Mouse and Rat NK and NK-T Cell Research Applied Reagents and Techniques

PharMingen Mouse and Rat NK and NK-T Cell Research Applied Reagents and Techniques BD Biosciences Clontech Discovery Labware Immunocytometry Systems PharMingen Transduction Laboratories

NK Cells Natural killer (NK) cells play an instrumental role in the innate immune responses against certain bacterial, parasitic and viral pathogens 1. Furthermore, NK cells have been shown to play an important role in suppressing tumor metastasis and growth 2. Activation of NK cells is primarily achieved by the viral induced interferons α/β (IFN-α and IFN-β) and to a lesser extent by IL-12 3 and IL-18 4. Once activated, NK cells produce large amounts of IFN-γ and a wide variety of other cytokines, combinations of which are thought to specifically tailor downstream adaptive immune responses against the invading pathogen 3. These important functions place NK cells at the interface between the innate and adaptive immune responses 5. For research use only. Not for use in diagnostic or therapeutic procedures. Not for Resale. i

NK Cell Development NK cells develop from pluripotent bone marrow derived hematopoietic stem cells and at one stage in their development share a common progenitor with T cells 6. However, unlike T cells, NK cells do not require receptor gene rearrangement for successful maturation. Primitive NK cells can be cultured from lineage negative, c-kit + (CD117), Sca-2 + (TSA-1, Ly-6E) multipotent progenitors in the presence of early acting cytokines such as IL-7, Stem Cell Factor, and flt3 ligand. However, further maturation of these primitive NK cells requires the presence of IL-15 7 (Figure 1). The crucial role of IL-15 has been demonstrated in mice that lack the IL-2/15Rβ chain or the IL-15Rα chain. These mice are deficient in NK and NK-T cells. In addition, removal of interferon regulatory factor 1 (IRF-1) results in reduced numbers of NK cells that appear phenotypically normal but lack cytolytic effector function 4. IRF-1 is a transcription factor essential for effective expression of IL-15 and perhaps other cytokines whose genes also contain IRF-1 response elements. Further development of NK cells requires the acquisition of inhibitory receptors, a process requiring the expression of MHC class I and as yet unidentified stromal derived factors 8. Terminal differentiation of NK cells into IFN-γ producing cells likely requires the action of IL-12 and IL-18, since loss of either results in significantly decreased NK cell cytotoxicity and IFN-γ production 4. The primary peripheral NK cell subset is present as a mature population of leukocytes, which can be easily identified in several compartments including peripheral blood, spleen and bone marrow. Progenitor NK Precursor Mature NK Cell SCF IL-7 flt-3l IL-15 IL-18 IL-12 CD34 +/- CD122 - c-kit + (CD117) Sca-2 + CD122 + NK1.1 - (CD161 - ) IL-15Rα + CD122 + NK1.1 + (CD161 + ) Ly49 + Figure 1. The role of cytokines in NK cell development. The basic steps of NK cell differentiation are shown together with the cytokines involved at each step and expression of selected surface antigens and receptors 4,7. 1

NK Cell Receptors NK cells are regulated by opposing signals from receptors that can either activate or inhibit effector function 5,9. Target cell specificity is not provided by the activating receptors that may be stimulated by a variety of signals, but rather by an array of inhibitory receptors that recognize MHC class I. This system is consistent with the missing-self hypothesis which postulates that NK cells survey tissues for normal expression of MHC class I and will become activated when in contact with cells that have down-regulated or do not express MHC class I 10. The last few years have seen a dramatic increase in the number of inhibitory receptors identified and a concurrent expansion in our understanding of their involvement in NK cell function. In contrast, many of the receptors involved in activation have been known for several years but only recently have their signaling pathways become more defined (Figure 2). MHC Class I Potential Target Cell Rae I H-60 Qa-1 b Ly-49 Activating & Inhibitory NKG2D Activating NK Cell CD94 NKG2A,C,E Activating & Inhibitory Figure 2. Activating and inhibitory C-type lectin superfamily receptors expressed by mouse NK cells. The ligands are shown above their respective receptors. 2

Activating Receptors Unlike T cells and B cells, which recognize antigen using clonally restricted receptors generated by gene rearrangement, NK cells use a variety of different nonrearranging receptors to initiate cytolytic activity and cytokine production. Many of these receptors are not unique to NK cells, but are also expressed by T cells. Receptors implicated in mouse and rat NK cell activation include CD2, CD16, CD28, Ly-6, CD69, NKR-P1, 2B4 (CD244), NKG2D, and CD94/NKG2A,C,E. Ligands for some of these receptors have also been identified: CD2:CD58, CD16:IgG, 2B4:CD48, NKG2D:RaeI and H-60, and CD94/NKG2A,C,E:Qa-1 b,11. It is believed that to successfully initiate effector function, signaling through one receptor is not sufficient. Additive or synergistic interactions between multiple activating receptors, which perhaps signal through a set of shared adaptor molecules or signaling pathways, is required 11. The well-known mouse NK cell antigen, NK-1.1 12, is a member of the NKR-P1 family of stimulatory receptors originally identified in the rat 13. These type II trans-membrane proteins are members of the C-type (Ca 2+ dependent) lectin superfamily and are expressed as dimers on the surface of most NK cells 9 in mice (Figure 3) and rats. Interestingly, expression of NKR-P1A in the rat has also been detected on activated peripheral blood granulocytes 14 and on activated monocytes 15 (Figure 4). Immunoreceptor tyrosine-based inhibition motifs (ITIMs) have been identified in rat and mouse NKR-P1B, suggesting that certain isoforms may be inhibitory rather than activating. NKR-P1 knockout studies and NKR-P1 inhibition studies suggest a role for this receptor in target cell recognition 16, 17. The physiological ligands of NKR-P1 have not been identified. NKG2D, also a C-type lectin family member, is expressed as a homodimer and associates with the signaling subunit DAP10 on the surface of NK cells. Two ligands, H-60 and RaeI, have been identified for NKG2D. Both are distant relatives of MHC class I. RaeI, while not expressed in normal tissue, is expressed on a large variety of tumor cells supporting the role of NK cells in tumor cell killing 18. Inhibitory Receptors The inhibitory receptor superfamily (IRS) 9 describes an expanding group of receptors that block activation of a number of different cell types in the immune system. By definition, IRS members must act in trans, be able to recruit phosphatases (such as SHP-1) through an ITIM, and must associate directly with an activating receptor 5. NK cell inhibitory receptors fall into two main structural groups, the calcium dependent lectin-like receptors and the immunoglobulin-like receptors. C-type lectin family members include the Ly-49 family identified in rodents, and CD94 and the NKG2 family identified in rodents and humans. These C-type lectins, predominantly expressed on NK cells, are encoded by genes located on distal mouse chromosome 6 and syntenic rat chromosome 4 and human chromosome 12 in a region called the NK gene complex. A B (negative control) C (DX5) D (2B4) (3A4) Figure 3. Co-expression of NK-1.1 with DX5, 2B4, and 3A4. C57BL/6 splenocytes were simultaneously stained with PK136-APC (anti-mouse NK-1.1) and DX5-PE (anti-mouse Pan-NK) (B), 2B4-PE (anti-mouse CD244) (C), or 3A4-Purified (antimouse NK cells) (D). DS-1-PE (antimouse IgM) was used to detect 3A4 and was used as a negative control (A). 3

SSC-Height SSC-Height Counts Counts 0 50 100 150 200 250 0 50 100 150 200 250 200C 0 40 80 120 160 A Granulocytes 0 50 100 150 200 250 B FSC-Height Mononuclear Cells (NKR-P1A) Mononuclear Cells 0 10 20 30 D (NKR-P1A) Granulocytes (NKR-P1A) Figure 4. Expression of NKR-P1A by rat granulocytes and monocytes. Lewis rat peripheral blood was stained with 10/78-PE (anti-rat NKR- P1A) and expression of the receptor was examined on different blood cell types. Granulocytes, distinguished by their high side scatter characteristics (A and B, in green) have an intermediate level of NKR-P1A expression (D). NK and NK-T cells (B, in blue and C) have a high level of NKR-P1A expression, while monocytes (B, in red and C) have an intermediate level of NKR-P1A expression. The Ly-49 receptor family includes at least 10 members 19 and is responsible for the recognition of polymorphic MHC class I molecules on potential target cells. Expression of the Ly-49 family of receptors is a relatively late event in NK cell development. At birth, significant numbers of NK cells are found in the spleen and bone marrow of the newborns, but they do not express Ly-49 receptors. Instead, acquisition of receptors occurs gradually over the first few weeks of life reaching a plateau at approximately one month of age 6. Successful recognition of MHC class I by Ly-49 requires the α1 and α2 domains of MHC class I as well as the presence of a peptide in the peptide binding groove 5. Diversity within the Ly-49 family is provided by alternate mrna splicing and allelic polymorphism. Expression of Ly-49 genes is monoallelic and each NK cell can co-express several Ly-49 receptors at one time (Figure 5). Figure 5. Co-expression of the Ly-49 receptors and NK-1.1. C57BL/6 splenocytes were simultaneously stained with PK136-APC (anti-mouse NK-1.1, all panels) and A1-FITC (anti-mouse Ly-49A) (A), 12A8-FITC (anti-mouse Ly-49A/D) (B), 5E6-FITC (anti-mouse Ly-49C/I) (C), 4E5-FITC (anti-mouse Ly-49D) (D), 4D11- FITC (anti-mouse Ly-49G2) (E), YLI-90-FITC (anti-mouse Ly-49I) (F), 14B11-Biotin (anti-mouse Ly-49C/F/H/I) followed by Streptavidin-FITC (G), or HBF-719-PE (anti-mouse Ly-49F) (H). 4 A D (Ly-49A B6 ) (Ly-49D) G B E (Ly-49C/F/H/I) (Ly-49A/D) (Ly-49G2) H C F (Ly-49F) (Ly-49C/I) (Ly-49I)

Acquisition of Ly-49 receptors is thought to be a stochastic process in which NK cells will continue to express different Ly-49 receptors in a cumulative manner until they express one that recognizes self MHC class I. The resulting diverse array of receptors displayed by each NK cell creates the divergent repertoire that monitors cells for the selective loss of class I molecules 20. Interestingly, two of the Ly-49 receptors Ly-49D and Ly-49H appear to be activating rather than inhibitory members of this family 19. The NKG2 family of C-type lectins includes NKG2A, NKG2C, NKG2D, and NKG2E. To be functionally active, NKG2A, C, or E form heterodimers with the invariant C-type lectin CD94 (Figure 6). These three heterodimeric receptors recognize the nonclassical MHC class I ligand Qa-1 b in mice. Successful recognition of Qa-1 b not only requires an intact CD94/NKG2 heterodimer but also the presence of a specific peptide which is derived from the signal sequence of some classical MHC class I molecules and presented by Qa-1 b,21. Therefore, while the Ly-49 family of receptors provides a direct method of MHC class I surveillance, the CD94/NKG2 receptors provide an indirect way of monitoring class I expression. Of the three NKG2 isoforms, only NKG2A contains an ITIM and has been shown to be inhibitory. In contrast, NKG2C and E bind the ITAM (immunoreceptor tyrosine based activation motif) -containing adaptor protein DAP12 and have been shown to be activating receptors 21. It should be noted that while the activating receptor NKG2D is grouped with the other three NKG2 isoforms by name, it bears very little sequence similarity to these proteins. It does not form hetrodimers with CD94 and it associates with DAP10. The only link between NKG2D and the other NKG2 proteins is the close proximity of these genes to each other within the NK gene complex. A less well-defined receptor implicated in NK cell inhibition was originally described as the mouse mast cell function-associated antigen (mmafa) by virtue of its high sequence similarity to rat MAFA. However, mouse MAFA is not expressed on mast cells, but is expressed on NK cells 22 (Figure 7) and virus-activated CD8 + T cells 23. This C-type lectin has an ITIM and has been renamed killer cell lectin-like receptor G1 (KLRG1 24 ). The physiological function of KLRG1 in NK cells and CD8 + T cells is not yet known. Unlike rodent NK cells that express the C-type lectin family of inhibitory receptors (Ly-49), human NK cells express KIRs (killer cell inhibitory receptors) which are members of the immunoglobulin superfamily 5. The closest relative in mice of human KIR is gp49, which was originally described on mast cells 25. It has now been established that the two isoforms, gp49a and gp49b, are expressed on activated NK cells, and gp49b, which contains an ITIM, can inhibit NK-1.1-mediated cytokine release 26. The KIR family is divided depending on the number of Ig domains expressed on the cell. The KIR3D is approximately 70 kda and has three extracellular Ig domains. The KIR2D is approximately 50-58 kda and has two extracellular Ig domains. These molecules recognize specific HLA-B and HLA-C alleles. (NKG2A/C/E) A B (CD94) C (NKG2A/C/E) (CD94) Figure 6. Co-expression of NK-1.1 and the CD94/NKG2A,C,E receptor subunits. C57BL/6 splenocytes were simultaneously stained with PK136-APC (anti-mouse NK-1.1, Panels A and B) and 18d3-Biotin (anti-mouse CD94) (A) or 20d5-FITC (anti-mouse NKG2A,C,E) (B). Panel C shows coexpression of 18d3-Biotin (anti-mouse CD94) and 20d5-FITC (anti-mouse NKG2A,C,E). 18d3-Biotin was detected using Streptavidin-PE. Note how the CD94 bright population is NKG2A,C,Epositive and the CD94 dim population is NKG2A,C,E-negative. 5

Counts 0 5 10 15 20 25 30 A Bone Marrow 43.3% Counts 0 140 280 420 560 700 A CD3 - CD3 + B CD3-70.3% C CD3 + 4.8% (CD3) (KLRG1) (KLRG1) Counts 0 80 160 240 320 400 B Liver 57.6% Counts 0 220 440 660 880 1100 D CD3 - CD3 + E CD3-67.3% F CD3 + 6.4% (CD3) (KLRG1) (KLRG1) Counts 0 80 160 240 320 400 C Spleen 3.9% Figure 8. Expression of NK-1.1 on CD3 + cells from the bone marrow, liver, and spleen. Lymphocytes were isolated from C57BL/6 bone marrow (A), liver (B) and spleen (C) and simultaneously stained with PK136-APC (anti-mouse NK-1.1) and 145-2C11- FITC (anti-mouse CD3). For analysis of cells from each tissue a gate was used to select only CD3 + cells. Expression of NK1.1 on CD3 + cells is shown in histograms A-C. The percent CD3 + cells that are also positive for NK1.1 is greater in the bone marrow (43.3%) and liver (57.6%) than in the spleen (3.9%). Figure 7. Co-expression of NK-1.1 and KLRG1 on freshly isolated and in vitro stimulated NK cells and T cells. Freshly isolated (A-C) or IL-2 activated (D-F) C57BL/6 splenocytes were simultaneously stained with PK136-APC (anti-mouse NK-1.1), 145-2C11-FITC (anti-mouse CD3e), and 2F1-biotin (anti-mouse KLRG1) followed by streptavidin-pe. Gating of the CD3 - or CD3 + populations is shown in panels A and D. KLRG1 is expressed on most unstimulated CD3 - /NK-1.1 + cells (B) but not CD3 + /NK-1.1 + cells (C). In vitro stimulation with IL-2 does not induce expression of KLRG1 on CD3 + / NK-1.1 + cells (F). NK-T cells It is well documented that several activating and inhibitory receptors found on NK cells are also expressed by subpopulations of T cells 27. NK-T cells in C57BL/6 mice are defined as lymphocytes expressing T cell receptor (TCR) in conjunction with NK-1.1 and other NK cell associated molecules 28. Classically, these cells were described as being either CD4 + CD8 - or CD4 - CD8 - and reacting with the non-classical MHC class I molecule CD1 through a restricted Vα14-Jα281 TCR chain associated with either a Vβ2, -7, or 8 TCR chain. These NK-T cells produce large amounts of IL-4 and are present in much higher frequencies in thymus, liver and bone marrow than in the spleen or lymph nodes (Figure 8), suggesting that they exert their functions in specialized locations. Speculations as to their physiological role include: the regulation of hematopoiesis in bone marrow, involvement in central and peripheral T cell tolerance, lysis of virus infected cells in the liver and influencing the differentiation of T helper precursors to T helper 2 cells 29. More recently it was shown that some CD8 + T cells also express NK cell markers including NK-1.1 and members of the Ly-49 family. Unlike classical NK-T cells, CD8 + NK-T cells do not express a restricted TCR repertoire but express a diverse one that is almost identical to the conventional CD8 + TCR repertoire. It has been shown that CD8 + NK-T cells have a memory phenotype, and it is therefore proposed that the expression of inhibitory receptors serves to dampen the immune response and/or inhibit T cell autoreactivity 29, 30. 6

NK Cell Associated Reagents Cat. No. New Cat. No. Specificity Clone Format Size Mouse 09941D 553855 Pan-NK cells DX5 Purified 0.5 mg 09942D 553856 Biotin 0.5 mg 09944D 553857 FITC 0.5 mg 09945B 553858 PE 0.2 mg 09949A 550991 APC 0.1 mg 01611A 557442 NK cells/3a4 3A4 Purified 0.1 mg 01701A 557451 CD244 (2B4 antigen) 2B4 Purified 0.1 mg 01702A 557452 Biotin 0.1 mg 01704D 553305 FITC 0.5 mg 01705B 553306 PE 0.2 mg 09111D 553675 Ly-49A B6 A1 Purified 0.5 mg 09112A 557423 Biotin 0.1 mg 09114D 553677 FITC 0.1 mg 09115A 557424 PE 0.1 mg 01620D 553273 Ly-49C and Ly-49I 5E6 NA/LE 0.5 mg 01621A 557417 Purified 0.1 mg 01622A 557418 Biotin 0.1 mg 01624D 553276 FITC 0.5 mg 01625B 553277 PE 0.2 mg 28530D 550985 Ly-49F HBF-719 NA/LE 0.5 mg 28531A 550984 Purified 0.1 mg 28532A 550986 Biotin 0.1 mg 28535A 550987 PE 0.1 mg 28461A 550579 Ly-49G2 B6 Cwy-3 Purified 0.1 mg 28464D 550580 FITC 0.5 mg 28465A 550581 PE 0.1 mg 28470D 550624 Ly-49I YLI-90 NA/LE 0.5 mg 28471A 550593 Purified 0.1 mg 28472A 550596 Biotin 0.1 mg 28474D 550594 FITC 0.5 mg 28475A 550595 PE 0.1 mg 28195A 555311 Ly-49A and Ly-49D 12A8 PE 0.1 mg 28201D 555312 Ly-49D 4E5 Purified 0.5 mg 28204D 555313 FITC 0.5 mg 28211D 555314 Ly-49G2 (LGL-1) 4D11 Purified 0.5 mg 28214D 555315 FITC 0.5 mg 28219A 555316 APC 0.1mg 28521A 550864 Ly-49C, F, H, and I 14B11 Purified 0.1 mg 28522A 550865 Biotin 0.1 mg 28441A 550518 NKG2A,C,E 20d5 Purified 0.1 mg 28442A 550519 Biotin 0.1 mg 28444D 550520 FITC 0.5 mg 01290D 553161 NK-1.1 (NKR-P1B and NKR-P1C) PK136 NA/LE 0.5 mg 01291D 553162 Purified 0.5 mg 01292D 553163 Biotin 0.5 mg 01294D 553164 FITC 0.5 mg 01295A 557391 PE 0.1 mg 01295B 553165 PE 0.2 mg 01299A 550627 APC 0.1 mg 0129TA 551114 PerCP-Cy5.5 0.1 mg 28371D 550081 NK-T/NK Cell Antigen U5A2-13 Purified 0.5 mg 28375B 550082 PE 0.2 mg 28491A 550772 CD94 18d3 Purified 0.1mg 28492A 550773 Biotin 0.1mg 28510D 550862 KLRG1 (MAFA) 2F1 NA/LE 0.5mg 28511A 550861 Purified 0.1 mg 28512A 550863 Biotin 0.1 mg 7

NK Cell Associated Reagents (continued from page 7) Cat. No. New Cat. No. Description Clone Format Size Rat 22641D 555006 CD161a (NKR-P1A) 10/78 Purified 0.5 mg 22642D 555007 Biotin 0.5 mg 22644D 555008 FITC 0.5 mg 22645B 555009 PE 0.2 mg Human 30620D 555403 CD16 3G8 NA/LE 0.5 mg 30621A 555404 Purified 0.1 mg 30622X 555405 Biotin 100 tests 30624X 555406 FITC 100 tests 30625X 555407 PE 100 tests 30628X 555408 Cy-Chrome 100 tests 31660D 555513 CD56 B159 NA/LE 0.5 mg 31661A 555514 Purified 0.1 mg 31662X 555515 Biotin 100 tests 31665X 555516 PE 100 tests 31668X 555517 Cy-Chrome 100 tests 31669X 555518 APC 100 tests 37421B 559043 CD56 NCAM16.2 Purified 0.2 mg 37491B 559049 CD56 MY31 Purified 0.2 mg 33251A 555618 CD57 NK-1 Purified 0.1 mg 33254X 555619 FITC 100 tests 33255X 555620 PE 100 tests 37471B 559048 CD57 HNK-1 Purified 0.2 mg 36814X 556062 CD158a HP-3E4 FITC 100 tests 36815X 556063 PE 100 tests 36811A 556061 Purified 0.1 mg 37754X 559784 CD158b CH-L FITC 100 tests 37755X 559785 PE 100 tests 37751A 559783 Purified 0.1 mg 37750D 559782 NA/LE 0.5 mg 36880D 559238 CD161 (NKR-P1A) DX12 Purified 0.5 mg 36881A 556079 Purified 0.1 mg 36884X 556080 FITC 100 tests 36885X 556081 PE 100 tests 37761A 559787 DNAM-1 DX11 Purified 0.1 mg 37764X 559788 FITC 100 tests 37765X 559789 PE 100 tests 36100D 555963 NKB1 DX9 NA/LE 0.5 mg 36101A 555964 Purified 0.1 mg 36102X 555965 Biotin 100 tests 36104X 555966 FITC 100 tests 36105X 555967 PE 100 tests 38081A 550810 LAIR-1 DX26 Purified 0.1 mg 38085X 550811 PE 100 tests 38101A 550814 2B4 2-69 Purified 0.1 mg 38104X 550815 FITC 100 tests 38105X 550816 PE 100 tests 36840D 559239 KIR-NKAT2 DX27 NA/LE 0.5 mg 36841A 556069 Purified 0.1 mg 36844X 556070 FITC 100 tests 36845X 556071 PE 100 tests 35151A 555887 CD94 (KP43) HP-3D9 Purified 0.1 mg 35154X 555888 FITC 100 tests 35155X 555889 PE 100 tests 35159X 559876 APC 100 tests 8

References 1. Trinchieri G. 1989. Biology of natural killer cells. Adv Immunol 47:187-376. 2. Kim S, Iizuka K, Aguila HL, Weissman IL, Yokoyama WM. 2000. In vivo natural killer cell activities revealed by natural killer cell-deficient mice. PNAS USA 97(6):2731-6. 3. Biron CA, Nguyen KB, Pien GC, Cousens LP, Salazar-Mather TP. 1999. Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu Rev Immunol. 17:189-220. 4. Liu CC, Perussia B, Young JD. 2000. The emerging role of IL-15 in NK-cell development. Immunol Today. 21:113-6. 5. Long EO. 1999. Regulation of immune responses through inhibitory receptors. Annu Rev Immunol. 17:875-904. 6. Raulet DH. 1999. Development and tolerance of natural killer cells. Curr Opin Immunol. 11:129-34. 7. Williams NS, Kubota A, Bennett M, Kumar V, Takei F. 2000. Clonal analysis of NK cell development from bone marrow progenitors in vitro: orderly acquisition of receptor gene expression. Eur J Immunol. 30:2074-82. 8. Held W, Dorfman JR, Wu MF, Raulet DH. 1996. Major histocompatibility complex class I-dependent skewing of the natural killer cell Ly49 receptor repertoire. Eur J Immunol. 26:2286-92. 9. Lanier LL. 1998. NK cell receptors. Annu Rev Immunol. 16:359-93. 10. Karre K, Ljunggren HG, Piontek G, Kiessling R. 1986. Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. Nature. 319:675-8. 11. Lanier LL. 2000. Turning on natural killer cells. J Exp Med. 191:1259-62. 12. Glimcher L, Shen FW, Cantor H. 1977. Identification of a cell-surface antigen selectively expressed on the natural killer cell. J Exp Med. 145:1-9. 13. Giorda R, Rudert WA, Vavassori C, Chambers WH, Hiserodt JC, Trucco M. 1990. NKR-P1, a signal transduction molecule on natural killer cells. Science. 249:1298-300. 14. Webster GA, Bowles MJ, Karim MS, Wood RF, Pockley AG. 1994. Activation antigen expression on peripheral blood neutrophils following rat small bowel transplantation. NKR-P1 is a novel antigen preferentially expressed during allograft rejection. Transplantation. 58:707-12. 15. Scriba A, Schneider M, Grau V, van der Meide PH, Steiniger B. 1997. Rat monocytes up-regulate NKR-P1A and down-modulate CD4 and CD43 during activation in vivo: monocyte subpopulations in normal and IFN-gamma-treated rats. J Leukoc Biol. 62:741-52. 16. Ryan JC, Niemi EC, Nakamura MC, Seaman WE. 1995. NKR-P1A is a target-specific receptor that activates natural killer cell cytotoxicity. J Exp Med. 181:1911-5. 17. Kung SK, Miller RG. 1995. The NK1.1 antigen in NK-mediated F1 antiparent killing in vitro. J Immunol. 154:1624-33. 18. Diefenbach A, Jamieson AM, Liu SD, Shastri N, Raulet DH. 2000. Ligands for the murine NKG2D receptor: expression by tumor cells and activation of NK cells and macrophages. Nature Immunol. 1:119-126. 19. Smith HR, Chuang HH, Wang LL, Salcedo M, Heusel JW, Yokoyama WM. 2000. Nonstochastic coexpression of activation receptors on murine natural killer cells. J Exp Med. 191:1341-54. 20. Dorfman JR, Raulet DH. 1998. Acquisition of Ly-49 receptor expression by developing natural killer cells. J Exp Med. 187:609-18. 21. Vance RE, Jamieson AM, Raulet DH. 1999. Recognition of the class Ib molecule Qa-1(b) by putative activating receptors CD94/NKG2C and CD94/NKG2E on mouse natural killer cells. J Exp Med. 190:1801-12. 22. Hanke T, Corral L, Vance RE, Raulet DH. 1998. 2F1 antigen, the mouse homolog of the rat mast cell function-associated antigen, is a lectin-like type II transmembrane receptor expressed by natural killer cells. Eur J Immunol. 28:4409-17. 23. Blaser C, Kaufmann M, Pircher H. 1998. Virus-activated CD8 T cells and lymphokineactivated NK cells express the mast cell function-associated antigen, an inhibitory C-type lectin. J Immunol. 161:6451-4. 24. Corral L, Hanke T, Vance RE, Cado D, Raulet DH. 2000. NK cell expression of the killer cell lectin-like receptor G1 (KLRG1), the mouse homolog of MAFA, is modulated by MHC class I molecules. Eur J Immunol. 30:920-30. 25. Rojo S, Burshtyn DN, Long EO, Wagtmann N. 1997. Type I transmembrane receptor with inhibitory function in mouse mast cells and NK cells. J Immunol. 158:9-12. 26. Wang LL, Chu DT, Dokun AO, Yokoyama WM. 2000. Inducible expression of the gp49b inhibitory receptor on NK cells. J Immunol. 164:5215-20. 27. Bendelac A, Rivera MN, Park SH, Roark JH. 1997. Mouse CD1-specific NK1 T cells: development, specificity, and function. Annu Rev Immunol. 15:535-62. 28. Assarsson E, Kambayashi T, Sandberg JK, Hong S, Taniguchi M, Van Kaer L, Ljunggren HG, Chambers BJ. 2000. CD8+ T cells rapidly acquire NK1.1 and NK cell-associated molecules upon stimulation in vitro and in vivo. J Immunol. 165:3673-9. 29. Vicari AP, Zlotnik A. 1996. Mouse NK1.1+ T cells: a new family of T cells. Immunol Today. 17:71-6. 30. Coles MC, McMahon CW, Takizawa H, Raulet DH. 2000. Memory CD8 T lymphocytes express inhibitory MHC-specific Ly-49 receptors. Eur J Immunol. 30:236-44. 9

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