Transient B-Cell Depletion with Anti-CD20 in Combination with Proinsulin DNA Vaccine or Oral Insulin: Immunologic Effects and Efficacy in NOD Mice

<div><p>A recent type 1 diabetes (T1D) clinical trial of rituximab (a B cell-depleting anti-CD20 antibody) achieved some therapeutic benefit in preserving C-peptide for a period of approximately nine months in patients with recently diagnosed diabetes. Our previous data in the NOD mouse demonstrated that co-administration of antigen (insulin) with anti-CD3 antibody (a T cell-directed immunomodulator) offers better protection than either entity alone, indicating that novel combination therapies that include a T1D-related autoantigen are possible. To accelerate the identification and development of novel combination therapies that can be advanced into the clinic, we have evaluated the combination of a mouse anti-CD20 antibody with either oral insulin or a proinsulin-expressing DNA vaccine. Anti-CD20 alone, given once or on 4 consecutive days, produced transient B cell depletion but did not prevent or reverse T1D in the NOD mouse. Oral insulin alone (twice weekly for 6 weeks) was also ineffective, while proinsulin DNA (weekly for up to 12 weeks) showed a trend toward modest efficacy. Combination of anti-CD20 with oral insulin was ineffective in reversing diabetes in NOD mice whose glycemia was controlled with SC insulin pellets; these experiments were performed in three independent labs. Combination of anti-CD20 with proinsulin DNA was also ineffective in diabetes reversal, but did show modest efficacy in diabetes prevention (p = 0.04). In the prevention studies, anti-CD20 plus proinsulin resulted in modest increases in Tregs in pancreatic lymph nodes and elevated levels of proinsulin-specific CD4+ T-cells that produced IL-4. Thus, combination therapy with anti-CD20 and either oral insulin or proinsulin does not protect hyperglycemic NOD mice, but the combination with proinsulin offers limited efficacy in T1D prevention, potentially by augmentation of proinsulin-specific IL-4 production.</p> </div

T-cells from combination therapy treated mice produce IL-4 in response to proinsulin peptide stimulation.

<p>At 60-days post anti-CD20 mono- (MT, open bars) or combination therapy (CT, filled black bars), CD4+ T-cells were purified from treated non-diabetic NOD mice. Simultaneously, T-depleted splenocytes (TDS) were obtained from 8–10 week old NOD mice and used as APCs. 250,000 purified CD4+ T-cells were incubated with proinsulin peptide or a mutated insulin B:9–23 (B16:A) peptide in the presence of 5×10⁴ APCs. Following 3-day incubation, IL-4 production was determined by ELISpot assay as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054712#pone.0054712-Fousteri1" target="_blank">[34]</a>. Background (media) subtracted spot numbers are shown on the Y-axis. Increased IL-4 production in response to proinsulin peptide was seen in combination therapy treated mice. Representative means ± SEM data from one of two independent experiments with similar results are shown. Statistical analysis was performed using unpaired students <i>t-</i>test using Graphpad Prism software.</p

Combination therapy increases numbers of CD4+ Foxp3+ cells in pancreas draining lymph node 60-days post treatment.

<p>Eight to ten week old prediabetic NOD mice were given 50 µg of anti-CD20 either alone or in combination with weekly administration of 50 µg proinsulin plasmid for four weeks. Splenocytes and PDLN cells from mono- or combination therapy treated mice were stained with anti-IgM, -B220 (B-cells), -CD4, -CD8, -CD25 and –Foxp3. B-cell frequencies were determined by gating on IgM+ B220+ cells. Frequency of Foxp3+ cells among CD4+ T-cells was determined by gating on CD4+ cells. Cumulative frequencies of B-cells (left panel), CD4+ T-cells (middle panel), and CD4+Foxp3+ cells (right panel) in PDLN (upper panels) or spleen (lower panels) are shown at 30 days and 60 days post combination therapy. Representative data from three independent experiments with similar results is shown. Statistical analysis was performed using unpaired students <i>t-</i>test using Graphpad Prism software. Each dot represents one mouse, with mean value indicated on the graph.</p

Combination therapy with anti-CD20 and proinsulin plasmid prevents T1D more efficiently.

<p>Eight to ten week old prediabetic NOD mice were treated with (<b>A</b>) weekly administration of 50 µg proinsulin plasmid only or, (<b>B</b>) one-time administration of anti-CD20 alone or, (<b>C</b>) anti-CD20 administered in combination with four weekly administrations of proinsulin plasmid. Statistical tests were performed by Kaplan-Meier analysis using Graphpad Prism software.</p

Priming and effector dependence on insulin B:9–23 peptide in NOD islet autoimmunity

NOD mice with knockout of both native insulin genes and a mutated proinsulin transgene, alanine at position B16 in preproinsulin (B16:A-dKO mice), do not develop diabetes. Transplantation of NOD islets, but not bone marrow, expressing native insulin sequences (tyrosine at position B16) into B16:A-dKO mice rapidly restored development of insulin autoantibodies (IAAs) and insulitis, despite the recipients’ pancreatic islets lacking native insulin sequences. Splenocytes from B16:A-dKO mice that received native insulin–positive islets induced diabetes when transferred into wild-type NOD/SCID or B16:A-dKO NOD/SCID mice. Splenocytes from mice immunized with native insulin B chain amino acids 9–23 (insulin B:9–23) peptide in CFA induced rapid diabetes upon transfer only in recipients expressing the native insulin B:9–23 sequence in their pancreata. Additionally, CD4+ T cells from B16:A-dKO mice immunized with native insulin B:9–23 peptide promoted IAAs in NOD/SCID mice. These results indicate that the provision of native insulin B:9–23 sequences is sufficient to prime anti-insulin autoimmunity and that subsequent transfer of diabetes following peptide immunization requires native insulin B:9–23 expression in islets. Our findings demonstrate dependence on B16 alanine versus tyrosine of insulin B:9–23 for both the initial priming and the effector phase of NOD anti-islet autoimmunity