mir-181A/B-1 controls thymic selection of treg cells and tunes their suppressive capacity

The interdependence of selective cues during development of regulatory T cells (Treg cells) in the thymus and their suppressive function remains incompletely understood. Here, we analyzed this interdependence by taking advantage of highly dynamic changes in expression of microRNA 181 family members miR-181a-1 and miR-181b-1 (miR-181a/b-1) during late T-cell development with very high levels of expression during thymocyte selection, followed by massive down-regulation in the periphery. Loss of miR-181a/b-1 resulted in inefficient de novo generation of Treg cells in the thymus but simultaneously permitted homeostatic expansion in the periphery in the absence of competition. Modulation of T-cell receptor (TCR) signal strength in vivo indicated that miR-181a/b-1 controlled Treg-cell formation via establishing adequate signaling thresholds. Unexpectedly, miR-181a/b-1–deficient Treg cells displayed elevated suppressive capacity in vivo, in line with elevated levels of cytotoxic T-lymphocyte–associated 4 (CTLA-4) protein, but not mRNA, in thymic and peripheral Treg cells. Therefore, we propose that intrathymic miR-181a/b-1 controls development of Treg cells and imposes a developmental legacy on their peripheral function

9 to 5 feces on fart and crass : subverting society's segregative structures

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] In resistance to oligarchical structures, which deny access and influence nearly every aspect of our society, I've created an inventory of work, which utilizes and embraces archetypes and pop culture motifs as a means to critique authoritative figures and institutions, and examine marginalizing structures. The works exist on paper, in zines, on posters, buttons, pins and other ephemera as a way of making the images affordable (and available) to the working and middle class. While these works are meaningful on their own, they have become secondary to the method in which they are displayed and sold. These images -- ranging from satirical repudiations of political and celebrity icons, to ambivalent reactions fueled by sports-based fandom -- reflect a desire for connectedness and well-being, concepts which are all summarized in the form of a transient, white cube.Includes bibliographical references (pages 124-128)

Decreased production of class-switched antibodies in neonatal B cells is associated with increased expression of miR-181b

The increased susceptibility to infections of neonates is caused by an immaturity of the immune system as a result of both qualitative and quantitative differences between neonatal and adult immune cells. With respect to B cells, neonatal antibody responses are known to be decreased. Accountable for this is an altered composition of the neonatal B cell compartment towards more immature B cells. However, it remains unclear whether the functionality of individual neonatal B cell subsets is altered as well. In the current study we therefore compared phenotypical and functional characteristics of corresponding neonatal and adult B cell subpopulations. No phenotypic differences could be identified with the exception of higher IgM expression in neonatal B cells. Functional analysis revealed differences in proliferation, survival, and B cell receptor signaling. Most importantly, neonatal B cells showed severely impaired class-switch recombination (CSR) to IgG and IgA. This was associated with increased expression of miR-181b in neonatal B cells. Deficiency of miR-181b resulted in increased CSR. With this, our results highlight intrinsic differences that contribute to weaker B cell antibody responses in newborns

Impaired class-switching in neonatal B cells.

<p>Sort-purified adult and neonatal B cell subsets (transitional 1 & 2 B cells: T1 & T2; naïve mature B cells: N) were stimulated with CpG alone or in combination with IL-4, IL-21, anti-CD40, and BAFF (SC) for 8 days. After this time, cells were harvested and immunoglobulin levels for <b>(A)</b> IgM, <b>(B)</b> IgG, and <b>(C)</b> IgA, were determined in the supernatant by ELISA; (n = 5). <b>(D)</b> Surface IgG expression in sorted and stimulated (5 d; ctrl = not stimulated, with CpG or SC alone; AB: n≥3, CB: n = 5) B cells were determined by flow cytometry. Students <i>t</i>-test; * P<0.05, ** P<0.01, *** P<0.0001.</p

Significant differences in miRNA profiles of adult and neonatal B cells.

<p><b>(A)</b> Analysis of miRNA expression of adult blood (AB) and neonatal cord blood (CB) (each n = 7; pooled) performed with QLUCORE analysis software and Multi Group Comparison of 1,105 human mature miRNAs; shown are differentially expressed miRNAs with a 3-fold difference between equivalent subsets; P<0.05. <b>(B)</b> QPCR analysis of miR-181b in B cell subpopulations (transitional 1 & 2 B cells: T1 & T2; naïve mature B cells: N) of neonatal and adult healthy donors; (n = 7); Students <i>t</i>-test; * P<0.05.</p

Neonatal B cell subpopulations show different responses in survival and proliferation upon stimulation.

<p>Analysis of <b>(A)</b> survival (live gate in FSC/SSC dot blot; %) and <b>(B)</b> proliferation of CFSE-labeled transitional 1 & 2 (T1 & T2) and naive mature (N) B cell subpopulations of adult blood (AB) and neonatal cord blood (CB) in response to stimulation with either CpG (n≥5), CpG+anti-Ig (n≥3) or to a stimulation cocktail (SC; IL-4, IL-21, anti-CD40, CpG and BAFF; n≥3) for 5 days. As control the cells were cultured with medium alone. <b>(C)</b> Distribution of cell cycle phases was analyzed by flow cytometry in adult and neonatal T1, T2 and mature naive B cells by using DAPI and PyroninY (n = 8). <b>(D)</b> The replication history of T1, T2, mature naive and memory B cells was determined by using the KREC assay and analyzing the ΔC_T of the coding joint and the signal joint PCR from three sorted donor samples. <b>(A-D)</b> Students <i>t</i>-test; * P<0.05, ** P<0.01.</p

High expression of <i>Aicda</i> mRNA and AID protein in stimulated neonatal B cells.

<p><b>(A)</b> QPCR analysis for <i>Aicda</i> transcripts in human B cells cultured for 12, 24, and 48h with IL4+ IL-21+antiCD40+CpG+BAFF. Expression is normalized to GAPDH and is calculated relative to expression in not stimulated control B cells at each time point. Mean and SEM of n ≥ 3 samples are shown. <b>(B)</b> AID protein expression after 48h and 72h of stimulation in sorted adult blood (AB) and neonatal cord blood (CB) B cell subpopulations analyzed by Western blot; β-Actin was used as housekeeping protein; 4 μg protein/sample was applied. One representative run is shown. <b>(C)</b> Normalized results of n ≥ 4 independent experiments. <b>(D)</b> Quantitative RT-PCR analysis for <i>Aicda</i> transcripts in adult murine spleen cells cultured and stimulated for 12, 24, and 48h with LPS+CpG+anti-CD40+IL-4. Expression is normalized to GAPDH and calculated relative to expression in not stimulated spleen cells. Mean and SEM of n ≥ 3 samples are shown. Statistical analysis was performed using for <b>(A)</b> Student´s <i>t</i> test and for <b>(C)</b> 1way ANOVA. * P < 0.05, ** P < 0.01. *** P < 0.001.</p

Relative distribution of B cell subpopulations (n = 8).

<p>Relative distribution of B cell subpopulations (n = 8).</p

MiR-181b plays a crucial role in the regulation of class-switching to IgG.

<p><b>(A, B)</b> QPCR analysis for mature miR-181b expression in <b>(A)</b> total splenocytes of adult and neonatal miR-181a/b^+/- het mice (adult: n = 11, neo: n = 4 (pooled from 18 mice)) and <b>(B)</b> sorted B cell subpopulations (transitional 1 &2 (T1 & T2), marginal zone (MZ), and follicular mature (FM); n = 5) of adult C57BL/6J mice. Relative expression was calculated by using the standard curve method. <b>(C)</b> IgM and IgG levels in supernatants from cultured and stimulated (5 d; ctrl = not stimulated, with LPS or CpG alone; adult n = 6, neonatal: n≥6) adult and neonatal splenic cells isolated from miR-181a/b^+/- and miR-181a/b^-/- mice were determined by ELISA. <b>(D)</b> Surface IgG expression in stimulated (5 d; ctrl = not stimulated, with LPS or CpG alone; adult n = 6; neonatal: n = 51(Het)/34(KO), pooled to n≥5(Het)/4(KO)) adult and neonatal splenic cells isolated from miR-181a/b^+/- and miR-181a/b^-/- mice were determined by flow cytometry. Students <i>t</i>-test; * P<0.05, ** P<0.01, *** P<0.001.</p

Neonatal and adult B cell subsets share similar phenotypic characteristics.

<p>Flow cytometric analysis of surface marker expression of <b>(A)</b> maturity (CD10 & CD22; n = 7), co-stimulation (CD40 & TLR9; n≥6/8) and activation (CD80 & CD86; n = 5/3), <b>(B)</b> the three BAFF receptors BAFFR (n = 8), BCMA (n = 8/9) and TACI (n = 8/9) and <b>(C)</b> of surface IgM (n = 4) and IgD (n = 4/6) on neonatal and adult B cell subpopulations (transitional 1 & 2 B cells: T1 & T2; naïve mature B cells: N), <b>(D)</b> Expression of discrete markers on the three populations in AB and CB. Representative of >4 independent experiments are shown. <b>(A-C)</b> 1way ANOVA; *** P<0.0001).</p