Autoreactive B Cell Regulation: Peripheral Induction of Developmental Arrest by Lupus-Associated Autoantigens

Anti-Sm and anti-ssDNA transgenic (Tg) mice were generated using the VH-D-JH rearrangement of an anti-Sm hybridoma of MRL/Mp-lpr/lpr origin. B cells of each specificity account for 15%–35% of the splenic repertoire, but no circulating anti-Sm or anti-ssDNA antibodies are detected. Most autoreactive cells exhibit an immature B cell phenotype and have short half-lives equivalent to those of non-Tg immature B cells. However, at least some anti-Sm B cells are functional, because immunization with murine snRNPs induces anti-Sm secretion. We propose that anti-Sm and anti-ssDNA are eliminated during the transition to mature B cells and that this late stage of tolerance induction is consequential to their spontaneous activation in murine lupus

Surrogate Antibodies That Specifically Bind and Neutralize CCL17 But Not CCL22

The chemokines CCL17 (TARC) and CCL22 (MDC) function through the same receptor, CCR4, but have been proposed to differentially affect the immune response. To better understand the role of the individual ligands, a panel of rat anti-mouse CCL17 surrogate antibodies was generated that can be used to differentiate CCL17 and CCL22 function in vitro and in vivo. We have successfully identified a panel of neutralizing antibodies by screening hybridomas for the ability to inhibit CCL17-mediated calcium mobilization. Chemotaxis in response to CCL17 is also inhibited, providing further evidence that the antibodies in this panel are antagonistic. Using a recombinant cell line expressing human CCR4, we show that the antibodies block ?-arrestin recruitment as evidence that the antibodies are specifically blocking CCL17 signaling through CCR4. The antibodies within this panel inhibit calcium mobilization with varying potency in the calcium flux assay, having apparent IC50 ranging from approximately 1 to >400?ng/mL. Although both CCL17 and CCL22 function through CCR4, only a single antibody was identified as having detectable binding to CCL22. This panel of CCL17-specific antibodies provides tools that can be used to differentiate CCL17 and CCL22 function through CCR4 interaction in vitro and in vivo.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140164/1/mab.2012.0112.pd

Multivalent RNA aptamers that inhibit CTLA-4 and enhance tumor immunity

The potency of cancer immunotherapy can be enhanced by administration of high-avidity ligands specific to receptors expressed on T cells. Antibodies or cytokines are the main agents used in such capacity. Antibody-mediated inhibition of cytotoxic T cell antigen-4 (CTLA-4) function in mice augments antitumor immunity and could serve as an important adjunct in cancer immunotherapy. However, antibody-based therapy used in the setting of chronic diseases such as cancer poses significant cost, manufacturing, and regulatory challenges. Here we describe the development of RNA aptamers that bind CTLA-4 with high affinity and specificity. These aptamers inhibit CTLA-4 function in vitro and enhance tumor immunity in mice. Moreover, assembly of the aptamers into tetrameric forms significantly enhances their bioactivity in vitro and in vivo. These results demonstrate that aptamers can be used to manipulate the immune system for therapeutic applications and that multivalent versions of aptamers may be particularly potent agents in vivo

Engagement of two distinct binding domains on CCL17 is required for signaling through CCR4 and establishment of localized inflammatory conditions in the lung.

CCL17 (TARC) function can be completely abolished by mAbs that block either one of two distinct sites required for CCR4 signaling. This chemokine is elevated in sera of asthma patients and is responsible for establishing inflammatory sites through CCR4-mediated recruitment of immune cells. CCL17 shares the GPCR CCR4, with CCL22 (MDC) but these two chemokines differentially affect the immune response. To better understand chemokine mediated effects through CCR4, we have generated chimeric anti-mouse CCL17 surrogate antibodies that inhibit function of this ligand in vitro and in vivo. The affinities of the surrogate antibodies for CCL17 range from 685 pM for B225 to 4.9 nM for B202. One antibody, B202, also exhibits weak binding to CCL22 (KD∼2 µM) and no binding to CCL22 is detectable with the second antibody, B225. In vitro, both antibodies inhibit CCL17-mediated calcium mobilization, β-arrestin recruitment and chemotaxis; B202 can also partially inhibit CCL22-mediated β-arrestin recruitment. Both B202 and B225 antibodies neutralize CCL17 in vivo as demonstrated by reduction of methacholine-induced airway hyperreactivity in the A. fumigatus model of asthma. That both antibodies block CCL17 function but only B202 shows any inhibition of CCL22 function suggests that they bind CCL17 at different sites. Competition binding studies confirm that these two antibodies recognize unique epitopes that are non-overlapping despite the small size of CCL17. Taking into consideration the data from both the functional and binding studies, we propose that effective engagement of CCR4 by CCL17 involves two distinct binding domains and interaction with both is required for signaling

Discovering Molecules That Regulate Efferocytosis Using Primary Human Macrophages and High Content Imaging

<div><p>Defective clearance of apoptotic cells can result in sustained inflammation and subsequent autoimmunity. Macrophages, the “professional phagocyte” of the body, are responsible for efficient, non-phlogistic, apoptotic cell clearance. Controlling phagocytosis of apoptotic cells by macrophages is an attractive therapeutic opportunity to ameliorate inflammation. Using high content imaging, we have developed a system for evaluating the effects of antibody treatment on apoptotic cell uptake in primary human macrophages by comparing the Phagocytic Index (PI) for each antibody. Herein we demonstrate the feasibility of evaluating a panel of antibodies of unknown specificities obtained by immunization of mice with primary human macrophages and show that they can be distinguished based on individual PI measurements. In this study ~50% of antibodies obtained enhance phagocytosis of apoptotic cells while approximately 5% of the antibodies in the panel exhibit some inhibition. Though the specificities of the majority of antibodies are unknown, two of the antibodies that improved apoptotic cell uptake recognize recombinant MerTK; a receptor known to function in this capacity <i>in vivo</i>. The agonistic impact of these antibodies on efferocytosis could be demonstrated without addition of either of the MerTK ligands, Gas6 or ProS. These results validate applying the mechanism of this fundamental biological process as a means for identification of modulators that could potentially serve as therapeutics. This strategy for interrogating macrophages to discover molecules regulating apoptotic cell uptake is not limited by access to purified protein thereby increasing the possibility of finding novel apoptotic cell uptake pathways.</p></div

Dexamethasone treatment impacts the ability of macrophages to engulf apoptotic cells in a dose-dependent manner.

<p>Macrophages were treated with varying doses of dexamethasone then imaged following 30 minute incubation with apoptotic Jurkat cells as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145078#pone.0145078.g002" target="_blank">Fig 2</a>. Phagocytic Index (PI) was calculated at each dose from 1 field/well collected using the Operetta High Content Imager. PI = (Total # engulfed cells/Total # counted MΦ) * (# MΦ’s containing engulfed cells/Total # counted MΦ) * 100 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145078#pone.0145078.ref049" target="_blank">49</a>].</p

Characteristics of SelecT generated macrophages and apoptotic status of staurosporine-treated cells.

<p>Macrophages were prepared by culture with M-CSF as described in materials and methods. Following treatment with IFN-γ, the majority of macrophages express markers found on both M1 and M2 populations (A). Cells were gated by scatter then expression of markers was assessed on live cells. M1-M2 intermediate phenotype macrophages are CD64⁺CD16⁺ (B). Jurkat cells were treated with staurosporine for 3 hours at 37°C as described in materials and methods. Cells were stained for active caspase 3 then quantitated using flow cytometry (C) or stained with Magic Red then measured using high content imaging (D).</p

The majority of antibodies enhance apoptotic cell phagocytosis through recognition of unidentified surface molecules.

<p>Antibodies having a PI ≥ average PI + 1 SD (standard deviations) for cultures with no antibody treatment are considered as enhancing. Those having a PI≤ average PI—1 SD are considered inhibitory. All other antibodies having PI within 1 SD of average calculated for untreated MΦ’s are considered as having no effect. Frequency of each category of antibody is calculated as percent of total number of antibodies in the panel (left). Binding to known molecules could be detected in a small number of clones (right). Antibodies were evaluated by ELISA and readings that were ≥ 5-fold over background were considered positive.</p

Phagocytic Index (PI) provides a direct measure of macrophage function.

<p>Comparison of data presented as either PI (A) or representing the frequency of macrophages containing apoptotic cells expressed as “percent (%) positive cells” (B) within the same population in each well of a 96 well plate. The PI was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145078#pone.0145078.g003" target="_blank">Fig 3</a>. Calculation of the % positive cells is based on the assumption that macrophages (APC⁺) containing apoptotic cells (Cy3⁺) appear fluorescent for both fluorophores and calculated as follows: % positive cells = (# APC⁺ Cy3⁺ cells / # APC⁺ cells) * 100. Ratios listed along the X-axis represent the (Target) T: MΦ ratios. Baseline engulfment was determined using MΦ’s co-cultured with live cells (Live); Negative controls consisted of cytochalasin D treated MΦ’s co-cultured with apoptotic cells (Neg). Test samples consisted of apoptotic cells co-cultured with MΦ’s (Apop).</p