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

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

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

NCR1 involvement in NK cells activation following infection of BMMQ/ BMDC with <i>S. pneumoniae.</i>

<p>(A-D) Six-day culture of BMMQ and BMDC grown from <i>Ncr1</i>^+/+ BM cells (C57BL/6) were stained with mNCR1-Ig, mNKG2D-Ig and LIR1-Ig fusion proteins. Shown are representative overlays of F4/80⁺CD115⁺ BMMQ (A, C) and CD11c⁺ BMDC (B, D) demonstrating staining with mNCR1-Ig (solid line, A-D), mNKG2D-Ig (dotted line, C–D), LIR1-Ig (dashed line, A–B) and with goat anti-human IgG (grey-filled, A–D). Shown is one representative experiment out of four to five independent experiments. In some experiments, NKG2D-Ig staining was higher compared to NCR1-Ig staining. (E–F) Six-day culture- BMMQ (E) and -BMDC (F) grown from <i>Ncr1</i>^+/+ BM cells (C57BL/6) were infected with 1 MOI of <i>S. pneumoniae</i> for 1.5 h, then bacteria were removed and antibiotics were added. In parallel, NK cells were isolated from splenocytes of <i>Ncr1</i>^+/+ (‘NK WT’) and <i>Ncr1</i>^gfp/gfp (‘NK KO’) C57BL/6 mice. Isolated NK were incubated with uninfected BMMQ or BMDC, or with <i>S. pneumoniae</i>-infected BMMQ or BMDC (‘BMMQ+SP’, ‘BMDC+SP’) after removal of bacteria for additional 18 hours. NK-BMMQ ratio was 1∶4 and NK-BMDC ration was 1∶10. IFNγ concentration levels in the co-culture supernatants were determined by ELISA. Results are from one representative experiment out of two. ** p<0.01 compared with IFNγ levels of NK WT incubated with infected BMMQ/BMDC, ± SD (ANOVA test).</p

NCR1 ligand expression on <i>S</i>. <i>pneumoniae</i> and naïve macrophages and DC.

<p>(A) Fixed bacteria were stained with mNCR1-Ig (left panel) and mNKG2D-Ig (right panel) fusion proteins. Shown are representative overlays of demonstrating staining with the fusion protein (bold line) and with goat anti-human IgG (plain line). Both lines overlapped. Cells from lung lavage (B, E, F), BM (C, E, F, G) and spleen (D) of C57BL/6 <i>Ncr1</i>^+/+ (WT), <i>Ncr1</i>^+/gfp (Het) and <i>Ncr1</i>^gfp/gfp (KO) naive mice were stained with mNCR1-Ig, mNKG2D-Ig, LIR1-Ig (B) or CD99-Ig (C, D) and with macrophages/DC markers (F4/80, CD115, CD11c). Panels B-F were gated on macrophages (F4/80⁺CD115⁺) and panel G was gated on DC (CD11c⁺). Panels B-D show overlays of mNCR1-Ig staining as follows: bold line for <i>Ncr1</i>^+/+ (B1, C1, D1) or <i>Ncr1</i>^gfp/gfp (B2, C2, D2), hairline for <i>Ncr1</i>^+/gfp (C1, D1); LIR1-Ig staining (grey line for B1, B2); CD99-Ig staining (dashed line for C1, C2, D1, D2); and goat anti-human IgG (grey line for B3, C3, D3). mNKG2D-Ig staining for all mice types is shown is the right panel (B3, C3 and D3: <i>Ncr1</i>^+/+ in bold line, <i>Ncr1</i>^+/gfp in dashed line and <i>Ncr1</i>^gfp/gfp in dotted line). The bar graphs (E–G) show the fraction of NCR1-ligand^high BAL and BM-gated macrophages for <i>Ncr1</i>^+/+ vs. <i>Ncr1</i>^gfp/gfp (E), the fraction of NCR1-ligand^high BAL and BM-gated macrophages for <i>Ncr1</i>^+/gfp vs. <i>Ncr1</i>^gfp/gfp (F) and NCR1-ligand^high BM-gated DC (G). Results in E are normalized to <i>Ncr1</i>^+/+ (n = 12 and 16 mice per group). Results in F are normalized to <i>Ncr1</i>^+/gfp (n = 12 and 15 mice per group). *** p<0.001 and * p<0.05 compared to WT/Het group +SD (two-tailed student t-test). NS, non significant compared to WT/Het group. Results (B–D) are from one representative experiment out of six to seven.</p

NK cells activation and cytokines mRNA levels in lungs following intranasal challenge with <i>S. pneumoniae</i>.

<p>Lungs were harvested from <i>Ncr1</i>^+/gfp (Het group) and <i>Ncr1</i>^gfp/gfp (KO group) C57BL/6 mice 3h after intranasal inoculation with 5×10⁷ CFU of <i>S. pneumoniae</i> strain WU2, and from non infected mice. (A) NK cells were gated as CD45⁺NK1.1⁺ and analyzed for the expression of membrane-associated CD107a by flow cytometry. The bar graphs show the fraction of CD107a positive NK cells (average of 3 mice per group, ±SD). ** p<0.01 compared to Het group in 3h-infected mice (ANOVA test). Total RNA was extracted from naïve lungs and from 3h-infected lungs of <i>Ncr1</i>^+/gfp (Het) and <i>Ncr1</i>^gfp/gfp (KO). Infection was performed with 5×10⁷ CFU of <i>S</i>. <i>pneumoniae.</i> Level of cytokines mRNA of IFNγ (n = 4), TNFα (n = 4) and IL-6 (n = 3) were analyzed by RT-PCR and calibrated to mRNA level of GAPDH. Results are from one representative experiment of two. *** p<0.001, ** p<0.01, compared with the Het group +SD (ANOVA test).</p

Bacterial load in lungs following <i>in vivo</i> depletion of NK cells.

<p><i>Ncr1</i>^+/+ and <i>Ncr1</i>^gfp/gfp C57BL/6 mice treated intraperitoneally either with 50 µl of anti asialo GM1 (WT-anti NK and KO-anti NK) or with 50 µl of PBS (mock treatment, WT-mock and KO-mock). 24 h after anti asialo GM1 or mock treatment, all mice were challenged with 5×10⁷ CFU of <i>S. pneumoniae</i> strain WU2 and lungs were harvested 3 h after challenge. (A) CFU of <i>S. pneumoniae</i> in lungs, 3 h after bacterial challenge (n = 3 to 5 per group). Results are presented as box plot of CFU. ** p<0.01 compared with WT-mock; * p<0.05, compared with WT-anti NK, ± SD (ANOVA test). (B) Flow cytometry analysis of spleen cells from depleted and non-depleted mice for CD3 negative-gated cell population showing NK1.1 versus GFP expression. Analysis was made 24 h after depletion and 3 h following bacterial challenge (representative mouse per each panel). Numbers indicate percentage of gated cells (LR+UR) from CD3⁻ cells. Results are from one representative experiment of two.</p