Bortezomib treatment significantly reduces anti-AAV IgG titre. a.

<p>Mice were infected with AAV2/8-EV and treated with bortezomib, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034684#s2" target="_blank">Materials and Methods</a> section. Sera were collected at the indicated times, and the levels of anti-AAV IgG were determined by titration. <b>b.</b> A subset of the same sera samples was assayed for the total amount of IgG using ELISA. Representative data from two separate experiments with similar outcomes are shown.</p

Bortezomib-mediated reduction in anti-AAV IgG titres is insufficient to facilitate reinfection with AAV2/8-DC190-alphaGal.

<p><b>a.</b> At twenty-four weeks following the administration of AAV2/8-EV (including 20 weeks of bortezomib treatment between weeks 4 and 24), mice were re-infected with 5×10¹¹ AAV2/8-DC190-alphaGal/mouse vector particles. Sera were collected at the indicated times, and the titres of anti-AAV IgG were determined. <b>b.</b> A subset of the sera samples was assayed for the total amount of IgG using ELISA. <b>c.</b> The level of α-galactosidase A in serum samples from mice infected with AAV2/8-DC190-alphaGal. Expression of α-galactosidase A was observed in mice that had not been previously infected with AAV2/8-EV. <b>d.</b> Immunohistochemistry was performed on liver sections from mice that were administered AAV2/8-DC190-alphaGal. The figures show representative data from two studies with similar outcomes.</p

Bortezomib treatment differentially affects T cells.

<p><b>a.</b> Mononuclear cells were prepared from spleens and analysed for the T cell markers CD4, CD8, and CD44 using flow cytometry. Plots depict CD44 expression gated on the CD4⁺ (left panel) and CD8⁺ (right panel) populations. <b>b.</b> A summary of the data from <b>a</b> is shown in a quantitative format. <b>c.</b> Mononuclear cells isolated from spleen were reactivated with AAV2/8-EV for 72 h <i>in vitro</i>. Supernatants were collected and assayed for IL-2 (left panel) or interferon-γ (right panel) levels using ELISA. The figures show representative data from two studies with similar outcomes.</p

A Bispecific Protein Capable of Engaging CTLA-4 and MHCII Protects Non-Obese Diabetic Mice from Autoimmune Diabetes

<div><p>Crosslinking ligand-engaged cytotoxic T lymphocyte antigen-4 (CTLA-4) to the T cell receptor (TCR) with a bispecific fusion protein (BsB) comprised of a mutant mouse CD80 and lymphocyte activation antigen-3 (LAG-3) has been shown to attenuate TCR signaling and to direct T-cell differentiation toward Foxp3⁺ regulatory T cells (Tregs) in an allogenic mixed lymphocyte reaction (MLR). Here, we show that antigen-specific Tregs can also be induced in an antigen-specific setting in vitro. Treatment of non-obese diabetic (NOD) female mice between 9–12 weeks of age with a short course of BsB elicited a transient increase of Tregs in the blood and moderately delayed the onset of autoimmune type 1 diabetes (T1D). However, a longer course of treatment (10 weeks) of 4–13 weeks-old female NOD animals with BsB significantly delayed the onset of disease or protected animals from developing diabetes, with only 13% of treated animals developing diabetes by 35 weeks of age compared to 80% of the animals in the control group. Histopathological analysis of the pancreata of the BsB-treated mice that remained non-diabetic revealed the preservation of insulin-producing β-cells despite the presence of different degrees of insulitis. Thus, a bifunctional protein capable of engaging CTLA-4 and MHCII and indirectly co-ligating CTLA-4 to the TCR protected NOD mice from developing T1D.</p></div

Longer-term treatment of NOD mice with BsB significantly delayed the onset of T1D in NOD mice.

<p>(A) The cumulative incidences of overt diabetes in BsB-treated (n = 16) and untreated mice (n = 16). BsB treatment significantly reduced the incidence of T1D compared with mice treated with saline (<i>p</i><0.01). (B) A histopathological analysis of pancreatic tissues from animals treated with saline or BsB. Panels a through c represent the sections from saline-treated mice that remained non-diabetic with H&E, an antibody to insulin (pink), or anti-CD3 (brown) and Foxp3 (pink), respectively. Similar observations were noted in BsB-treated NOD mice that remained disease-free. No evidence of infiltration or insulitis was noted in any of the sections; a few Foxp3⁺ Tregs may be present (arrows in panel c). Panels d through f represent the pancreatic sections from diabetic NOD animals. Invasive insulitis was clearly evident, and the insulin-producing β-cells were completely destroyed (e). Several CD3⁺ T cell infiltrations were also detected along with a few Tregs and many non-T cell leukocytes with blue nuclei (f). Panels g through i show that the islets of BsB treated animals that remained non-diabetic but exhibited characteristic peri-insulitis. Leukocyte infiltrations were noted but were restricted to the periphery of the islets. Moreover, there was no notable destruction of the insulin-producing β-cells. Most of the leukocytes at the periphery were non-T cells (with blue nuclei). The enlarged inset (panel j, represents the red square in i) indicated Foxp3⁺ Tregs (yellow arrow head) were intermixed with other CD3⁺ T cells and non-T cell leukocytes (with blue nuclei) at the periphery of islets. The images were acquired with a 40× objective; the inset was acquired with a 60× objective, which was then further enlarged 3× digitally.</p

Treatment of NOD mice with BsB modestly delayed the onset of T1D in a late prevention treatment paradigm.

<p><b>(</b>A) The levels of Foxp3⁺ Tregs in the blood of BsB-treated (closed circles, n = 15) and saline-treated control mice (closed triangles, n = 14). There was a moderate but significant increase in the number of Tregs in the BsB-treated animals over the number of Tregs noted in the control animals. (B) The cumulative incidences of overt diabetes in animals treated with BsB (filled circles) or saline (filled triangles).</p

BsB-mediated induction of antigen-specific Tregs in vitro.

<p>(A) The in vitro induction of Ova_233–339-specific Tregs. Naïve OT-II T cells were mixed with LPS-activated and irradiated syngeneic APCs in the presence of 0.5 µg/ml Ova_233–239 peptide. Control mIgG2a, BsB, and BsB plus an anti-TGB-β antibody (αTGF-β) were then added and tested as indicated (left panels). The cells were cultured for 5 days and then labeled with anti-CD25 and anti-Foxp3 antibodies before being analyzed by flow cytometry. IL-2, IL-10 and TGF-β levels in the culture media were assayed by ELISA. (B) The monitoring of induced Tregs proliferation. The studies were conducted as in (A) except naïve OT-II T cells were pre-labeled with CFSE before being mixed with APCs. The cells were gated on Foxp3 and CFSE fluorescent channels.</p

Analysis of asparagine-linked glycosylation on BsB.

<p>The amino acid sequence of BsB was submitted to the NetNGlyc 1.0 Server for the prediction of asparagine-linked glycosylation sites. A total of 10 asparagine-linked glycosylation sites (denoted N) were predicted; other amino acids are presented as dots. The monosaccharide composition of BsB was also performed to determine the composition of the glycans. Fucose (Fuc), N-acetylglucosamine (GlcNAc), galactose (Gal), mannose (Man), sialic acid, N-acetylneuraminic acid. A sialic acid:galactose ratio of 0.68 indicates that approximately one third of the galactose residues are available for binding to the ASGPR. Numbers represent mean ± std.</p

Pharmacokinetics of BsB in vivo and biochemical analysis.

<p>(A) The pharmacokinetic profiles of BsB in mice. Normal C57BL/6 mice (n = 5) were dosed intraperitoneally with 20 mg/kg of BsB. Blood samples were collected at the different time points indicated, and the levels of BsB were determined using an ELISA. Data represent mean ± sem. (B) Comparison of the binding of BsB and mouse IgG2a to FcRn. The FcRn were immobilized to a Biacore chip as described in the material and methods. BsB or control mouse IgG2a was loaded onto the chip at various concentrations, and the signals were then recorded.</p

Image_2_IL11-mediated stromal cell activation may not be the master regulator of pro-fibrotic signaling downstream of TGFβ.tif

Fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF) and systemic scleroderma (SSc), are commonly associated with high morbidity and mortality, thereby representing a significant unmet medical need. Interleukin 11 (IL11)-mediated cell activation has been identified as a central mechanism for promoting fibrosis downstream of TGFβ. IL11 signaling has recently been reported to promote fibroblast-to-myofibroblast transition, thus leading to various pro-fibrotic phenotypic changes. We confirmed increased mRNA expression of IL11 and IL11Rα in fibrotic diseases by OMICs approaches and in situ hybridization. However, the vital role of IL11 as a driver for fibrosis was not recapitulated. While induction of IL11 secretion was observed downstream of TGFβ signaling in human lung fibroblasts and epithelial cells, the cellular responses induced by IL11 was quantitatively and qualitatively inferior to that of TGFβ at the transcriptional and translational levels. IL11 blocking antibodies inhibited IL11Rα-proximal STAT3 activation but failed to block TGFβ-induced profibrotic signals. In summary, our results challenge the concept of IL11 blockade as a strategy for providing transformative treatment for fibrosis.</p