At least 10 days after the third s.c. injection mice were challenged

by aerosolized OVA 1% in phosphate-buffered saline three times every third day. Airway responsiveness to increasing doses of methacholine GSK-3 cancer was measured 24 h after the last challenge; thereafter, mice were dissected, bronchoalveolar lavage was performed and blood and lung samples were taken. Clinical grade CTLA-4–Ig (Abatacept; Bristol-Myers, Woerden, the Netherlands) was used in the experiment using IDO-KO mice. In other experiments CTLA-4–Ig was obtained as described previously [26, 27]. CTLA–Ig (280 μg/injection) or control IgG (280 μg/injection) were mixed with OVA-SIT (100 μg/injection) and injected s.c. Airway reactivity to methacholine was evaluated by direct measurement of airway resistance in response to increasing doses of methacholine, as explained previously [23]. In brief, anaesthetized mice (by i.p. injection of ketamine 100 mg/kg; Pfizer, New York, NY, USA and medetomidine 1 mg/kg; Pfizer) were tracheotomized (20-gauge intravenous: i.v. cannula; Becton Dickinson, Alphen a/d Rijn, the Netherlands), attached to a computer-controlled small-animal ventilator (Flexivent; Scireq, Montreal, Quebec, Canada), then paralysed (i.v. injection of pancuronium bromide: Pavulon, 50 μg/kg; Merck Sharp & Dohme, Rahway, NJ, USA).Ventilation was adjusted at a breeding frequency of 300 breaths/min and a tidal volume of 10 ml/kg. Tidal volume was pressure

limited at 300 mm H2O. An i.v. cannula was inserted through the jugular vein for the administration of methacholine. until Airway resistance in response to i.v. methacholine (acetyl-b-methylcholine selleck kinase inhibitor chloride; Sigma-Aldrich, Dordrecht, the Netherlands) was calculated from the pressure response to a 2-s pseudorandom pressure wave. Serum levels of OVA-specific IgE were determined by enzyme-linked immunosorbent assay (ELISA), as described previously [28], and results are expressed as experimental unit/ml. Animals were lavaged five times through the tracheal cannulae with 1-ml aliquots of saline. Broncho-alveolar lavage (BAL) cells

were pooled, counted, and cell types were identified using flow cytometry, as described elsewhere [29]. Homogenates were made from the cardiac lobe of lung, as described elsewhere [30]. The levels of interleukin (IL)-4, IL-5, IL-10, interferon (IFN)-γ and transforming growth factor (TGF)-β in the lung homogenates were determined by commercially available ELISA kits, according to the manufacturer’s instructions (BD Pharmingen, Franklin Lakes, NJ, USA). Peridinin chlorophyll (Per-CP)-anti-CD4 (BD Pharmingen), fluorescein isothiocyanate (FITC)-anti-T1ST2 (also known as IL-33Ra) (MD-Biosciences, Zurich, Switzerland), phycoerythrin (PE)-anti-forkhead box protein 3 (FoxP3) and eFluor450-anti-CD25 (eBioscience, San Jose, CA, USA) were used for fluorescence activated cell sorting (FACS). Data are expressed as mean ± standard error of the mean (s.e.m.).

In addition to stimulating factors such as cytokines which can provoke NK cells function, it should be noted that viral infections Ponatinib supplier may be as other potent

stimulators of NK cells in AD. It has been shown that CNS infections by herpes simplex virus type 1, picornavirus, Borna disease virus, and other microorganisms such as Chlamydia pneumonia, Helicobacter pylori, and spirochaete could be possible aetiological agents in the development of AD [62, 63]. AD is a progressive and neurodegenerative disorder that accounts for 50–60% of all dementia patients. It is characterized by language difficulties, impairments in decision-making and cognitive dysfunction. The responsible mechanisms in degeneration of cerebral neurons and synapses in AD remain an enigma.

The AD patients’ brain shows some degree of cerebral atrophy, but the extent of neuronal loss varies among patients. The Beta amyloid has been found outside senile plaques and within cerebral blood vessels in AD cases [64]. Increased production of cytokines, such as IL-1α, IL-1β, IL-2, IL-3, IL-6, and TNF-α in senile plaques in the hippocampus and cortex of Alzheimer’s brain has been reported [3]. NK cells are granular lymphocytes that have cytotoxic activities. They can affect adaptive immune responses selleck chemical and control immune cell homoeostasis in humans PIK3C2G and they can produce cytokines, such as IFN-γ, IL-3, IL-5, IL-10, IL-12 and IL-13 [13]. Recent findings show that NK cells are involved in AD and could be a target for evaluating the immunopathogenesis of this disease and/or approaching to prevention and treatment of AD [9]. Regarding the results of various studies, it seems that although the frequency of NK cells in AD is not affected, however, their functional potential shows some degree of defects [7, 32]. Surprisingly, it has been shown that this anergic behaviour of NK cells in AD patients is not permanent and their NK cells

can be a potent cytotoxic and cytokine secreting cells following cytokine-mediated stimulation [7]. The cause of this behaviour is unknown but it is suggested that dysregulation in signalling pathways is in part involved in this fashion [27]. The precise role of NK cells as protective or deleterious factors in AD immunopathogenesis is also matter of debate. However, knowing this matter that NK cells can be as a therapeutic target in AD therapy requires to more investigation in both human and its animal experimental models in the future. “
“During the past decade, it has been firmly established that IL-23 is essential for disease development in several models of autoimmune disease, including psoriatic skin inflammation, inflammatory bowel disease (IBD), and experimental autoimmune encephalomyelitis (EAE).

To examine this possibility, CFSE splenocytes from LPS-treated mice were transferred into recipient mice treated with Dex after the inflammatory process was triggered by LPS (Fig. 2F). Interestingly, in this experimental condition the entrance of peripheral cells into the thymus occurred. Similar data were observed when T. cruzi infected mice were used instead of LPS-treated mice NVP-BKM120 (data not shown). Overall, these data indicate that space is necessary but not sufficient for the entrance of cells into the thymus and we hypothesize that specific signals that recruit peripheral cells into the organ are also required. To characterize the phenotype of cells that

enter the thymus during Th1-inflammatory/infectious processes, we analyzed the expression of markers that discriminate between naïve, recently act-ivated or memory T cells (CD44, CD62L, Sotrastaurin and CD69). Data shown in Fig. 3A demonstrate that cells that enter the thymus exhibited high expression of CD44 and CD62L but low expression of CD69. Together, cells migrating to the thymus exhibited surface expression markers compatible with a central memory phenotype. It has been demonstrated that traffic of peripheral B and T cells to the thymus in AKR mouse is mediated by the expression of L-selectin

on immigrating lymphocytes [11]. Thus, we analyzed CD62L expression in all the cell types recruited to the thymus in LPS-treated and T. cruzi infected mice. As shown in Fig. 3B, CD62L was expressed by most immigrating B and CD4+ T cells and about 70% of CD8+ lymphocytes,

suggesting that the integrin could represent an important pathway for cells to extravasate into the thymus. However, data presented in Fig. 3C demonstrate that CD62L is not involved not in cell migration to the thymus since splenocytes from LPS-treated mice incubated with an anti-CD62L neutralizing Ab before the adoptive transfer did not affect migration of either mature T or B cells to the thymus (Fig. 3C), but highly diminished the entrance of transferred cells to popliteal LNs (data not shown) [28]. Similar results were found in the LPS model (data not shown). did not participate in the entry of mature lymphocytes into the thymus, we focused our attention on other integrin/chemokines candidates. We found that the expression of the chemokine MCP-1 was highly upregulated in the thymi of LPS-treated, C. albicans, or T. cruzi infected mice compared with that of controls (Fig. 4A). Ex vivo treatment of thymocytes from T. cruzi infected mice with Brefeldin A for 4 h and then intracellular staining with an anti-MCP-1 Ab demonstrated a low but consistent detection of MCP-1+ cells (Supporting Information Fig. 1). The expression of MCP-1 was mainly restricted to B and CD4+ and CD8+ CD44lo resident thymocytes, but not to CD44hi peripheral T-cell counterparts or CD11b+ and CD11c+ subsets (Supporting Information Fig. 1).

Valsartan has a high affinity for the AngII receptor. The ddY mice were treated with 10 mg/kg bodyweight of valsartan from 20–60 weeks of age (group I; early treatment group) or with the same dose of valsartan from 30–60 weeks of age (group II; late treatment group), or were observed only from 20–60 weeks of age (group III; control group). There were no significant changes in the levels of systemic blood pressure among the three groups.

The levels of urinary albumin excretion in groups I and II were lower than those in group III, but there was no statistical significance. The degree of glomerular fibrosis in groups I and II was significantly lower than that Small molecule library in group III (P < 0.01 and P < 0.01). These findings were

independent of the antihypertensive effect of valsartan. It appears that the AngII AT1 Selleck Belnacasan receptor antagonist valsartan may influence glomerular fibrosis in IgA nephropathy of ddY mice. Mizoribine, an immunosuppressant developed in Japan, was shown to prevent the proliferation of lymphocytes in vitro and to possess immunosuppressive action in vivo. Mizoribine has been used successfully in the treatment of IgA nephropathy, including steroid-resistant disease. Because mizoribine also has a suppressive effect on antibody formation through direct inhibition of B-cell function, it has beneficial effects in patients with chronic glomerulonephritides, lupus nephritis, nephrotic syndrome and renal transplantation. Shimizu et al.22 examined the clinical and immunopathological effects of mizoribine in ddY mice. The ddY mice were treated with 0.05 mg/mL (low dose) or 0.1 mg/dL (high dose) Fossariinae of mizoribine for 35 weeks. After 20 weeks of treatment, levels of urinary protein excretion in the ddY mice treated with the high dose of mizoribine were lower than those treated with the low dose. Expansion of glomerular mesangial areas in ddY mice treated with the high dose of this drug was significantly decreased

compared with the low dose or with the drinking water control. Numbers of B cells and IgA-bearing B cells among the spleen cells of ddY mice treated with the low or high dose of mizoribine were lower than in those given only drinking water. It appeared that treatment with mizoribine affects B cells, especially IgA-bearing B cells, and improves glomerular injury in IgA nephropathy of ddY mice. Although glucocorticoids are effective in IgA nephropathy patients associated with minor to moderate glomerular injuries, it is necessary to use large doses of the drug for long periods. There are severe adverse effects such as diabetes, peptic ulcers and aseptic necrosis of the bones.

We recently observed immunostimulatory properties in the root extracts of chemotypes NMITLI-101,

NMITLI-118, NMITLI-128 and pure withanolide, Withaferin A. In the present study, we evaluated the potential immunoprophylactic efficacies of these extracts against an infective pathogen. Our results show that administration of aqueous ethanol extracts (10 mg/kg) and Withaferin A (0.3 mg/kg), 7 days before and after challenge with human filarial parasite Brugia malayi offer differential protection in Mastomys coucha with chemotype 101R offering best protection (53.57%) as compared to other chemotypes. Our findings also demonstrate that establishment of B .malayi larvae was adversely affected by pre-treatment with Withaferin A as evidenced PD98059 solubility dmso by 63.6% reduction in adult

worm establishment. Moreover, a large percentage of the established female worms (66.2%) also showed defective embryogenesis. While the filaria-specific immunological response induced by Withaferin A and NMITLI-101 showed a mixed Th1/Th2 phenotype, 118R stimulated production of IFN-γ, and 128R increased levels of IL-4. Taken together, our findings reveal potential immunoprophylactic properties of Withania somnifera and further studies are needed to ascertain the benefits of this plant against other pathogens as well. 2011 Blackwell Publishing Ltd “
“Over the last decade, live cell imaging has revealed the surprisingly complex orchestration of antigen receptor inhibitor signalling at the immunological synapse. The imaging studies showed that one of the earliest steps in antigen receptor activation GSK2126458 ic50 is the formation of submicroscopic clusters, which regulate the early signalling events. However, the molecular mechanisms operating inside these microclusters have remained beyond the resolution of optical microscopy. Recent development of imaging techniques that approach molecular resolution in intact cells offers a first view of the molecular processes inside these structures. Here I review the contributions

of molecular imaging of the immunological synapse to our understanding of antigen receptor clustering, binding to antigens, and recruitment of signalling molecules. Finally, I provide an outlook on the future prospects of this rapidly advancing technology. Activation of antigen receptors, the T-cell receptor (TCR) and the B-cell receptor (BCR), is a highly regulated process that sets in motion the adaptive immune response. In accord with their pivotal role in immune responses, antigen receptors are tuned to an unusually high degree of ligand discrimination and sensitivity. Each lymphocyte clone responds specifically to high-affinity interactions with the cognate antigen, which potentially signifies an infection, but disregards low-affinity interactions, which occur with self structures.

Rapamycin enhanced the T cell stimulatory capacity of TLR-7-activated PDC by stimulating the up-regulation of the co-stimulatory molecule CD80. Apparently, CD80 is less important in stimulating expansion of CD8+ T cells and generation of CD8+ Treg. Rapamycin also enhanced

CD252 (OX40-ligand; ligand for the secondary co-stimulatory molecule CD134) and CCR7 expression on TLR-7-activated PDC (data not shown). We do not know why mTOR-inhibition has opposite effects on CD40 and CD80/CD252/CCR7 expression. PDC maturation, resulting in up-regulation of co-stimulatory molecules, is thought to be mediated by nuclear factor kappa B (NFκB) signalling [33], which is inhibited in PDC by rapamycin [16]. PDC utilize an autocrine IFN-α feedback loop that further enhances INF-α production [34] after stimulation with CpG or loxoribine.

We tested if mTOR inhibition is involved in this autocrine IFN-α Kinase Inhibitor Library screening feedback loop to explain the reduced IFN-α production of the PDC after rapamycin treatment. This was performed by blocking the IFN-α-receptor2 with neutralizing antibodies during TLR-9 or TLR-7 activation. Blocking the IFN-α-receptor reduced IFN-α production by PDC, but did not influence the effects of rapamycin on IFN-α production, nor on IL-6 production. In addition, blocking of the IFN-α-receptor had no effect on CD40, CD80 and CCR7 expression on PDC (data not shown). These data indicate that rapamycin does not affect the autocrine IFN-α feedback loop in PDC, and that this KPT 330 loop is not involved in the differential regulation of CD40 and CD80/CD252/CCR7 expression. While rapamycin enhanced the capacity of loxoribine-activated PDC to stimulate CD4+ T cell proliferation, we found no effect of rapamycin on the T cell stimulatory capacity of CpG-A-stimulated PDC. Accordingly, rapamycin did not up-regulate CD80 expression on TRL-9-activated PDC. In contrast, Cao et al. [16] reported that rapamycin suppresses the capacity of CpG-A-stimulated mouse PDC to stimulate antigen-specific proliferation by CD4+ T cells.

Apart from the species difference, it should be realized that Cao et al. used a more artificial system by adding T cells which expressed a transgenic T cell receptor Farnesyltransferase specific for an ovalbumin peptide to the PDC, while we used primary T cells. Currently, we do not know how rapamycin inhibits the capacity of TLR-activated PDC to stimulate cytokine production by T cells. Neither blocking of CD80 nor blocking of IFN-αR2 abrogated the difference in cytokine production of T cells that were stimulated by PDC-activated loxoribine in the presence or absence of rapamycin. Previously, we have reported that corticosteroids induce apoptosis of resting human PDC and suppress the functions of activated PDC [35].

Nguyen et al. reported the capacity of healthy donors’ sera to bind and kill human

leukemic cells and activated T cells that were exogenously fed with Neu5Gc, but in these studies the detected cell death was mediated only by a complement-mediated mechanism [12]. The antibodies that recognized NeuGcGM3-expressing cells were of the IgM isotype. The IgM fraction isolated from one of the healthy donor’s sera retained the capacity to induce complement-independent death of the tumor cells. To our knowledge, this is the first report of anti-NeuGcGM3 antibodies that are able to induce the oncotic cell death of MI-503 antigen-expressing tumor cells without the necessity of any other immune component. These results suggest the existence of antibodies with antitumor potential, which could contribute to tumor immune surveillance. It is interesting to observe that the levels of anti-NeuGcGM3 RXDX-106 antibodies decreased as the age of the donors increased. Not only is the level of anti-NeuGcGM3 antibodies lower

in elderly donors, but also the percentage of responding donors decreases with age. An age-associated decrease in antibody levels against foreign antigens was first reported more than 70 years ago [35], supporting the idea of an immune deficiency state in the elderly. However, this seems to be a phenomenon dependent on the nature of the antigen and the cells involved in the different responses, since other studies have shown that the concentration of serum antibodies against a variety of self-antigens such as thyroglobulin, DNA, and IgG, increases with age [36]. In fact our results demonstrate that the total amount of IgG and IgM

did not decrease with age, suggesting that it is not the amount of antibodies but the those antibody repertoire that changes with age. One possible explanation for the decrease in antibody levels with increasing age involves an impaired capacity of T cells to facilitate the maturation of B cells in the periphery and the generation of a diverse B-cell repertoire from precursors within the bone marrow [37]. According to this theory, the response against T-independent antigens should not be affected by age [38]. However, the antibody response against not only NeuGcGM3 but also against other tumor related gangliosides (T-independent antigens), significantly decrease with increasing donor age [19]. Another possibility could be a reduction in the B-cell population responsible for the production of naturally occurring antibodies. Recently, Griffin et al. described a human B-cell population equivalent to mouse B1 cells [39], the main source of murine natural antibodies [40]. These researchers showed that human B1 cells decline with age.

Associations between polymorphism (rs1799964, rs1799724, rs1800630) and immune-mediated diseases such as rheumatoid arthritis and Crohn’s disease (CD) have been reported [14, 15]. Limited PD98059 reports are available showing that variants (rs1800629 and rs361525) are involved in the regulation of cytokine production [16]. The rs1799964 polymorphism has been associated with extra intestinal manifestations of CD including uveitis, erythema nodosum and large joint arthropathy [17] and Crohn’s disease itself [16]. It is clear that TNF enhancer polymorphism is implicated

in several case–control studies. In the present review, the literature regarding the role of TNF-α polymorphism has been studied with respect to different human diseases and different populations. Several single nucleotide polymorphisms (SNPs) in TFBS of different TFs have been

predicted computationally. The purpose of this review is to provide an overview of what is currently known about the role of gene level polymorphism of TNF and susceptibility/resistance to human diseases and to highlight directions that are Y 27632 likely to see major advances. Pulmonary tuberculosis. Mycobacterial tuberculosis is the leading cause of mortality in India as well as in the world. Approximately one-third of the world’s population is suffering from Mycobacterial diseases [18, 19]. Pulmonary tuberculosis, caused by M. tuberculosis, is a granulomatous disease of the lungs. The host genetic factor plays a significant role in determining susceptibility to developing the active form of the disease [20, 21]. A number of genes have been identified, which are important in tuberculosis [22–24]. Elevated serum tumour necrosis factor-α (sTNF-α) levels have been reported in patients with advanced tuberculosis Ceramide glucosyltransferase in comparison with those with mild tuberculosis and healthy controls. Several

polymorphisms within the promoter region of TNF-α and the intron 1 of LT-α have been associated with altered circulating levels of TNF-α [25, 26]. Some of these polymorphisms have been determine susceptibility or resistance to tuberculosis in several ethnic groups [27–33]. Sharma et al. [34] carried out a case–control study, including patients with pulmonary tuberculosis and controls in North India. In this study, five promoter SNPs in TNF-α gene and one SNP rs909253 in LTα gene were detected in patients with tuberculosis and controls samples collected from North India (Fig. 2). No significant differences in allele frequencies between the patients with tuberculosis and controls were reported. Serum TNF-α levels showed a significant difference between patients with tuberculosis and controls, and none of the polymorphism affects the serum TNF levels. Ates et al.

We have recently shown that mycobacteria-specific Th17 cells are also detectable in peripheral blood of M.tb-exposed humans 20. This population was distinct from specific Th1 cells. No data on the induction of specific T cells expressing IL-17 or GM-CSF after TB or other vaccination in humans have been published. Six candidate TB vaccines are currently undergoing clinical trials 21. Modified vaccinia Ankara-expressing Ag85A (MVA85A), a recombinant strain of modified vaccinia Ankara-expressing Ag85A from M.tb22, is the most advanced in the clinical development process. This vaccine, designed to enhance

BCG-induced immunity, was found to be safe and highly immunogenic in healthy adults from the UK 23, The Gambia 24 and South Africa 25. KU-57788 price MVA85A is the first novel TB vaccine to be tested in children, who are an important target population for vaccination. As

part of an age de-escalation strategy in a TB-endemic region, we evaluated and compared the safety of MVA85A R428 manufacturer vaccination and characterized the induced T-cell responses in healthy, M.tb-naïve adolescents and children. Twenty six adolescents and 56 children were screened between November 2006 and January 2008. Twelve adolescents and 24 children, none infected with M.tb, were found eligible for vaccination. Demographic characteristics are presented in Table 1 and reasons for exclusion in Supporting Information Table 1. All adolescents completed the 364-day follow-up period. One participant missed a single scheduled visit. Two of the 12 adolescents did not enter a record on their AZD9291 datasheet diary cards for all of the first 7 days post-vaccination, as had been requested. These two participants were able to recall symptoms during scheduled visits on days 2 and 7 after vaccination, when specifically questioned for possible adverse events, including those recorded on the diary cards. Among adolescents, 64 adverse events were recorded (Table 2): 61 (95%) were classified as mild and 3 (5%) as moderate; no severe or serious adverse events were recorded. The moderate events were all skin reactions at the vaccination

site. There was a median of six adverse events per participant. Fifty (78%) adverse events were local reactions at the vaccination site, which occurred in all participants. The reactions were most pronounced 2 days after vaccination; by day 7 post-vaccination 31 (62%) had resolved. Of the 19 (38%) local events that had not resolved by day 7, 15 (30%) had resolved by day 14, and the remainder by day 28. The majority of these more persistent events were desquamation and swelling. Systemic adverse events were infrequent, and comprised primarily arthralgia, headache and feeling feverish. There were no significant abnormalities in hematology or biochemistry parameters, measured 7 and 84 days after vaccination.

1 channels at the rear part of cells induces localized cell shrinkage and retraction of this cell part thereby promoting cell migration [9]. Moreover, the migratory activity of macrophages infiltrating atherosclerotic lesions and exhibiting an enhanced KCa3.1-expression was sensitive to the blockade of KCa3.1 [10]. Recently, it has been shown that KCa3.1 is also involved in the migration of lung DCs towards CCL19 or CCL21 using a transwell CFTR modulator system [11]. We here explored the role of KCa3.1 channels in LPS-induced DC migration. Additionally, cell volume changes of DCs upon stimulation with LPS were monitored since cell swelling has been described as a crucial event for cell migration

in leukocytes and DCs [12, 13]. BMDCs were obtained from 8- to 12-week-old female C57BL/6 N

(Charles River, Sulzfeld, Germany), TLR4−/− mice (on the C57BL/6 background), KCa3.1−/− mice (on the C57BL/6 background) as previously described [14]. KCa3.1-deficient mice (KCa3.1−/−) were generated Epigenetics inhibitor as described [15]. TLR4−/− mice [3] were kindly provided by Tilo Biedermann (Department of Dermatology, University of Tübingen). Briefly, immature BMDCs were generated from bone marrow-derived cells by cultivating them in RPMI 1640 medium (Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum (Sigma, Taufkirchen, Germany), 2 mM L-glutamine (Invitrogen, Darmstadt, Germany), 100 U/mL penicillin, 100 µg/mL streptomycin, 1% (vol/vol) nonessential amino acids, 1 mM sodium pyruvate (all from Biochrom), 50 µM β-mercaptoethanol (Sigma), and 200 U of GM-CSF/mL produced by mouse myeloma cells P3 × 63. On Day 8 of culturing BMDCs were seeded in uncoated 6-well plates (Greiner Bio-One, Frickenhausen, Germany) at a density of 1 × 106 cells in supplemented RPMI 1640 medium and stimulated or not with 500 ng/mL LPS (ultra pure, from Salmonella minnesota) (Calbiochem 437628, Darmstadt, Germany) up to 4 hr. At the indicated time points, 1.25 × 105 cells were harvested and analyzed by

flow cytometry. As a measure of cell size the mean of the forward scatter of BMDCs were analyzed by flow cytometry on a FACSCalibur (BD Biosciences, Heidelberg, PAK6 Germany) using WinMDI version 2.8 software (J. Trotter, The Scripps Institute, La Jolla, CA). As a control, aqua bidest (20%) to induce oncotic cell swelling, and staurosporine (4 µM, Sigma) to induce cell shrinkage, respectively, were added to the cell culture medium. On Day 8 of culturing 5 × 105 BMDCs in supplemented RPMI 1640 medium were seeded per insert of a BD Falcon™ FluoroBlok™ 24-Multiwell Insert System (Heidelberg, Germany) containing a membrane with 6.5 mm diameter and 3 µm pore size. The bottom wells of this transwell system were filled with supplemented RPMI medium with or without 100 ng/mL CCL21 (PeproTech, Hamburg, Germany), a chemoattractant and ligand for CCR7.