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Figure 3.

Blocking antigen access, but not Tfh cell help, prevents initiation of PC differentiation in the GC. (A) Schematic of antigen engagement and reception of cognate CD4 Tfh cell help by SW B cells in unmanipulated GCs (top), plus the mAb-based approaches used to specifically block Tfh cell help (bottom left) or block engagement of intact antigen by the SW BCR (bottom right). Both HyHEL10* and HyHEL9 bind to HEL, but only HyHEL10* blocks access to the antigen by the SW BCR. (B) Experimental design for mAb-blocking experiments. SW. B cells were permitted to form GCs in response to HEL-SRBCs and recipients and then given a single injection of mAbs 2 or 3 d before spleen harvest and analysis on day 9. (C and D) Impact of 3 d of mAb treatment on IgG1 PC-lineage cells in GCs derived from SW. donor B cells. Representative flow cytometry profiles are shown (C), as well as enumeration of early (-GFP IgG1) and late (-GFP IgG1) PC-lineage populations in individual recipients (D). (E and F) Impact of 2 d of HyHEL10* treatment analyzed as for C and D. Data from each mAb treatment are representative of two to four independent experiments of five mice per group. Flow cytometry plots are concatenated data from five recipient mice. P-values were calculated using an unpaired Student’s test. **, 0.001 ≤ P < 0.01.

Treatment with either HyHEL10* anti-CD4 or CD40L for 3 d led to similar (40–60%) reductions in the overall size of the GC response, with the DZ compartment most impacted in each case (Fig. S3 A). However, only antigen blockade with HyHEL10* abolished PC differentiation, removing both early ( Blimp1 -GFP lo IgG1 hi ) and late ( Blimp1 -GFP hi IgG1 lo ) PC-lineage cells ( Fig. 3, C and D ). The fact that both anti–Tfh cell treatments reduced GC size without preventing PC differentiation indicated that the abrogation of PC differentiation by HyHEL10* cannot be attributed to impaired antigen presentation and reduced Tfh cell help. Instead, HyHEL10* must act by preventing delivery of signals normally imparted to LZhi GC B cells directly upon antigen engagement. The specific impact of antigen blockade on the induction of PC differentiation was also evident after 2 d of HyHEL10* treatment (from days 7 to 9), which selectively depleted early PC-lineage cells ( Fig. 3, E and F ) including all LZ phenotype cells (not depicted). Analysis of the IgG1 (predominantly IgG2b + ) PC-lineage cells in the GC showed similar results (not depicted), indicating that the triggering of the PC differentiation pathway by antigen engagement is a mechanism that is likely to apply generally to IgG-switched GC responses.

Although the overall numbers of PC-lineage cells were largely unchanged by abrogation of Tfh cell help, anti-CD4 (but not anti-CD40L) treatment had a pronounced qualitative impact. Thus, in direct contrast to antigen blockade, Tfh cell depletion resulted in the relative enrichment of early PC-lineage cells ( Fig. 3, C and D ). Accordingly, anti-CD4 (but not anti-CD40L) treatment also increased the fraction of PC-lineage cells that exhibited an LZ phenotype ( Fig. 4, A and B ). To determine whether this shift in the phenotype of PC-lineage cells was associated with physical retention in the LZ, immunofluorescence histology was undertaken. PC-lineage cells were identifiable by their strong cytoplasmic expression of IgG1 and localized primarily within the DZ of control GCs before appearing to exit through the base of the DZ ( Plus Size Casual Hollow Out Flat Heel Slingback Sandals GOLD 2018 Cheap Online TWkW2hXPv
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; Meyer-Hermann et al., 2012 ), many PC-lineage cells (30–40% by our enumeration) are found in small clusters in or just outside the GC, suggesting that ongoing cell division may occur during early PC differentiation. LZ PC-lineage cells were in natural proximity to LZ-resident FDCs ( Fig. 4 C ), raising the possibility that they may access antigen via the immune complexes displayed on the FDC surface. Strikingly, LZ PC-lineage cells were greatly enriched in GCs from mice treated with anti-CD4 ( Fig. 4 C ). LZ retention of PC-lineage cells was not seen upon treatment with anti-CD40L (Fig. S3 C), confirming that Tfh cell–derived signals other than CD40L are required to drive the LZ egress and phenotypic maturation of LZhi GC B cells that have been previously licensed to initiate PC differentiation upon direct engagement of antigen.

Fig 4. EBOV GP activates the TLR4 pathway that leads to T cell death.

A, B. Western blot analysis of proteins involved in TLR4 signaling pathway TRAM1, p-TRAM1, MyD88, IRAK4, p-IRAK4, Pyk2, p-Pyk2, p38, p-p38 in SupT1 cells and monocytes (A) or THP-1, THP-1 MyD88-/- and SupT1 cells (B) following stimulations with LPS, poly I:C, HPIV3/EboGP, HPIV3/ΔF-HN/EboGP, HPIV3 or EBOV (A) or empty beads or EBOV GP beads (B) in the presence or absence of the TLR4 inhibitor CLI-095. C , D , E . Western blot analysis of p65 phosphorylation in SupT1 cells (C), monocytes (D) or THP-1, THP-1 MyD88-/- and SupT1 cells (E) following stimulation with CD3/CD28 beads, LPS (+, 100 ng/ml, ++; 500 ng/ml), VLP (+, 100 μl; ++, 250 μl) and HPIV3/ΔF-HN/EboGP (+, MOI 0.1 PFU/cell; ++, MOI 1 PFU/cell), empty beads or EBOV GP beads, as indicated, with or without CLI-095. Western blots in panels A-E are representative of two independent experiments. F . Percentages of dead (Live/Dead + ) cells, cell positive for caspase 8 and 9 and proliferated CD4 + T lymphocytes following a 4 day-long incubation with EBOV or LPS with or without CLI-095. Mean values ±SE based on triplicates from one of two independent experiments with P values * P <0.05, ** P <0.01, n.s., non-significant (Student T-test).

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We next examined activation of NFκB, a downstream transcription factor known to be activated following TLR4 stimulation [ 36 ], by stimulating SupT1 cells, monocytes, THP-1 and THP-1 MyD88 -/- cells with GP delivered by HPIV3/ΔF-HN/EboGP, virus-like particles (VLPs) or GP beads ( Fig 4C–4E ). Cell lysates were examined for the phosphorylation status of the p65 subunit of NFκB, which plays a major role in immune and inflammatory responses and whose phosphorylation is indicative of NFκB signaling activation [ 43 ]. Peak phospho-p65 (p-p65) was detected 2 h after stimulation ( S3B Fig ). Stimulation of SupT1 cells, monocytes, THP-1 and THP-1 MyD88 -/- cells with EBOV, HPIV3/ΔF-HN/EboGP, VLPs, GP beads or LPS, as well as control CD3/CD28 activation beads, resulted in a marked increase in phosphorylated p65 ( Fig 4C–4E ). Treatment of cells with CLI-095 for 1 h prior to stimulation reduced the levels of phosphorylated p65 associated with EBOV, HPIV3/ΔF-HN/EboGP, VLPs, GP beads or LPS stimulation. As expected, CLI-095 did not affect phosphorylation of p65 in control CD3/CD28 activated cells ( Big Discount Cheap Price Zanellato stud detail tote bag Free Shipping Pay With Paypal Eastbay For Sale Outlet Store Locations Free Shipping Wiki AtE0zCqO
). These findings further confirm that EBOV stimulation results in TLR4-mediated signal transduction in primary CD4 + T cells by both MyD88-dependent and MyD88-independent signaling pathways.

The Wounded Walking Ones

When a baby starts walking , game on. Just because a child can walk doesn't mean he's good at it. But your little walker won't be the only one in the house who's feeling a little worse for wear from practicing walking. You'll need a chiropractor and a deep tissue massage from the hours spent hunched over trying to keep that little guy from walking into yet another inanimate object.

Terror-Alert Twos

Forget the terrible twos ! Two-year-olds are little terrors who thrive on destroying things, touching things, eating things, peeing on things, climbing on things and jumping off of anything possible. Once you have a 2-year-old in the house, your home's terror threat level is permanently on high. Anything can happen with a terror-alert two in the house. Well, anything but quiet, that is.

Threenagers

Three-year-old toddlers are like little teenagers minus the braces. They have all the attitude and eye-rolling of a teen, minus the moments when teens remember to be nice. The most loving thing your threenager will say to you is, "Mommy, you have a big tummy." This will be much better than her usual comment to you: "You're the worst Mommy ever!"

The 'Eff You' Fours

Four-year-olds see the world through 'eff you!'-colored lenses and they're always looking for ways to tell you that you're wrong, don't know what you're talking about, or how they'd be better off living in another family. This is particularly deceptive because 4-year-olds are absolutely adorable … when they're sleeping. Don't be fooled, 4-year-olds are brutal. Trust me, I have one.

The Fantastic Fives

On your child's 5th birthday, you'll hear harps play and everything will seem to move in slow motion. That's because, for the first time in your parenting career, your child is a real human being. Gone are the diapers, cribs and car seats that are more difficult to unlock than Fort Knox. In their place will be a small, but lovely human being who doesn't pee his pants or throw a temper tantrum every time he enters the local Target. Enjoy age five while it lasts. Six is coming.

The Sassy Sixes

Six-year-olds are the Google of young children, or so they think. That's because 6-year-olds are experts in everything. And just when you got used to your fantastic 5-year-old, she had a birthday and opened up a gift box filled with sass. So get ready for car rides filled with, "Mommy, you're wrong!" and "Mommy, you don't know anything about fairies." One thing you will know a lot about is sass, because your 6-year-old will deliver it each and every day.

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The 7 Going on 30s

Three words: Drop-off playdate! Boo-ya! You made it. Your 7-year-old will have sleepovers and drop-off birthday parties, and will start to see his friends as his primary entertainment rather than you. This will seem amazingly liberating for the first 27 seconds until you realize your 7-year-old is growing up. And in a few short years—just 23 to be exact—he'll be 30. That'll feel like it's almost tomorrow. Suddenly, those days of having a "Is This All You Do?" 3-month-old will seem like paradise.

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