Soluble Antigen Arrays for Selective Desensitization of Insulin-Reactive B Cells

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Molecular Pharmaceutics, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.molpharmaceut.8b01250.Autoimmune diseases are believed to be highly dependent on loss of immune tolerance to self-antigens. Currently, no treatments have been successful clinically in inducing autoantigen-specific tolerance, including efforts to utilize antigen-specific immunotherapy (ASIT) to selectively correct the aberrant autoimmunity. Soluble antigen arrays (SAgAs) represent a novel autoantigen delivery system composed of a linear polymer, hyaluronic acid (HA), displaying multiple copies of conjugated autoantigen. We have previously reported that Soluble Antigen Arrays proteolipid protein (SAgAPLP) induced tolerance to a specific multiple sclerosis (MS) autoantigen, proteolipid peptide (PLP). Utilizing SAgA technology, we have developed a new ASIT as a possible type 1 diabetes (T1D) therapeutic by conjugating human insulin to HA, known as Soluble Antigen Array Insulin (SAgAIns). Three types were synthesized: low valency lvSAgAIns (2 insulins per HA), medium valency mvSAgAIns (4 insulins per HA) and, high valency hvSAgAIns (9 insulins per HA) to determine if valency differentially modulates the ex vivo activity of insulin-binding B cells (IBCs). Extensive biophysical characterization was performed for the SAgA molecules. SAgAIns molecules were successfully used to affect the biologic activity of IBCs by inducing desensitization of the B cell antigen receptors (BCR). SAgAIns bound specifically to insulin-reactive B cells without blocking epitopes recognized by antibodies against the Fc regions of membrane immunoglobulin or CD79 transducer components of the BCR. Pre-incubation of IBCs (125Tg) with SAgAIns, but not HA alone, rendered the IBCs refractory to re-stimulation. SAgAIns induced a decrease in BCR expression and IP3R-mediated intracellular calcium release. Surprisingly, SAgAIns binding to BCR on the surface of IBCs induced the observed effects at both high and low SAgAIns valency. Future studies aim to test the effects of SAgAIns on disease progression in the VH125.NOD mouse model of T1D.NIH T32 GM00854

Endogenous HAQ STING is strongly impaired in mounting a type I IFN and proinflammatory cytokine responses against <i>Legionella</i> infection or stimulation with DNA or CDNs.

<p>(A-D) PBMCs from healthy volunteers (N = 4 for WT and N = 4 for HAQ) were isolated by density gradient centrifugation. 7 d after isolation cells were infected for 6 h with <i>L</i>. <i>pneumophila</i> at MOIs 10 and 50 or stimulated for the same period with 1 and 5 ug/ml 2´-3´cGAMP or either bacterial or synthetic DNA at a concentration of 0.2 or 1 ug/ml. RNA was isolated and the expression of <i>IFNB</i> (A), <i>IL1B</i> (B), <i>IL6</i> (C) and <i>TNFA</i> (D) was determined by qRT-PCR. Data are shown as the RQ of specified mRNAs. Data represent the mean ± SEM of 4 independent experiments carried out in triplicates. Differences were assessed with the Mann-Whitney U Test. Comparisons with a p < 0.05 were considered significant.</p

The cGAS/STING axis contributes to the production of pro-inflammatory cytokines during <i>L</i>. <i>pneumophila</i> infection.

<p>(A-F) WT, <i>Tmem173</i>^<i>-/-</i> <i>and cGas</i>^<i>-/-</i> BMDMs were infected for 6 h with <i>L</i>. <i>pneumophila</i> WT at MOI 10 and relative cytokine expression was determined by qRT-PCR. (G-J) Cytokine protein concentrations in whole lung homogenates from <i>L</i>. <i>pneumophila</i>-infected mice were quantified by sandwich ELISA. Data are shown as mean ± SEM. (A-F) Data representative of 3 to 4 independent experiments carried out in duplicates. (G-J) Data representative of 6 o 7 mice per group. Data were analyzed through the Mann-Whitney U Test. Comparisons with a <i>p</i> < 0.05 were considered significant.</p

Endogenous R232H STING is partly deficient in sensing bacterial CDN but responds normally to <i>Legionella</i> infection or stimulation with DNA.

<p>(A-D) PBMCs from healthy volunteers (N = 3 for WT and N = 3 for R232H) were isolated by density gradient centrifugation. 7 d after isolation cells were infected for 6 h with <i>L</i>. <i>pneumophila</i> at MOI 10 or stimulated for the same period with 1 ug/ml 2´-3´cGAMP, Rp-c-diAMPSS, cGMP or either bacterial DNA at a concentration of 1 ug/ml. RNA was isolated and the expression of <i>IFNB</i> (A), <i>IL1B</i> (B), and <i>IL6</i> (C) and <i>TNFA</i> (D) was determined by qRT-PCR. Data are shown as the RQ of specified mRNAs. Data represent the mean ± SEM of 3 independent experiments carried out in triplicates. Differences were assessed with the Mann-Whitney U Test. Comparisons with a p < 0.05 were considered significant.</p

STING contributes to the antibacterial defense in mice infected with <i>L</i>. <i>pneumophila</i>.

<p>WT, cGAS- and STING-deficient mice were intranasally infected with 1×10⁶ <i>L</i>. <i>pneumophila</i> WT and the bacterial loads in the lungs were assessed 144 h p.i. Data represent mean ± SEM of 6–13 mice per group. Comparisons were performed with the Mann-Whitney U Test. Comparisons with p < 0.05 were considered significant.</p

Type I IFN responses during <i>L</i>. <i>pneumophila</i> infection are mediated by the cGAS/STING pathway.

<p>(A-C) WT and <i>Tmem173</i>^-/- mouse BMDMs were left untreated or stimulated with 1 ug/ml <i>L</i>. <i>pneumophila</i> DNA (JR32 DNA) or 5 ug/ml 2´3-cGAMP (A) or were infected with <i>L</i>. <i>pneumophila</i> JR32 WT and 130b WT, or mutant strains deficient for <i>dotA</i> or <i>sdhA</i> at MOI 10 for 6 h (B, C). Expression of <i>Ifnb</i> (A, B) or <i>Irg1</i> (C) was measured by qRT-PCR. (D-G) WT and cGAS-deficient BMDMs were stimulated with <i>L</i>. <i>pneumophila</i> DNA or 2´3-cGAMP or infected with <i>L</i>. <i>pneumophila</i> JR32 WT, and expression of <i>Ifnb</i> and <i>Irg1</i> was quantified by qRT-PCR (D-F) or production of IP-10 was measured by ELISA (G). (H-N) WT, STING- and cGAS-deficient mice were intranasally infected with 1×10⁶ <i>L</i>. <i>pneumophila</i> JR32 WT or instilled with PBS as control (H-J). <i>Ifnb</i> and <i>Irg1</i> expression in the lungs was assessed 48 (H, I) or 144 h p.i. (K-N) by qRT-PCR, or IP-10 production was measured at 48 h (J). Data are represented as the relative quantification (RQ) of specified mRNAs. Data are shown as the mean + SEM of three to four independent experiments, measured in technical duplicates (Fig. 1A-G) or 6 to 7 mice per group (Fig. H-N). Analyses were performed through the Mann-Whitney U Test. Comparisons with a <i>p</i> < 0.05 were considered significant.</p

Distribution of <i>TMEM173/STING</i> HAQ and R232H in German patients and healthy controls.

<p>Distribution of <i>TMEM173/STING</i> HAQ and R232H in German patients and healthy controls.</p

<i>L</i>. <i>pneumophila</i> infection and stimulation with DNA or cGAMP induce weak cGAS-dependent type I IFN responses in THP-1 cells.

<p>WT THP-1 or cGAS-/- THP-1 clones A5 and B5 were allowed differentiation prior to stimulation with either cGAMP or synthetic DNA (A) or infection with two different strains of <i>L</i>. <i>pneumophila</i> (B). <i>IFNB</i> expression was determined by qRT-PCR. Data represent mean ± SEM of 2 independent experiments carried out in duplicates. Analyses were performed by employing the Mann-Whitney U Test. Comparisons with a p < 0.05 were considered significant.</p