NK Cells

Experimental Basis of Immunology

January 17, 2007 

W.H. Chambers, Ph.D.

G.17e Hillman Cancer Center

623-3218

chamberswh@msx.upmc.edu

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