCR or cells were harvested to obtain cell lysates. DCs were derived from BALB/c BM as previously described 27.

For example, the capillary network in a normal human placenta is estimated to be 550 km in length and 15 m2

in surface area [13]. Both branching (the formation of new vessels by sprouting) and nonbranching (the formation of capillary loops through elongation) angiogenesis have been described in the placenta, with a major switch around the last third of gestation. Specifically, normal human placental development is characterized by branching angiogenesis prior to 24-week post-conception, followed by nonbranching angiogenesis that occurs thereafter to term [58]. There is compelling evidence to suggest that vasculo-genesis and angiogenesis are sequentially regulated selleck chemical by different growth factors. VEGF is critically required for all steps of placental vascular formation and Selleckchem PF2341066 development. Targeted inactivation of a single VEGF allele [17, 37] or disruption of genes encoding VEGF receptors such as VEGFR1 [108] and VEGFR2 [40] as well as neuropinin-1 and -2 [112] causes embryonic lethality due to abnormal blood vessel formation during embryogenesis, suggesting a pivotal role of

VEGF/VEGFRs in vasculogenesis. FGF2 has a particular role in the formation of hemagiogenic progenitor cells (angioblasts) early during embryonic development [96]. PlGF seems to play a synergistic role with VEGF for the formation of the vascular network with the development of the villous tree [72]. During the third trimester of gestation, placental expressions of many other growth factors (see below) increase substantially to facilitate the coordinated development of the vascular system via sprouting and elongation in the placental villi (Figure 1). Extensive neovascularization in the placenta is accompanied with periodic increases in uterine and placental blood flows during gestation. Blood flows to the maternal, fetal, and placental

Osimertinib units are established during implantation and placentation when the maternal–fetal circulations connect within the placenta, gradually increases until mid-gestation, then substantially increases at the last one-third portion of gestation, essentially keeping pace with the rate of the growing fetus [100]. Animal studies have clearly shown that angiogenesis and vasodilatation of the uterine and placental vessels are the two key mechanisms to increase placental (umbilical cord) blood flow during late gestation, which is imperative for normal fetal growth and survival and is also directly linked to the well-being of the fetus, newborn, and the mother during pregnancy and postpartum [99]. Endothelial cells are in close contact with the trophoblast cells in the placenta; trophoblast-derived factors are expected to have a significant role in the regulation of placental vascular formation and morphogenesis. For example, the Esx1 gene encodes a homeobox transcription factor that is expressed solely in trophoblast cells of the labyrinth [73, 74].

The proportion of Tregs was evaluated. To elucidate possible differences in functional properties of Tregs, MFI of FoxP3 and intracellular regulatory cytokines IL-10 and TGF-beta were tested. Differences in Treg proportions and their functional properties were found between the groups. Using our gating strategy (Fig. 1) and antibodies against CD4, CD25, CD127 and FoxP3, we did not find significant differences in the proportion of Tregs in the cord blood of children of healthy and allergic mothers, although the trend towards an increased number of Tregs in the CD4+ lymphocyte population from the allergic group was obvious (P = 0·07) (Fig. 2a). A significantly

increased proportion of Tregs in cord blood of children of allergic mothers was observed when selleck screening library Tregs were considered only as CD4+CD25+ cells BMS-354825 solubility dmso (P = 0·0117) (Fig. 2b). Different gating strategies together with using different Treg markers may account for variation among the results of different research groups. Transcription factor FoxP3 is considered to be a master marker for identifying Tregs[24] (as CD25 can be expressed on other activated CD4+ T lymphocytes and CD127 is present on various cell types). The values of MFI of

FoxP3 in cord blood of children of allergic mothers followed an opposite trend to the proportion of Tregs. A significantly higher MFI of FoxP3 (P = 0·0159) in cord blood Tregs of children of healthy mothers was detected in comparison to children Rebamipide of allergic mothers (Fig. 3). To evaluate the possible differences in functional characteristics of Tregs, the presence of regulatory cytokines IL-10 and TGF-beta was estimated by intracellular staining. A significantly

higher number of IL-10+ Tregs in cord blood of children of healthy mothers was detected in comparison to children of allergic mothers (P = 0·0012) (Fig. 4). Similarly, a significantly higher proportion of TGF-beta+ Tregs in cord blood of children of healthy mothers is documented in Fig. 5 (P = 0·0174). The importance of Tregs in immune regulations consists mainly in their role in induction of peripheral tolerance against autoantigens and harmless food and environmental antigens [25]. An insufficiency of Tregs can result in autoimmunity and allergy development [26–29]. We followed the status of newborn Tregs as a possible prognostic marker for future allergy manifestation. It is possible to assume that changes of immune regulation in allergy-prone infants can be evident prior to development of the clinical signs of allergy. We found differences in immune characteristics of Tregs in the cord blood of children of allergic mothers in comparison to children of healthy mothers. Tregs were assessed on the basis of their cell surface markers (CD4, CD25high and CD127low), typical transcription factor FoxP3 and intracellular regulatory cytokines IL-10 and TGF-beta.

Purified CT (Sigma-Aldrich, St. Louis, USA) was administered as described previously 16, 35, with some modifications: 8 wk after transplantation, mice with

mLNtx or pLNtx were selleck products immunized orally with 10 μg of CT (in 50 μL of 0.01 M PBS containing 0.2% gelatine) on days 0, 8 and 14. On day 19, the mice were exsanguinated and cell suspensions were made (n=4–5). Analysis via flow cytometry was performed as described below. Eight wk after transplantation mice were fed with 25 mg OVA (Grade III; Sigma-Aldrich) in 200 μL PBS or PBS only as a control on day 0, 3, 6, and 8 by gavage. On day 16 mice were immunized by subcutaneous injection of 300 μg OVA (Grade VI; Sigma-Aldrich) in 200 μL PBS emulsified in complete Freud’s adjuvant (CFA; Sigma-Aldrich). On day 34 mice were challenged by subcutaneous selleck kinase inhibitor injection of 50 μg OVA (Grade VI) in 10 μL PBS into the right ear and PBS only into the left ear. Ear swelling was measured before challenge and 48 h later. DTH response was calculated as described previously

12. Based on the protocol for ot induction, tolerance in the periphery by the skin draining LN (pLN-pt) was induced as follows: 4.2 mg OVA (Grade III; Sigma-Aldrich) in 10 μL PBS or PBS only as a control on day 0, 3, 6 and 8 by subcutaneous injection into the forepaw of C57BL/6 mice. On day 16 mice were immunized by subcutaneous injection of 300 μg OVA (Grade VI; Sigma-Aldrich) in 200 μL PBS/CFA emulsion. On day 34 mice were challenged L-gulonolactone oxidase by subcutaneous injection of 50 μg OVA (Grade VI) in 10 μL PBS into the right ear and PBS only into the left ear. Ear swelling was measured before challenge and 48 h later and the DTH response was calculated. ot as well as pt were induced as described above. The mice were immunized by subcutaneous injection of OVA and CFA emulsion,

and on day 34 one group of mice was tested with the DTH reaction against OVA to verify that tolerance had been induced (n=3). The other mice were killed, the mLN or the pLN were removed and IgG+ cells or CD4+cells were isolated using the MACS technique following the instructions provided by Miltenyi (Bergisch-Gladbach, Germany). The purity of IgG+ cells was 90–97% and of CD4+ cells 90–93%. IgG+cells and CD4+ cells from mLN-ot or pLN-pt were injected intravenously (12–26×106 IgG+ cells/mouse; 7×106 CD4+ cells/mouse) into naïve wt mice. The recipients were immunized 1 day after cell transfer and the DTH response was measured 20 days later.

For infection with S. ratti, approximately 8 to 10-week-old female C57BL/6 mice were inoculated with indicated numbers of purified iL3 in 30 μL sterile PBS into the hind footpad. Twenty-four hour faeces samples were collected for the detection of L1. For L. major infection, 3 × 106 (high dose) or 3 × 103 (low

dose) L. major stationary phase promastigotes in a final volume of 30 μL PBS were injected subcutaneously into one of the hind footpads of mice. Re-infection with 3 × 106 promastigote parasites was performed at indicated time points after primary infection. The course of disease was monitored daily, and this website the footpad thickness was measured weekly. The relative increase in footpad thickness in per cent was calculated by employing the following form: (thickness of infected foot × 100: thickness of non-infected foot) − 100. For analysis of parasite-specific serum Ig, blood from mice was collected at indicated time points by puncture of the tail vein. The blood was allowed

to coagulate for 1 h at 4°C. Serum was collected after centrifugation at 12 000 × g for 15 min at RT and stored at −20°C for further analysis. For analysis of cellular responses, mice were killed at the indicated time points and mesenteric (mes) LN as GDC-0973 concentration well as popliteal (pop) LN were prepared. In experimental infections with S. ratti, the egg and L1 output was analysed by collecting faeces over 24-h periods. DNA was extracted from 200 mg representative stool samples using the QIAamp DNA stool kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. The DNA was eluted in 200 μL, Amino acid and finally 2 μL of a 1 : 10 dilution in water was used as template for the qPCR. The S. ratti 28S ribosomal RNA gene was quantified by qPCR as described (10). Briefly, a 180 bp fragment of the S. ratti 28S RNA gene was amplified by qPCR in duplicates. For each run, a melting curve analysis was performed to guarantee

the specificity in each reaction tube. Comparative quantification (efficiency-corrected Ct method) was used to transform the difference in Ct values between the test samples and the calibrator sample into a copy number ratio. To count the number of adult nematodes in the gut, mice were killed at the indicated time points post-infection (p.i.) The small intestine was sliced open longitudinally and incubated at 37°C for 3 h in a Petri dish with tap water. The released adult females were collected, centrifuged for 5 min at 300 × g at RT and counted. Microscopic analysis of the small intestine revealed that no significant numbers of adults remained in the intestine. To measure the L. major parasite burden, DNA was isolated from footpads at days 10 and 31 p.i. The concentration of mouse ß-actin DNA was quantified by 5′ nuclease PCR (20).

Recent studies have shown that IgG4 concentrations in serum are elevated and that plasmacytic cells infiltrating the salivary glands are positive for IgG4 in chronic sclerosing sialadenitis but not in Sjogren’s syndrome [3, 4], suggesting that the former involves

inflammatory processes distinct from those of the latter. A dense IgG4-positive plasma PI3K Inhibitor Library order cell infiltration has also been found in Mikulicz’s disease, chronic sclerosing pancreatitis (or autoimmune pancreatitis) [5], IgG4-related sclerosing cholangitis [6] and other sclerosing lesions. Steroids are very effective in treating these IgG4-related disorders, and autoimmune mechanisms may play a role in their development [7]. Analysis of the immunoglobulin heavy chain gene is helpful in clarifying the characteristics of B cells infiltrating inflammatory autoimmune lesions. In this study, we analysed immunoglobulin heavy chain gene rearrangement and somatic hypermutation of SS and IgG4-related sclerosing sialadenitis, using sialolithiasis

selleck chemicals as a control. Case selection.  Typical cases of primary SS (n = 3), IgG4-related sclerosing sialadenitis (n = 3) and sialolithiasis (n = 3) were recruited. None of these cases showed evidence of virus-associated hepatitis or tuberculosis. Clinicopathological data were obtained from the medical records, and the study was approved by the institutional review board of Nagoya City University. For SS cases, biopsy specimens of the minor RG7420 cell line salivary gland of the lower lip were obtained to histologically confirm the diagnosis (focus scores for three SS cases were 4, 4 and 5, respectively) [8], and small germinal centres were present in all cases), which was further supported by the increased levels of serum anti-SS-A/Ro antibody, anti-SS-B/La antibody and rheumatoid factor. The diagnosis of SS was made

according to revised Japanese criteria for SS [9]. The lip biopsy specimens were used for this study. Patients with sclerosing sialadenitis presented with painless swelling of the submandibular glands. Cryptogenic tumours were suspected, and the patients underwent surgical resection of the submandibular glands, which were subjected to examination in this study. Typical cases of sialolithiasis of the submandibular glands were resected and used as a control. Immunohistochemical techniques.  The sections were immunostained for IgG (Eu-N1; Dako, Tokyo, Japan) and IgG4 (MCO11, Binding-Site, Birmingham, UK). Infiltration of IgG-positive or IgG4-positive plasma cells was evaluated by counting the number of positive cells in ten high-power fields (×400), and the percentage of the IgG4-positive cells/IgG-positive cells was calculated in each case. Percentages of memory B and plasma cells to total B and plasma cells were calculated using immunohistochemical techniques in each case. CD27-positive B cells have been considered as memory B cells, and CD27 is positive for T, B and plasma cells [10].

After removal of cell surface CD4 and LAG-3 with pronase treatment, cells were incubated with colchicine (tubulin polymerization inhibitor) or cytochalasine D (actin polymerization inhibitor) for 3 h and the restoration of cell surface CD4 and LAG-3 was measured. Brefeldin A, which was shown above to block the restoration Selleck MG 132 of LAG-3/CD4 expression, was included as a positive control. Surprisingly, actin and tubulin depolymerization did not affect restoration of cell surface CD4 and LAG-3 (Fig.

4). To confirm disruption of actin and tubulin after inhibitor treatment, we stained actin and tubulin in inhibitor-treated cells and verified disruption of actin and tubulin by confocal microscopy (data not shown). We then incubated pronase-treated T cells with different vesicular acidification/function inhibitors (NH4Cl, chloroquine, concanamycin A). Interestingly, all three inhibitors decreased CD4 and LAG-3 cell surface restoration in

T cells (Fig. 4), suggesting that vesicular acidification/function was required for the restoration of both molecules. To assess the subcellular location of LAG-3 and CD4, we treated activated T cells with pronase, and then EPZ-6438 order permeabilized and stained with anti-CD4 or anti-LAG-3 in conjunction with Ab against different subcellular markers for analysis by confocal microscopy. A significant proportion of LAG-3 appeared to colocalize with the microtubule organizing center (MTOC), using γ-tubulin as a marker (Fig. 5A). While the colocalization of γ-tubulin with LAG-3 was statistically greater than with CD4, as determined using Pearson coefficient analysis (Fig. 5C), some CD4/γ-tubulin colocalization was still evident. A significant proportion of both intracellular CD4 and LAG-3 appeared to colocalize with the early and recycling endosome marker, early endosomal antigen 1 (EEA1), which likely represents newly synthesized protein that is on route to the cell surface and/or Y-27632 2HCl in the process of recycling (Fig. 5B and D). To further investigate subcellular location and possible intracellular trafficking pathway of CD4 and LAG-3, we used Rab11b, which is a marker of the endosomal recycling compartment, and Rab27a, which plays a critical role

in secretory lysosome-dependent exocytosis. In the staining of both markers, a significantly higher proportion of LAG-3 appeared to colocalize with Rab11b and Rab27a than CD4, although this was most evident with Rab11b:LAG-3 colocalization (Fig. 6). These observations suggest that CD4 and LAG-3 have partially overlapping but distinct patterns of intracellular location and trafficking mechanisms that might play an important role in regulating LAG-3 membrane expression in activated T cells. Finally, we asked which domains of CD4 and LAG-3 are important for their differential intracellular retention. We generated a panel of LAG-3/CD4 chimeric constructs that were transduced into a LAG-3−/CD4− 3A9 T-cell hybridoma (Supporting Information Fig. 1A).

Previous reports demonstrated CD70-triggered down-modulation of CD27 expression on haematopoietic progenitor cells 28 and T cells 29. Therefore, we first examined CD27 expression on the cell membrane of NK cells in CD70-Tg mice. Over-expression of the CD70

ligand resulted in severe down-regulation of CD27 receptor expression on NK cells in BM, spleen and liver. BM located NKP cells showed reduced CD27 expression as well. The down-modulation of CD27 in NKP and NK cells was already established at 4 wk of age and persisted up to the last Hydroxychloroquine cell line time point analysed, i.e. 15 wk of age (Fig. 1A, Supporting Information Fig. 1 and data not shown). To study whether continuous CD27 triggering affects NK cell numbers, NK cell number kinetics were analysed in BM, spleen and liver of CD70-Tg and their WT counterparts. At 4 wk of age all tested organs contained equal NK numbers in CD70-Tg versus WT mice, but gradually, a significant reduction of CD70-Tg NK cells was observed. At 15 wk of age a nearly complete NK cell depletion had occurred in CD70-Tg BM, spleen and liver (Fig. 1B and 3). As 15-wk-old CD70-Tg

mice had so few remaining NK click here cells, all further experiments were conducted in 4- to 8-wk-old mice. NK cells mainly develop in the BM, where successive differentiation stages have been defined. Figure 2A (and Supporting Information. Fig. 1) shows that no or only minor reductions in absolute cell number were found in NKP and iNK cell subpopulations of CD70-Tg mice. Conversely, a major reduction was observed in the mNK cell subpopulation. To examine whether this decrease in cell number of mNK cells in CD70-Tg mice was due to apoptosis, cells were labelled with annexin-V and 7-amino-actinomycin D (7-AAD) to distinguish early (annexin-V+7-AAD−) from late (annexin-V+7-AAD+) apoptotic cells. Interestingly, NK cells from BM, spleen and liver of CD70-Tg mice

displayed significant higher percentages of early apoptotic cells compared with WT mice (Fig. 2B). Percentages of late apoptotic NK cells followed the same tendency, but differences between CD70-Tg and WT were smaller (Fig. 2B), presumably because of the fast removal of dead cells in vivo. Although cell numbers MTMR9 of NKP and iNK subpopulations were not or only marginally reduced in BM of CD70-Tg mice (Fig. 2A), both NKP and iNK cells are only minor subpopulations compared with mNK cells. As a result, it was not unexpected that also percentages of early and late apoptotic cell numbers were increased in the total NK cell population in BM of CD70-Tg mice. Furthermore, expression of CD95 was up-regulated on NK cells of CD70-Tg BM, spleen and liver (Fig. 2C), which might indicate that CD95-mediated cell death is involved in the decrease in NK cell numbers in these mice. However, when we treated CD70-Tg mice from 3 wk of age, when NK cell numbers are still normal, with blocking anti-mouse CD95 ligand mAb versus isotype control, NK cell numbers were not rescued after 4 wk of treatment (data not shown).