Targeting Natural Killer Cell Reactivity by Employing Antibody to NKp46: Implications for Type 1 Diabetes

<div><p>Natural killer (NK) cells belong to the innate lymphoid cells. Their cytotoxic activity is regulated by the delicate balance between activating and inhibitory signals. NKp46 is a member of the primary activating receptors of NK cells. We previously reported that the NKp46 receptor is involved in the development of type 1 diabetes (T1D). Subsequently, we hypothesized that blocking this receptor could prevent or hinder disease development. To address this goal, we developed monoclonal antibodies for murine NKp46. One mAb, named NCR1.15, recognizes the mouse homologue protein of NKp46, named Ncr1, and was able to down-regulate the surface expression of NKp46 on primary murine NK cells following antibody injection <i>in vivo</i>. Additionally, NCR1.15 treatments were able to down-regulate cytotoxic activity mediated by NKp46, but not by other NK receptors. To test our primary assumption, we examined T1D development in two models, non-obese diabetic mice and low-dose streptozotocin. Our results show a significantly lower incidence of diabetic mice in the NCR1.15-treated group compared to control groups. This study directly demonstrates the involvement of NKp46 in T1D development and suggests a novel treatment strategy for early insulitis.</p></div

Prolonged multiple treatments with NCR1.15 down-regulates the surface expression of NKp46.

<p>Mice were injected i.p. 8 doses of PBS, 100 μg of NCR1.15 or cIgG1,κ every other day; 3 days after the last inoculation mice were sacrificed for further analysis. (A) C57BL/6 WT PBMCs and splenocytes were harvested and stained to detect the levels of CD3^-NK1.1⁺ NK cells. Data from one representative of three independent experiments are shown. (B) PBMCs drained from <i>NCR1</i>^<i>gfp/+</i> mice were analyzed for GFP expression following the prolonged repeated treatments. (C, D) Membrane associated NKp46 and NKG2D levels were analyzed on gated CD3ε^-NK1.1⁺ NK cells. (E) <i>NCR1</i> transcript levels from treated mice splenocytes were assessed by qRT-PCR. Data from one representative of two independent experiments are shown. * <i>p</i><0.05, ** <i>p</i><0.01 by Student's unpaired <i>t</i>-test. Bars, ±SD.</p

Single dose treatment with NCR1.15 inhibits NKp46-mediated activity on NK cells.

<p>(A) 3 days following i.p. injection of NCR1.15, isotype control or PBS purified splenic NK cells were co-incubated for 2–4 hours with YAC-1 target cells in the presence of anti-CD107a antibody. CD107a surface levels on NK cells were assessed by FACS. (B) Labeled YAC-1 and RMA cells were injected into the tail vein of 5 NCR1.15-, c-IgG1 κ- or PBS-treated mice. Cells in the lungs were quantified 3–4 hours following the injection using FACS analysis. Data from one of two independent experiments are shown. (C) 3 days following mice i.p. injection c-IgG1 κ-treated <i>UBC-GFP</i> and NCR1.15-treated C57BL/6 WT purified splenic NK cells were co-incubated with PD1.6 target cells in 1:1:1 ratio (E₁:E₂:T) with the presence of anti-CD107a antibody for 3–4 hours. CD107a surface levels on NK cells were assessed by FACS. (D) Purified splenic NK cells harvested from control- or NCR1.15-treated mice 3 days after the treatment were co-incubated with Ba/F3-Rae1ε target cells in 1:1 E:T ratio for 3–4 hours. CD107a surface expression levels were analyzed using FACS. Data from one representative of three independent experiments are shown. ** <i>p</i><0.01 by Student's unpaired <i>t</i>-test. Bars, ±SD.</p

Single dose treatment with NCR1.15 down-regulates the surface expression of NKp46 on NK cells, but not the NKp46 transcript or spleen/blood NK levels.

<p>3 days following i.p. injection of NCR1.15, isotype control or PBS splenocytes (A, B) and PBMCs (B) were stained and analyzed for CD3^-NK1.1⁺ NK cells using flow cytometry. Data from one representative of five independent experiments are shown. The membrane associated NKp46 (C, D) and NKG2D (C) on NK cells were stained and analyzed using flow cytometry. (E) Representative confocal images of purified splenic NK cells stained for extra- and intracellular expression of the NKp46. (F) qRT-PCR of NKp46 transcripts in splenocytes harvested from treated mice. (G) GFP intensity (GeoMFI) expressed by NK cells from <i>NCR1</i>^<i>gfp/+</i> following the various treatments. Data from one representative of three independent experiments are shown. ** <i>p</i><0.01 by Student's unpaired <i>t</i>-test. Bars, ±SD.</p

Short term treatment with NCR1.15 down-regulates NKp46-mediated activity on pancreatic β-cells and inhibits LD-STZ induced diabetes.

<p>(A) Purified splenic NK cells from treated cells were co-incubated for 2–4 hours with purified C57BL/6 β-cells in the presence of anti-CD107a antibody. CD107a surface levels on NK cells were assessed by FACS. * <i>p</i><0.05 by Student's unpaired <i>t</i>-test. (B) LD-STZ induced diabetes development in C57BL/6 mice following 50μg injections at days-2 and 5. (<i>n</i> = 8). Animals were considered diabetic when blood glucose was ≥300mg/dl. *** <i>p</i><0.005 Kaplan-M log-rank (Mental-Cox). (C) Body weight of mice in the indicated groups following the LD-STZ administration; body weight baselines at day 0 of the control and NCR1.15 groups were 19.2±0.86, 19.8±1 grams, respectively. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.005 by Student's unpaired <i>t</i>-test. Bars, ±SD. (D) Representative H&E sections of the pancreas from sex&age-matched C57BL/6 mice treated with NCR1.15 or mock-treated (IgG1, κ) in LD-STZ induced diabetes model (magnification X40). (E) Summary of insulitis score over the days calculated as described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118936#pone.0118936.ref037" target="_blank">37</a>]. Score is quantified from 0 (low) to 4 (most severe).</p

Treatment with NCR1.15 reduces NKp46-mediated NK activity on β-cells and inhibits T1D development.

<p>(A) 8 weeks after the first treatment NOD PBMCs drained from mice within the indicated groups were stained and analyzed for NK levels. NK cell levels were normalized to mock (PBS) treatment. (B) Representative histogram of membrane associated NKp46 levels on gated CD3ε-CD49b+ NK cells from the indicated groups. (C) Normalized GeoMFI of membrane associated NKp46 on gated NK cells, ** p<0.01 by Student's unpaired t-test. GeoMFI values were normalized to mock (PBS) treatment. (D) T1D development in NOD female mice in the indicated groups. (n = 14–15). Animals were considered diabetic when blood glucose was ≥250mg/dl. * p<0.05 Kaplan-M log-rank (Mental-Cox). Bars, ±SD.</p