Our results demonstrate that while UVL and LVL asymptomatic Tx patients exhibit NK-cell phenotype and function comparable to HC, patients with PTLD display critical changes in NK-cell phenotype paralleled by impaired function and accumulation of unusual NK-cell subsets. In addition, NK cells from asymptomatic HVL patients who are at higher risk

of EBV complications, demonstrated similar phenotypic trends as PTLD patients in addition to a selective decrease in cytotoxicity. NK-cell subset characterization was performed on peripheral blood CD3−CD19− cells, out of the lymphocyte gate, as shown in Fig. 1A. NK cells were defined based on CD56 and CD16 expression, and four subsets were further identified as follows: CD56brightCD16±, CD56dimCD16+, CD56dimCD16− and CD56−CD16+ populations (Fig. 1A). While the overall frequencies RAD001 datasheet (%) of all NK cells were not different among groups (data not shown), the analysis of NK-cell subsets revealed that pediatric thoracic Tx patients (including patients with PTLD) displayed significantly lower levels of the CD56dimCD16+ NK subset (mean±SD: UVL: 52±20%; Carfilzomib concentration LVL: 55±14%;

HVL: 55±15%; PTLD: 34±26%), a subset previously described to be the most abundant NK-cell subset in peripheral blood of HC (77±4%) (Fig. 1B). In addition, asymptomatic pediatric thoracic Tx patients displayed a trend of higher percentages of circulating CD56brightCD16± NK cells (UVL: 25±20%; LVL: 22±13%) as compared with HC (6±3%) (Fig. 1C). Conversely, PTLD patients displayed increase in peripheral blood CD56dimCD16− subset (PTLD:

43±7% versus HC: 10±6%) and CD56−CD16+ NK subset (PTLD: 19±20%; HC: 7±2%) (Fig. 1D and E). We next investigated the levels of triggering receptor expression on NK cells. Previous reports have documented that the activating receptors are expressed at highest levels on CD56brightCD16± and CD56dimCD16+ NK subsets in healthy subjects 8. Our results show significant down-modulation of NKp46 expression on total NK cells from PTLD patients (mean±SD=42±23%) Protein tyrosine phosphatase as compared with those from asymptomatic pediatric Tx patients (UVL: 70±24%; LVL: 84±13%) or HC (85±5%) (Fig. 2A). Similar decrease in NKp46 expression was detected on all four NK-cell subsets, including the CD56brightCD16± and CD56dimCD16+ (Fig. 2B and C). Similar to NKp46, the NKG2D expression was also significantly decreased on all NK cells from PTLD patients (4±4%) as compared with NK cells from asymptomatic Tx patients (LVL: 21±12%) or HC (22±5%) (Fig. 2D). Similar findings were also observed on CD56bright CD16± and CD56dimCD16+ NK-cell subsets (Fig. 2E and F) as well as on the unusual CD56dimCD16− and CD56−CD16+ subsets (data not shown).

Selective T-cell depletion in CD70-Tg peripheral lymph nodes is also not caused by IFN-γ 30. Higher apoptosis percentages and LBH589 order up-regulated CD95 expression in CD70-Tg

NK cells indicate that the observed NK cell depletion is at least partly due to apoptotic events and that these are mediated via CD95. However, we performed an in vivo CD95 ligand blocking experiment in CD70-Tg mice and we did not observe rescue of NK cells. Therefore, we have no evidence that the increased expression of CD95 on NK cells from CD70-Tg mice leads to their death. Taken together, our results show that although CD27 cross-linking initially induces activation of lymphocytes, continuous stimulation results in severe homeostatic changes of the lymphocyte population, Nutlin3a including activation-induced cell death of

NK cells. Residual NK cells in CD70-Tg mice exhibited decreased, but not absent expression of CD11b and CD43. This demonstrates that continuous CD27 triggering does not induce a total blockade of NK cell differentiation. In addition, it has been evidenced that under some circumstances CD11blowCD43− NK cells express Ly49 receptors and are lytic, which demonstrates their acquired effector functions 37. This stresses that the CD11blowCD43− phenotype already might be an important checkpoint in the functional differentiation of NK cells. This hypothesis is supported by the findings that only CD11blowCD43− NK cells are present in mice deficient for several different transcription factors, such as GATA-3, IRF2 or T-bet, and in mice bearing constitutively active NFκB. In all these models, the CD11blowCD43− NK cells exhibit normal cytotoxic capacities 38–41. In our study, we found that splenic NK cells from CD70-Tg mice, whether stimulated through the IL-12/IL-18 receptor or through the NK1.1 receptor, produced less IFN-γ compared with WT NK cells, whereas in liver no differences were demonstrated. Regarding cytotoxicity, both liver and splenic NK cells from CD70-Tg mice showed increased activity. Hence, we evidenced opposite effects of CD70 triggering on the major NK cell effector functions. Different outcomes of those NK cell effector functions

upon the same triggering have been described before, for example in the GATA-3 deficient mouse model 38. On the other hand, as several hours are required for ifn-γ HAS1 transcription and translation, the NK cells were incubated for 6 h in the IFN-γ assay, during which the higher apoptosis in the mNK cell population of CD70-Tg mice can have an important impact. Conversely, the outcome of the cytotoxicity assay is probably less influenced by the increased apoptosis of the CD70-Tg NK cell effector population as the trigger to induce NK cell-mediated cytotoxicity of YAC-1 targets requires only 20 min 42. Since YAC-1 lysis is known to be NKG2D-mediated 33, NK cell expression of this receptor was measured in CD70-Tg mice. As expected, enhanced NKG2D expression confirmed the observed up-regulated cytotoxicity in CD70-Tg NK cells.

Flow cytometry.  Neutrophil cell surface adhesion molecule expression was determined by flow cytometry. Isolated neutrophils (10 × 106/ml) were incubated in RPMI with anti-CD11b-AlexaFluor488 and anti-CD62L-PE or anti-CD11a-PE, for 30 min, 4 °C, protected from light. Subsequently, cells were washed with PBS and fixed with 1% paraformaldehyde until analysis. Cells were analysed at 488 nm on a FACScalibur (BD Biosciences, Heidelberg, Germany) and CellQuest Software was used for acquisition. Data were expressed as mean fluorescence intensities (MFI) and % of positive cells (% gated) compared to a negative isotype control. Real-time PCR.  Extraction of mRNA

selleck compound and synthesis of cDNA: For extraction of neutrophil RNA, neutrophils (5 × 106 cells minimum) were pelleted at 4800 g for 20 min and RNA extracted using TRIzol, according to the manufacturer’s instructions (Invitrogen Corp., Carlsbad, CA, USA).

Complementary DNA (cDNA) was synthesized and verified as previously described [19]. Amplification and quantification of gene expression: Synthetic oligonucleotide primers were designed to amplify cDNA for conserved regions of the CD62L, alpha subunit of CD11a and alpha subunit of CD11b (PrimerExpress™; Applied Biosystems, Foster City, CA, USA). For primer PLX4032 nmr sequences, see Table 1. Primers were synthesized by Invitrogen (São Paulo, Brazil) and ACTB and GAPDH were used as control genes. All samples were assayed in a 12 μl volume containing 5 ng cDNA, 6 μl SYBR Green Master Mix PCR (Applied Biosystems) and adhesion molecule gene primers as well as GAPDH and ACTB primers in 96-well reaction plate (StepOne Plus – Applied Biosystems). To confirm accuracy and reproducibility of real-time PCR, the intra-assay precision was calculated according Amobarbital to the equation: E(−1/slope) [20]. The dissociation protocol was performed at the end of each run to check for non-specific amplification. Two replicas were run on the plate for each sample. Results were expressed as the arbitrary units (A.U.) of gene expression when compared with the

control genes. Measurement of serum sL-selectin, IL-8 and ENA-78.  Peripheral blood was collected in glass tubes without anti-coagulant and serum separated by centrifugation and stored frozen (−80 °C) until ELISA. Serum sL-selectin, ENA-78 and IL-8 were determined by high sensitivity ELISA (R&D Systems, Minneapolis, MN, USA and BD Biosciences, San Jose, CA, USA, respectively), according to the manufacturers’ instructions. Statistical analysis.  All data are expressed as means ± SEM. Differences between groups were evaluated by ANOVA followed by Bonferroni’s test or by the Kruskal–Wallis test followed by Dunns test, as appropriate, unless otherwise specified. A P-value of ≤0.05 was considered statistically significant.

Wet tail-blood films of the infected mice were examined microscopically at 2-day intervals to estimate the parasitaemia (15). When the parasitaemia reached between 107 and 108 trypanosomes/mL, tail-blood was collected and diluted with Phosphate buffer Saline Glucose (PSG) to achieve a concentration of 105 parasites in a total

volume of 0·2 mL. This volume was injected AZD1208 in vitro I.P. in six OF1 mice for each strain. A group of six mice, injected I.P. with 0·2 mL of PSG, was used as control. For each strain, the prepatent period (number of days between the inoculation and the first appearance of parasites in the blood) and the survival time were recorded up to 60 days post-infection. Mortality in infected and control mice was recorded daily. An animal was considered parasitologically

negative when no trypanosomes were detected in at least 50 microscopic fields. Animal ethics approval for the experimental infections was obtained from the Ethics Commission of the Institute of Tropical Medicine, Antwerp, Belgium (Refs DG001-PD- M-TTT and DG008-PD-M-TTT). The median mice survival time of the infected mice was estimated in parametric survival models using a log-normal HM781-36B purchase hazard distribution in Stata 10. The strains for which none of the infected mice died during an observation period >60 days were discarded from the analysis. In a first model, the strains were used as discrete explanatory variables. In a second model, transmission cycle type (domestic or sylvatic) was used as explanatory variable. Data clustering in relation to the different isolates was taken into account using the frailty option (shared for strains). Strains were subsequently allocated to three virulence classes according to their estimated median survival time (<10 days, 10–50 days and >50 days). Strains for which none of the infected mice died during an observation period of more than 60 days were allocated

to the last class. An ordered Loperamide multinomial regression was applied on the data using the cycle type as explanatory variable. The virulence of a total of 62 T. congolense strains was tested and compared. Median survival time of infected mice differed substantially between strains with mice infected with the most virulent strains having a median survival time of <5 days and mice infected with the least virulent strains surviving for more than 50 days. An overview of the median survival time (95% C.I.) of mice infected with 60 of the 62 strains (survival time could not be calculated for two strains because survival was more than 60 days) is presented in Figure 1. Based on the distinction made by Masumu et al. (9), strains were grouped into a high virulence (median survival time <10 days), a medium virulence (median survival time between 10 and 50 days) and a low virulence (median survival time between >50 days) category.

Briefly, 2 × 106 target 721.221 cells were labelled with 5 μl of the DiO Vybrant cell-labelling solution (Molecular Probes, Carlsbad, CA) in 2 ml PBS for 15 min at room temperature. Target cells were washed twice and plated in R-10 at a final concentration of 25 000 cells per well in U-bottom 96-well plates. Effector cells (either cytokine-treated selleck compound PBMCs or sorted CD8α+ and CD8α− NK

cells) were added at the indicated E : T ratios to a final volume of 200 μl. Plates were incubated at 37° for 4 h. After incubation, cells were labelled with 0·2 μl per well of the far red Live/Dead fixable dead cell stain kit (Invitrogen). Plates were washed twice with PBS and finally fixed in 200 μl of a 2% PBS-paraformaldehyde solution. Labelled cells were stored at −4° until acquisition on a FACSCalibur (BD Biosciences). At least 5000 target cells (FL1-DiO+ events) were acquired. Specific target cell killing was measured by incorporation of the far red LIVE/DEAD

amine dye (FL4) in the DiO+ population. Target cells alone CHIR-99021 in vivo were used as controls to correct for background levels of cell killing. CD4+ T lymphocytes, to be used as target cells, were purified from naive macaque PBMCs using a non-human primate CD4+ T-cell isolation kit (Miltenyi Biotec), labelled with DiO (as described for the 721.221 killing assay), and then coated with 15 μg SIV251 gp120 (ABL) at room temperature for 45 min in RPMI-1640. CD4+ target cells were then washed twice and plated in R-10 at a final concentration

of 10 000 cells per well in U-bottom 96-well plates containing serial dilutions of macaque sera (known to mediate ADCC activity) and incubated for 15 min at room temperature to allow antibody–antigen interaction. Effector cells (autologous PBMCs or sorted CD8α+ and CD8α− NK cells) were added at a 25 : 1 (PBMCs) or 12 : 1 (sorted cells) E : T ratios to a final volume of 200 μl. Plates were centrifuged for 3 min at 400 g to promote cell-to-cell IMP dehydrogenase interactions and then incubated at 37° for 4 hr. After incubation, cells were labelled and analysed as indicated for the 721.221 killing assay. SIV251 gp120-coated target cells alone, ADCC-negative pre-immunization sera from the same macaques, and a no-serum target plus effector cell mixture were used as negative controls. To calculate results, non-specific killing (from target cells alone and from a no-serum target plus effectors mixture) was subtracted from all wells and an ADCC cut-off value was calculated as the mean of values from all dilutions of negative pre-immune sera plus three standard deviations. The ADCC killing was considered positive when killing percentages were higher than the cut-off value. To assess phenotypic stability of macaque NK cell subsets, PBMCs or sorted CD8α+ and CD8α− NK cells were left untreated or were stimulated with IL-2 (400 ng/ml), IL-15 (150 ng/ml), or a combination of both for different time periods.

[19] By 1998, immunoglobulin and TCR genes were fully identified and sequenced. There are seven major loci, which undergo somatic learn more recombination in developing B and T cells during the formation of antigen receptors. These are immunoglobulin heavy chain (IgH), light chain κ (IgK) and light chain λ (IgL) in B cells and TCR-α (TCRA), TCR-β (TCRB), TCR-γ (TCRG) and TCR-δ (TCRD) in T cells. Each of

these is further divided into subexons, which undergo the recombination (Fig. 1). A fairly conserved DNA sequence known as recombination signal sequence (RSS) resides adjacent to each subexon and consists of a palindromic heptamer (CACAGTG) and an A/T-rich nonamer (ACAAAAACC)[14, 22-24] (Fig. 2a,b). The first three nucleotides of the heptamer

are crucial for the recombination activity.[25, 26] Though the nonamer binding domain of RAG1 is well characterized, the region of the RAG complex that recognizes the heptamer is yet to be deciphered.[27, 28] The heptamer and nonamer are separated by a spacer DNA sequence of either 12 bp (12RSS) or 23 bp (23RSS) (Fig. 2a). Although the length of the spacer is conserved, its sequence is not of much importance.[12, 24] Generally, a 12RSS recombines only with a 23RSS and vice versa, a restriction termed as the ‘12/23 RO4929097 nmr rule’ (Fig. 2b), which prevents non-productive rearrangements. The coupled cleavage of a 12RSS and 23RSS requires Mg2+, whereas

Mn2+ supports RAG-mediated nicking of a single RSS.[29] Recently, the ‘beyond 12/23’ rule has been proposed to explain the exclusion of direct TCRBV to TCRBJ joining in the TCR-β region, in spite of the incidence of appropriately oriented pairs of 12RSS and 23RSS.[30] The exclusion was enforced during the DNA cleavage step of the V(D)J recombination and was attributed to several factors, like relatively slow nicking of the TCRB substrates and poor synapsis of the TCRBV and the TCRBJ.[31] Extrachromosomal V(D)J recombination assays could recapitulate the ‘beyond 12/23 rule’ in the TCRBV, ZD1839 price implying that it is solely the RAG proteins and RSSs, which play a role in establishing this restriction.[32] In contrast, with respect to TCRDV locus, the involvement of other factors was also suggested.[33] RAG1 and RAG2 initiate recombination by introducing a single-strand nick in DNA precisely at the border between the heptamer of RSS and the coding segment.[34] The 3′-OH group of the nick at the coding end then becomes covalently linked to the opposing phosphodiester bond of antiparallel strand by a transesterification reaction resulting in hairpin structure at the coding end and blunt signal end.[35] The signal ends remain associated with RAG proteins resulting in a transitory structure referred to as a ‘post-cleavage complex’.

However, a role of p53 in regulation of T-cell responses or apoptosis has been poorly Ponatinib defined. TCR-mediated signaling in the absence of CD28 costimulation induces both apoptosis and proliferation of naïve T cells from WT mice. In this report we show that, in response to TCR stimulation, T cells from naïve p53-deficient mice exhibited higher proliferation and

drastically reduced apoptosis than WT T cells. CD28 costimulation enhanced the proliferation of TCR-stimulated WT and p53−/− T cells, suggesting that p53 uncouples CD28-mediated antiapoptotic and proliferative signals. To evaluate the physiological significance of these findings, we transplanted OVA expressing-EG.7 tumor cells into WT and p53−/− mice. Unlike WT mice, p53−/− mice exhibited a robust tumor-resistant phenotype and developed cytotoxic T-cell responses against OVA. Collectively, these data support the hypothesis that p53 is an essential factor in negative regulation of T-cell responses and have implication for immunomodulation during treatment of cancers and other inflammatory conditions. Transformation related protein 53 (Trp53 or p53) is a member of the p53 transcription factor family that regulates LDE225 ic50 DNA repair,

genomic integrity, DNA replication, cell proliferation and apoptosis 1–3. It contains an N-terminal transactivation domain, a C-terminal tetramerization domain and a central DNA binding domain. Under normal conditions p53 is expressed at low levels in a variety of cell types. Exposure of cells to ionizing radiation, DNA damage, or certain cellular or physiological stresses leads Exoribonuclease to stabilization and activation of p53 and its pathway 2. Once activated, p53 binds to target

DNA and initiates transcription of target genes that directly or indirectly inhibit the cell cycle or induce cell death 4, 5. Lack of p53 expression or function is related to development of a vast variety of tumor types and a role for p53 in apoptosis of cells has been the subject of numerous studies for many years. Traditionally, increased expression p53 has been reported in conditions that favor tumoroigenesis, e.g. ionizing radiations. However, p53 expression is also upregulated during inflammation and infections. Synovia from rheumatoid arthritis patients exhibit dominant negative mutations of p53 and expression of p53 is also upregulated in the joints of these patients 6. This increased level of p53 in arthritic synovium joints can be seen in the early stages of disease development 7. Further, lymphocytes from rheumatoid arthritis patients express lower levels of p53 mRNA and protein, and have an impaired ability to induce p53 expression after exposure to gamma radiation, which correlated with increased survival of CD4+ and CD8+ T cells after exposure to gamma radiation 8.

mansoni actin 1.1 gene (23) was constructed and transfected into schistosomes

by electroporation of larval stages together with mRNAs encoding the piggyBac transposase. The activity of piggyBac was determined by plasmid excision assays, and the recovery of excised plasmids from tissues of transformed schistosomules in these assays indicated that piggyBac was ACP-196 active in the worm. Southern blot hybridization analysis of genomic DNAs from populations of schistosomules transformed with donor constructs plus helper transposase mRNA detected numerous variable length luciferase-positive signals. These findings further indicated piggyBac transposon insertions into the schistosome chromosomes. piggyBac integration sites were detected by a PCR technique. Numerous piggyBac integrations were detected and, after cloning, the fragments sequenced

ranged in size from approximately 1·5 to 4 kb. Sequence analysis indicated that integration of piggyBac took place at numerous loci in the schistosome genome at target TTAA sites. The discovery of sequence-specific Rapamycin purchase gene silencing in response to double-stranded RNAs (dsRNA) has had an enormous impact on molecular biology by uncovering an unsuspected layer of gene regulation. The process, also known as RNA interference (RNAi) or RNA silencing, involves complementary pairing of dsRNAs with their homologous messenger RNA targets, thereby preventing their expression, and leading ultimately to their degradation, or interfering with protein translation (33). Since its discovery, RNAi technology has been used widely as a reverse genetics tool in C. elegans, Drosophila and many other organisms, including zebrafish, plants, human, mouse and mammalian cell culture. The ability to inhibit gene activity on a post-transcriptional

level allows generation CHIR-99021 mw of loss-of-function mutants to study gene function, or identification and validation of novel therapeutic targets [reviewed in ref. (34)]. In C. elegans, silencing was found to have high potency and specificity, and was activated throughout the treated animal (35,36), even in cells that did not encounter the double-stranded RNA. It has now been revealed that a complex protein machinery is involved in the transport of the silencing signal. This raises the possibility that animals can communicate gene-specific silencing information between cells (37). In schistosomes, the presence of transcripts encoding dicer and RISC-associated proteins (piwi/argonaute orthologs) was relatively recently described (6,38,39). SmDicer was later shown to contain all domains that are characteristic of metazoan dicers including an amino terminal helicase domain, DUF283, a PAZ domain, two RNAse III domains and an RNA binding domain. An examination of the available S.

In contrast, IL-17A deficiency had a profound effect on the development of severe disease as determined in prospective survival experiments, whereas the lack of IFN-γ signaling did not significantly influence the course of DCM development (Fig. 5B). To assess to which extent the concerted action of IL-17A

and IFN-γ impinges on the development of myocarditis, IFNGR-KO mice were treated every other day between weeks 4 and 8 with a neutralizing learn more anti-IL-17A antibody. The effect of this treatment was a further drastic reduction of severe myocarditis in IFNGR-KO mice, that is, none of the antibody-treated mice developed a severity grade higher than 2 (Fig. 5A). Furthermore, TCR-M mice were crossed onto the IL-6-deficient background to assess the contribution of a pro-inflammatory cytokine check details in the transition from myocarditis to DCM. Here, the effect of the cytokine deficiency was important both for myocarditis and DCM, most likely because of the strong attenuation of the initial cardiac inflammation

(Fig. 5A and B). Assessment of cytokine production by heart-infiltrating CD4+ T cells following peptide restimulation (Fig. 5D) confirmed that IFN-γ was the major effector cytokine of the pathogenic CD4+ T cells in TCR-M mice lacking IL-6, IL-17A, or the IFNGR. Taken together, these data indicate that IFN-γ functions mainly as an effector molecule in the initiation of myocarditis, whereas IL-17A is critical for the progression toward the more severe disease. Collectively, our results clearly demonstrate a cooperative role of IFN-γ and IL-17 in the transition from myocarditis to DCM. In this study, we analyzed the pathogenic mechanisms of spontaneous autoimmune myocarditis and the progression to DCM in a novel TCR transgenic model. We found that

lack of expression of cardiac myosin alpha in the thymus prevented negative selection of high-avidity mhyca614–629-specific CD4+ Th and PLEKHB2 resulted in the egress of TCR transgenic cells to secondary lymphoid organs. Activation of mhyca614–629-specific TCR-M cells occurred within the heart-draining lymph node and was followed by accumulation of pathogenic Th cells in the heart muscle leading to progressive heart inflammation. The activity of the self-reactive Th cells was highest between weeks 4 and 8, whereas the progression to lethal DCM occurred in the age of 8 to 12 weeks. The finding that 40% of the TCR transgenic mice did not progress to DCM suggests that either a particular threshold of T-cell activation has to be reached or that negative regulatory circuits such as peripheral co-inhibitory molecules [29, 30] or regulatory T cells [31] had been activated.

Furthermore, Foxo1f/fCd19Cre mice had markedly fewer LN B cells and an increase in peripheral blood B cells (Supporting Information Fig. 1D). The paucity of LN B cells correlated with reduced surface expression of CD62L (L-selectin), the LN homing receptor (Supporting Information Fig. 1E). The mice also had a reduced percentage of CD5+ B cells in the peritoneal cavity (Supporting Information Fig. 1F). The report from Dengler et al. did not examine the developmental status or function of peripheral B220+IgM+ cells in Foxo1f/fCd19Cre mice 10. We stained splenocytes from our Foxo1f/fCd19Cre mice and

controls with antibody combinations that distinguish two mature subsets (FO, MZ) and four transitional Compound Library cell line B-cell subsets (T1, T2, T3 and MZ precursor (MZP)) 13. When compared with control Foxo1f/+Cd19Cre mice, Foxo1f/fCd19Cre mice displayed a consistent and statistically significant increase in the percentage of MZ cells, defined as B220+AA4.1−IgMhiCD21hiCD23lo (Fig. 1A). In contrast, the percentage of FO cells (B220+AA4.1−IgMloCD21intCD23hi) was reduced (Fig. 1A). A normal percentage of MZP cells was present in Foxo1f/fCd19Cre mice, despite reduced percentages of T1 and T2 cells; this suggests that immature transitional cells might commit preferentially to the MZP stage. The absolute numbers of splenocytes were equivalent between Foxo1f/fCd19Cre mice and control mice (data not shown). Increased abundance of B220+ cells

in the splenic MZ and other extrafollicular regions selleck screening library was also apparent by immunofluorescent staining of spleen sections (Fig. 1B). The

percentages of mature FO and MZ cells were comparable in the two control groups (Foxo1f/+Cd19Cre and Foxo1f/f) (Fig. 1A), and other experiments showed a consistently greater population of MZ cells (B220+CD21hiCD23lo) in Foxo1f/fCd19Cre compared with Foxo1f/f mice (data not shown). Therefore, we used Foxo1f/f mice as controls in Fig. 1B and in other experiments to simplify breeding schemes. The altered balance of FO and MZ cells in Foxo1f/fCd19Cre mice Cepharanthine was not observed in analyses of mice with Foxo1-deficient B cells generated using Cd21Cre10. A likely explanation is that Cd21Cre drives deletion of Foxo1 at a time point after transitional B cells commit to either the FO or the MZ lineage, whereas Cd19Cre deletion is complete by this stage. Interestingly, Foxo1f/fCd21Cre mice 10 shared the reduced LN B-cell population and CD62L expression observed here in Foxo1f/fCd19Cre mice. This could be explained by a requirement for Foxo1 in CD62L gene expression in mature B cells, after Cd21Cre-mediated deletion is completed. We purified splenic B cells and activated them in vitro with titrated doses of either a BCR stimulus (anti-IgM) or a TLR stimulus (LPS). We measured cell proliferation and survival by cell division tracking using CFSE. B cells from Foxo1f/fCd19Cre proliferated more weakly to anti-IgM, compared with B cells from Foxo1f/f mice (Fig. 2A).