The results demonstrated significant differences in selected serum inflammatory mediators during the ligation phase of the study related to the time-point see more of the study and associated with ligation of teeth in two quadrants (MP) or four quadrants (D). Interestingly, the profile of inflammatory mediators at the various time-points of disease was not associated consistently with increasing disease, with only IL-6 levels demonstrating a significant increase after 6 months of periodontal disease. The results suggested that although there were variations in systemic analyte measures related to periodontitis, individual

variation in the clinical responses of the animals may have a substantial impact upon interpreting the direct link between oral disease and systemic responses. PLX4032 mouse Moreover, while previous studies in human periodontitis have suggested local involvement of a range of mediators, including IL-1β and TNF-α, expression of these proinflammatory response molecules were not observed in the systemic responses of the baboons to periodontal disease progression. This is consistent with differences in local versus systemic cytokine/chemokine response profiles observed with this disease in humans [13]. Therefore, we evaluated changes in the inflammatory mediators through the 6-month ligation in subsets of the animals based upon clinical presentation

at baseline. These results demonstrated consistent patterns of systemic

inflammation related to progressing periodontitis. PGE2 levels increased significantly by MP and remained elevated throughout the entire pregnancy. Similarly, BPI levels were also increased significantly by MP in most of the animals and generally decreased substantially by delivery. LBP levels were elevated generally at baseline and decreased significantly throughout the disease process. As was noted with the population as a whole, IL-6 levels were increased significantly by delivery, irrespective of the baseline clinical characteristics of the animals. Both IL-8 and Idoxuridine MCP-1 decreased from baseline throughout the study, with the lowest levels of IL-8 in serum samples obtained at delivery, unrelated to the clinical presentation of the animals at baseline. A summary of these outcomes was that the clinical presentation at baseline had less impact on the systemic inflammatory mediator levels than the effect of the continued disease over 6 months induced by ligation and creation of chronic periodontitis in the animals. Finally, based upon these findings, we evaluated response differences in subsets of animals as they progressed through the experimental challenge during pregnancy. Thus, at baseline, stratification of the animals related to naturally occurring oral health/disease showed some distinct differences in serum inflammatory mediators that differentiated the healthy from gingivitis from the periodontitis groups.

4A). Interestingly, the majority of mice vaccinated with the subdominant GP283 epitope survived the LCMV infection as did the control mice vaccinated with the control P. berghei CS252 epitope. As previously observed,

the majority of mice vaccinated with the dominant NP118 epitope succumbed to the LCMV infection (Fig. 4B and 1E). Importantly, the NP118- and the GP283-specific memory CD8+ T cells exhibited similar memory phenotype and function (CD127hi, KLRG-1lo, CD27hi, CD43lo, and high frequencies of these cells buy MK-8669 produce IL-2 and TNF upon specific peptide restimulation) at the time of LCMV infection (Fig. 4C) suggesting the difference in outcome was not an issue of memory quality. However, a statistically significant difference

(p = 0.03) in total number of NP118- and GP283-specific memory CD8+ T cells in the spleen of vaccinated PKO mice prior to LCMV challenge was observed (Fig. 4D). To determine if the difference in the starting number of memory CD8+ T cells of different Ag-specificity controls the difference in susceptibility to the LCMV challenge, groups of naïve PKO mice were immunized with different numbers of peptide-coated DC to equalize the number of memory CD8+ T cells. At day 124 following DC immunization, the frequency of GP283-specific memory CD8+ T cells was approximately equal to that of NP118-specific memory CD8+ find more T cells (Fig. 4E). More importantly, the magnitude of expansion was also similar between GP283- and NP118-specific CD8+ T cells at days 5 and NADPH-cytochrome-c2 reductase 7 after LCMV infection (Fig. 4E). However, we observed 100% mortality in DC-NP118-vaccinated mice but 0% mortality in DC-GP283- or DC-CS252- vaccinated groups of mice (Fig. 4F). Thus, PKO mice containing memory CD8+ T cells against a dominant epitope, but not a subdominant epitope, are predisposed to LCMV-induced mortality, under conditions where the starting number and magnitude of expansion of memory CD8+ T cells are similar. These results suggested that the epitope specificity dictates vaccination-induced mortality in BALB/c-PKO mice following LCMV challenge. Since

vaccination of naïve PKO with the subdominant epitope did not result in mortality following LCMV challenge, we also sought to determine whether these vaccinated mice showed enhanced resistance against LCMV infection. Similar to the DC-NP118-vaccinated PKO mice, the DC-GP283-vaccinated mice had significantly reduced viral load at day 5 post-LCMV infection compared with the nonimmunized mice. However, the viral load reduction was not sustained by day 7 post-LCMV (Fig. 5). Thus, although CD8+ T-cell-mediated LCMV-induced mortality can be avoided by vaccination of PKO mice with the subdominant instead of the dominant epitope, this immunization did not provide sustained virus control. In general, CD8+ T cells exhibit tight regulation of cytokine production and do not produce IFN-γ directly ex vivo unless they receive Ag-stimulation.

One unlabelled mAb was adsorbed

to the microtitre plate well and used as a capture antibody, and the other labelled mAb served as the probe. For the multicytokine assay, we have used Multiflex Biomarker Immunoassay (Millipore, Billerica, MA). To determine T-cell proliferation, TCR-Tg spleen T cells were labelled in vitro with the intracellular dye carboxyfluorescein DNA Methyltransferas inhibitor succinimidyl ester (CFSE) by using the Vybrant CFSE SE tracer kit (Molecular Probes, Eugene, OR.). Carboxyfluorescein succinimidyl ester (CFSE; 0·5 mm) was added to the cell suspensions, and the mixture was incubated for 10 min at 37°. The labelling reaction was stopped by repetitive washing with ice-cold RPMI-1640 medium containing 10% fetal calf serum. Labelled cells (4 × 106/ml) were cultured with various concentrations of antigen for 2, 3 or 4 days. Cultured

cells were harvested, washed and labelled with anti-TCR Vβ11 antibodies. The number of cell divisions was determined by Nutlin-3 dilution of the intracellular dye CFSE in TCR Vβ11+ gated T cells by flow cytometry. In some experiments, Bodipy-FL (Invitrogen, Carlsbad, CA) was used for the cell proliferation assay instead of CFSE. To measure the effect of HBeAg on effector T-cell activation in vitro, we cultured splenocytes from naive 7/16-5 TCR-Tg and 7/16-5 × HBeAg dbl-Tg mice in the presence of 1 μg/ml of the HBeAg-derived peptide p120–140 and analysed CD69 expression by FACS. There was a dramatic difference between T-cell activation in 7/16-5 TCR-Tg mice versus 7/16-5 × HBeAg dbl-Tg mice. A high percentage (59·30%) of T cells derived from 7/16-5 TCR-Tg mice expressed CD69 after 3 days in culture, which was sustained after 6 days. In contrast, only 18·56% of T cells derived from HBeAg × 7/16-5 dbl-Tg mice expressed CD69 at 3 days of culture and the expression of CD69 remained low (11·17%) at day 6 (Fig. 1a). Similarly, in vitro IL-2 production by 7/16-5 × HBeAg dbl-Tg splenic T cells cultured with either HBeAg or HBcAg was significantly reduced

compared with T cells from 7/16-5 single TCR-Tg mice (Fig. 1b). It is notable that the tolerance exhibited in 7/16-5 × HBeAg dbl-Tg mice is not the result of clonal deletion of 7/16-5 TCR-Tg T cells either Sorafenib in the thymus or in the spleen (data not shown,30). CFSE-labelled splenocytes from 7/16-5 TCR-Tg and 7/16-5 × HBeAg dbl-Tg mice and 7/16-5 × HBcAg dbl-Tg mice were cultured in vitro in the presence of HBeAg peptide p120–140 to examine CD4+ and CD8+ effector cell proliferation. As shown in Fig. 2, after 4 days in culture both CD4+ and CD8+ Vβ11+ TCR-Tg T cells from 7/16-5 mice and 7/16-5 × HBc dbl-Tg mice proliferated. In contrast, the vast majority of the proliferating Vβ11+ TCR-Tg T cells from 7/16-5 × HBeAg dbl-Tg mice were neither CD4+ cells nor CD8+ cells (Fig. 2, middle column) and represented a DN, Vβ11+ population.

Another effect mediated by Ab–FcR interactions is the induction of reactive oxygen and nitrogen species in macrophages, neutrophils, and other phagocytic cells. The resulting oxidative burst, mediated by these short-lived molecular species, plays an important role in the control of viruses, bacteria, and parasites 10. Ab–FcR interactions have a number of additional functions such as cell activation, the induction of cytokine production, receptor-mediated endocytosis, targeting www.selleckchem.com/products/ulixertinib-bvd-523-vrt752271.html of immune complexes for degradation, storage of immune complexes in germinal centers

of secondary lymphoid organs, and the augmentation of MHC-restricted Ag presentation. In this review, we will focus on the role this website of these functions in immune responses against intracellular bacteria and parasites, and in invasive fungal infections. Four different classes of FcγRs have been identified

in mammals, known as FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIV, which bind the different IgG subclasses with varying affinity and specificity. Functionally, FcγRs can be divided into activating (FcγRI, FcγRIIA/C, FcγRIII, and FcγRIV) and inhibitory (FcγRIIB) receptors, which transmit signals via immunoreceptor tyrosine-based activation (ITAM) or inhibitory motifs (ITIM), respectively. Activating signals through ITAM-containing FcRs involve a number of kinases and ultimately lead to a large variety of effector responses in innate immune effector cells, such as oxidative burst, cytokine release, phagocytosis, ADCC, and the degranulation of mast cells. On the contrary, the inhibitory receptor FcγRIIB acts as a negative regulator of immune complex-triggered activation as it counteracts effector cell functions (-)-p-Bromotetramisole Oxalate triggered through activating receptors. It also plays an important role in the selection of affinity-matured B cells and the modulation of Ab production 11. Most cell types express activating as well as inhibitory

FcγRs and simultaneous engagement sets thresholds for cell activation and ensures a balanced immune response 12. In contrast to FcR-independent phagocytosis involving interactions between the cell-surface receptors and the corresponding ligands on a particulate Ag, FcR-mediated phagocytosis involves FcR activation and downstream ITAM signaling 13. The ratio of local concentrations of activating to inhibitory FcγRs recruited during phagocytosis determines whether an IgG-opsonized particle is ultimately taken up or not, and differential recruitment of FcγRs is mainly achieved by their different affinities for IgG subclasses 14. Furthermore, the density of IgG on the particle correlates with the magnitude of early FcR signals and results in an all or none response of uptake 15.

These populations were then co-cultured with MSC (1·5 × 105/ml) for 72 h in cRPMI. PBMC or sorted CD4+ T cells were recovered from culture by gentle aspiration from adherent MSC and examined by flow cytometry. Cells were washed in PBS, surface-stained for CD4 APC and CD25 phycoerythrin (PE) where required. Cells were then fixed in 2% (v/v) paraformaldehyde, permeabilized in PBS/Tween

and blocked using normal rat serum. Following this, cells were incubated with anti-human FoxP3 fluorescein isothiocyanate (FITC) (eBioscience) for 30 min at 4°C. Cells were washed, fixed in 1% (v/v) formaldehyde/PBS and analysed by flow cytometry within 4 h. Regulatory T cell (Treg) induction in vivo was Decitabine examined in the aGVHD model described above with either IFN-γ-stimulated MSC (4·4 × 104 g−1) administered

i.v on day 0 or non-stimulated MSC (4·4 × 104 g−1) on Nutlin-3 nmr day 7 post-PBMC transfusion. On day 12, the day of aGVHD pathology manifestation, the lungs, livers and spleens of NSG mice were harvested and a single-cell suspension prepared. The surface expression of human CD4 APC, CD25 PE and intracellular expression of human FoxP3 FITC was determined by flow cytometry. Statistical analysis was performed using GraphPad Prism™ software (GraphPad, San Diego, CA, USA). The Student’s paired t-test was used when statistical analysis was required between two experimental groups. Sorafenib in vitro One-way analysis of variance (anova) was used to test for statistically significant difference when multiple experimental groups were compared. Kaplan–Meier curves (log-rank test) were used to compare survival between treatment groups. Data are presented as ± standard error of the mean (s.e.m.). P-values

of P < 0·05 (*), P < 0·01 (**) or P < 0·001 (***) were considered statistically significant. A robust and reproducible model of aGVHD was established in NSG mice by delivery of human PBMC. This was adapted from Pearson et al. [29], and reproducibility achieved by (i) normalizing PBMC dose to murine body weight, (ii) use of freshly isolated PBMC from healthy donors and (iii) preconditioning of mice by exposure to 2·4 Gy irradiation prior to PBMC delivery. On day 7 post-PBMC transfusion, human MSC allogeneic to the PBMC donor were given i.v. as a cell therapy. NSG mice that received PBS alone did not develop any signs of aGVHD, whereas mice that received PBMC developed aGVHD consistently between days 12 and 15, with weight loss, hunched posture, ruffled fur and reduced locomotion (Fig. 1a,b). Delivery of non-stimulated human MSC on day 0 had no detectable beneficial effect (data not shown); however, MSC therapy on day 7 significantly extended the survival of NSG mice with aGVHD (P < 0·0001), with some mice surviving for more than 30 days (Fig. 1c).

Histamine-mediated signals affect the ability of DC to induce the maturation of T cells along Th1 or Th2 pathways. Histamine appears to be involved in the Th-switch: Th1 cells express H1R, while H2R is found on both Th1 and Th2 cells, as well as DC. H2R also appears to play a critical role in the induction of immune tolerance. Histamine has many important, but still poorly understood immune-related functions, highlighting the need for additional animal models, including histamine receptor gene knockout

mice. Mast cells play critical, but undefined, immunoprotective roles in bacterial and helminth check details infections. Studies from the laboratory of Richard Stevens (Boston, MA) led to the identification of two major serine mast cell tryptases, mouse mast cell protease (mMCP)-6 and mMCP-7, that are critical factors in protection from bacterial and helminth infection. Dr. Stevens and colleagues demonstrated that mast cell-deficient

W/Wv mice can successfully combat a Klebsiella pneumoniae pulmonary infection when pre-treated with physiologic amounts of recombinant mMCP-6 or its human ortholog hTryptase-β 17. Dr. Stevens and Dr. Adachi created transgenic mice that lack both mMCP-6 and mMCP-7 18. They then showed that these tryptase-deficient animals have a markedly reduced ability to combat K. pneumoniae infection of the peritoneal cavity and an impaired ability to combat Trichinella spiralis infections. The mechanisms by which mast cell-restricted tryptases selleck chemical are beneficial in varied infections remain to be determined at the molecular level, but it appears that they play important roles in orchestrating the accumulation of granulocytes in tissues. K. Frank Austen (Boston, MA) addressed an unexpected

role of mast cell proteases in the response to ischemia reperfusion injury. In mouse models of ischemia reperfusion injury, the heightened exposure of self-Ag leads to Ag recognition by natural IgM and subsequent complement activation. This results in immune mediated injury that depends on specific mast cell-derived proteases, as evidenced by the fact that mast cell-deficient mice are protected from injury. In hind limb ischemia reperfusion injury, mice Resveratrol lacking the elastase mMCP-5 are significantly protected. The same mechanistic principles apply to a second-degree burn model in which mice deficient in mast cell chymase/elastase (mMCP-4/5), but not tryptase (mMCP-6/7), are protected from ulceration and scarring. Dr. Austen proposes that mast cell-derived proteases such as mMCP-4/5 play a critical role in the tissue damage following injury. Stephan C. Bischoff (Stuttgart, Germany) observed that much of the mast cell literature is based on data obtained in animal species that, in nature, do not suffer from mast cell-mediated allergic diseases.

The primers used were as follows: HIF-1α (predicted length 343 bp) sense: 5′-TGCTCATCAGTTGCCACTT-3′, antisense: 5′-TGGGCCATTTCTGTGTGTA-3′; HIF-2α used were sense: 5′-GACGGTGACATGATCTTTCTGTC-3′, antisense: 5′-CACTTCATCCTCATGAAGAAGTCAC-3′; VEGF (predicted length; VEGF165: 535 bp and VEGF121: 403 bp) sense: 5′-CCAAGTGGTCCCAGGCTGCACC-3′, antisense: 5′-GGTTAATCGGTCTTTCCGGTGAG-3′, and GAPDH (predicted length 609 bp) sense: 5′-GCCATCAACGACCCCTTCATTGAC-3′, antisense: 5′-ACGGAAGGCCATGCCAGTG AGCTT-3′. PCR reactions were performed in a thermocycler (GeneAmp® PCR System 2400, Applied Biosystems, Foster City, CA, USA).

Quantitative RT-PCR analysis was performed using the LightCycler® FastStart DNA Master SYBR Green I (Roche click here Diagnostics, Mannheim, Germany). The ΔCT-method was used for the calculation of relative changes of mRNA by

LightCycler 480® Multiple Plate Analysis Software (Roche Diagnostics) 55. The data were normalized to the expression of β-actin and was confirmed by quantitative real-time RT-PCR to be ubiquitously and consistently expressed gene among all groups analyzed. The sequences of primers used were as follows: HIF-1α sense: 5′-TGCTCATCAGTTGCCACTT-3′, antisense: 5′-TGGGCCATTTCTGTGTGTA-3′; HIF-2α used were sense: 5′-GACGGTGACATGATCTTTCTGTC-3′, Roxadustat molecular weight antisense: 5′-CACTTCATCCTCATGAAGAAGTCAC-3′; ZD1839 research buy VEGF sense: 5′-CCAAGTGGTCCCAGGCTGCACC-3′,

antisense: 5′-GGTTAATCGGTCTTTCCGGTGAG-3′, and β-actin sense: 5′-CAGATCATGTTTGAGAC CTTC-3′ and antisense: 5′-ACTTCATGATGGAATTGAATG-3′. PI3K enzyme activity was measured as described previously 33. The amount of PIP3 produced was quantified by PIP3 competition enzyme immunoassays according to the manufacturer’s protocol (Echelon, Salt Lake City, UT, USA). An inhibitor of HIF-1α, 2ME2 (50 or 100 mg/kg body weight/day), was suspended in 0.5% carboxymethylcellulose (Sigma-Aldrich) and administered by oral gavage six times at 24-h interval on days 19–24, beginning 2 days before the first challenge 56. Cyclopeptidic vascular endothelial growth inhibitor, CBO-P11 (Flt-1; IC50=700 nmol/L, Flk-1/KDR; IC50=1.3 μmol/L, D-Phe-Pro (79–93); Calbiochem-Novobiochem) was used to inhibit VEGF activity. CBO-P11 (2 mg/kg body weight/day) was administered i.p. three times at 24-h interval, beginning at 1 h before the first inhalation. IC87114 (0.1 or 1.0 mg/kg body weight/day) or vehicle control (0.05% DMSO) diluted with 0.9% NaCl was administered in a volume of 50 μL by intratracheal instillation two times to each animal, once on day 21 (1 h before the first airway challenge with OVA) and the second time on day 23 (3 h after the last airway challenge with OVA) 33. Protein expression levels were analyzed by Western blot analysis as described previously 48.

The FLS lack NALP3 protein expression despite the presence of NALP3 mRNA, and activators of the NALP3 inflammasome were unable

to induce functional IL-1β secretion. Finally, the pattern of expression of known NLRs are comparable in RA and OA synovium, suggesting that NLRs are not a critical determinant of the pathology of these two diseases. This work was supported by grants from the Fonds National Suisse de la Recherche Scientifique (K-32K1-116460 to N.B. and 320000-120319/1 to G.P.) and by the Jean and Linette Warnery foundation. We are indebted to Monica Azevedo for excellent technical Palbociclib support. The authors declare that they have no competing interests. L.K. was responsible for the majority of the practical work and for the writing of the manuscript. The study was originally designed by A.S. and N.B. G.P., D.T.

and V.C. were involved in different methodological parts and interpretation of the data. A.S. and N.B. were involved in interpretation of the results and manuscript writing. All authors read and approved the final manuscript. “
“Citation MAPK Inhibitor Library Ohel I, Levy A, Zweig A, Holcberg G, Sheiner E. Pregnancy complication and outcome in women with history of allergy to medicinal agents. Am J Reprod Immunol 2010; 64: 152–158 Problem  Pregnancy outcome in women with a previous history of drug allergy and the role of drug allergies in adverse pregnancy outcomes is unclear. Method of study  A retrospective cohort GPX6 study comparing pregnancies of women with and without history of drug allergy was conducted. Data were collected from the computerized perinatal database. A multiple logistic regression model, with background

elimination, was constructed to control for confounders. Results  Of 186,443 deliveries, 4.6% (n = 8647) occurred in patients with a history of drug allergy. The following conditions were significantly associated with a history of drug allergy: advanced maternal age, recurrent abortions, fertility treatments, hypertensive disorders, and diabetes mellitus. Using multivariate analysis, with background elimination, history of drug allergy was significantly associated with intrauterine growth restriction (OR = 1.52, CI = 1.3–0.8, P < 0.001) and with preterm delivery (OR = 1.26, CI = 1.14–1.38, P < 0.001). Conclusion  A history of drug allergy is an independent risk factor for intrauterine growth restriction and preterm delivery. Further prospective studies are needed to investigate the nature of this association. "
“Thrombophilia is associated with pregnancy complications. Treatment with low molecular weight heparin (LMWH) improves pregnancy outcome, but the underlying mechanisms are not clear. We analyzed Treg frequency in blood from thrombophilic pregnancies treated with LMWH (n = 32) or untreated (n = 33) and from healthy pregnancies (n = 39) at all trimesters.

Previously we found that stone formers developed significant proteinuria and high oxidative stress. Currently we aimed to investigate the proteinuria and oxidative stress in their family members. Methods: Twenty-eight post-calculi removal stone formers (SF) and their disease-free children were recruited, and 30 non-stone forming healthy adult (NSF) and their children who lived in the same region were enrolled as the control. Blood and 24-hours urine

were collected. Plasma creatinine, total urine proteins (UP), microalbuminuria (MA), plasma protein carbonyl (PC) and urinary total antioxidant status (TAS) were measured. Results: Age, gender and BMI were matched between SF and NSF control. Age and gender between SF’s children and NSF’s children Alvelestat mw were matched as well. SF had significantly higher UP (436.6 ± 117.8 mg/day) and MA (223.2 ± 73.0 mg/day) than any groups. Nephrolithiasis

children had significantly increased UP (78.4 ± 8.6 mg/day) than NSF and NSF’s children (34.8 ± 7.7 and 23.2 ± 3.7 mg/day, respectively). MA was not different between SF’s children, NSF and NSF’s children (6.3 ± 2.1, 7.7 ± 2.0, 0.4 ± 0.2 mg/day, respectively). Plasma creatinine, PC and urinary TAS were not significantly ICG-001 cell line different between each groups. Conclusion: The present study demonstrated that approximately 21.4% (6/28) of stone formers had marked proteinuria (>500 mg/day) and microalbuminuria (>150 mg/day), indicating both glomerular and tubulointerstitial injury. This is against the traditional beliefs that renal stone is corresponded with isolated tubulointerstitial inflammation. The precise pathophysiology of glomerular proteinuria in nephrolithiasis is not yet established, but might be associated with hyperoxaluria or diminished sulfated glycosaminoglycans. As disease-free nephrolithiasis children had elevated proteinuria compared with many the normal population, this might indicate an asymptomatic

tubulointerstitial injury. This injury was not correlated with the current oxidative status, since we could not demonstrated the increased oxidative stress in neither SF nor their children. We hypothesized that SF’s children who commonly had hypocitraturia and low urinary glycosaminoglycans level might form small urinary crystals that could initiate the tubular inflammation. This hypothesis needs to be elucidated in further. LAI LINGYUN1, LI HUIXIAN1, AZRAD MARIA2, ZHONG JIANYONG1, HAO CHUAN-MING1, NOVAK JAN2, JULIAN BRUCE A.2, NOVAK LEA2 1Division of Nephrology, Huashan Hospital, Fudan University, Shanghai, China; 2University of Alabama at Birmingham, Birmingham, AL, USA Background: Manifestation of HSPN in Chinese adults is not very well known. We evaluated histopathological changes in renal biopsy specimens and assessed clinical data of 114 adult HSPN patients.

6A). However, the percentage of LMP7−/−-derived CD4+ T cells (3.89±0.21%) was clearly decreased in VV-WR-infected WT mice, compared with immunoproteasome expressing CD4+ T cells (7.62±0.4%), LMP2−/−

or MECL-1−/− CD4+ T cells (Supporting Information Fig. 6B). So far, we had mainly used Gefitinib supplier CD8+ T cells to study a requirement of immunoproteasomes during antiviral immune responses. To investigate other leukocyte populations, we investigated the development of adoptively transferred LMP7−/− CD4+ T cells (CD4+), B cells (CD19+), DC (CD11c+) and NK cells (NK1.1+) in naïve and LCMV-WE infected WT hosts compared with the corresponding endogenous cell types. Six days after transferring total splenocytes of LMP7−/− (CD45.2+) or C57BL/6 mice (CD45.2+), the numbers of donor-derived CD4+, CD8+, CD19+, CD11c+ and NK1.1+ cells in CD45.1 recipient mice were determined.

In the absence of LCMV infection, the numbers of cells lacking or expressing LMP7 were equal for all cell types analyzed (Fig. 3A). On the contrary, in LCMV-WE-infected host mice, the percentage of LMP7−/− cells was markedly reduced compared with C57BL/6 cells with CD4+, CD8+ and CD11c+ cells being hardly detectable (Fig. 3B). The loss of CD11c+ cells does most likely not represent a loss of DC but rather T cells which have been shown to upregulate CD11c expression during LCMV infection 17. Almost all remaining donor LMP7−/−-derived cells were B cells and also these were significantly reduced compared with WT SB203580 research buy Phosphatidylinositol diacylglycerol-lyase donor B cells. The almost complete loss of LMP7-deficient CD4+ and CD8+ T cells in the infected mice in face of a relative persistence of B cells argues by itself against an MHC class I-dependent rejection phenomenon being the cause of the loss of LMP7−/−

T cells because flow cytometric analysis of transferred B cells and CD8+ T cells showed a similar cell surface expression of H-2Kb and a slightly higher expression of H-2Db on B cells. To better document this finding, we simultaneously transferred sorted B220+ B cells and CD8+ T cells from CD45.2+ WT or LMP7−/− donor mice into CD45.1+ WT recipient mice and monitored the survival of B cells and T cells up to day 8 post-transfer. Although the LMP7−/−CD8+ T cells had almost completely disappeared by day 8, LMP7−/− B cells survived in the same mouse (Fig. 3C) which is inconsistent with a rejection based on different peptide/MHC I complexes displayed on the surface of LMP7−/− T cells. Instead, this finding points at a function of immunoproteasomes for the expansion and/or survival in the virus-infected host which is particularly crucial for T cells. As immunoproteasome-compromised T cells fail to expand in response to LCMV-WE infections, we crossed LMP7−/− and MECL-1−/− mice with P14 mice, which are TCRtg for the LCMV-WE MHC class I epitope GP33 (glycoprotein derived, aa 33–41). With these mice, we were able to track the in vivo expansion of virus-specific CD8+ T cells that lack LMP7 or MECL-1, respectively.