bovine natural killer cells

Veterinary Immunology and Immunopathology 130 (2009) 163–177

Review paper

Bovine natural killer cells

Preben Boysen *, Anne K. Storset

Norwegian School of Veterinary Science, Department of Food Safety and Infection Biology, PO Box 8146 Dep, NO-0033 Oslo, Norway

Contents lists available at ScienceDirect

Veterinary Immunology and Immunopathology

journal homepage: www.e lsev ier .com/ locate /vet imm

A R T I C L E I N F O

Article history:

Received 4 November 2008

Received in revised form 11 February 2009

Accepted 20 February 2009

Keywords:

Natural killer cells

Lymphocytes

Cattle

Bovine

NKp46

CD335

Interferon-gamma

Innate immunity

Immunity

Cellular

Immunogenetics

Cytotoxicity

Natural cytotoxicity

Antibody-dependent cell cytotoxicity

Lymphokine-activated killer cells

A B S T R A C T

Natural killer (NK) cells have received much attention due to their cytotoxic abilities, often

with a focus on their implications for cancer and transplantation. But despite their name,

NK cells are also potent producers of cytokines like interferon-gamma. Recent discoveries

of their interplay with dendritic cells and T-cells have shown that NK cells participate

significantly in the onset and shaping of adaptive cellular immune responses, and

increasingly these cells have become associated with protection from viral, bacterial and

parasitic infections. Furthermore, they are substantially present in the placenta,

apparently participating in the establishment of normal pregnancy. Consequently, NK

cells have entered arenas of particular relevance in veterinary immunology. Limited data

still exist on these cells in domestic animal species, much due to the lack of specific

markers. However, bovine NK cells can be identified as NKp46 (CD335) expressing, CD3(�)

lymphocytes. Recent studies have indicated a role for NK cells in important infectious

diseases of cattle, and identified important bovine NK receptor families, including multiple

KIRs and a single Ly49. In this review we will briefly summarize the current understanding

of general NK cell biology, and then present the knowledge obtained thus far in the bovine

species.

� 2009 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

2. NK cell biology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

2.1. Mechanisms of NK cell activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

2.1.1. Cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165

2.1.2. Cytokine production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

2.2. Definition of NK cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

3. NK cells in veterinary species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

4. The search for NK cells in cattle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168

4.1. Natural cytotoxicity by bovine lymphocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168

4.2. ADCC activity by bovine lymphocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168

4.3. LAK activity by bovine lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168

4.4. Innate IFN-g production by bovine lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168

5. Characteristics of bovine NK cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

* Corresponding author. Tel.: +47 22964817.

E-mail address: [email protected] (P. Boysen).

0165-2427/$ – see front matter � 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetimm.2009.02.017

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P. Boysen, A.K. Storset / Veterinary Immunology and Immunopathology 130 (2009) 163–177164

5.1. NKp46+/CD3� lymphocytes are bovine NK cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

5.2. Subsets and localization of NK cells in cattle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

5.3. Relationship to other innate lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

6. NK cell receptors in cattle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172

6.1. Killer immunoglobulin-like receptors (KIRs). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172

6.2. Killer cell lectin-like receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172

6.2.1. CD94/NKG2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172

6.2.2. NKG2D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

6.2.3. Ly49 and other lectin-like receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

7. The role of bovine NK cells in infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

7.1. NK response to Mycobacteria spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

7.2. NK response to protozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

7.3. Impact for veterinary host-pathogen research and vaccinology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

8. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

1. Introduction

Natural killer (NK) cells were first discovered in theearly 1970s as ‘‘null cells’’ that could spontaneously lysetumor cells without prior immunization (Trinchieri, 1989).At first often disregarded as disturbing sources of back-ground effects in cancer immunology (Hokland andKuppen, 2005), increasing interest during the next fifteenyears culminated with the formulation of the ‘‘missingself’’ hypothesis by Ljunggren and Karre, suggesting that aprinciple function of NK cells is to eliminate cells that lackself-markers (Karre et al., 1986; Ljunggren and Karre,1990). Such situations may occur in cancer, viral infectionsand transplantation, indicating a prominent role for NKcells in these conditions. Following this shift of paradigms,studies of NK cells exploded in the 1990s, leading to thediscovery of the inhibitory receptors responsible for thisphenomenon, and later also activating receptors.

Biased by their name ‘‘killers’’, the majority of thisresearch was occupied with their cytotoxic abilities, butthe view on these cells is now shifting towards anorchestrating role in many immune responses (Degli-Esposti and Smyth, 2005; Strowig et al., 2008). Inparticular, NK cells interact with dendritic cells (DCs)and other myeloid cells to direct immune responsestowards the elimination of damaged and/or infected cells.There is evidence that NK cells polarize T-cells in the ‘‘T-helper cell 1 (Th1)’’ direction in the lymph nodes (LNs)(Martin-Fontecha et al., 2004; Laouar et al., 2005), andearly during an infection, the contribution of NK cells andother innate lymphocytes appear crucial for the rapid andappropriate quality of the downstream immune response(Reschner et al., 2008; Strowig et al., 2008). It has been welldocumented that NK cells play a role in the early defenseagainst not only viral, but also parasitic and bacterialinfections (Korbel et al., 2004; O’Connor et al., 2006;Roetynck et al., 2006; Newman and Riley, 2007).

NK cells even appear in the field of reproductivephysiology, as they are present and highly active in boththe pregnant and the non-pregnant uterus of most studiedspecies (Croy et al., 2006; Moffett and Loke, 2006). Theirrole in this setting is much debated, but they appearinvolved in remodelation of vascular tissue during theimplantation of the fetus, as well as following fetal death.

Furthermore, they may participate in the immunologicalprotection against uterine infections, by way of cytotoxi-city as well as their cytokine-producing abilities (Hannaand Mandelboim, 2007; Le Bouteiller and Piccinni, 2008).However, since the physiology of placentation vary greatlybetween species, and since limited data exist from speciesother than rodents and humans, both of which have ahaemochorial placentation, great care should be taken inextrapolating these conclusions into veterinary specieslike cattle.

2. NK cell biology

The current understanding of NK cell functions havebeen comprehensively reviewed in two special journalissues (Immunological Reviews 2006, vol. 214, no. 1, 5–263and Nature Immunology 2008, vol. 9, no. 5, 471–511). Mostof what is known derives from studies in rodents andhumans, briefly sketched in the following.

2.1. Mechanisms of NK cell activity

In contrast to B and T cells, NK cells work throughgermline encoded receptors, and thus belong to the innateimmune system. They probably co-evolved with T cells,with which they share a common progenitor as well asseveral functional parallels. NK cells express receptors thatrecognize MHC class I molecules, as a rule not involving thebound peptide, and in contrast to the T-cell receptor, suchrecognition leads to inhibition rather than activation of thecell. NK cells share killing mechanisms with CD8+ cytotoxicT-cells, and they produce cytokines in a way resemblingCD4+ helper T cells. However, instead of leaning on onedominant activating receptor like T- and B-cells, theresponse of NK cells is determined by multiple activatingand inhibitory receptors. Most NK cell receptors are alsofound on subsets of T-cells and other leukocytes. Theprevailing view has been that NK cells circulate in a ‘‘ready-to-go’’ state, although this view may need modification asrecent findings indicate that also NK cells start out in anaıve, relatively unresponsive state and need priming,induced by cytokines from APCs following microbialstimulation, to become fully reactive (Lucas et al., 2007;Fehniger et al., 2007; Chaix et al., 2008).

P. Boysen, A.K. Storset / Veterinary Immunology and Immunopathology 130 (2009) 163–177 165

The effector functions of NK cells can be broadlycategorized in two; cytotoxicity and cytokine production,often but not always simultaneously present in the sameNK cell.

2.1.1. Cytotoxicity

The major paradigm of NK cell recognition, the ‘‘missingself’’ theory, is still regarded an appropriate model tounderstand NK cytotoxicity, despite some modifications

Table 1

NK cell receptors.

Receptor CD name Species*

Immunoglobulin-like

KIR family

KIRs, long tail CD158 HS, BT***

KIRs, short tail CD158 HS, BT***

NCRs

NKp46 CD335 HS, MM, BT

NKp44 CD336 HS

NKp30 CD337 HS

CD2 family

CD2 CD2 HS, MM, BT

2B4 CD244 HS, MM

NTB-A HS

FcgRIIIA CD16 HS, MM, BT

N-CAM CD56 HS

ILT/LILR CD85

DNAM-1 CD226 HS

Lectin-like

L-selectin CD62L HS, MM, BT

CD69 CD69 HS, MM

CD94/NKG2A/B CD94/CD159ab HS, MM, BT***

CD94/NKG2C CD94/CD159c HS, MM

NKR-P1 family CD161 HS, MM

NKG2D CD314 HS, MM

Ly49 family MM, BT***

KLRJ1 BT***

NKp80 HS

Integrins

b2-integrins/LFA-1 CD11a/b/c + CD18 HS, MM, BT

DX5 CD49b MM

TNF superfamily

CD27 CD27 HS, MM

FasL CD95 HS, MM

TRAIL CD253 HS. MM

Pattern-recognizing receptors

TLR family CD281-291 HS

Cytokine receptors

IL-2Ra CD25 HS, MM, BT

IL-2/IL-15Rb CD122 HS, MM

IL-21R HS, MM

Common g-chain CD132 HS, MM

IL-12R CD212 HS, MM

IL-18Ra/b CD218 HS, MM

IFNRa/b (IFNAR) HS, MM

Chemokine receptors

Various (Ref. Gregoire et al., 2007)

Table lists NK cell receptors currently considered of major importance, and is not

(BT) listed.* Species where molecule has been associated with NK cells.** Function refers to information from HS and MM, except CD16, CD25 and C*** mRNA demonstration only.

(Lanier, 2005). Essentially, if the NK cells cannot recognizea self MHC class I molecule on the target cell, no inhibitorysignal is delivered, and the target is killed. This may occur ifthe MHC molecules are absent or (severely) downregu-lated, like during certain viral infections, or of a typeforeign to the host, as in transplanted tissues. However,normal red blood cells or neural tissues, which carry no orlittle MHC class I molecules are spared by self NK cells,illustrating that engagement of activating receptors is also

Function** Ligands

Inhibition MHC class 1 (HLA variants)

Activation MHC class 1 (HLA variants)

Activation** Unkown/virus-encoded proteins

Activation Unknown/virus-encoded proteins

Activation BAT-3/virus-encoded proteins

Adhesion/Activation CD58 (LFA-3)/CD48

Activation/Inhibition CD48

Activation NTB-A (homophilic)

Activation** Fc-region of IgG

Unknown Unknown

Inhibition/Activation MHC class 1 (HLA-A, B, C, E, F, G)

Activation PVR (CD155)/Nectin-2 (CD112)

Adhesion GlyCAM, MadCAM, CD34, others

Activation Unknown

Inhibition HLA-E (HS), QaIb (MM)

Activation HLA-E (HS), QaIb (MM)

Inhibition/Activation LLT-1/C-type lectins

Activation MICA, MICB, ULBPs

Inhibition/Activation MHC class 1 (H-2 variants) or m157 from

murine cytomegalovirus

Unknown Unknown

Activation AICL

Adhesion iC3b, ICAMs, fibrinogen

Adhesion Collagen, laminin

Activation CD70

Apoptosis induction Fas

Apoptosis induction TRAIL-receptors

Activation PAMPs (pathogen patterns)

Activation** IL-2

Activation IL-2/IL-15

Activation IL-21

Activation IL-2/IL-15/IL-21

Activation IL-12

Activation Type I interferons (IFN-a/b)

exhaustive. Only Homo sapiens (HS), Mus musculus (MM) and Bos taurus

D335, which have been studied in BT (see text).


necessary. As a rule, inhibitory NK cell receptors arecharacterized by immunotyrosine inhibitory motifs(ITIMs) in their cytoplasmatic tails. Activating receptorsrecruit positive signalling adapter molecules like FcgeRIg,CD3z and DAP12, which bear activating cytoplasmaticelements like immunotyrosine activation motifs (ITAMs)(Lanier, 2005). Cytokines like interleukin (IL)-2, IL-15, IL-21 and type I interferons (IFN-a/b) enhance NK cytotoxi-city. In the effector phase, the NK cell forms animmunological synapse with the target and releasespreformed cytotoxic lysosomes, containing perforin orgranzymes (Bryceson et al., 2006a). NK cells can also killtarget cells by induction of apoptosis through FAS ligandand other mechanisms, although with less efficiency(Screpanti et al., 2005).

Recent studies have shown that two or more activatingreceptors must work in synergy to trigger resting cells, butthat some dominant activating receptors are sufficient ontheir own when the NK cells have been activated with IL-2

Fig. 1. NK cytotoxic phenomena. The release of cytotoxic granula and subsequen

Natural cytotoxicity is the spontaneous lysis of a target cell by the NK cell. This re

overrule activating (+) signals. (B) LAK (lymphocyte-activated killer) activity

interleukin-2. (C) ADCC (antibody-dependent cell-mediated cytotoxicity) involv

to structures on the target cell. This reaction may also involve other recognition

called redirected lysis, the two cells are also linked by antibodies, but in a reve

another species) directed towards an activating receptor on the NK cell, while th

(derived from the same species that raised the antibody). A simultaneous linki

(Bryceson et al., 2006b). Most activating receptors aredependent on actin rearrangements in the cytoskeleton,which is continuously blocked as long as inhibitoryreceptor signalling is present (Stebbins et al., 2003).Because of this, the inhibitory signals are very potent,most often overruling even strong or multiple activatingsignals. In sum, a complex interplay of activating andinhibitory signals determines the cytotoxic function of NKcells. Some of the individual NK receptors are listed inTable 1, and will be discussed in some detail below in thecontext of bovine NK cells.

The records of NK research mainly refer to four types ofcytotoxic phenomena (Fig. 1): (1) killing in the absence ofstimulation, referred to as ‘‘natural cytotoxicity’’ (NC), (2)killing by cytokine-stimulated lymphocytes (typically byIL-2), generating so-called ‘‘lymphokine-activated killer(LAK) cells’’, (3) killing following antibody opsonisation oftarget cells, called ‘‘antibody-dependent cell-mediatedcytotoxicity (ADCC)’’ and (4) redirected lysis, or reverse

t lysis of a target cell may occur from different experimental settings: (A)

sults when inhibiting signals (�) to the NK cell are absent or insufficient to

is the result of pre-activation of the NK cell by cytokines, most often

ed the cross-linking of Fcg receptors on the NK cell, by antibodies directed

events between NK cells and target cells. (D) In the experimental assay

rsed manner, as the assay involves an introduced antibody (produced in

e Fc region of the antibody is bound to an Fcg receptor on the target cell

ng and NK cell activation occurs.

Fig. 2. Relationship between NK cells in vivo and experimental LAK cells.

(A) Recent evidence has shown that in the living organism, NK cells are

initially naıve and poorly responsive, but following microbial exposure,

priming by cytokines (IL-15 and IL-18) render the NK cells responsive.

Primed NK cells become activated and fully functional in response to

activating cytokines and ligands on the target cells. (B) ‘‘Resting’’ isolated

NK cells normally show poor or moderate cytotoxic capacity in in vitro

assays, and it may not be clear whether these are naıve or primed.

Stimulation of resting NK cells with IL-2 for several days result in LAK-

cells, which are strongly cytotoxic.

Fig. 3. Cytokine production by NK cells. Several types of stimuli result in

the NK cell production of cytokines like IFN-g and TNF. Such stimuli can

be: (A) damaged or infected cells display altered cellular ligands and

release cytokines like IFN-a/b, both recognized by NK cells. (B) Microbial

stimulation of accessory cells lead to their production of IL-12 and IL-18,

that work in synergy to activate the NK cell. (C) Pathogens may also

directly act on pattern-recognising receptors on the NK cells. The

cytokines released from NK cells trigger several mechanisms of cellular

immunity, most importantly: (D) Assistance to DCs to produce ‘‘Th1’’

cytokines, which promote cellular T-cell responses. (E) Prompting of

macrophages to eliminate microbes (DC = dendritic cell).


ADCC, where an antibody against an activating NK cellreceptor is introduced in an experimental assay using Fcg-receptor bearing targets (like the murine P815), thuscausing activation as well as linking of the two cells. Itshould be noted that many works refer to these functionsin mixed cell populations, where various interactionsbetween cell types may take place, and where the exactnature of the effector cell may be ill-defined. Notably, IL-2is predominantly T-cell derived and probably not sig-nificantly present in the early phases of an immuneresponse, although this is subject to debate as DCs can alsoproduce IL-2 under certain conditions (Schartz et al., 2005;Granucci et al., 2008). LAK cells may represent anexperimental shortcut from resting to activated NK cells,a process that seems to involve several sequential steps inthe living organism (Fig. 2) (Fehniger et al., 2007),involving IL-15 which binds to receptor chains sharedwith IL-2 (Table 1).

2.1.2. Cytokine production

The best-characterized non-cytotoxic function of NKcells is production of interferon-gamma (IFN-g) andtumour necrosis factor (TNF) (Fig. 3). Unlike T cells, NKcells do not secrete IL-2. For cytokine production to takeplace, the main understanding has been that NK cellsmust be activated by accessory cells; thus when DCs ormacrophages recognize pathogenic structures, theyrelease IL-12 that stimulate NK cells in synergy with IL-2, IL-15 and/or IL-18 (Degli-Esposti and Smyth, 2005;Newman and Riley, 2007). Such cytokines from accessorycells were thought to be an absolute requirement untilit was demonstrated that NK cells also can directlyrecognize pathogen-associated molecular patterns(PAMPs) through toll-like receptors (TLRs) (Beckeret al., 2003; Sivori et al., 2004; Schmidt et al., 2004) andother pattern-recognition receptors (PRRs) (Athie-Mor-ales et al., 2008). Consequently, it appears that the extentof NK cell activation and function is determined by thesum of direct and indirect recognition of pathogens(O’Connor et al., 2006).

2.2. Definition of NK cells

NK cells were initially defined based upon theirfunction (spontaneous lysis) and morphology (largegranular lymphocytes; LGLs). In a meeting in 1988, aconsensus on the phenotypic definition was reached: NKcells were defined as CD3� and TCR� lymphocytes that inthe human express CD56 and most often CD16 (FcgRIIIA)(Hercend and Schmidt, 1988; Trinchieri, 1989). However,species as closely related to man as baboons, rhesusmacaques and chimpanzees, express CD56 on either noneor only subsets of NK cells (Malyguine et al., 1996; Carteret al., 1999; Stone et al., 2000). Mice do not express CD56on their NK cells, which are instead identified as NK1.1+

cells (NKR-P1C), but since this molecule has proven to beabsent in some mouse strains, CD49b (DX5) has beenintroduced as a more widely applicable marker (Araseet al., 2001). In the rat, the most commonly used NKidentifier is NKR-P1 (Giorda et al., 1990).

Human NK cells are commonly divided into two mainsubsets; the principally cytotoxic CD56dim/CD16+ cells,dominating in blood, and the cytokine-producingCD56bright/CD16� cells, sparse in blood, but enriched inlymphoid tissues as well as in the uterus (Cooper et al.,2001; Fehniger et al., 2003; Ferlazzo et al., 2004b; Hannaand Mandelboim, 2007). Most likely, the former subsetrepresents an end stage following differentiation from thelatter (Romagnani et al., 2007; Chan et al., 2007). In themouse, three major subsets have been described based onCD11b and CD27 expression (Hayakawa and Smyth, 2006),and recent observations indicate that CD27 may be a bettersubset marker even in humans (Vossen et al., 2008; Silvaet al., 2008).

3. NK cells in veterinary species

Curiously, the first observation of cytotoxic lympho-cytes was made in dog cells (Govaerts, 1960). Yet, incontrast to many other aspects of immunology, NK cellshave not been well characterized and described in


domestic and pet animals. Precise markers for NK cellshave been elusive in most species, since most NK receptorsare also expressed by other cells, and since the CD56definition appears only to be relevant in man. The lack ofCD56 expression in NK cells has been observed in dogs andcattle at the gene transcription level (Bonkobara et al.,2005; Endsley et al., 2006). In the absence of appropriateantigen markers, identification had to be based on theabsence of other cell lineage markers, together withfunctional characteristics. Generally, NK cells weresearched within non-B and non-T lymphocytes showingeither ADCC, NC or LAK activity. Cells resembling NK cellshave thus been described in several domestic speciesincluding dogs, cats and horses (Evans and Jaso-Fried-mann, 1993; Viveiros and Antczak, 1999; Tekin andHansen, 2002), and in more distant species like bony fishand the Xenopus frog (Horton et al., 2003; Yoder, 2004). Apicture is starting to emerge in pigs (Krog et al., 2003;Gerner et al., 2008) and chicken (Gobel et al., 1994;Viertlboeck et al., 2008). Finally, in cattle, significantprogress has been made in the understanding of NK cellssince the search began.

4. The search for NK cells in cattle

4.1. Natural cytotoxicity by bovine lymphocytes

NC activity by non-adherent leukocytes was initiallydescribed against target cells infected with virus, likebovine herpesvirus 1 (BHV-1) and parainfluenza 3 virus(PI3V) (Rouse and Babiuk, 1977; Campos et al., 1982;Chung and Rossi, 1987; Palmer et al., 1990), or with theprotozoan Theileria parva (Emery et al., 1981). Studiesusing uninfected target cells observed limited NC activity(Leibold et al., 1980; Campos et al., 1982). One of the firstapproaches to characterize the bovine NC cell in moredetail, attributed it to the monocyte-macrophage lineage,as depletion of such cells reduced the effect dramatically(Bielefeldt et al., 1985). However, in the light of today’sknowledge this could be explained as omission ofactivating stimuli to the NK cells, like IL-12 and IL-18from accessory cells (Newman and Riley, 2007). Incontrast, a subsequent study found a non-T, non-B, non-monocyte, large lymphocyte to be the most prominent NCcell against BHV-1 infected targets (Cook and Splitter,1989). As antibodies against homologues to human CDmarkers started to appear, NK cell candidates appearedwithin the CD4� CD8� T-cell populations (Baldwin et al.,1988), and Theileria-transformed CD2+ or CD8+ cell clonesshowed NC (Goddeeris et al., 1991). In common, all of thesestudies noted that the putative bovine NK cells werevirtually inactive in the absence of infection or otherstimuli.

4.2. ADCC activity by bovine lymphocytes

Bovine lymphoid cells with ADCC activity againstchicken erythrocytes were demonstrated already in1976 (Rouse et al., 1976). Later, similar activity wasshown against other targets, including PI3V-infectedtargets (Grewal and Rouse, 1979; Belden and Peng,

1987; Bradford et al., 1992; Bradford et al., 2001).However, in neither of these works nor later has thebovine lymphoid ADCC effector cell been characterized indetail.

4.3. LAK activity by bovine lymphocytes

Following the demonstration of the LAK phenomenonand its association with NK cells in mice and humans(Herberman et al., 1987) (Trinchieri, 1989), studies incattle showed that LAK cells could be generated frombovine lymphocytes in response to IL-2 (Campos and Rossi,1986; O’Reilly and Splitter, 1990; Jensen and Schultz,1990). Despite several phenotypical investigations of themain cytotoxic lymphocyte in the LAK population throughthe 1990s, no unambiguous phenotypic definition wasreached. Some reported it to be CD2� (Amadori et al.,1992a,b), others CD2+ (Campos et al., 1992) and yet othersCD2 variable (Li and Splitter, 1994). One report noted that aputative NK cell antibody raised against natural cytotoxicfish cells (anti-FAM clone 5B6) could identify ruminant andhorse NK cells within fresh blood and LAK cultures (Harriset al., 1993), but a subsequent study could not detect FAM+

cells in ovine blood (Tekin and Hansen, 2002), and we havefound no reactivity of this antibody above backgroundwith either bovine PBMC or isolated NK cells (unpublishedresults). Bovine IL-2 was cloned and expressed in 1986(Cerretti et al., 1986), but recombinant human IL-2 wasused in most of the above studies. It should be noted thatrecent experiments have indicated that bovine NK cellsproliferate much more efficiently to bovine IL-2 comparedto the human cytokine (Storset et al., 2004).

4.4. Innate IFN-g production by bovine lymphocytes

At the time of these first searches for bovine NK cells,the IFN-g producing abilities of NK cells were still not wellrecognized. Interferon production by bovine NC cells wasreported in 1985 (Bielefeldt et al., 1985), but the type ofinterferon or the exact nature of the NC cell was still notestablished. In 1990, an ELISA-based IFN-g detectionmethod was introduced to diagnose mycobacterial infec-tion in ruminants (Wood et al., 1990). After some years ofroutine use of this test, it became clear that some animals,especially young, responded falsely positive to the test(McDonald et al., 1999; Olsen and Storset, 2001). Mean-while, the importance of innate cells, and particularly NKcells, in T-cell independent IFN-g production had beenacknowledged (Bancroft et al., 1991; Biron et al., 1999),renewing attention to the elusive bovine NK cells. Thus,using the now available cytokine detection methods, non-Tlymphocytes responding unspecifically with IFN-g pro-duction to Bacille Calmette-Guerin (BCG) and Babesia

infections were demonstrated (Hope et al., 2002; Goffet al., 2003). These cells were CD3� and CD2+ and/or CD8+,and were described as ‘‘NK-like’’ cells since no NK-specificmarkers had yet been identified. Since these and otherworks (described below) show that CD8 is a highly relevantNK cell marker, it is important to bear in mind that CD8+

cells, often interpreted as T-cells (Antonis et al., 2006;Hagiwara et al., 2008), may also contain significant


numbers of NK cells, being a possible cause for ‘‘non-specific’’ IFN-g production.

5. Characteristics of bovine NK cells

5.1. NKp46+/CD3� lymphocytes are bovine NK cells

Following the new millennium’s exploding informationof genomic and expressed genes in a variety of species,major bovine NK receptor gene families have beendescribed (McQueen et al., 2002; Storset et al., 2003).One of the sequenced molecules, NKp46 (CD335) stood outas a particularly appropriate NK definition marker, beinghighly restricted to, and present on all or the vast majorityof NK cells in man, mouse, rat and monkeys (Pessino et al.,1998; Falco et al., 1999; Biassoni et al., 1999; De Mariaet al., 2001; Westgaard et al., 2004; Walzer et al., 2007a). Ithas consequently been proposed as the most precise pan-species NK cell marker (Moretta et al., 2005; Walzer et al.,2007b). NKp46 is a strongly activating receptor, whichtogether with NKp44 (CD336) and NKp30 (CD337),belongs to a receptor family called ‘‘natural cytotoxicityreceptors (NCRs)’’, whose ligands are mostly unknown, butappear to be present on both tumor-transformed cells ofthe host and xenogeneic cells (Moretta et al., 2001; Bottinoet al., 2005). Virally encoded or controlled ligands can alsobe recognized by NCRs (Arnon et al., 2004; Vieillard et al.,2005; Arnon et al., 2005).

Employing a mAb against bovine NKp46 (Storset et al.,2004), this molecule was found to be expressed on a minorCD3�, non-gdT and non-B, lymphocyte subset, while it wasabsent from other leukocyte subsets and correlatedstrongly with CD16 expression (Storset et al., 2004; Boysenet al., 2008). Cells positively isolated using this mAb wereLGLs, and proliferated vigorously in the presence of bovinerecombinant IL-2, resulting in strongly cytotoxic cells withtypical NK cell characteristics as known from other species(Storset et al., 2004). These cells were able to kill human,murine and bovine target cells (Storset et al., 2004; Boysenet al., 2008). Like in other species, fresh ex vivo bovine NKcells have very limited cytotoxicity, except when using anNKp46-mediated redirected lysis method (Klevar et al.,2007; Boysen et al., 2008). Bovine NKp46+ cells respondedwith IFN-g production in response to cytokines and/ormicrobial structures (Olsen et al., 2005; Boysen et al.,2006a,b, 2008). Later, a gross correlation in characteristicsand phenotype was shown between these NKp46+ cellsand an alternatively defined NK cell population obtainedby negative selection strategy (Endsley et al., 2006). Thisgroup had previously cloned bovine granulysin and NK-lysin (Endsley et al., 2004), and showed that the negativelyselected NK cells expressed granulysin and NKp46 at themRNA level, and contained intracellular perforin. Intra-cellular perforin was subsequently also detected in NKp46+

cells (Boysen et al., 2008).The finding that NKp46 can be used to identify NK cells

in histological sections in cattle (Maley et al., 2006; Boysenet al., 2008) and mice (Walzer et al., 2007a) opens up newpotentials in NK cell biology, where the understanding ofthese cells may have been strongly biased due to the lack ofreliable methods to detect such cells in tissues (Yokoyama,

2008). Taken together, NKp46 has proven to be an accurateand highly useful marker for bovine NK cells.

5.2. Subsets and localization of NK cells in cattle

The phenotype of bovine NK cells suggests a hetero-geneous cell population, as several markers vary withinand between body compartments (Storset et al., 2004;Endsley et al., 2006; Boysen et al., 2008). Particularly, CD2stands out as a distinct subset marker for bovine NK cells,since only a minor fraction of NK cells in bovine bloodwas found to be CD2�, while conversely, this subsetappeared to dominate in LNs. The CD2�NK cells appearedmore activated, proliferated better in IL-2 culture, andwere more capable of producing IFN-g than CD2+ NK cells(Boysen et al., 2006b). However, their cytotoxic capabil-ities appear equal (Boysen et al., 2006b). In terms ofcytokine-producing and proliferative abilities and loca-lization in LNs, the bovine CD2� subset resembles thehuman CD56bright subset; they also share a CD44bright andCD25+/variable phenotype. While the human subsets differin NC (Cooper et al., 2001), they do not differ in LAKactivity, which was the only method tested with bovinecells (Boysen et al., 2006b). However, CD2 expressionappears not to correlate with the human functionalsubsets, CD11c expression would be opposite in thesespecies, and while CD16 is absent on human CD56bright

NK cells, this receptor was equally expressed on allbovine NK cells in blood as well as LNs (Cooper et al.,2001; Boysen et al., 2008). In conclusion, it seems clearthat the main NK cell subsets described in the human andbovine species do not represent directly comparablepopulations.

The number of NK cells in bovine blood shows asubstantial variation, and relative blood levels usually varyfrom 2 to 10% of lymphocytes in apparently healthyanimals, generally highest early in life (Kulberg et al., 2004;Endsley et al., 2006; Kampen et al., 2006). Levels>20% mayoccasionally be seen (Kampen et al., 2006), but this mightbe a result of rapid mobilization due to subclinicalinfections, as shown for Neospora caninum infection(Klevar et al., 2007), discussed below.

Similar to mice and humans (Gregoire et al., 2007),NK cells have been detected and characterized in thebovine spleen, lung, liver, a variety of LNs (Storset et al.,2004; Goff et al., 2006; Boysen et al., 2008) as well as inbone marrow (our unpublished results). In bovine LNs,NK cells were found residing in the paracortex and themedulla, corresponding well to the gross localization inmice and humans. Recent studies have shown that NKcells need priming to become reactive (Lucas et al., 2007;Long, 2007; Fehniger et al., 2007; Chaix et al., 2008).According to this model, summarized in Fig. 4, microbialexposure will attract naıve NK cells to lymph nodes,where they acquire the ability to rapidly respond toinfections. Laboratory mice are kept in highly protectedand hygienic conditions, possibly limiting such priming,whereas the human body is more exposed to minorinfections and commensal bacteria, leading to anenduring state of primed NK cells in the circulation(Long, 2007). It is conceivable that cattle, which host

Fig. 4. Body compartments for NK cell priming of human/murine NK cells. (A) Pre-microbial exposure, NK cells are mainly present in blood and spleen, in a

‘‘naıve’’ state. Few such cells are present in lymph nodes. This may represent the ‘‘steady state’’ in individuals or species kept free from microbial exposure.

(B) Priming phase. Following microbial invasion and infection of somatic cells, microbial PAMPs (and other danger signals) are recognized by macrophages

and DCs, which undergo maturation. Mature DCs migrate to lymph nodes, where they recruit naıve NK cells from the blood. Trans-presentation of IL-15, as

well as other signals like IL-12 and IL-18, prime the recruited NK cell, which subsequently re-enter the bloodstream. This may represent the ‘‘steady state’’ in

individual/species living under moderate microbial exposure. (C) Effector phase. Upon significant microbial attack, primed NK cells may re-enter the lymph

nodes, where they are activated by DCs to produce interferon-gamma, which participate in the polarization of Th1-cells. In addition, primed NK cells are

recruited into inflamed tissues, where they are activated by damaged/infected cells as well as accessory cells, triggering effector functions like killing of

damaged cells and release of cytokines. Unlike T-cells, these NK effector responses are not restricted by microbe-specific antigens. In individuals or species

heavily exposed to microbes, this may be illustrative of a ‘‘steady state’’ situation, and comparably more primed NK cells will be present in lymph nodes and

circulation (DC = dendritic cell; TLR = toll-like receptor; PAMP = pathogen-associated microbial patterns).


more commensal microbes and live under less hygienicconditions than humans, could harbour an even higherproportion or degree of primed NK cells under normalhousing conditions. Several findings in cattle could beinterpreted through this priming model: Cattle have atleast comparable numbers of LN-residing NK cells ashumans, and much higher than mice (Fehniger et al.,2003; Ferlazzo et al., 2004b; Bajenoff et al., 2006; Walzeret al., 2007a; Boysen et al., 2008). A majority of bovineLN-residing NK cells showed phenotypic signs ofactivation, they contained perforin and were able tokill targets, unlike in humans, where these abilities areonly aquired following activation. In mice, the few NKcells found in LNs under the steady state seem to besolitary (Bajenoff et al., 2006; Walzer et al., 2007a),whereas in humans, they are found in close proximitywith DCs and T-cells (Fehniger et al., 2003; Ferlazzoet al., 2004a). Interestingly, such cross-talk does occureven in mice following microbial challenge. It isimportant to stress that data is still too limited toconclude firmly about comparative differences, but thepriming concept should nevertheless be kept in mindwhen interpreting data across species.

5.3. Relationship to other innate lymphocytes

The view that innate lymphocytes may play a criticalrole in the early phases of an immune response isemerging, but the differential functions between NK cells,NK-T cells, gdT-cells and other minor subsets are still badlyunderstood (Reschner et al., 2008).

NK-T cells are T cells with NK-like functions, whichpredominantly recognize self and microbial glycolipidsthrough the non-polymorphic CD1d receptor. Cattle lackfunctional CD1d molecules, and it has been speculated thatcattle may not have homologues to NK-T cells at all (VanRhijn et al., 2006). NKp46 it is not expressed on NK-T cellsin humans or mice (Walzer et al., 2007a) and is thereforenot a likely marker for bovine NK-T cells.

gdT-cells are evolutionary conserved lymphocytes,whose roles are poorly understood. Although gdT-cellsdo rearrange their TCR genes, they also show several innatetypes of recognition (Born et al., 2007; Beetz et al., 2008).These cells have multiple functions comprising bothcytotoxicity, secretion of multiple cytokines (includingIFN-g), antigen presentation and immune suppression(Hayday, 2000; Moser and Brandes, 2006; Born et al.,


2007); thus sharing functions with NK cells as well asseveral other leukocytes. While gdT-cells constitute only aminority of lymphocytes in most species, around 20–30%of circulating lymphocytes in young ruminants are gdT-cells, suggesting that they play a critical role in thesespecies (Hein and Mackay, 1991; Davis et al., 1996;Kulberg et al., 2004). Innate recognition by bovine gdT-cells has been demonstrated, at least as an early primingevent (Jutila et al., 2008). They have been suggested toconstitute an early innate source of IFN-g in mycobacterialand other intracellular infections, leading to Th1 inductionand macrophage activation (Pollock and Welsh, 2002).However, our group found that the majority of non-specificIFN-g response to mycobacterial antigens was attributableto NK cells, not gdT-cells (Olsen and Storset, 2001; Olsenet al., 2005). Since earlier studies lacked specific reagentsfor bovine NK cells, these questions need to be read-dressed. It must be noted that under certain experimentalcircumstances, overlapping receptor repertoire may blurthe discrimination between NK cells and other lympho-cytes (Lanier, 2007). A recent study found that bovinesplenic gdT cells started to express NKp46 after prolongedactivation by human recombinant IL-15 (Johnson et al.,2008). Similar findings have been reported in mice strains,where the germline (but not rearranged) TCR-deltareceptor gene is transcribed in developing NK cells, andwhere a small fraction of gdT-cells express moderate levelsof NKp46 and low levels of TCR, probably representingchronically activated gdT-cells (Stewart et al., 2007).Nevertheless, the same researchers point out that inperipheral blood of normal mice and primates, NKp46expression remains strictly reserved for bona fide NK cells(Walzer et al., 2007a), and in fresh bovine leukocytes, thereseems to be no overlap between NKp46 and bovine gdT-cell markers (Storset et al., 2004). Consequently, theavailability of specific markers for bovine NK and gdT, butnot NK-T cells, enables future studies of their differentialcontributions in the innate immune responses.

6. NK cell receptors in cattle

In primates, inhibitory receptors that recognize selfMHC class I molecules (mediating the ‘‘missing self’’phenomenon) belong to the killer immunoglobulin-likereceptors (KIRs), encoded in the leukocyte-receptor com-plex (LRC), while interestingly, the Ly49 family, highlydifferent lectin-like receptors from the NK gene complex(NKC) has exactly the same function in rodents (Yokoyama,2008) (Table 1). Both these receptor families also containactivating receptors, apparently recognizing very similarligands as their inhibitory counterparts. This may appearparadoxical, but could be a way to overcome molecularmimicry by viruses that ‘‘disguise’’ themselves by encodingfor MHC-like gene products (Lanier, 2005). Indeed, genomeanalyses indicate that KIR and Ly49 genes show a very rapidevolution compared to other genes – perhaps as acompetition with rapidly evolving microbial adaptation– and that activating receptors derive from inhibitoryvariants, since in humans these variants are very similar(Abi-Rached and Parham, 2005). With this in mind, careshould be taken in inferring functions to such receptors

solely based on their structure. Immunogenetic studiesin cattle have allowed identification and cloning ofmembers of both NKC and LRC; NKC is divided into twoparts located on chromosomes 1 and 5, while LRC is locatedon chromosome 18 (Storset et al., 2003; Hao et al., 2006),but few functional studies of bovine NK receptors havebeen made, except for NKp46 and CD16. The divergencebetween rodents and humans make the study of cattle NKreceptors comparatively interesting:

6.1. Killer immunoglobulin-like receptors (KIRs)

It was believed that only primates had KIR genes untilthis gene family was shown in cattle (Storset et al., 2003;Dobromylskyj and Ellis, 2007), where it has expandedthrough gene duplication and diversification (Guethleinet al., 2007). Although several cattle KIR genes have nowbeen fully sequenced, it is yet not known how manydifferent KIR genes are present in cattle. But, as inhumans, cattle KIR haplotypes seem to differ in genecontent and combination of alleles, and have bothactivating and inhibitory receptors (Dobromylskyj andEllis, 2007).

While the majority of the inhibitory human KIRs havetwo extracellular immunoglobulin (Ig) domains and twoITIM sequences in the cytoplasmic tail, the majority of thecattle KIRs comprise three Ig-domains and have only oneITIM sequence. However, the bovine KIR sequences have acysteine residue in the transmembrane region that may beutilized to form homodimers with two ITIMs (Storset et al.,2003; Dobromylskyj et al., 2007). Unlike in humans, wherethe inhibitory and activating KIRs are very similar, theactivating cattle KIR genes described this far has acompletely different sequence in the cytoplasmic domain(Dobromylskyj et al., 2007). It has been suggested thatcattle KIR genes comprise two lineages, and that thelineage that is related to the highly diversified human KIRfamily has limited diversity while the highly diversifiedcattle KIR lineage is related to a conserved single copyprimate KIR with unknown function (Guethlein et al.,2007). It has not yet been shown if cattle KIR receptors bindto MHC class I molecules.

6.2. Killer cell lectin-like receptors

6.2.1. CD94/NKG2

In humans the non-polymorphic CD94/NKG2 hetero-dimer receptor recognises HLA-E. It may be activating(CD94/NKG2C) or inhibitory (CD94/NKG2A). The role ofHLA-E is to present peptides derived from the leadersequences of classical MHC class I molecules (O’Callaghanet al., 1998), so in humans this system may allow asurveillance of the overall MHC expression. In cattle thereis a family of different NKG2A genes but only one NKG2C

gene (Birch and Ellis, 2007). Hence the inhibitory cattleCD94/NKG2A heterodimer receptors may bind severaldifferent ligands while the CD94/NKG2C will probably nothave the same ability. The ligands for the CD94/NKG2receptors have not yet been described in cattle; howeverthe differences in gene structure may imply that thesereceptors have other functions in cattle than in humans.


6.2.2. NKG2D

In humans and rodents NKG2D is an activating receptorthat is expressed as a homodimer and is signalling throughDAP10 or DAP12 molecules. Human NKG2D binds withhigh affinity to stress-induced self-ligands ligands like MICand ULBP that is encoded in the MHC complex, and thismay lead to NK-cell killing of damaged cells (Lanier, 2005).NKG2D have been described in cattle, and as in mice thecell surface expression of the receptor is dependent oneither DAP10 or DAP12 (Fikri et al., 2007). MIC genes havealso been found in the cattle genome, located close to non-classical MHC class I genes. In total four MIC genes havebeen described with one gene consistently present in theanimals studied so far (Birch et al., 2008). In contrast tohumans who have two ULBP genes, cattle have a genecluster containing several ULBP loci with nine genes andseveral pseudogenes (Larson et al., 2006). As neither theligands for bovine NKG2D nor the role of bovine MIC andULBP in signalling cell stress has been determined, it is yetpremature to discuss the implications of this geneduplications in cattle.

6.2.3. Ly49 and other lectin-like receptors

A single Ly49 gene has been demonstrated in cattle, andstructural analysis indicate that it may be potentiallyfunctional (McQueen et al., 2002). Like for bovine KIR, itsligand is as yet unknown. So far, MHC class 1 is the onlyligand identified for these receptors in humans androdents, respectively, but it has never been shown thatboth receptor classes share this function in one and thesame species. Another bovine lectin-like receptor is theKLRJ, which neither appear to be present in other species,nor to have related genes in cattle (Storset et al., 2003).Transcripts for bovine homologues of CD69 (Ahn et al.,2002) and partially, NKP-R1 (Govaerts and Goddeeris,2001) have also been reported.

7. The role of bovine NK cells in infections

7.1. NK response to Mycobacteria spp.

Studies of mycobacterial infections in cattle hadsuggested that NK-like cells were likely to be significantreactors during Bacille Calmette-Guerin (BCG) infection(Hope et al., 2002) as well as impairing the IFN-g test inyoung animals (Olsen and Storset, 2001). To this end, usingthe novel NKp46+ selection strategy, the contribution of NKcells in the IFN-g response to mycobacterial antigens wasmeasured in animals free from previous mycobacterialinfection (Olsen et al., 2005). ESAT-6 from the Mycobacter-

ium tuberculosis-complex and MPP14 from M. avium subsp.paratuberculosis induced an IFN-g response from NK cellsin some animals, mediated through accessory cells andpartly IL-12, while no response was seen by anotherantigen (MPB70). Depletion of NK cells abrogated thisresponse completely. In subsequent studies, negativelyisolated NK cells (Endsley et al., 2006) and NKp46+ cells(Denis et al., 2007) were shown to significantly reduce thereplication of M. bovis inside autologous macrophages in acontact-dependent manner. The mechanism behind thisremains unclear as the microbicidal effect seemed not to

be mediated by either IFN-g or killing of the infectedmacrophages, although NK cells did induce IL-12 produc-tion from the macrophages (Denis et al., 2007). Conversely,a recent study showed that bovine NK cells werestimulated by BCG-infected DCs both to produce IFN-gand to kill infected target cells, again requiring direct cellcontact (Bastos et al., 2008). Taken together, these studieshave provided evidence that bovine NK cells respond toMycobacteria or their components, explaining the frequentnon-specific diagnostic results in young animals testedwith the IFN-g assay, which harbour the highest numbersof such cells.

7.2. NK response to protozoa

While Mycobacteria-induced NK cell responses seem tobe mediated via accessory cells, we detected direct NK cellrecognition of another intracellular pathogen, the proto-zoan Neospora caninum (Boysen et al., 2006a). Purified, IL-2stimulated NK cells produced IFN-g in the absence of IL-12,although addition of IL-12 greatly increased the response,and infected fibroblasts were killed by NK cells. In asubsequent study, calves experimentally infected with N.

caninum responded with increased blood levels of NK cells(Klevar et al., 2007), in agreement with reports of early NKcell mobilization during infections (Gregoire et al., 2007).In the infected animals, these cells were also shown to beamong the early IFN-g producing cells responding tosonicated N. caninum antigens. The response to thesesonicates did not occur directly, as seen previously withintact parasites, but required accessory cells (Boysen et al.,2006a; Klevar et al., 2007).

An immunohistochemical study demonstrated infiltra-tion of NK cells to the placenta following experimental N.

caninum-infection, suggesting a role in the induction of aTh1-response that lead to fetal rejection (Maley et al.,2006). On the other hand, NK cells were only sparselypresent in the placenta of uninfected cows, raising thequestion if these cells participate in normal placentalphysiology like in humans and mice (Croy et al., 2006;Moffett and Loke, 2006). In another protozoal infection ofcattle, Babesia bovis, NK cells were shown to be the majorearly IFN-g producing cell, dependent on IL-12 and IL-18stimulation from accessory cells (Goff et al., 2003, 2006). Inaddition, NK cells stimulated by Babesia-exposed DCsincreased their cytotoxic ability, supported by detection ofDC-NK cell contact in tissues early in B. bovis infection(Bastos et al., 2008).

7.3. Impact for veterinary host-pathogen research and

vaccinology

These recent studies of infections, together with theearly studies of NK-like and LAK activity against virus-infected cells (discussed in Section 4), indicate a role forbovine NK cells in the early defence against infections.However, only two reports have so far studied their role in

vivo (Maley et al., 2006; Klevar et al., 2007). Even inhumans, limited in vivo studies of NK cells and infectionexist, and since experimental trials are not possible inhumans, much has to be learned from NK cell deficiencies


and susceptibility to various infections. Unfortunately,there exist only a few, rare types of NK deficiencies inhumans (Orange, 2002), perhaps in itself a testimony of theimportance of these cells. Moreover, human data oftenseem to diverge from experimental studies in murinemodels, perhaps explained by the theory of a low degree ofmicrobial priming of NK cells in laboratory mice (discussedabove). Thus, that an outbred, microbially exposed specieslike the cow can offer an important complementary modelfor immunology (Hein and Griebel, 2003) certainly holdstrue for the field of NK cell biology.

NK cells and other innate lymphocytes like gdT-cellshave recently been proposed as crucial mediators ofadjuvant effects (Woo et al., 2006; Martino and Poccia,2007; Brilot et al., 2008; Karimi et al., 2008). Theobservations that mycobacterial antigens stimulate bovineNK cell activation in an innate (adjuvant-like) manner(Olsen et al., 2005), and that young individuals have morenumerous (and perhaps more primed) circulating NK cellsthan adults, could partly explain the increased efficacy ofBCG vaccines in neonatal individuals compared to adults, asnoted in both cattle and humans (Hope and Villarreal-Ramos, 2008). Furthermore, whole mycobacteria like BCGhave been shown to contain NK-cell triggering structures(Schierloh et al., 2007; Esin et al., 2008; Marcenaro et al.,2008), while individual antigens may not always be NK-cellstimulating (Olsen et al., 2005); these findings should beconsidered when investigating why whole-bacteriumvaccines like BCG induce better protection than subunitvaccines (Hope and Villarreal-Ramos, 2008). Appropriatecellular responses to vaccines may require the simultaneoustriggering of several pattern-recognizing receptors (Trinch-ieri and Sher, 2007), and NK cells also need multiple signalsto be activated (Bryceson et al., 2006a). Thus, vaccineformulations against intracellular infections may need to becomplex, and the study of an isolated vaccine componentmay not reach a response threshold on its own. Further-more, synergistic adjuvant effects should be targeted notonly towards ‘‘classical’’ immune sensors like DCs, but alsoencompass innate lymphocytes like NK cells.

8. Concluding remarks

The recent characterization of bovine NK cells has leadto findings that suggest a role of these cells duringinfections. An emerging understanding of these cells asregulators of adaptive immune responses underscores whyNK cells should be considered in the studies of host-pathogen interactions, vaccinology and other aspects ofveterinary immunological research. As discussed in thisreview, observations made in cattle not only can helpdevelopment in the veterinary context, but also maypotentially open up new ways to understand innateimmunity in general.

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