NKp30 is a functional activation receptor on a subset of rat natural killer cells - PubMed (original) (raw)

NKp30 is a functional activation receptor on a subset of rat natural killer cells

Christine L Hsieh et al. Eur J Immunol. 2006 Aug.

Abstract

NKp30 is a stimulatory receptor on human NK cells implicated in tumor immunity, and is capable of promoting or terminating dendritic cell maturation. To gain a better understanding of NKp30 biology, we have investigated the expression and function of rat NKp30 (rNKp30). We generated stable transfectants of rNKp30 in RNK16 cells, a rat NK lymphoma line, and used a novel panel of mAb against rNKp30 to study this receptor. Using agonistic rNKp30 mAb, we demonstrated that rNKp30 mediates robust IFN-gamma production and cytolytic responses from rNKp30-transfected RNK16 cells. We determined by flow cytometry that rNKp30 is expressed by a subset of primary NK cells isolated from the blood and spleen, and to a lesser extent also on liver NK cells. Stimulation of rNKp30 on primary NK cells led to IFN-gamma production. Liver NK cells expressed low levels of NKp30 and had reduced rNKp30-mediated IFN-gamma responses. During an alloimmune response in vivo, the proportion of the rNKp30(+) NK cell subset in the peripheral blood significantly increased, suggesting that rNKp30 may play an important role during alloactivation. Thus, our data demonstrate that NKp30 is indeed expressed in rodents and is a functional stimulatory receptor in a subset of rat NK cells.

PubMed Disclaimer

Figures

Fig. 1

Anti-rNKp30 mAb specifically detect rNKp30 on transfected cells. (A) Anti-rNKp30 pAb, CA1071, anti-rNKp30 mAb CLH-1 through CLH-9, and isotype control antibodies followed by PE-conjugated secondary antibodies were used to stain parental RNK16 cells (filled histograms) and stably transfected rNKp30+ RNK16 Clone F11 cells (open histograms). (B) Immunoblot analysis of lysates from parental RNK16 and rNKp30+ Clone F11 cells were performed and blots were probed with anti-rNKp30 antibodies as specified and reprobed for β-actin. Molecular masses (kDa) of a protein size ladder are indicated on the left by arrowheads. rNKp30 antibodies detect two prominent bands between 25 and 30 kDa specifically in the rNKp30+ RNK16 clone F11 lysates (_n_=3).

Fig. 2

rNKp30 activation in rNKp30+ RNK16 transfectants induces IFN-γ production and cytolytic activity. (A) rNKp30+ RNK16 clone 2B3 cells express rNKp30 as detected by flow cytometry (right). Anti-rNKp30 antibody, CA1071 (open histogram), followed by PE-conjugated secondary antibodies, and not isotype control antibodies (filled histogram), positively stained clone 2B3 cells. On the left, clone 2B3 cells were treated with immobilized antibodies (10 µg/mL) as indicated on the y-axis for 24 h (_n_=3). Harvested supernatants were analyzed for IFN-γ by ELISA. (B) rNKp30+ RNK16 clone 2B3 and parental RNK16 cells were analyzed for cytolytic activity in 4-h redirected lysis assays against [3H]thymidine-labeled P815 targets (_n_=8). On the left, rNKp30+ RNK16 cells stimulated with rNKp30 antibody, CA1071 (black triangles), showed increased cytotoxicity with increasing E:T ratios. Target cells were resistant to killing when control IgG or parental RNK16 cells were used (white diamonds, white squares, and white circles). (C) Redirected lysis assays were performed with titrated amounts of rNKp30 antibodies (CA1071) or control antibodies at an E:Tof 100:1 (_n_=2). Symbols used in (B) and (C) represent the same effector cells and antibody treatments. (D) Redirected lysis assays were performed to compare the anti-rNKp30 antibodies, CA1071 pAb (black triangles) and CLH-9 mAb (black circles). Both anti-rNKp30 antibodies induce killing as compared to control antibodies (white triangles and white circles). A symbol legend for (D) is boxed on the right. (E) Antibodies specific for activation receptors triggered cytotoxicity from rNKp30+ RNK16 cells. Antibodies (2 µg/106 effector cells) against rNKp30 (black triangles), NKRP1A (black circles), CD30 (black squares), CD8 (asterisks), and control mIgG1 (white circles) and control rabbit IgG (white diamonds) were analyzed in redirected killing assays (_n_=2). A symbol legend for (E) is boxed on the right.

Fig. 3

rNKp30 is expressed on a major subset of F344 spleen NK cells and activation induces IFN-γ production. (A) rNKp30 and rNKp46 expression were analyzed on F344 rat spleen NK cells (NKRPl-FITChi, αβTCR-PerCP−) and T cells (αβTCR-FITC+) by flow cytometry. NKp30 expression was detected using CLH-9-biotin (middle, open histograms) and NKp46 expression was identified with WEN23-biotin (right, open histograms). NCR histograms were overlayed on isotype controls (filled histograms). (B) Splenocytes or PBMC (2.5 × 106/mL, 5 × 105 cells/ well) were cultured in media alone (untreated), or with immobilized antibodies (12 µg/mL). Splenocytes were treated with antibodies against rNKp30 (CLH-8 or CA1071), or control antibodies (mIgG1 or rb IgG) for 11 h. As a control for NK-mediated IFN-γ secretion, PBMC were treated with the anti-rNKp46 mAb, WEN23. Harvested supernatants were assessed for IFN-γ production by ELISA (_n_=5). (C) Treated mononuclear cells were permeabilized and analyzed for IFN-γ with a PE-conjugated anti-IFN-γ mAb, and costained with a FITC-conjugated anti-NKRPl antibody (_n_=3). Each dotplot contains a small box (right) indicating the percent of IFN-γ+ NK cells, and a large box (left) gating on the fraction of non-NK cells expressing IFN-γ. (Top row) Splenocytes were treated with PMA (10 ng/mL) and ionomycin (0.5 µg/mL) (left), mIgG1 isotype control (12 µg/mL) (middle), and anti-rNKp30 mAb CLH-8 (12 µg/mL) (right). (Bottom row) PBMC were treated with mIgG1 isotype control antibodies (12 µg/mL) or anti-rNKp46 mAb WEN23 (12 µg/mL). rNKp30 and rNKp46 activation induced increased IFN-γ production from a subset of NK cells.

Fig. 4

Peripheral blood and liver NK cells express rNKp30. (A) F344 rat PBMC and liver mononuclear cells were analyzed by flow cytometry for rNKp30 (left columns, open histograms) or rNKp46 (right columns, open histograms) using CLH-9 or WEN23 mAb, respectively. Isotype controls (filled histograms) and PE-conjugated secondary reagents were used. Gates were set on NK cells (NKRP1-FITChi αβTCR-PerCP−, upper panels) and T cells (αβTCR-FITC+, lower panels). Representative data from three to four separate experiments are shown. (B) The proportion of rNKp30+ cells in the total NK cell population was quantitated (_n_=3). rNKp30+ NK cells comprise 63.1 ± 4.4% of spleen NK cells, 24.6 ± 7.2% of peripheral blood NK cells, and 14 ± 15.5% of liver NK cells. (C) PBMC (left) and liver mononuclear cells (right) were cultured in media alone (untreated), or treated with platebound rNKp30 antibodies (CLH-9 or CA1071), or control antibodies (mIgG1 or rb IgG) for 24 h and supernatants were assessed for IFN-γ by ELISA.

Fig. 5

The percent of rNKp30+ NK cells in the peripheral blood increases during allo-activation. PBMC were isolated from normal Lewis animals or from recipients of syngeneic (Lewis to Lewis) and allogeneic (DA to Lewis) liver grafts 3 and 7 days post transplant. rNKp30 expression on peripheral blood NK cells (NKRPlhi αβTCR−) was examined by flow cytometry. (A) NK cells from normal Lewis rat PBMC (left), and peripheral blood NK cells from syngeneic graft recipients (middle column) and allograft recipients (right column) were stained with the CLH-9 mAb (open histograms), and compared to staining with the isotype control antibody (filled histograms). Representative data are shown. (B) The mean percentage of rNKp30+ NK cells in the peripheral blood NK cell population was quantified and is reported ± SEM. The mean percent of rNKp30+ NK cells in normal Lewis rats is 23 ± 3% (white bar) (_n_=2). In syngeneic transplant recipients (striped bars), the mean percent of rNKp30+ NK cells is 29.9 ± 4.7% on day 3 (syn day 3, _n_=3), and 21.5 ± 8.5% on day 7 (syn day 7, _n_=2). In the allogeneic transplant group (gray bars), the mean percent of rNKp30+ NK cells increases to 41.9 ± 1.76% by day 3 (allo day 3, _n_=6), and decreases to 19 ± 4.9% by day 7 (allo day 7, _n_=3). Fisher's PLSD analysis determined significance as _p_≤ 0.05 (*_p_=0.0155).

References

1. Cerwenka A, Lanier LL. Natural killer cells, viruses and cancer. Nat. Rev. Immunol. 2001;1:41–49. -PubMed
1. Raulet DH. Interplay of natural killer cells and their receptors with the adaptive immune response. Nat. Immunol. 2004;5:996–1002. -PubMed
1. Salazar-Mather TP, Hamilton TA, Biron CA. A chemokine-to-cytokine-to-chemokine cascade critical in antiviral defense. J. Clin. Invest. 2000;105:985–993. -PMC -PubMed
1. Obara H, Nagasaki K, Hsieh CL, Ogura Y, Esquivel CO, Martinez OM, Krams SM. IFN-gamma, produced by NK cells that infiltrate liver allografts early after transplantation, links the innate and adaptive immune responses. Am. J. Transplant. 2005;5:2094–2103. -PMC -PubMed
1. Martin-Fontecha A, Thomsen LL, Brett S, Gerard C, Lipp M, Lanzavecchia A, Sallusto F. Induced recruitment of NK cells to lymph nodes provides IFN-gamma for T(H)1 priming. Nat. Immunol. 2004;5:1260–1265. -PubMed

NKp30 is a functional activation receptor on a subset of rat natural killer cells - PubMed (original) (raw)