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 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 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

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

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

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

Time course of changes in HBECC baseline mucin secretion following a Careful Wash.

<p>HBECCs were Carefully Washed and returned to the incubator under air:liquid luminal conditions. The cultures were then removed as a function of time and luminal mucus harvested with minimal perturbation. Cultures were sampled just once; different sets of cultures were used at the different time points (n = 4). <i>Inset</i>: Data plotted to show the total mucins harvested from the cultures over time. Mucins in all samples were determined by subunit ELISA.</p