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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
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 lysisLarge granular lymphocytesCD3-, 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
p-NK
c-T/NKPCLPHSC p-T/NK p-T/NK
c-kit+
Thy-1-
CD25-
CD161c-
c-kit+
Thy-1-
CD25-
CD161c-
CD19-B220lo
Fetal liver
c-kit+
Thy-1+
CD25-
CD161c+
Fetal bloodp-T
NK .. ..
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
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
NK .. ..HSC CLP NKP p-NK NK .. ..Lin-
c-kitlo
Thy-1-
IL7R+
Sca-1lo
CD122+
CD161c-
CD49b-
CD122+
CD161c+
CD49b-
CD122+
CD161c+
CD49b+
Ly49+
Bone marrowstroma
Modified from Lian and Kumar, 2002
p-T/NK
p-NK
p-T
NK .. ..
TT
Thymus
CD122+
CD161c+
CD49b+
Ly49+
NKNK
NK
NK
NK .. ..
NK .. ..
NK .. ..
NK .. ..
NKP
T/NKP
CLP
HSC
Modified from Yokoyama, et al, 2004
Cytokines: IFN GM-CSF TNF
Bone Marrow
Periphery
EnhancedCTX
Stimulus
pB
pT
MY X
CTX
NKNK
NK
NK
NK .. ..
Cytokines: IFN GM-CSF TNF
Periphery
EnhancedCTX
Stimulus: Hrs
IL2IL12IL15IL23IL27IFN, -
NK Progenitors: Adult
NKNK
NK
NK
NK .. ..
Cytokines: IFN GM-CSF TNF
Periphery
EnhancedCTX, with broader specificity
Stimulus: Days
IL2IL12IL15IL23IL27IFN, -
Proliferation
NK Progenitors: Adult
Knockouts/Transgenics: Transcription Factors
Gene Deleted Effect ReferenceIkaros NK cells absent Georgopoulos, 1994
Wang, 1996PU.1 NK cell number decreased, Colucci, 2001
normal lytic functionEts-1 NK cell number decreased, Barton, 1998
decreased lytic functionId2 NK cells decreased or absent, Yokota, 1999
reduced lytic function Ikawa, 2001TCF-1 Altered acquisition of Ly49s Held, 1999
Kunz,2001IRF-1 NK cell number decreased, Duncan, 1996
lytic function impaired Ogasawara, 1998IRF-2 NK cell number decreased, Lohoff, 2000
lytic function impaired
Knockouts/Transgenics: Receptors
Gene Deleted Effect Reference
LTr NK cells severely decreased Wu, 2001
LT12 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/LTR (P)
Sgnl. Trnsdcr. Jak3 CD3e tg
FcR1 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 CellVirus-infected Cell
CytokineReceptor
MHC Class IInhibitoryReceptor
MHC Class IInhibitoryReceptor
ActivatingReceptor
MHC ClassIHomolog
SelectiveExpression
Down RegulatingActivating Ligand
ActivatingReceptorAntogonist
Cytokine BindingProtein
CytokineReceptor
CytokineAntogonist
NK CellInfection
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
Characteristics Innate AdaptiveSpecificity inherited in the genome
Yes No
Expressed by all cells of a particular type
Yes No
Trigger immediate response Yes NoRecognize broad classes of pathogens
Yes No
Encoded in multiple gene segments
No Yes
Require gene rearrangement No YesClonal distribution No YesAble to recognize a wide variety of molecular structures
No Yes
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 Ligandp58.1 KIR2DL1 HLA-Cw2,4,5,6p58.2 KIR2DL2 HLA-Cw1,3,7,8p70 KIR3DL1 HLA-Bw4p140 KIR3DL3 HLA-A3, -A11p49 KIR2DL4 HLA-GLIR1 ILT2/LILRB1 HLA-G HCMV-UL18LIR2 ILT4/LILRB2 HLA-FCD94* KLRD1 HLA-E** NKG2A KLRC1/CD159A HLA-ENKR-P1B, D CD161B, D Clrbp40 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
SHP-1
COOH
NH3
Inhibition of lytic function
NK Cell membrane
Target Cell membrane
I/VxYxxL
Cytoplasm
IRp60
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 LigandNKp46 Ly94/NCR1 ? SV-HA, IV-HANKp30 IC7/NCR3 ?NKp44 Ly95/NCR2 ? SV-HA, IV-HA2B4 CD244 CD48NTB-A KALI ?NKp80 KLRF1 ?CD16 FcRIII IgGCD2 LFA-2 CD58, LFA-3DNAM-1 CD226 PVR/CD155, Nectin-2/CD112 NKG2D D12S2489E/CD159D MICA, MICB, MULT1 ULBP1-4NKR-P1A CD161A [IC-21]*NKR-P1C CD161C ?NKR-P1F CD161F ClrgP40 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
ZAP70 SYK
NKp46:SV-HA or IV-HA
Cytoplasm
IgC2
IgC2
R * D
FcR1CD3
ITAM
ITAM
ITAM
ITAM
Activation
NH3
COOH
NK Cell membrane
NKp44:SV-HA or IV-HA
Cytoplasm
IgV
D*
DAP12
ITAM
Activation
K
ITAM
NH3
COOH
ZAP70 SYK
NK Cell membrane
NKp30:? [iDCs and some tumors]
Cytoplasm
IgV
R *
CD3
ITAM
ITAM
Activation
NH3
D
ITAM
ITAM
ITAM
ITAM
COOH ZAP70 SYK
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, CD3-CD3 homodimers, CD3-Fcr1 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
Cytokine secretionCytotoxicity
NK Cell membrane*R
CTLD CTLD
NKG2D:MICA, MICB, ULBPs
D
DAP10
YxxM
PI3K
COOH COOH
NH3 NH3
Cytoplasm
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 ModifiersIL2, IL12, IL15, IL21, IFN, IFN, IFNPolyI:C, -glucan
Adoptive Cellular ImmunotherapyFreshly isolated NK cells – autologous/allogeneic-alloreactiveBRM/Cytokine activated NK cells – autologous/allogeneicLong term established NK cell lines (NK-92)
NK Cells as Vehicles for Delivery of Therapeutic AgentsChemotherapeutic agents - doxorubicinCytokines – 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 invivo Expansion
NK-92-CD20
In vivo controlof proliferationthrough suicidegene binding
NK-92-Her2/neu
NK-92-CD38
NK-92-CD19
IL2
Epithelial tumors breast ovarian
Myeloma B-cell precursorleukemia
Prolongedin vivo activity
Improved cytolytic efficacyAccessibility to resistant tumors Modified from Suck, G. 2006