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    • NK Cells
      • Experimental Basis of Immunology
      • January 17, 2007
      • W.H. Chambers, Ph.D.
      • G.17e Hillman Cancer Center
      • 623-3218
      • [email_address]
    • I. Introduction
      • Natural Killer (NK) Cells were first described in the early 1970’s by R. Herberman; R. Kiessling; and G. and E. Klein
      • Defined as a functional entity, i.e. cell capable of recognizing and killing tumor cells without prior exposure
      • Represent a component of the non-adaptive immune system
      • Defined in the early 1980’s as having a large granular lymphocyte (LGL) morphology (Reynolds, et al., 1981)
      • Represent a heterogeneous population of cells with diverse functions
      • Can be best defined phenotypically as CD3 - , CD16 + , CD56 + , CD122 + , CD158 + , CD161 +
    • Innate capacity of lysis Large granular lymphocytes CD3 - , CD16 + , CD56 + , CD122 + , CD158 + , CD161 +
    • II. Pathway of NK Cell Differentiation: Topics
      • Differentiation of NK cells in the fetus
      • Differentiation of NK cells in adults
      • Terminal differentiation of mature
      • NK cells
    • NK Cell Differentiation
      • Derive from, and require normal, intact bone marrow for functional maturation
      • Represent one of the major lymphocyte populations [T, B, NK, NK-T] – ~5% of cells among PBLs
      • Present in athymic [nude] mice and rats
      • Present in scid mice, and in RAG-1 and RAG-2 knockout mice
      • Can be distinguished from other lymphocytes by the absence of clonally distributed, receptors derived via gene rearrangements
    • c-kit + Thy-1 - CD25 - CD161c - c-kit + Thy-1 - CD25 - CD161c - CD19- B220 lo Fetal liver c-kit + Thy-1 + CD25 - CD161c + Fetal blood c-kit + Thy-1 + CD25 - CD161c + c-kit - Thy-1 +/- CD25 - CD161c + c-kit + Thy-1 + CD25 + CD161c - T Fetal thymus NK Progenitors: Fetus Modified from Lian and Kumar, 2002 p-NK c-T/NKP CLP HSC p-T/NK p-T/NK p-T NK . . . .
    • Differentiation of NK Cells: In Vitro Requirements for Growth and Maturation
      • - Stroma from normal animals
      • estrogen- or strontium-treated mice have functionally impaired NK cells
      • stroma from LT  -/- mice have functionally impaired NK cells
      • - Cytokines for growth and Differentiation
      • c-kit ligand; IL7; Flt3 ligand [stem cell factor]; IL15
      • - Cytokines and direct contact with stroma are required for differentiation of phenotypically and functionally mature NK cells – LY49 - NK cells develop in cultures with cytokines but no stroma
    • NK Progenitors: Adult Lin - c-kit lo Thy-1 - IL7R + Sca-1 lo CD122 + CD161c - CD49b - CD122 + CD161c + CD49b - CD122 + CD161c + CD49b + Ly49 + Bone marrow stroma Modified from Lian and Kumar, 2002 Thymus CD122 + CD161c + CD49b + Ly49 + NK . . . . HSC CLP NKP p-NK NK . . . . p-T/NK p-NK p-T NK . . . . T T
    • NKP T/NKP CLP HSC Modified from Yokoyama, et al, 2004 Cytokines: IFN  GM-CSF TNF  Bone Marrow Periphery Enhanced CTX Stimulus pB pT MY X CTX NK NK NK NK NK . . . . NK . . . . NK . . . . NK . . . .
    • Cytokines: IFN  GM-CSF TNF  Periphery Enhanced CTX Stimulus: Hrs IL2 IL12 IL15 IL23 IL27 IFN  , -  NK Progenitors: Adult NK NK NK NK NK . . . .
    • Cytokines: IFN  GM-CSF TNF  Periphery Enhanced CTX, with broader specificity Stimulus: Days IL2 IL12 IL15 IL23 IL27 IFN  , -  Proliferation NK Progenitors: Adult NK NK NK NK NK . . . .
    • Knockouts/Transgenics: Transcription Factors
      • Gene Deleted Effect Reference
      • Ikaros NK cells absent Georgopoulos, 1994
      • Wang, 1996
      • PU.1 NK cell number decreased, Colucci, 2001
      • normal lytic function
      • Ets-1 NK cell number decreased, Barton, 1998
      • decreased lytic function
      • Id2 NK cells decreased or absent, Yokota, 1999
      • reduced lytic function Ikawa, 2001
      • TCF-1 Altered acquisition of Ly49s Held, 1999
      • Kunz,2001
      • IRF-1 NK cell number decreased, Duncan, 1996
      • lytic function impaired Ogasawara, 1998
      • IRF-2 NK cell number decreased, Lohoff, 2000
      • lytic function impaired
    • Knockouts/Transgenics: Receptors
      • Gene Deleted Effect Reference
      • LT  r NK cells severely decreased Wu, 2001
      • LT  1  2 NK cells severely decreased, Iizuka, 1999
      • reduced lytic function Smyth, 1999
      • Ito, 1999
      • IL15R  NK cells severely decreased Lodolce, 1998
      • IL2/15R  NK cells absent Gilmour, 2001
      • Suzuki, 1997
      • c-kit NK cells decreased, impaired Colucci, 2000
      • lytic function
    • Knockouts/Transgenics: Cytokines
      • Gene Deleted Effect Reference
      • IL15 NK cells absent; no lytic Puzanov, 1996
      • function Kennedey, 2000
      • Flt3-ligand NK cells severely decreased, McKenna, 2000
      • impaired lytic function
    • NK Cell Differentiation Pathway: Informative Gene Knockout and Transgenic Mice
      • HSC >> CLP >> T/NKP >> NKP >> NK
      • Trnscrptn. Fctr./DBP Ikaros Ets1 IRF-1
      • PU.1 (P)* Id2 IRF-2
      • STAT5a/b (P)
      • MEF (P)
      • Cytokine/Rcptr. Flt3L IL15
      • IL15R 
      • IL2/IL15R 
      • LT  /LT  R (P)
      • Sgnl. Trnsdcr. Jak3 CD3e tg
      • Fc  R1  tg
    • III. NK Cell Function as Anti-tumor and Anti-Viral Effector Cells
      • NK cells were initially described as being cells important for surveillance against tumor development, or more importantly, against tumor metastases
      • NK cells were also found to be important as anti-viral effector cells, particularly against Herpes virus infection.
    • NK cells and Anti-tumor Activity
      • What is the evidence of NK cell
      • anti-tumor function?
      • In vitro – many tumor cells are
      • susceptible to lysis by NK cells
      • depending upon how you assess killing (Kashii,
      • Y., et al. J. Immunol . 163:5358-66 [1999]).
      • In vivo…..
    • Putative Evidence for Immunosurveillance by NK Cells Using Transplantable Tumor Models
      • Elimination of NK cells resulted in increased tumor growth
      • Elimination of NK cells resulted in increased numbers of metastastic lesions in lungs
      • Adoptive transfer of NK cells, into immunodeficient animals challenged with tumors, results in tumor clearance in metastases models
      • Best results almost always were derived in models of metastatic disease (Barlozzari, T., et al., J. Immunol . 134:2783-2789, 1985)
    • This evidence did not initially garner robust support for NK cell participation in immune surveillance – Why?
      • There has been a growing belief that transplantable tumor models have little value in assessing tumor immunity, and particularly for “immune surveillance” of tumors
      • The only report providing evidence for disease in individuals with reduced NK cells is for recurrent Herpes virus infections (Biron, C.A., et al., NEJM 322:1731-1735, 1989)
      • Identification of receptors on NK cells with coordinate tumor cell ligand was lacking
    • Studies Supporting Increased Incidence of Cancer in Immunosuppressed Individuals
      • An 11 year follow-up study of immune function and cancer incidence in a general population of 3625 individuals was carried out (Imai, K., et al., The Lancet 356:1795-1799, 2000)
      • Immune function, i.e. NK cell lytic activity, was assessed at baseline and cancer incidence
      • Medium and high cytolytic function was associated with reduced cancer risk; low cytolytic function was associated with increased cancer risk
    • Support for NK Cells Providing a Mechanism for Immune Surveillance of Cancer
      • Families of NK cell receptors (e.g. NKG2s) with activating and inhibitory function have been defined
      • Tumor associated ligands similar to MHC Class I have been defined, e.g. Rae-1 [mice], MICA/B [humans]
      • Binding of MHC Class I and Class I-related proteins (e.g. Rae-1(  ); ULBP-1, -2, -3; H60) by NKR has been demonstrated
      • In mice, binding of NKG2D to Rae1  (Cerwenka, A., et al., PNAS USA 98:11521-11526, 2001) or Rae1  (Diefenbach, A., et al., Nature 413:165-171, 2001) has been demonstrated to activate anti-tumor lytic function
      • Human orthologs of Rae-1 genes, e.g. ULBP-1 also are bound by NKG2D; and this activates NK lytic function
    • NK Cells as Anti-viral Effector Cells: Evidence for a role as anti-virus effector cells
      • Natural defects in NK cells
      • Recurrent Herpes virus infections [Biron, 1989]
      • Expansion of NK cells during viral infections
      • LCMV infections
      • Viral antigens as ligands for NK cell receptors
      • ULBP1-4
    • NK-mediated Response to Virus Infection
    • NK Cells as Anti-viral Effector Cells: Mechanisms of Evasion of NK Cell Function by Viruses
      • Expression of virally encoded MHC class I protein homologs
      • Selective modulation of MHC Class I expression by viral proteins
      • Virus-mediated inhibition of activating receptor function
      • Production of virally encoded cytokine-binding proteins or cytokine-receptor agonists
      • Direct viral effects on NK cells – infection/envelope ligation of inhibitory receptors
    • NK Cell Virus-infected Cell Cytokine Receptor MHC Class I Inhibitory Receptor MHC Class I Inhibitory Receptor Activating Receptor MHC ClassI Homolog Selective Expression Down Regulating Activating Ligand Activating Receptor Antogonist Cytokine Binding Protein Cytokine Receptor Cytokine Antogonist NK Cell Infection 1 2 3 4 5 Virus
    • IV. NK Cell Recognition Receptors
      • “Missing Self” Hypothesis
      • Activation and Inhibition via Receptors
      • Recognition of “Self”
      • Recognition of Tumor Cells
      • Recognition of Virus-infected Cells
    • “ Missing Self” Hypothesis
      • NK cells do not require expression of MHC Class I determinants for recognition of target cells.
      • There is, in fact, an inverse relationship between expression of MHC Class I and susceptibility to lysis by NK cells, i.e. less Class I equals more lysis.
      • Led to the hypothesis* that NK cells surveyed the surface of target cells for “self”. If it was present, the cell was presumed to be normal and not lysed. If self was absent, as is often the case in tumor cells and virus-infected cells, NK cells could be activated to lyse the “abnormal” cell.
      • *Ljunggren, H.G. and K. Karre, 1990. Immunology Today 11:237-244.
    • Receptors in Innate and Adaptive Immunity Yes No Able to recognize a wide variety of molecular structures Yes No Clonal distribution Yes No Require gene rearrangement Yes No Encoded in multiple gene segments No Yes Recognize broad classes of pathogens No Yes Trigger immediate response No Yes Expressed by all cells of a particular type No Yes Specificity inherited in the genome Adaptive Innate Characteristics
    • Recognition – NK cells
      • There is no evidence supporting clonally restricted recognition molecules expressed by NK cells, nor for recombinatorial events being important for development of an NK cell repertoire
      • NK cells recognize MHC determinants, but these structures, nor peptides expressed by MHC, are target antigens for activation of NK lytic function
      • Some NK cells express CD8 homodimers, but it is unclear whether binding to MHC Class I affects activation
      • NK cell recognition of targets involves a balance between inhibitory signals and activation signals
      • Receptor:ligand pairs providing inhibitory signals are fairly well defined
      • Receptor:ligand pairs providing activation signals are rapidly being defined
    • NK Cell Gene Complex (NKC)
      • The NKC is a genomic region, first described on NK cells, encoding structurally related receptors
      • NKC maps to Chromosome 12p13, 6 and 4 in man, mouse and rat, respectively
      • Type II integral membrane proteins with external domain similar to C-type (Ca ++ -dependent) lectins. However, they lack amino acid residues that coordinate binding of Ca ++ , and do not bind carbohydrates in the same manner as conventional C-type lectins. Can be expressed homo- or heterodimers.
      • Highly conserved evolutionarily – found in sea squirt and several poxviruses
      • Activating and inhibitory receptors for immune cells; can be either primary or co-stimulatory receptors.
    • NK Cell Gene Complex (NKC) - Contains genes encoding C type lectin related receptors - Disease resistance elements mapped to this locus, e.g. Cmv1 - Conserved across species Human – Chromosome 12 Mouse – Chromosome 6 Rat – Chromosome 4
    • Leukocyte Receptor Cluster (LRC) LRC is a ~1 mb region located on chromosome 19q13.42
    • NK Cell Inhibitory Receptors: CLRR and KIR
      • Name Alternative Name[s] Cellular Ligand Viral Ligand
      • p58.1 KIR2DL1 HLA-Cw2,4,5,6
      • p58.2 KIR2DL2 HLA-Cw1,3,7,8
      • p70 KIR3DL1 HLA-Bw4
      • p140 KIR3DL3 HLA-A3, -A11
      • p49 KIR2DL4 HLA-G
      • LIR2 ILT4/LILRB2 HLA-F
      • CD94* KLRD1 HLA-E**
      • NKG2A KLRC1/CD159A HLA-E
      • NKR-P1B, D CD161B, D Clrb
      • p40 LAIR1 ?
      • IRC1 IRp60/CMRF35H ?
      • p75AIRM1 Siglec-7 Sialylated sugars
      • *CD94 forms heterodimers with NKG2A, -C and –E
      • **CD94/CD159A heterodimer is specific for HLA-E
    • IgV COOH NH 3 Inhibition of lytic function NK Cell membrane I/VxYxxL Cytoplasm IRp60 SHP-1 Target Cell membrane
    • ITIM
      • Immunoreceptor tyrosine-based inhibitory motif
      • Based upon the amino acid motif: I/VxYxxL
      • Commonly expressed in signaling receptors in lymphocytes
      • Recruits SHP-1/SHP-2 phosphatases
      • Linked to inhibition of function in lymphocytes
      • Name Alternative Name[s] Cellular Ligand Viral Ligand
      • NKp46 Ly94/NCR1 ? SV-HA, IV-HA
      • NKp30 IC7/NCR3 ?
      • NKp44 Ly95/NCR2 ? SV-HA, IV-HA
      • 2B4 CD244 CD48
      • NTB-A KALI ?
      • NKp80 KLRF1 ?
      • CD16 Fc  RIII IgG
      • CD2 LFA-2 CD58, LFA-3
      • DNAM-1 CD226 PVR/CD155, Nectin-2/CD112
      • NKG2D D12S2489E/CD159D MICA, MICB, MULT1 ULBP1-4
      • NKR-P1A CD161A [IC-21]*
      • NKR-P1C CD161C ?
      • NKR-P1F CD161F Clrg
      • P40 LAIR1 ?
      • IRC1 IRp60/CMRF35H ?
      • p75AIRM1 Siglec-7 Sialylated sugars
      • *Rat NKR-P1A binds an undefined determinant on IC-21 tumor cells
      NK Cell Activating Receptors
    • NK cell activating receptors
      • Loss of the inhibitory signal does not, in and of itself, provide signals to kill target cells
      • Some receptors able to activate NK cells to kill target cells have been defined – NKG2D, Ly49D, Ly49H, NKp30, NKp44, NKp46, CD161A
      • Some activating receptors are members of the C-type lectin [e.g. NKG2D] and IgSF [NKp30] superfamilies
      • IgSF members often referred to as KARs
      • Associate with an adaptor molecule [e.g. DAP12] containing an ITAM. Associate via a charged residue in the TM domain
      • Some ligands for activating receptors have been defined, e.g. RAE-1 for NKG2D
    • NK Cell membrane NKp46:SV-HA or IV-HA Cytoplasm IgC2 IgC2 R * NH 3 COOH ZAP70 SYK D Fc  R1  CD3  I T A M I T A M I T A M I T A M Activation
    • NK Cell membrane NKp44:SV-HA or IV-HA Cytoplasm IgV D * DAP12 I T A M Activation K I T A M NH 3 COOH ZAP70 SYK
    • NK Cell membrane NKp30:? [iDCs and some tumors] Cytoplasm IgV R * CD3  I T A M I T A M NH 3 D I T A M I T A M I T A M I T A M COOH ZAP70 SYK Activation
    • ITAM
      • Immunoreceptor tyrosine-based activating motif
      • Based upon the amino acid motif: …YxxL/Ix 6-8 YxxL/I…
      • Serves as a signaling partner to transmembrane receptors with a charged residue in the transmembrane region which allows docking of signal transducers such as DAP12, CD3  -CD3  homodimers, CD3  -Fc  r1  heterodimers
      • Activation of cells either via PI3 kinase, or ZAP70 or Syk tyrosine kinases
    • NKG2D
      • Single gene
      • Distantly related to other NKG2 family members
      • Alternatively spliced isoforms (short and long) in mice
      • NKG2D-s and NKG2D-l, short from binds both DAP10 and DAP12
      • Expressed in NK cells, CD8 + cells and macrophages
    • NK Cell membrane * R CTLD CTLD NKG2D:MICA, MICB, ULBPs COOH COOH NH 3 NH 3 Cytoplasm Cytokine secretion Cytotoxicity D DAP10 Y x x M PI3K Grb2 ERK1/2 MAPK
    • Ligands for NK Cell Activating Receptors
      • MICA, MICB : Stress-inducible molecules encoded within the human MHC, also can be induced by some infections. Normally expressed by gastrointestinal epithelium, but also by some epithelial, lung, breast, kidney, ovary, prostate and colon tumors, and by some melanomas. Transmembrane with  1,  2, and  3 domains; but do not associate with  2m and do not bind peptides.
      • ULBP1-4 : 1-3 are GPI-linked, cell surface molecules which bind human cytomegalovirus UL-16; ULBP-4 is a cell surface molecule with transmembrane and cytoplasmic domains. ULBPs have  1 and  2 MHC Class I-like domains.
      • Rae1  : Retinoic acid inducible protein, in mice, that shares sequence homology with ULBPs. Expressed in early embryogenesis and in some tumors, but generally absent in normal tissues.
      • H60 : Minor histocompatibility antigen expressed by Balb/c mice, target for alloreactivity responses by C57Bl/6 mice.
      • DCs: Known that NKp30 is required for recognition of immature DCs by activated NK cells.
      • IC-21: Known that rat CD161A is required for recognition of IC-21 tumor cells to mediate their lysis.
    • Signal Transduction Pathway for NK cells* Modified from Vely and Vivier, 2005, www.stke.org/cgi/content/full/sigtrans;2005/292/cm6 (NKG2D) (DAP12) (NKp44) (KIR2DL1)
    • V. Non-adaptive vs. Adaptive Function
      • - Mediators of non-adaptive immunity
      • - Interface between non-adaptive and adaptive immunity –
      • “ Passive” interaction – antibody dependent cellular cytotoxicity
      • “ Active” interaction – reciprocal co-activation of NK cells and DCs to induce adaptive responses
      • - New hypothesis regarding NK cells as mediators of adaptive immunity is the topic of the journal club article
    • Interactions with Dendritic Cells to Promote Adaptive Immune Responses
    • VI. Therapeutic Applications of NK Cells
      • Biological Response Modifiers
      • IL2, IL12, IL15, IL21, IFN  , IFN  , IFN  PolyI:C,  -glucan
      • Adoptive Cellular Immunotherapy
      • Freshly isolated NK cells – autologous/allogeneic-alloreactive
      • BRM/Cytokine activated NK cells – autologous/allogeneic
      • Long term established NK cell lines (NK-92)
      • NK Cells as Vehicles for Delivery of Therapeutic Agents
      • Chemotherapeutic agents - doxorubicin
      • Cytokines – IL2
      • Trials for:
      • melanoma, renal cell carcinoma, lung carcinoma, ovarian cancer,
      • Glioblastoma – variable results
    • Utilization of Modified NK-92 NK-92 Modification of functional activity Targeting specific tumor types Control of in vivo Expansion NK-92-CD20 In vivo control of proliferation through suicide gene binding NK-92-Her2/neu NK-92-CD38 NK-92-CD19 IL2 Epithelial tumors breast ovarian Myeloma B-cell precursor leukemia Prolonged in vivo activity Improved cytolytic efficacy Accessibility to resistant tumors Modified from Suck, G. 2006