[33]. Cells were washed

three times in media and counted. PBMC from cutaneous leishmaniasis patients and non-infected individuals were used for in vitro culture TCR-usage analysis. Cultures were set up using a concentration of 2·5 × 105 cells in 96-well plates in the presence or absence of SLA (10 µg/ml final concentration) and were incubated for approximately 20 h. During the last 4 h of culture, Brefeldin-A (Sigma-Aldrich) (1 µg/ml), which impairs protein secretion by the Golgi complex, was added to the cultures. After the incubation period, cultures were harvested and submitted to flow cytometric analysis to evaluate T cell repertoire, surface markers and cytokine profile. The antibodies used for staining were immunoglobulin fluorescein isothiocyanate (FITC) and phycoerythrin (PE) controls (PharMingen, San learn more Diego, CA, USA), anti-Vβ2-biot, anti-Vβ3-biot,

anti-Vβ5·1-biot, anti-Vβ5·2-biot, anti-Vβ11-biot, anti-Vβ17-biot, anti-Vβ 24-biot (Immunotech, Burlingame, CA, USA) anti-Vβ8-FITC, anti-Vβ 12-FITC (Immunotech), SA-FITC (PharMingen), anti-CD69 PE (Ebioscience, San Diego, CA, USA), selleck screening library anti-HLA-DR-PE, anti-CD45RO-PE (PharMingen) and anti-CD4-PE-Cy5 (Ebioscience). The anti-cytokines antibodies used were PE-labelled anti-IFN-γ, anti-TNF-α (PharMingen) and anti-IL-10 (Caltag, Carlsbad, CA, USA). PBMC were analysed for their repertoire, surface markers and intracellular cytokine expression pattern. Briefly, 2·5 × 105 PBMC were cultured in 96-well plates in

200 µl cultures for 20 h with either media alone or SLA (at 10 µg/ml final concentration). Brefeldin-A (1 µg/ml) was added during the last 4 h of culture to impair protein secretion, allowing for cytokine intracellular staining, as performed previously by us [11]. The cells were then stained for T cell receptor Vβ repertoire and surface markers, and fixed using 4% formaldehyde (Sigma-Aldrich). Cells were then permeabilized with a solution of saponin (Sigma-Aldrich) and stained, for 30 min at 4°C, using anti-cytokine monoclonal antibodies directly Adenosine conjugated with PE. PE-labelled immunoglobulin control antibodies and a control of unstimulated PBMC were included in all experiments. Preparations were washed and fixed as described in the previous section and analysed using fluorescence activated cell sorter analysis (FACS), selecting the total lymphocyte population (Fig. 1). In all cases both cytokine and surface marker staining were associated with T cell receptor Vβ repertoire staining for studying the expression of cytokines and surface markers together and the phenotype of the cells that produced them. At least 40 000 gated events were acquired for later analysis.

These FcγR form hetero-oligomeric complexes with the same FcR γ-chain, which contains an ITAM sequence required for cell activation and cell surface expression. Most FcγR-triggered responses are balanced by the signaling of inhibitory ITIM-bearing FcγRII. Via the simultaneous triggering of activating

and inhibitory signaling pathways, FcγR control a wide array of Maraviroc cellular responses, including phagocytosis, antibody-dependent cell-mediated cytotoxicity and the release of inflammatory mediators, which ultimately lead to the amplification of normal and pathological immune reactions in vivo8–11. FcγR are expressed on many inflammatory cell types involved in allergic airway inflammation. It is, therefore, likely that FcγR, as well as polymorphisms in genes encoding FcγR, play a pivotal role in allergic airway disease 12. Allergen-specific IgG is present in the serum of allergic individuals and sensitized mice 13, and a specific role has been postulated for FcγRIII signaling in the regulation of optimal Th2-cell differentiation in allergy. This augmented Th2 differentiation was found to be independent of FcR-mediated antigen uptake and processing 14. Others 13 suggested that expression of FcγRI is important

during the sensitization phase of the development of allergic airway inflammation and airway hyperresponsiveness. selleck chemicals llc In our study, we used a mouse model of experimental asthma to verify the impact of FcγR on antigen uptake and presentation by DC. We hypothesized that activating FcγR control the strength and characteristics of airway hyperresponsiveness and inflammation, and before sought to demonstrate that IC can potentiate acute airway inflammation after sensitization, mediated by augmented T-cell proliferation after challenge. To compare the inflammatory response to inhaled antigens

in B6 and FcγR-deficient mice, the animals were sensitized with OVA+alum as described and challenged with OVA by inhalation. On day 3 after challenge, B6 mice revealed the characteristic perivascular and peribronchiolar infiltrate of mononuclear cells, whereas the allergic airway inflammation was reduced in FcγR-deficient mice (Fig. 1A). We observed significant eosinophilia in the BALF of B6 mice, which was virtually absent in FcγR-deficient (Fig. 1B). Control experiments of sensitized but non-challenged mice confirmed absence of eosinophilia and neutrophils in the BALF of all mice (data not shown). Both B6 as well as FcγR-deficient mice mounted a strong OVA-specific IgE response after sensitization, which resulted in equivalent mean OVA-specific IgE levels in both groups. No OVA-specific IgE responses were detectable in non-sensitized mice (data not shown). In order to confirm the constitutive FcγR expression on murine splenic and lung DC, cells from B6 mice were enriched by density gradient centrifugation and cell sorting of CD11c+MHC class II+ cells (Fig. 2A).

From a vaccination standpoint, regulation of T-cell responses by B cells must be better understood to better design effective vaccines. In our hands, the use of CpG as an adjuvant for peptide immunizations is superior to other

TLR ligands for reasons that are not clear. Strategies for avoiding stimulation of B cells with CpG in peptide-based vaccinations could make these approaches more effective. Female BALB/c mice 5–8 wk Bortezomib cost of age were purchased from Taconic Farms and housed in microisolater cages. TCR-Tg mice expressing a TCR specific for H2Kd-SYVPSAEQI have been previously described 5. B-cell-deficient mice (JHT) were purchased from Taconic Farms. For adoptive transfer, indicated numbers of TCR-Tg CD8+ T cells (TCR-Tg) from whole splenocytes were injected intravenously into naïve

BALB/c mice. Experiments involving mice were approved by the Institutional Care and Use Committee of the Johns Hopkins University. Vybrant CFDA-SE Cell Tracer Kit (Molecular Probes) was used to label cells to track proliferation according to the manufacturer’s instructions. Briefly, spleen cell suspensions were suspended in CFSE solution (5 μM in PBS) at 107 cells per mL for 6 min at room temperature. The reaction was then quenched by five-fold dilution of suspension with media containing 10% serum. Selleckchem BMS 354825 Cells were then washed in cold media and transferred into mice. Synthetic peptide representing the immunodominant epitope of P. yoelii CS protein and cognate antigen of the TCR-Tg cells (SYVPSAEQI) was diluted in PBS and Rebamipide injected subcutaneously at the base of the tail in 100 μL. When peptide was emulsified in IFA, peptide stock is diluted to in sterile PBS and emulsified 1:1 with IFA. CpG oligodinucleotide 1826 was synthesized by Integrated DNA Technologies and solubilized in sterile PBS (5′-TCC-ATG-ACG-TTC-CTG-ACG-TT-3′).

Intranucleotide bonds were phosphorothioated to enhance stability in vivo. CpG stock solution was diluted to 0.3 mg/mL in sterile PBS just prior to immunization and mice were injected subcutaneously at the base of the tail with 30 μg CpG. Spleens and draining LN were removed following euthanasia and placed in cold media on ice. Single-cell suspensions of lymphocytes were obtained by grinding organs between the frosted ends of two microscope slides and filtering twice through 100 μm pore size nylon mesh. Cells were washed and resuspended in fresh media containing 10% serum. LN cells were pooled among mice of the same group and spleens were analyzed individually for statistical analyses. All antibodies for flow cytometry were purchased from eBioscience unless otherwise noted. Frequency of parasite-specific TCR-Tg T cells was determined by staining of single cell suspensions with anti-CD8-APC and either anti-Thy-1.1-PE (BD Biosciences) or PE-conjugated H2Kd-CS260 tetramer, as previously described 5.

Briefly, a mouse was placed into the main chamber of the plethysmograph. The mouse was exposed to nebulized PBS and methacholine (Sigma-Aldrich) in PBS using an ultrasonic nebulizer. As an index of in vivo airway obstruction, Daporinad purchase enhanced pause (Penh) values were measured and expressed as relative values compared to baseline Penh values following PBS exposure for each methacholine concentration (1–25 mg/ml). Levels of plasma OVA-specific IgE

(OVA-IgE) in challenged mice were measured by enzyme-linked immunosorbent assay (ELISA), as described previously [16]. Th1 and Th2 cytokine levels (IL-4, IL-5, IL-13, IFN-γ) were measured in BALF by ELISA (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. To estimate OVA-specific T cell proliferation in vivo, we used OTII CD4+ cells labelled with CFSE; Molecular Probes, Eugene, OR, USA). Single-cell spleen suspensions from OTII mice were depleted of dendritic cells (DCs) using CD11c microbead and automatic magnetic-activated cell sorting (autoMACS) system

(Miltenyi Biotech, Auburn, CA, USA). The purity of CD4+ cells was estimated to be over 90% using a flow cytometer. Cells were incubated with 5 µM CFSE, according to the manufacturer’s instructions. CFSE-labelled OTII cells (5 × 106 cells) were transferred intravenously into each IgG or PBS-administered wild-type mouse. After injection, mice were challenged with OVA for 30 min a day for 2 days. Seventy-two hours after the OTII cell transfer, mononuclear cells from the thoracic lymph nodes were stained with anti-CD4-magnetic-activated Parvulin cell sorting MK-2206 in vitro (BD Biosciences, Franklin Lakes, NJ, USA) to analyse transferred CD4+ OTII cell proliferation using a flow cytometer. Data were analysed using Cellquest (BD Biosciences) and FlowJo

software (Treestar, Ashland, OR, USA). To analyse the function of lung CD11c+ antigen-presenting cells (APCs), they were collected 24 h after the mice were administered with 1 mg of IgG or PBS, as described previously [17]. Briefly, mouse lungs were minced and then incubated in the digestion medium consisting of RPMI-1640 (Sigma-Aldrich), 5% fetal bovine serum (Sigma-Aldrich), 1 mg/ml collagenase type 4 (Roche Diagnostics, Indianapolis, IN, USA) and deoxyribonuclease I (bovine pancreas; Wako). Lung CD11c+ APCs were isolated using the CD11c microbeads and autoMACS system according to the manufacturer’s instructions. The purity of CD11+ cells was estimated to be over 80% using a flow cytometer. OTII CD4+ cells were isolated from OTII mouse spleens using the MACS system. OTII CD4+ cells (2·5 × 105 cells/well) were co-cultured in a 96-well plate in complete medium with lung CD11c+ APCs (2·5 × 104 cells/well) from naive WT mice after PBS or IgG administration. Cultures were stimulated in vitro with an OVA323–339 peptide (5 µg/ml; GenWay Biotech, San Diego, CA, USA) or medium for 6 h.

Patients with renovascular disease Selleck Erastin are at high risk of poor cardiovascular outcomes. Optimal control of hypertension in patients with renovascular disease often requires the combined use of multiple blood pressure-lowering medications. Kidney Disease Outcomes Quality Initiative: No recommendation. UK Renal Association: No recommendation. Canadian Society of Nephrology: No recommendation. European Best Practice Guidelines: No recommendation.

American College of Cardiology/American Heart Association, 200552 1 AEC inhibitors are effective medications for treatment of hypertension associated with unilateral renal artery stenosis. (Level of evidence: A) 1 Consideration should be given to performing a large multicentre RCT of blockade of the renin–angiotensin system vs dihydropyridine calcium channel blocker in patients with proven renovascular disease. Patients enrolled should have high grade (>70%) renal artery stenosis and not have other definite indications or contraindications to renin–angiotensin system blockade. The proposed primary outcome would be composite cardiovascular events. Important secondary outcomes include blood pressure control and the risk of acute renal failure. Peter Mount has a Level II b conflict of interest according

Panobinostat to the conflict of interest statement set down by CARI. “
“Aim:  Long-term peritoneal dialysis (PD) may lead to peritoneal fibrosis and ultrafiltration failure. It had been demonstrated that the renin–angiotensin system (RAS) plays a key role

in the regulation of peritoneal function in rats on PD. We investigated the effects of angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB) on long-term PD patients. Methods:  We analyzed data from 66 patients treated with PD therapy at our centre for at least 12 months retrospectively, during which time at least two peritoneal equilibration tests (PET) were performed. Thirty-eight patients were treated with ACE/angiotensin II (AII) inhibitors (ACE/ARB group); BCKDHA the other 28 received none of the above drugs during the entire follow up (control group). The expression of fibronectin, transforming growth factor-β1 (TGF-β1), Aquaporin1 (AQP1) and vascular endothelial growth factor (VEGF) in the overnight effluent were examined by enzyme-linked immunosorbent assay. Results:  The demographic data of the two groups showed no difference during the study. No difference between the groups was found with respect to residual renal function (RRF) at the start for both groups by the end of follow up, decreased in the vast majority of patients from both groups (P = 0.014). After 12 months, a significant difference in ultrafiltration was found between the two groups: in the control group it had decreased, while it had not changed in the ACE/ARB group (P < 0.05).

9a,b) in a STIM1-dependent manner and by CD28-dependent store-independent activation of Ca2+ entry, potentially in a STIM2/ORAI1 or ORAI3-dependent manner. The CD28-dependent Ca2+ entry can occur in the context of the IS formation. If only CD28 is expressed, we would therefore not expect differences in

Ca2+ signals between CD80 or CD86 costimulation because CD28 is recruited to the IS independent of the type of costimulation (Fig. 9a). This is the case in Jurkat E6-1 and naïve primary T cells. However, if both CD28 and CTLA-4 are present at high concentrations, as in the case of effector T cells, it is expected that CD80 should preferentially bind to CTLA-4 and not so much to CD28, with the opposite being true for CD86.37 Therefore, CD86 should enhance CD28 recruitment to the IS and CD80 should inhibit CD28 recruitment by recruiting CTLA-4 instead. Through an unknown mechanism find more CD86, but not CD80, somehow enhances the store-independent activation of the CRAC channel,21,53 most likely in a STIM2/ORAI1 and/or ORAI3-dependent manner (Fig. 9b). In this model, the negative effect of CTLA-4 on T-cell activation is caused by the inhibition of CD28 recruitment to the IS. The knowledge of the fine-tuned difference in T-cell activation mediated by costimulatory molecules is of utmost importance not only to understand the underlying biology, but may also lead to novel therapeutic strategies that aim to activate the immune system against infectious

and malignant diseases. Super-agonistic antibodies targeting costimulatory molecules and activating T cells

by bypassing C646 the first signal have been developed in recent years.58 These super-agonistic antibodies bind to receptor domains that are not physiologically recognized by naturally occurring ligands, Methocarbamol circumvent the need for TCR specificity and, most importantly, are no longer regulated by the human immune system. This last issue has recently gained significant attention because a clinical trial using a CD28 super-agonistic antibody (TGN1412) confirmed in a dramatic manner that no model systems exists today that can predict immune mechanisms induced by super-agonistic molecules.58 In an early clinical trial performed in healthy volunteers, it was expected that activation of regulatory T cells by TGN1412 would further suppress the immune system and that the antibody would, eventually, be developed to treat patients with autoimmune diseases. As the CD28 antigen is expressed on the vast majority of T cells and not only on the small proportion of regulatory T cells, a broad T-cell activation pattern was observed resulting in a life-threatening cytokine release syndrome requiring treatement in the intensive-care unit. This clinical experience has demonstrated that the nature of super-agonistic, non-physiological ligands is unpredictable when tested in vivo. Along that line, a CTLA4–immunoglobulin has been developed for blockage of the CD28-CD80/CD86 pathway.

5%) vs. the control (35.7%) group (P = 0.02). The numbers of patients demonstrating clinical or radiological response were selleck chemical also significantly higher in the itraconazole group (P = 0.016 and 0.01

respectively). Adverse events were noted in eight patients in the itraconazole group, however, none was serious or led to discontinuation of the study drug. Itraconazole was found to be superior to standard supportive treatment alone in stabilising cases of CCPA. (clinicaltrials.gov; NCT01259336). The fungus Aspergillus commonly colonises the human respiratory tract and can lead to variety of diseases such as acute invasive pulmonary aspergillosis (IPA), subacute IPA [also called chronic necrotising pulmonary aspergillosis (CNPA)], allergic bronchopulmonary aspergillosis (ABPA) and chronic pulmonary aspergillosis (CPA). CPA is further classified as aspergilloma, chronic cavitary pulmonary aspergillosis (CCPA) and chronic fibrosing pulmonary aspergillosis Tigecycline research buy (CFPA).[1, 2] Pulmonary aspergilloma is the term given to colonisation of preexisting lung cavities with Aspergillus species, and formation of a conglomerate of fungal mass. It may be

further divided into simple and complex aspergilloma (or CCPA).[3] Simple aspergilloma is associated with a single fungal ball in a single cavity, and no invasion of surrounding lung tissue by the organism. CCPA is characterised by the presence of multiple aspergillomas in multiple thick walled cavities with or without presence of underlying parenchymal and pleural fibrosis or both with no or little tissue invasion by Aspergillus.[4] In contrast, CNPA (better termed subacute IPA) occurs in patients with mild degree of immune compromise, and is characterised by formation of lung cavities, cavitary Phosphoglycerate kinase consolidation and nodules with or without a fungal ball.[1, 2] In CNPA, there is evidence of invasion of lung tissue by Aspergillus. Many cavitary lung diseases are complicated by aspergilloma or CCPA including tuberculosis, sarcoidosis, bronchiectasis, bronchial

cysts, chronic obstructive lung disease, ankylosing spondylitis and pulmonary infection.[5] Of these, tuberculosis is probably the most common association especially in developing countries.[6] The symptoms and signs of CPA can range from incidentally detected chest radiographic findings to a situation with life-threatening haemoptysis.[4] Patients with CCPA/CFPA commonly present with chronic cough, expectoration, haemoptysis, malaise, weight loss, fatigue and progressive loss of lung function. CNPA presents in a subacute fashion with pulmonary or systemic symptoms in an ill patient in contrast to simple aspergilloma and CCPA where patients may be asymptomatic.[7] In patients with simple aspergilloma, treatment is not associated with significant improvement in symptoms and/or radiology, with rates of spontaneous complete radiological resolution being approximately 5% over 3 years.

The use of splenic MDSCs from tumor-bearers throughout this study is in line with the central importance of this organ for inducing tolerance to tumor antigens [19]. IFN-γR−/− and STAT-1−/−, but not IFN-γ−/−, splenic MO-MDSCs induced by EG7-OVA largely lost their antiproliferative capacity, illustrating that suppression is entirely dependent on IFN-γ-mediated triggering by activated T cells, but not on IFN-γ production by the MDSC — as claimed before [31] — or IFN-γ priming in vivo. Interestingly, IRF-1-deficiency uncovers the existence of parallel IRF-1-dependent and -independent suppressive mechanisms in MO-MDSCs, both of which

are needed to maximize suppression. IRF-1-dependent NO production is responsible for at least 50% of the suppression, but the IRF-1-independent Alvelestat mechanism remains unknown. Remarkably, also the PMN-MDSC-mediated

suppressive mechanism is heterogeneous, with a minor IFN-γ/STAT-1/IRF-1-dependent component and a major IFN-γ-independent mechanism. Since different pathological conditions — including different tumor types [12] — preferentially expand one or the other MDSC subset, these data suggest that different intervention strategies might be needed to ablate suppression in different settings. In the case of EG7-OVA, the total splenic MDSC population contains approximately ICG-001 order 40% MO-MDSCs (both before and after purification), but these cells appear to dominate since the suppressive mechanism of unseparated MDSCs largely depends on NO (Supporting Information Fig. 14). Finally, it should be noted that these findings are not confined to the EG7-OVA model. Indeed, RMA-OVA-induced splenic MO-MDSCs from WT mice suppress T-cell proliferation in a dose-dependent and largely NO-dependent fashion, while IFN-γR−/− MO-MDSCs lack this activity (Supporting Information Fig. 15). PMN-MDSCs display a lower T-cell antiproliferative capacity in this model, which is partly dependent on IFN-γ signaling and independent from NO. many Proliferation is a relatively late event in the course of CD8+ T-cell

activation, preceded by the secretion of cytokines such as IL-2 and IFN-γ, and the expression of early activation markers such as CD69 and CD25 [3]. Our data now demonstrate that MDSCs manipulate early activation events in an intricate way — suppressing some aspects, while stimulating others — to optimize T-cell suppression. Most literature, with some exceptions [31], suggests that MDSCs suppress IFN-γ production, but those data are often confounded by the antiproliferative effect of MDSCs resulting in lower T-cell numbers. Via intracellular IFN-γ staining, we demonstrated that IFN-γ production by CD8+ T cells is enhanced on a per cell basis in the presence of splenic PMN-MDSCs already before the initiation of proliferation, and the percentage of IFN-γ+CD8+ T cells remains enhanced throughout each division cycle. This makes sense from the MDSC point of view, since IFN-γ initiates their antiproliferative program.

Alternatively, a single subtype was detected in the 26OB5 and 26OB6 clusters in the MLVA, whereas five and three subtypes were detected in the 26OB5 and 26OB6 clusters, respectively, in the PFGE analysis. Nevertheless, most of these results were consistent with each other, as in the case of O111OB3, where all the isolates exhibited 100% similarity in both the analyses. Genotyping is a powerful and useful tool for epidemiological investigation;

for example, during outbreaks of infectious diseases. MLVA is a newly developed genotyping method for bacterial infectious diseases and is based on differences between the isolates with regard to the repeat copy numbers Selleckchem LBH589 in certain genomic loci. Dozens of bacterial species, including EHEC Selleck RXDX-106 O157, have been studied using this method (6, 7). Owing to its simplicity and discriminating power, it is considered one of the methods of the next generation to PFGE, which is currently the golden method of genotyping. MLVA can be accomplished through PCR and electrophoresis. The results are converted to digitalized

repeat copy numbers, which can be clearly evaluated for each isolate. MLVA is also a rapid method—the results can be obtained within several hours after isolation (16). MLVA, however, requires high-quality electrophoresis facilities, such as an automatic sequencer, which has a high cost of implementation. Further, for the start-up process, genome sequences of target bacterial agents are required, and the efficacy of an MLVA system can be affected by information on the genome sequences analyzed. That is, increasing availability of the genome sequences of a given bacterial species increases the efficiency of MLVA. In the present study, we developed and evaluated the efficiency of an expanded MLVA system that was designed for analyzing the EHEC O26 and O111 isolates as well as the EHEC O157 isolates. The three serogroups account for more than 95% of

the EHEC isolated in Japan (5). The results of evaluation of the MLVA system that is now being routinely used for analyzing EHEC O157 isolates (7) indicate that it is not applicable to the EHEC O26 and O111 isolates. Most loci were not amplified by PCR, even if any amplification occurred, Dichloromethane dehalogenase the repeat copy numbers exhibited less variation among the EHEC O26 and O111 isolates (Fig. 1). Comparison and re-inspection of the genome sequences also resulted in correction of interpretation of the O157-34 locus (Fig. 2). By modifying the O157-9 primer and including nine additional loci, six of which were newly developed in the present study, we finally developed an improved MLVA system that can be used for genotyping EHEC O157, O26, and O111. All the loci adopted in this study exhibited D values of more than 0.

5a). In addition, the percentage and total number of switched GC B cells were also enhanced after late stage Treg-cell disruption. These data indicate that Treg cells participate in the control of GCs throughout the entire response, and not just at the induction phase. Given the observation that Treg cells participate in the control of GC reactions, it was of interest to explore the frequency and phenotype of the splenic Treg-cell population after immunization with SRBC. To monitor Treg cells, Foxp3-GFP reporter mice were used.47 As shown in Fig. 6(a), CD4+ Foxp3+ T cells are readily detected in the spleens of these mice, allowing for enumeration and phenotypic

characterization. Of interest, the proportion of Foxp3+ Treg cells within the splenic CD4+ compartment was unaltered throughout the GC response mTOR inhibitor (Fig. 6b), although total cellularity of the spleen increased modestly at days 8 and 12 (data not shown). As iTreg cells are probably activated to control the humoral IWR-1 supplier response to novel antigens,

a range of surface markers were examined in an attempt to identify an activated iTreg-cell sub-set. When comparing naive with SRBC-challenged mice, no differences were found in the proportion of Treg cells expressing CD103, CD45RB, CD62L, CD178, GITR or PD-1 at any time-point (data not shown). Several reports have demonstrated the presence of Treg cells within the GCs of human and mouse secondary lymphoid tissue,44,45,60,61 indicating their ability to migrate into activated follicles.62 Accordingly, CXCR5 and CCR7 expression was examined on CD4+ Foxp3+ T cells from naive and immunized mice. As shown in Fig. 6(a), the splenic Treg-cell population consists of four sub-sets defined as CXCR5− CCR7+, CXCR5lo CCR7lo, CXCR5 CCR7− and CXCR5+ CCR7−. CXCR5− CCR7+ Treg cells would be expected to reside in T-cell zones with CXCR5lo CCR7lo Treg cells positioned at the borders of T-cell : B-cell

areas. CXCR5− CCR7− Treg cells would probably be found in red pulp tissue. Importantly, CXCR5+ CCR7− Treg cells should have the ability to migrate into B-cell follicles with the potential to control B-cell activity locally. In naive mice (day 0), the CXCR5− CCR7+, CXCR5lo CCR7lo, CXCR5− CCR7− and CXCR5+ CCR7− sub-sets composed 29%, 14%, 30% Vasopressin Receptor and 27% of the Treg-cell compartment, respectively. It is of interest that all four sub-sets exist in unimmunized mice, suggesting that Treg cells patrol all areas of the spleen under steady-state conditions. The four Treg-cell sub-sets were similarly enumerated in SRBC-immunized mice at days 8, 12 and 18 post-challenge. Figure 6(c) shows no change in the frequency of CXCR5− CCR7+ and CXCR5+ CCR7− Treg cells during the course of the response, indicating no major shift of Treg cells from the T-cell zone into activated follicles. Percentages of CXCR5lo CCR7lo and CXCR5− CCR7− Treg cells were also unchanged (data not shown).