1 A hyporesponsive subset of rat NK cells negative for Ly49s3 and NKR-P1B are precursors to the functionally mature NKR-P1B+ subset.
Amanda Sudworth*,†, John T. Vaage†,‡, Marit Inngjerdingen*,†,1, and Lise Kveberg†, ‡,1
*Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
†Department of Immunology, Oslo University Hospital, Oslo, Norway
‡University of Oslo, Institute of Clinical Medicine, Oslo, Norway
1Shared last authorship
Summary sentence: In this paper we show that functional competence of hyporesponsive rat NK cells is associated with acquisition of the inhibitory receptor NKR-P1B.
Running title: NKR-P1B confer function to hyporesponsive NK cells
Full correspondence:
Dr. Marit Inngjerdingen, Department of Immunology, Oslo University Hospital, P.O.Box 4950 Nydalen, N-0424 Oslo, Norway.
Tel: +47 23 07 35 00, Fax: +47 23 07 35 10, E-mail: [email protected]
Keywords: NKR-P1B, Ly49, Eomes, NK cells, CD11b
Total character count: 22428. Total number of figs: 8. Total number of color figs: 0. Total number of references: 33. Total number of words in Abstract: 187. Total number of words in summary sentence: 22.
2 Abbreviations
Clr, C-type lectin-related molecules; Eomes, Eomesodermin; IFN-γ, interferon gamma;
IL, interleukin; MHC, major histocompatibility complex I.
3 Abstract
Rat NK cells are divided into major subsets expressing either Ly49 receptors or the inhibitory NKR-P1B receptor in conjunction with NKG2A/C/E receptors. A minor subset of NKp46+ cells lacking expression of both Ly49 receptors and NKR-P1B is present in blood and spleen, and associated with decreased functional competence. We hypothesized that this subset may represent precursors to Ly49+ and/or NKR-P1B+ NK cells. When cultured in vitro in IL-2 and IL-15, or adoptively transferred to syngeneic hosts, a portion of NKR-P1B-Ly49s3- cells transformed to express NKR-P1B but very little Ly49s3. Acquisition of NKR-P1B by NKR-P1B-Ly49s3- cells coincided with increased degranulation. Also, while NKR-P1B-Ly49s3- cells highly proliferate, proliferative activity was reduced upon acquisition of NKR-P1B at comparable levels to bona fide NKR-P1B+ NK cells. A fraction of NKR-P1B-Ly49s3- cells remained negative for NKR-P1B both in vitro and after adoptive transfer in vivo. The majority of NKR-P1B- Ly49s3- cells expressed the transcription factor Eomesodermin and NK cell markers, indicating that these cells represent conventional NK cells. Our findings suggest that the NKR-P1B-Ly49s3- NK cells are precursors to NKR-P1B single positive cells, and that functional competence is acquired upon expression of NKR-P1B.
4 Introduction
NK cells target infected, malignant, or allogeneic cells. These responses are controlled by activating or inhibitory receptors. The Ly49 receptors (rodents), killer immunoglobulin- like receptors (KIRs; human), or CD94/NKG2 receptor families recognize MHC class I, and lack of MHC class I recognition leads to target cell lysis due to lack of engagement of inhibitory receptors for MHC class I. This process is known as “missing self”[1]. In addition, stress-induced ligands may be expressed, leading to engagement of activating receptors, such as NKG2D, the natural cytotoxicity receptors NKp30, NKp44, and NKp46, or activating members of the Ly49, CD94/NKG2, or KIR receptor families. The NKR-P1 receptors, expressed by both rodent and human NK cells, may perform a parallel missing self-reaction upon interactions with their ligands C-type lectin-related molecules (Clr (rodents); LLT1 (human)) [2-4]. In particular, Clr-b expression has been shown to decrease during viral infection or tumor development in the mouse similar to MHC class I [5], and result in loss of inhibitory signal to NK cells via the inhibitory NKR-P1B receptor [6]. Inhibitory receptor/MHC class I interactions are also important for developing functional NK cells through the process of education [7-9], where increasing numbers of inhibitory receptors on NK cells correlate with increased functional ability [10]. Without this interaction, NK cells remain anergic or hyporesponsive [7,8]. Recently, it was suggested that NKR-P1B and its ligand Clr-b serve a similar function in the mouse [6].
NK cell receptors are expressed at different stages during development. In the rat, activating NKR-P1A is the first receptor detected in NK cells from newborns, followed by the inhibitory NKR-P1B, while expression of Ly49 receptors is modest at birth
5 [11,12]. Similarly, NKR-P1s are expressed before Ly49s in the mouse [13,14], while human inhibitory NKR-P1A is one of the earliest receptors expressed by immature human NK cells [15].
Circulating NK cells consists of subsets of cells of varying degrees of maturity. In the mouse, it was previously demonstrated that immature and mature NK cells are distinguished by differential expression of CD27 and CD11b, where NK cells mature in the following sequence: CD11blowCD27low → CD11blowCD27hi → CD11bhighCD27high → CD11bhighCD27low [16,17]. Different developmental stages of rat NK cells have not yet been characterized, but the circulating pool of rat NK cells can be divided based on almost mutually exclusive expression of NKR-P1B or Ly49s3 [11,18]. The CD94/NKG2A receptor is co-expressed within the NKR-P1B+ subset and the Ly49 receptors are restricted to the Ly49s3+ subset, thus dividing the major MHC I-binding receptors into distinct subsets. This partial separation of NKR-P1B versus Ly49 expression is also observed in the mouse [13], and in humans where subsets of CD56dim NK cells express either NKG2A or KIR though not on a mutually exclusive basis [19- 21]. Rat NKR-P1B and Ly49s3 single positive cells both show similar levels of interferon-γ (IFN-γ) production and cytotoxicity, except towards allogeneic targets, which are only recognized and killed by Ly49s3+ NK cells [11,18].
We previously identified a subset of rat NK cells lacking expression of both NKR-P1B and Ly49 receptors. This subset is present in all tissues, and we showed that in the blood, this subset was hyporesponsive in terms of IFN-γ production and cytotoxicity [22]. Here, we have characterized this subset further, and show that its functional capacity is linked to induced expression of NKR-P1B. We further show that established
6 maturation markers for mouse NK cells, CD27 and CD11b, are not optimal markers to distinguish immature from mature rat NK cells.
7 Materials and Methods
Animals
Eight to 12-week-old rats of the strains PVG (RT1c, CD45.1), PVG.7B (RT1c, CD45.2), PVG.1U (RT1u, CD45.1), and PVG.R23 (RT1u-a-av1, CD45.1) were used. PVG.1U and PVG.R23 are genetically identical to PVG with the exception of the MHC complex, express the same frequencies of NK cell receptors, and are used interchangeably. The rats have been maintained at the Department of Comparative Medicine, Institute of Basic Medical Sciences, University of Oslo for more than 20 generations. The Department of Comparative Medicines institutional veterinarian has established the rules for feeding, monitoring, handling, and sacrifice of animals in compliance with regulations set by the Ministry of Agriculture of Norway and "The European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes". The institutional veterinarian has delegated authority from the Norwegian Animal Research Authority (NARA). The laboratory animal facilities are subject to a routine health- monitoring program and tested for infectious organisms according to a modification of Federation of European Laboratory Animal Science Associations (FELASA) recommendations. The use of animals for this study was approved by NARA, license number 6010 (in vitro) and 6060 (in vivo). Rats were euthanized by asphyxiation with CO2 in a chamber allowing controlled input of gas, to reduce suffering.
Antibodies and reagents
Antibodies against NKp46 (Wen23-Pacific blue (PB)), NKR-P1A (3.2.3-PB), Ly49s5/i5 (Fly5-biotin), KLRH1 (STOK9-biotin), CD62L (OX85-biotin), CD25 (OX39-biotin),
8 CD93 (Lov3-biotin), CD8 alpha (OX8-biotin), CD90 (OX7-biotin), NKR-P1B (STOK27-Alexa 647), Ly49s3 (DAR13-FITC, -biotin), CD107a (SIM1-FITC) and CD45.2 (His41-FITC, biotin) were generated in our laboratory and conjugated according to standard protocols. Ab against NKG2A/C/E (Wen28-biotin) was a kind gift from Professor E. Dissen, Oslo (manuscript in preparation). Abs against CD3 (G4.18-FITC), NKR-P1A (10/78-PE), CD11b (WT.5-biotin), CD27 (LG.3A10-PE), NKG2D (9C1164- PE), IFN-γ (DB1-PE), FITC hamster IgG polyclonal isotype, CD195 (C34-3448-biotin;
isotype control), mouse IgA isotype (M18-254-biotin) and streptavidin-PerCP were from BD Biosciences (Franklin Lakes, NJ). Anti-Eomes (Dan11mag-PE) and Rat IgG2a isotype control (eBR2a-PE) were from eBioscience (United States). Anti-CD117 (RA14132) was from Neuromics (Edina, MN), FITC anti-rabbit IgG from Jackson Immunoresearch Laboratories (Suffolk, UK), and anti-CD127 (FAB5607P-PE) from R&D Systems (Minneapolis, MN). Anti-KLH (G1-2-2-biotin; rat IgG1 isotype control) was from GeneTex (Irvine, CA) and Pyk2 (17H4L19-unconjugated; rabbit IgG isotype control) was from Thermo Fisher Scientific (Waltham, MA). Rat recombinant (rr) IL-2 was obtained from dialyzed cell culture supernatants of a CHO cell line stably transfected with a rat IL-2 expression construct. rrIL-12 was purchased from Biosource (Cammarillo, CA), rrIL-15 from PeproTech (Rocky Hill, NJ), and rrIL-18 from R&D Systems.
Flow cytometry and cell sorting
Spleens were crushed through 70 µM filters, and mononuclear cells isolated using Lymphoprep (Axis-Shield, Oslo, Norway) overlays. Remaining red blood cells were lysed, and further incubated in nylon wool (Polysciences, Warrington, PA) columns at
9 37°C to remove B cells and myeloid cells. For intracellular IFN-γ and sorting experiments, NK cells were further enriched by depleting T cells, B cells and myeloid cells using Pan-mouse IgG Dynabeads (ThermoFisher Scientific) coupled to anti-SIRPα, (OX41), anti-TCRαβ (R73), anti-CD5 (OX19), anti-Ig κ chain (OX12), and anti- CD45RA (OX33), resulting in 50-70 % NKR-P1A+CD3- NK cells. Cell sorting was performed with a FACS Aria (BD Biosciences), while all other flow cytometry analyses were performed using FACSCanto, LSR II, or FACSCalibur (all BD Biosciences). Data analyses were performed by the FlowJo 7.6.5 software (TreeStar, Ashland, OR). NK cells were gated as either NKR-P1A+CD3-, NKp46+, or NKR-P1Abright after prior gating on lymphocytes based on forward and side scatter characteristics (Suppl. Fig. 3). NK cell gating strategy is indicated in the Figure legends.
Cell culture
Sorted NKR-P1B-Ly49s3-, Ly49s3-NKR-P1B+ and NKR-P1B-Ly49s3+ (all NKR- P1A+CD3-) NK cell subsets were cultured in complete RPMI (cRPMI; RPMI-1640 supplemented with 10 % heat inactivated FBS, 1 % streptomycin/penicillin, 1 mM sodium pyruvate, 50 µM 2-Mercaptoethanol) containing 10 ng/ml IL-15 and 1/100 IL-2 for various time points. The mouse T cell lymphoma cell line YAC-1 (ATCC TIB-160) was maintained in cRPMI.
Adoptive transfer
NKR-P1B-Ly49s3-, NKR-P1B+Ly49s3-, and NKR-P1B-Ly49s3+ NKp46+ NK cell subsets were sorted from PVG.7B rats (CD45.2+) and injected into PVG rats (CD45.1+) that had
10 been irradiated at 6 Gy 24 hrs before transfer. Similarly, non-irradiated PVG hosts were infused with sorted CD11blowCD27low, CD11blowCD27high, CD11bhighCD27high, and CD11bhighCD27low NKp46+ NK cells from PVG.7B rats. Two weeks after transfer, spleen and blood lymphocytes were isolated from the hosts and analyzed by flow cytometry.
Intracellular IFN-γ staining
Enriched NK cells were incubated with IL-12 (0.02 ng/ml), IL-15 (0.1ng/ml), IL-18 (0.25 ng/ml), and IL-2 (1/100) for 18 hrs. IL-2 and IL-15 were used as a negative control.
Brefeldin A was added for the last three hours of the experiment at a final concentration of 10 µg/ml. Cells were washed and surface receptors were stained with the appropriate antibodies. The cells were fixed in 2% paraformaldehyde for 10 min, permeabilized with 0.5% saponin in PBS for 20 min, and then stained and analyzed by flow cytometry for intracellular levels of IFN-γ in NKp46+ cells. When co-staining for Eomes and IFN-γ, enriched NK cells were incubated with IL-12 (0.2 ng/ml), IL-18 (2.5 ng/ml), IL-15 (1 ng/ml), and IL-2 (1/50) for 6 hrs, with Brefeldin A added after 3 hours. Staining of Eomes and IFN-γ were done using the FoxP3/Transcription Factor Staining Buffer Set from eBioscience (San Diego, CA) according to the manufacturer’s instructions.
Degranulation assay
Sorted NKR-P1B-Ly49s3-, NKR-P1B+Ly49s3-, and NKR-P1B-Ly49s3+ NKp46+ NK cell subsets were incubated with YAC-1 target cells at a 1:1 ratio in 50 µl cRPMI in the presence of anti-CD107a antibody for 4 hrs. For plate-bound antibody stimulation, NK cells were cultured in the presence of CD107a antibodies in flat bottom 96-well plates
11 pre-coated with 10 µg/ml of anti-NKR-P1A. Golgistop (BD Biosciences, Franklin Lakes, NJ) was added for the last 3 hrs of incubation to prevent degradation of internalized CD107a. Cells were analyzed by flow cytometry.
CFSE proliferation assay
Sorted NKR-P1B-Ly49s3-, NKR-P1B+Ly49s3- and NKR-P1B-Ly49s3+ NKp46+ NK cell subsets were stained with 5 µM CFSE (Sigma-Aldrich, St.Louis, MO) for 10 minutes at 37°C and washed with PBS and 2% FBS. The cells were cultured in IL-15 (10 ng/ml) and IL-2 (1/100) for 4 to 7 days, and analyzed by flow cytometry.
Statistical analysis
Graphics and statistical analysis were performed with the GraphPad Prism software. Data are presented as the mean ± standard error of the mean (SEM). Statistical significance was calculated using an unpaired t-test with Welch’s correction. P values less than 0.05 were considered statistically significant.
12 Results
NKR-P1B-Ly49s3- NK cells are hyporesponsive.
Rat NK cells consist of two major, fully functional subsets expressing either Ly49s3 or NKR-P1B. We previously observed a small subset lacking expression of both NKR-P1B and Ly49s3 in all tested tissues (Suppl. Fig. 1), and we have previously shown decreased IFN-γ production and cytotoxicity by blood NKR-P1B-Ly49s3- NK cells compared to the two other NK cell subsets [22]. Here, we recapitulated these findings, showing lower IFN-γ production from splenic NKR-P1B-Ly49s3- NK cells after overnight stimulation with a cocktail of IL-2, IL-12, IL-15, and IL-18 as compared to the NKR-P1B+ and Ly49s3+ single-positive subsets (Figs. 1A and B). Also, lower degranulation by splenic NKR-P1B-Ly49s3- NK cells in response to the sensitive tumor target YAC-1 was observed (Figs. 1C and D). In contrast, cytolytic activity of sorted NKR-P1B-Ly49s3- NK cells was not lower than that of Ly49s3+ NK cells, but only of NKR-P1B+ NK cells (Fig.
1E). The reason for this apparent discrepancy between the degranulation and cytotoxicity assays is currently unresolved.
NKR-P1B-Ly49s3- cells differentiate to express NKR-P1B.
Lack of NKR-P1B or Ly49 expression by NKR-P1B-Ly49s3- NK cells prompted us to test whether expression of these receptors could be induced. Sorted NKR-P1B-Ly49s3- cells were cultured in IL-15 and IL-2 for up to 28 days. After 6 days, the majority expressed NKR-P1B, while only a minor fraction expressed Ly49s3 (Fig. 2A). Sorted Ly49s3+ and NKR-P1B+ NK cells remained stable in culture until the end-point at day 28, with only minor subsets co-expressing either NKR-P1B or Ly49s3 respectively in
13 each culture (Fig. 2A). The expression of NKR-P1B by NKR-P1B-Ly49s3- cells was detected at as early as 1 hour of culture (Fig. 2B). By 24 hours the expression of NKR- P1B was comparable to the levels observed by day 6. Many cells remained NKR-P1B- Ly49s3- after culture, indicating that the population is heterogeneous. To test if the remaining NKR-P1B-Ly49s3- cells could be induced to express NKR-P1B, we purified the remaining NKR-P1B-Ly49s3- cells after day 7 of culture. The remaining NKR-P1B- Ly49s3- cells were unable to up-regulate NKR-P1B in vitro (data not shown), indicating there are at least two distinct subsets within the NKR-P1B-Ly49s3- subset.
To determine if the NKR-P1B-Ly49s3- cells can be induced to express NKR-P1B also in vivo, splenic NKR-P1B-Ly49s3- cells were sorted from a CD45.2 donor rat and injected into an irradiated CD45.1 host (Fig. 3A). In parallel, the NKR-P1B+ and Ly49s3+ subsets were also sorted and injected into separate hosts. After two weeks, the spleen and blood were harvested and the donor CD45.2+ cells were identified with an antibody against CD45.2 (Fig. 3B; Suppl. Fig. 2). A major proportion of the transferred donor CD45.2+ NKR-P1B-Ly49s3- cells recovered from the spleen had changed to express NKR-P1B, but remained negative for NKR-P1B and Ly49s3 in peripheral blood (Fig.
3B). Transferred NKR-P1B+ and Ly49s3+ cells remained stable in the spleen, but in the blood some NKR-P1B+ cells had developed into an NKR-P1Bbright phenotype. Overall, the data suggest the NKR-P1B-Ly49s3- subset can develop into NKR-P1B+ NK cells and may represent less differentiated NK cells.
14 Up-regulation of NKR-P1B coincides with increased degranulation
We next tested whether newly formed NKR-P1B single positive cells gained similar functional properties as bona fide NKR-P1B+ cells in degranulation assays in response to YAC-1 targets or plate bound anti-NKR-P1A. NKR-P1B-Ly49s3- and NKR-P1B+ cells were sorted and cultured overnight in IL-15 and IL-2. Degranulation was measured by gating separately on cells that remained NKR-P1B-Ly49s3-, and cells that up-regulated NKR-P1B. Cells that changed to express NKR-P1B showed increased degranulation after stimulation with plate-bound NKR-P1A, compared to cells that remained negative (Fig.
4A and B). The level of degranulation was comparable to that of bona fide NKR-P1B+ cells indicating that the newly formed NKR-P1B+ cells gain similar functional potential.
In contrast, no differences in degranulation against YAC-1 targets between NKR-P1B- Ly49s3- or newly formed NKR-P1B+ NK cells were observed (Fig. 4C and D). As responses to YAC-1 are mainly facilitated via NKG2D, this discrepancy could reflect different responses of NKR-P1A and NKG2D.
Cells remaining double negative have increased proliferative capacity
We next compared the proliferative activity of remaining NKR-P1B-Ly49s3- cells versus newly formed NKR-P1B+ NK cells. CFSE-stained, sorted NKR-P1B-Ly49s3-, NKR- P1B+, and Ly49s3+ cells were cultured in IL-2 and IL-15 for up to 7 days. After 7 days, cells from the NKR-P1B-Ly49s3- cell culture had higher proliferative capacity than Ly49s3+ or NKR-P1B+ NK cells (Fig. 5A). When comparing cells within the NKR-P1B- Ly49s3- subset that expressed NKR-P1B after culture or not, we observed that
15 proliferation was higher for cells that remained negative for NKR-P1B, while newly formed NKR-P1B+ cells were similar to the bona fide NKR-P1B+ cells (Fig. 5B).
NKR-P1B-Ly49s3- cells represents conventional NK cells
As the NKR-P1B-Ly49s3- NK cells were heterogeneous in ability to up-regulate NKR- P1B, we tested whether this subset uniformly represent conventional NK cells.
Phenotypic analysis revealed similar expression levels of NKp46 and CD8α, and slightly reduced levels of NKG2D compared to Ly49s3+ or NKR-P1B+ NK cells (Fig. 6). The transcription factor Eomesodermin (Eomes) that is expressed by conventional NK cells but not by other innate lymphoid cells [23], was also expressed by the majority of NKR- P1B-Ly49s3- cells (Fig. 6). Interestingly, distinct populations of Eomesbright and Eomesdim subsets was clearly observed, which potentially could explain some of the heterogeneity of NKR-P1B-Ly49s3- cells. Moreover, a third of the NKR-P1B-Ly49s3- cells expressed NKG2A/C/E receptors. Unfortunately, we were unable to determine whether NKG2A/C/E expression was localized within the Eomesbright or the Eomesdim subset as the NKG2A/C/E antibody is sensitive to fixation. Overall, the data indicates that NKR- P1B-Ly49s3- cells represent conventional NK cells.
NKR-P1B-Ly49s3- NK cells express a phenotypic profile associated with mature cells As the NKR-P1B-Ly49s3- NK cells can further differentiate, we tested whether this subset expressed markers associated with immature NK cells, such as CD117 and CD127 [24,25]. A small fraction of NKR-P1B-Ly49s3- cells expressed CD117, in contrast to NKR-P1B+ or Ly49s3+ subsets, while being negative for CD127 (Fig 7A). Further, the
16 combined staining of CD27 and CD11b showed that most NKR-P1B-Ly49s3- NK cells were CD11b single positive indicative of a mature phenotype (Fig. 7B), which were surprising as our data suggested the cells were less differentiated.
CD11b and CD27 have been characterized as NK cell maturation markers in the mouse [16,17], and we tested whether these markers are applicable for rat NK cells.
Subsets of splenic NK cells expressing CD27 and/or CD11b were sorted from CD45.2+ donor rats and injected into separate CD45.1+ hosts. We observed increased expression of CD11b by the transferred CD11blowCD27low cells (Fig. 8A and B), but the staining intensity was rather low when compared to the intensity of CD11b by host NK cells (Fig.
8C), and a large fraction remained CD11blowCD27low. Transferred CD11bhighCD27low cells remained remarkably stable, suggesting that these cells are unable to express CD27 (Fig. 8A and B). For CD27highCD11blow/high cells the patterns were more complex. The transferred CD11blowCD27high and CD11bhighCD27high appeared to predominantly retain CD27, although a significant fraction of CD11b single positive cells developed from CD11bhighCD27high cells in blood (Fig. 8A and B). We conclude that the combined use of CD27 and CD11b are not useful to define stages of maturing rat NK cells. Based on available markers we can therefore not clearly define whether NKR-P1B-Ly49s3- NK cells are mature or not.
17 Discussion
In this study, we have characterized an NK cell subset defined by the absence of NKR- P1B andLy49s3 that has the characteristics of a less differentiated cell with potential for further differentiation to functionally competent cells linked to up-regulation of NKR- P1B.
A large fraction of NKR-P1B-Ly49s3- NK cells were induced to express NKR- P1B but very little Ly49s3. Up-regulation of NKR-P1B coincided with increased functional competence and reduced proliferative activity. Both human and mouse NK cells express NKR-P1s at the precursor stage, activating NKR-P1C in the mouse and inhibitory NKR-P1A in the human [26]. Activating NKR-P1A and inhibitory NKR-P1B are also present early in NK cell development in the rat [12]. Expression of Ly49s and KIRs are expressed later in ontogeny in both rodents and human, implying that in these species NKR-P1s are expressed before MHC class I binding receptors [14,15]. NKR- P1B-Ly49s3- cells follow this expected line of differentiation, with early NKR-P1B expression followed by Ly49 at later stages in vitro. Expression of NKR-P1B was detected after only 1 hour culture in IL-2 and IL-15. Semi-quantitative RT-PCR studies on sorted NKR-P1B-Ly49s3- blood NK cells showed presence of NKR-P1B transcripts (data not shown) further rationalizing the hypothesis that some of the cells may have intracellular NKR-P1B protein and are pre-programmed to express NKR-P1B.
NKR-P1B was recently shown to have a possible role in education/licensing [6].
With no Ly49 or NKR-P1B expression, NKR-P1B-Ly49s3- NK cells may be hyporesponsive due to a limited ability to interact with self and thereby become educated through inhibitory receptors for self. Induction of NKR-P1B expression coincided with
18 increased degranulative ability in response to NKR-P1A, which may be the result of an education process occurring when NKR-P1B interacts with its ligand Clr11. Gene transcripts for Clr11 are found in most hematopoietic cells, including activated natural killer cells [27,28]. This indicates that the NKR-P1B-Ly49s3- cells may express Clr11 allowing the purified population to become educated in vitro upon expression of NKR- P1B. However, it was surprising that no difference in degranulation was measured between the newly expressing NKR-P1B+ cells and the cells that remained NKR-P1B- Ly49s3- when YAC-1 targets were used as a stimulus. However, responses to YAC-1 are mainly facilitated via NKG2D, and it may be that NKG2D-dependent responses are uncoupled from the putative Clr-dependent licensing of NKR-P1B-Ly49s3- cells.
Upon adoptive transfer of NKR-P1B-Ly49s3- cells, we recovered cells that expressed NKR-P1B in the spleen, but not in the blood. Possibly, the NKR-P1B+ cells that develop from NKR-P1B-Ly49s3- NK cells are retained in certain tissues such as the spleen. We have previously shown that a subset of NK cells expressing a NKR-P1Bbright phenotype is preferentially found in certain tissues such as the blood, liver, Peyer’s patches, mesenteric lymph nodes, and thymus [22]. Possibly, the tissue microenvironment plays an important role for migration to or retention of the newly expressing NKR-P1B cells within the spleen. The remaining NKR-P1B-Ly49s3- cells may not be as tissue-restricted as the newly expressing NKR-P1B+ cells and thereby found in both the blood and the spleen. Even though we normally find NKR-P1B+ cells both in the spleen and blood, we speculate that the newly expressing NKR-P1B+ cells can have different properties than the bona fide NKR-P1B+ found in the blood.
19 It is interesting that Ly49s3+ NK cells emerged from culturing NKR-P1B-Ly49s3- NK cells in vitro but not in vivo, although the numbers of Ly49s3+ NK cells in vitro was low. We find it unlikely that the population of Ly49s3+ NK cells arose from a small contaminating population of Ly49s3+ NK cells during sorting, as we would expect that such contamination also would have been evident after in vivo adoptive transfers. The rats were irradiated prior to adoptive transfer, and we have previously observed that irradiation leads to efficient expansion of Ly49s3+ NK cells [29]. In light of the few Ly49s3+ NK cells that do develop, we speculate that there may be precursors to Ly49s3+ NK cells within the NKR-P1B-Ly49s3- subset, but that there is a lack of factors necessary for driving expression of Ly49 receptors under our experimental conditions. In the mouse, Ly49 up-regulation has been demonstrated in vitro when grown on bone marrow stromal cells [30-32]. We tried growing NKR-P1B-Ly49s3- cells on rat mesenchymal stem cells but were unable to achieve biological levels of Ly49s3 expression (data not shown).
The NKR-P1B-Ly49s3- subset was heterogeneous with respect to expression of the transcription factor Eomes that is shown to distinguish conventional NK cells from other ILCs [23]. Such varied expression of Eomes has also been observed within the CD56dim subset of human NK cells, and different expression levels linked to maturation status [33]. We have found that after four days of culture of sorted NKR-P1B-Ly49s3- cells with IL-2 and IL-15 all cells become Eomesbright similarly to bona fide NKR-P1B+ NK cells (data not shown). This indicates that Eomes expression in rat NK cells may vary depending on differentiation level or stimulus.
20 In the mouse, expression of CD27 and CD11b has been shown to change as the NK cell develops from an immature CD11blowCD27low cell progressing to CD11blowCD27high, then CD11bhighCD27high, and finally to a mature CD11bhighCD27low cell [17]. Therefore, it was surprising that NKR-P1B-Ly49s3- cells expressed high levels of CD11b. In the mouse, NK cell function has been shown to peak in the CD11bhighCD27high subset and decrease in the CD11bhighCD27low cells [16]. Since a major fraction of NKR-P1B-Ly49s3- cells are CD11bhighCD27low it could be plausible that the low function of the NKR-P1B-Ly49s3- cells is due to the large CD11bhighCD27low population within. However, this is opposed by the fact that in culture the newly formed NKR-P1B+ cells gain increased functional capability while maintaining the CD11bhighCD27low phenotype (data not shown). By adoptively transferring the different CD11b/CD27 subsets we observed that NK cell maturation defined by CD27 and CD11b expression is not clear cut in the rat and conclude that CD27 and CD11b cannot be used as maturation markers for rat NK cells. Even though we could not define maturation status of the NKR-P1B-Ly49s3- subset based on phenotypic markers, it is clear that this subset is a precursor to the NKR-P1B+ subset due to its induced expression of NKR-P1B both in vitro and in vivo. A recent study in mouse demonstrated that sorted NKR-P1B negative cells changed to express NKR-P1B when cultured in IL-2 suggesting that this developmental pathway takes place in the mouse as well [6].
In conclusion, we report that a subset of rat NK cells lacking expression of NKR- P1B and Ly49s3 with decreased function that develop into functionally mature NKR- P1B+ NK cells both in vivo and in vitro. Future studies are required to further understand the development of distinct subsets of NK cells in different tissues.
21 Authorship
AS, JTV, LK, and MI conceived and designed the study; AS acquired data; AS, JTV, MI, and LK analyzed and interpreted data; AS, MI, and LK drafted the manuscript; AS, JTV, MI, and LK approved the final version of the manuscript.
22 Acknowledgements
This work was supported by the Institute of Basic Medical Sciences, University of Oslo, the South-Eastern Norway Regional Health Authority, Anders Jahres Fond til Vitenskapens Fremme, Legat for fremme av kreftforskningen, and Legatet til Henrik Homans Minde. The authors thank Ulla Heggelund and Dr. Ke-Zheng Dai for technical assistance.
23 Conflict of interests
The authors declare no conflict of interests.
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30 Figure legends
Figure 1. Splenic NKR-P1B-Ly49s3- NK cells are hyporesponsive.
Enriched NK cells were cultured overnight with IL-2, IL-15, IL-18, and IL-12 or with IL- 2 and IL-15 as a control. Intracellular IFN-γ was measured gating on NKR-P1B-Ly49s3-, Ly49s3+ or NKR-P1B+ NKp46+ NK cells. One representative experiment of 4 independent experiments is shown in (A), and the mean and standard deviation from 4 experiments shown in (B). Sorted NKR-P1B-Ly49s3- (80-95 % purity), Ly49s3+ (84-97%
purity), or NKR-P1B+ (99% purity) NKp46+ NK cells subsets were cultured with or without YAC-1 target cells and an antibody against CD107a. Data from one representative experiment is shown in (C), and the mean and standard deviation of 3 independent experiments shown in (D). (E) Cytotoxicity was measured using sorted NKR-P1B-Ly49s3- (74-76% purity), Ly49s3+ (81-82% purity), or NKR-P1B+ (70-87%
purity) NKp46+ NK cell subsets, incubated with YAC-1 target cells in a 4 hour 51Cr release assay. Data represent the mean values of triplicates ±SEM from two experiments.
Statistical analysis was performed with an unpaired t-test with Welch’s correction;
significant p-values are indicated in the figures.
Figure 2. NKR-P1B-Ly49s3- cells express NKR-P1B and Ly49s3 upon culture in IL- 2 and IL-15.
(A) NKR-P1B-Ly49s3-, Ly49s3+, and NKR-P1B+ NKp46+ NK cells were sorted and cultured in IL-2 and IL-15 for the indicated times, and NKR-P1B and Ly49s3 expression was measured by flow cytometry. (B) NKR-P1B-Ly49s3- NKp46+ NK cells were sorted and cultured in IL-2 and IL-15 for the indicated time points, and NKR-P1B and Ly49s3
31 expression was measured by flow cytometry. The 0-hr time point represents the sort purity. Data shown are from one representative experiment of 1-5 independent experiments.
Figure 3. NKR-P1B-Ly49s3- cells express NKR-P1B after adoptive transfer in vivo.
(A) Purity of sorted NKR-P1B/Ly49s3 subsets from CD45.2+ donor rats. (B) Sorted NKR-P1B-Ly49s3-, NKR-P1B+ and Ly49s3+ CD45.2+ donor NK cells were adoptively transferred into an irradiated CD45.1 host. Two weeks after transfer, the spleen and blood were isolated and donor cells were identified with an antibody against CD45.2, and NK cells further gated as NKR-P1A+CD3- cells. Data shown are from one experiment of 3 independent experiments*. (*Two experiments lack data for one of the transferred subsets, but all subsets are represented by at least two comparable data sets).
Figure 4. Expression of NKR-P1B cells is associated with increased degranulation.
NKR-P1B-Ly49s3- (71-88% purity) and NKR-P1B+ (89-99% purity) NKp46+ NK cell subsets were sorted and cultured overnight in IL-2 and IL-15. The following day the subsets were stimulated with (A and B) plate bound anti-NKR-P1A or (C and D) YAC-1 target cells. In panels B and D, the mean ±SD from 6 independent experiments is shown, and statistical significance calculated with an unpaired t-test with Welch’s correction;
significant p-values are indicated in the figure.
32 Figure 5. NKR-P1B-Ly49s3- NK cells has high proliferative capacity that is reduced upon acquisition of NKR-P1B.
(A) NKR-P1B-Ly49s3- (73-83% purity), NKR-P1B+ (80-97% purity), and Ly49s3+ (84- 97% purity) NKp46+ NK cells were sorted and stained with CFSE. The number of dividing cells was determined by flow cytometry after 4 and 7 days of culture in IL-2 and IL-15. Data shown are from one experiment representative of 3-4 independent experiments. (B) Proliferation of NKR-P1B+ and NKR-P1B- cells after culture of sorted NKR-P1B-Ly49s3- NKp46+ NK cells (purity 73-83%) for 4 days in IL-2 and IL-15 as measured by CFSE dilution. Data shown are from one experiment representative experiment of three.
Figure 6. NKR-P1B-Ly49s3- NK cells represent conventional NK cells.
White histograms show expression of NK cell receptors and markers by NKR-P1B- Ly49s3-, NKR-P1B+, and Ly49s3+ NK cells. Gray histograms represent isotype controls.
NK cells were gated as NKp46+ NK cells, except when assessing NKp46 expression, where NK cells were gated as NKR-P1A+CD3- NK cells. Data shown are from one representative experiment of 3 independent experiments.
Figure 7. NKR-P1B-Ly49s3- NK cells are mainly CD11b+.
(A) Expression of CD117 and CD127 by NKR-P1B-Ly49s3-, NKR-P1B+, and Ly49s3+ NKp46+ NK cells (white histograms; isotype control staining in gray histograms). (B) Expression of CD27 and CD11b by NKR-P1B-Ly49s3-, NKR-P1B+, and Ly49s3+
33 NKp46+ NK cells. Data in (A) and (B) are representative of three independent experiments.
Figure 8. CD27 and CD11b are not clear markers for maturing NK cells in the rat.
(A) Splenic CD11blowCD27low, CD11blowCD27high, CD11bhighCD27high, and CD11bhighCD27low NKp46+ NK cells were sorted from CD45.2+ donor rats, and injected into separate CD45.1+ hosts. After two weeks, the spleen and blood were harvested and the donor cells were identified by an antibody against CD45.2. Day 0 represents the sort purity. Percentage of donor CD11b and CD27 subsets in spleen and blood is shown, and represents one of three independent experiments. (B) Data depicted in (A) are presented as the mean ±SD from 3 independent experiments. (C) Expression of CD11b and CD27 in bulk NK cells from either blood or spleen, representative of 3 independent experiments.
Figure 1
NKR-P1B Ly49s3+
NKR-P1B- Ly49s3-
Figure 2
0 hr Day 6Day 6 Day 28Day 28
-
0 hr
+
NKR-P1B
Ly49s3
NKR-P1B- Ly49s3-
B A
Ly49s3 NKR-P1B
Day 6 24 hr 0 hr 1 hr 6 hr
CD45.2+(donor) Sort
Purity
NKR-P1B
NKR-P1B-Ly49s3Ly449s3-49s3 Ly49s3Ly49s3+
Before sort Before sort
NKR-P1B
Ly49s3
Figure 3
Blood Spleen
NKR P1B
NKR-P1B-Ly49s3Ly499s3- Ly49s3Ly49s3+
CD45.1+ (host)
NKR-P1B
Ly49s3
NKR-P1B+ NKR-P1B+
B A
Figure 4
% CD107a+ cells% CD107a+cells Remaining
NKR-P1B- Ly49s3-
Newly formed NKR-P1B+
Sorted NKR-P1B+
Remaining NKR-P1B -Ly49s3-
Newly formed NKR-P1B +
Sorted NKR-P1B+ 0
10 20 30
NKp46 NKp46
CD107a
Newly formed NKR-P1B+
Remaining NKR-P1B- Ly49s3-
Sorted NKR-P1B+
NKp46
CD107a
Isotype
Isotype Anti-NKR-P1AAnti NKR P1A
Eff. + YAC Effectors
Remaining NKR-P1B
-Ly49s3
-
Newly formed NKR-P1B +
Sorted NKR-P1B+ 0
20 40 60
p=0.0019 p=0.0065
A B
C D
NKRLy49
Ly49
Day 7
1
Ly49s3 2
NKR-P1B
1 2
Day 7
R-P1B- 9s3-
9s3+
CFSE NKRR-P1B+
Figure 5
A
B
CFSE 60% 61% 1%
63% 71% 2%
78% 52% 4%
46% 45% 10%
29% 66% 6%
6% 78% 15%
Day 4 Day 4
78% 20%
57% 40%
Figure 6
NKR-P1B- Ly49s3-
NKR-P1B+
Ly49s3+
NKp46 NKG2D NKG2A/C/E Ly49s5/i5 KLRH1 CD8alpha Eomes
NKR-P1B+
Ly49s3+
B+
+
CD27
CD11b
Figure 7
NKR-P1B- Ly49s3-
NKR-P1B+
Ly49s3+
A B
NKR-P1B- Ly49s3-
CD117 CD127
d=0 Spleen Blood d=14
72DC
CD11b
CD27
CD11b Host NK
Spleen
Blood
0 20 40 60 80
0 20 40 60 80 100
0 50 100 150
0 20 40 60 80
0 50 100 150 0
20 40 60 80
0 20 40 60 80
0 20 40 60 80 100
Spleen Blood
% positive cells
CD11blow CD27low
CD11blow CD27high
CD11bhigh CD27high
CD11bhigh CD27low
CD11blow CD27low
CD11blow CD27high
CD11bhigh CD27high
CD11bhigh CD27low
CD11blowCD27low CD11blowCD27high CD11bhighCD27high CD11bhighCD27low
A
B
C
Figure 8
Nomenclature
Ly49s3+
NKR-P1B-
Ly49s3- NKR-P1B+ NKR-P1Bbright
Spleen Blood
NKR-P1B
Ly49s3
Supplementary figure 1
Spleen
NKR-P1B- Ly49s3- NKR-P1B+
Ly49s3+
Blood
CD45 RT7.2 CD3 CD45 RT7.2 CD3
FSC
SSC NKR-P1ASSC SSC SSC NKR-P1A
FSC
Supplementary figure 2
Supplementary Figure 2. Gating strategy for identifying adoptively transferred NKR- P1B-Ly49s3-, NKR-P1B+, and Ly49s3+ NK cell subsets in spleen or blood. NKR- P1A+CD3- donor NK cells were identified within the CD45.2+ cell gate.
NKp46
NKR-P1A NKR-P1B
Ly49s3
NKR-P1B
Ly49s3
CD3 Ly49s3
NKR-P1B NKR-P1A
SSC
A
B
Supplementary Figure 3. Different gating strategies for identifying NK cells during flow cytometric analysis. A) After gating on lymphocytes, NK cells were either gated as
NKp46+ or NKR-P1A+CD3-, and NKR-P1B-Ly49s3- NK cells identified by co-staining for Ly49s3 and NKR-P1B. B) After gating for lymphocytes, NKR-P1A+ cells were gated as NK cells, and NKR-P1B-Ly49s3- NK cells identified by co-staining for Ly49s3 and NKR- P1B.
Supplementary figure 3
