NK Cells

1
NK Cells

• Experimental Basis of Immunology
• January 17, 2007
•  
• W.H. Chambers, Ph.D.
• G.17e Hillman Cancer Center
• 623-3218
• chamberswh_at_msx.upmc.edu

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I. Introduction

• Natural Killer (NK) Cells were first described in
  the early 1970s 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 1980s 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

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Innate capacity of lysis
Large granular lymphocytes
CD3-, CD16, CD56, CD122, CD158, CD161
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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

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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

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NK Progenitors Fetus
Fetal thymus
T
Fetal liver
Fetal blood
c-kit Thy-1 CD25 CD161c-
c-kit Thy-1 CD25- CD161c
c-kit Thy-1- CD25- CD161c-
c-kit Thy-1- CD25- CD161c- CD19- B220lo
c-kit Thy-1 CD25- CD161c
c-kit- Thy-1/- CD25- CD161c
Modified from Lian and Kumar, 2002
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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 LTa-/- 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

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NK Progenitors Adult
Bone marrow
stroma
CD122 CD161c CD49b Ly49
Lin- c-kitlo Thy-1- IL7R Sca-1lo
CD122 CD161c CD49b-
CD122 CD161c CD49b Ly49
CD122 CD161c- CD49b-
Thymus
Modified from Lian and Kumar, 2002
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HSC
pB
CLP
X
MY
Bone Marrow
T/NKP
NKP
pT
Periphery
CTX
Enhanced CTX
Stimulus
Cytokines IFNg GM-CSF TNFa
Modified from Yokoyama, et al, 2004
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NK Progenitors Adult
Periphery
Cytokines IFNg GM-CSF TNFa
Stimulus Hrs
IL2 IL12 IL15 IL23 IL27 IFNa, -b
Enhanced CTX
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NK Progenitors Adult
Periphery
Stimulus Days
Cytokines IFNg GM-CSF TNFa
IL2 IL12 IL15 IL23 IL27 IFNa, -b
Proliferation
Enhanced CTX, with broader specificity
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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

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Knockouts/Transgenics Receptors

• Gene Deleted Effect Reference
• LTbr NK cells severely decreased Wu, 2001
• LTa1b2 NK cells severely decreased, Iizuka, 1999
• reduced lytic function Smyth, 1999
• Ito, 1999
• IL15Ra NK cells severely decreased Lodolce, 1998
• IL2/15Rb NK cells absent Gilmour, 2001
• Suzuki, 1997
• c-kit NK cells decreased, impaired Colucci, 2000
  
• lytic function

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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

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NK Cell Differentiation Pathway Informative
Gene Knockout and Transgenic Mice

• HSC gtgt CLP gtgt T/NKP gtgt NKP
  gtgt NK
• Trnscrptn. Fctr./DBP Ikaros
  Ets1 IRF-1
• 
  PU.1 (P) Id2
  IRF-2
• 
  
  STAT5a/b (P)
• 
  
  MEF (P)
• Cytokine/Rcptr. Flt3L
  IL15
• 
  
  IL15Ra
• 
  
  IL2/IL15Rb
• 
  
  LTa/LTbR (P)
• Sgnl. Trnsdcr. Jak3
  CD3e tg
• 
  FceR1g tg

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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.

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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. 1635358-66
  1999).
• In vivo..

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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. 1342783-2789, 1985)

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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 3221731-1735, 1989)
• Identification of receptors on NK cells with
  coordinate tumor cell ligand was lacking

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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 3561795-1799, 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

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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(a-e) ULBP-1, -2, -3 H60)
  by NKR has been demonstrated
• In mice, binding of NKG2D to Rae1a (Cerwenka, A.,
  et al., PNAS USA 9811521-11526, 2001) or Rae1b
  (Diefenbach, A., et al., Nature 413165-171,
  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

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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

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NK-mediated Response to Virus Infection
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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

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NK Cell
Virus-infected Cell
MHC Class I Inhibitory Receptor
MHC ClassI Homolog
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Selective Expression
MHC Class I Inhibitory Receptor
2
Activating Receptor Antogonist
Activating Receptor
3
Down Regulating Activating Ligand
Virus
Cytokine Binding Protein
Cytokine Receptor
4
Cytokine Receptor
Cytokine Antogonist
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NK Cell Infection
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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

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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 11237-244.

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Receptors in Innate and Adaptive Immunity
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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
• Receptorligand pairs providing inhibitory
  signals are fairly well defined
• Receptorligand pairs providing activation
  signals are rapidly being defined

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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.

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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
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Leukocyte Receptor Cluster (LRC)
LRC is a 1 mb region located on chromosome
19q13.42
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NK Cell Inhibitory Receptors CLRR and KIR

• Name Alternative Names 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
• LIR1 ILT2/LILRB1 HLA-G HCMV-UL18
• 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

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NH3
IRp60
IgV
NK Cell membrane
Cytoplasm
COOH
Inhibition of lytic function
I/VxYxxL
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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

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NK Cell Activating Receptors

• Name Alternative Names 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 FcgRIII 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

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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

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NH3
NKp46SV-HA or IV-HA
IgC2
IgC2
NK Cell membrane

R
Cytoplasm
COOH
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NKp44SV-HA or IV-HA
NH3
IgV
DAP12
NK Cell membrane

K
D
Cytoplasm
I T A M
I T A M
ZAP70 SYK
COOH
Activation
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NKp30? iDCs and some tumors
NH3
IgV
CD3z
NK Cell membrane

R
D
I T A M
I T A M
Cytoplasm
I T A M
I T A M
ZAP70 SYK
COOH
I T A M
I T A M
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ITAM

• Immunoreceptor tyrosine-based activating motif
• Based upon the amino acid motif
  YxxL/Ix6-8YxxL/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, CD3z-CD3z
  homodimers, CD3z-Fcer1g heterodimers
• Activation of cells either via PI3 kinase, or
  ZAP70 or Syk tyrosine kinases

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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

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NKG2DMICA, MICB, ULBPs
COOH
COOH
CTLD
CTLD

NK Cell membrane
R
Cytoplasm
NH3
NH3
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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 a1, a2, and a3 domains but do
  not associate with b2m 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 a1 and a2 MHC Class I-like domains.
• Rae1b 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.

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Signal Transduction Pathway for NK cells
(NKG2D)
(NKp44)
(KIR2DL1)
(DAP12)
Modified from Vely and Vivier, 2005,
www.stke.org/cgi/content/full/sigtrans2005/292/cm
6
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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

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Interactions with Dendritic Cells to Promote
Adaptive Immune Responses
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VI. Therapeutic Applications of NK Cells

• Biological Response Modifiers
• IL2, IL12, IL15, IL21, IFNa, IFNg, IFNb,
  PolyIC, b-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

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Utilization of Modified NK-92
NK-92
Control of in vivo Expansion
Modification of functional activity
NK-92-CD20
Targeting specific tumor types
IL2
In vivo control of proliferation through
suicide gene binding
Prolonged in vivo activity
NK-92-Her2/neu
NK-92-CD19
NK-92-CD38
Epithelial tumors breast ovarian
Myeloma
B-cell precursor leukemia
Improved cytolytic efficacy Accessibility to
resistant tumors
Modified from Suck, G. 2006
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