1. Experimental Basis of Immunology
NK Cells
Experimental Basis of Immunology
January 17, 2007
W.H. Chambers, Ph.D.
G.17e Hillman Cancer Center

2. 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+

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

4. 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

5. 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

6. NK Progenitors: Fetus . Fetal thymus Fetal liver Fetal blood p-T T HSC
c-kit+
Thy-1+
CD25+
CD161c-
HSC
CLP
c-T/NKP
p-T/NK
p-T/NK
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+
p-NK NK .
c-kit-
Thy-1+/-
CD25-
CD161c+
Modified from Lian and Kumar, 2002

7. 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

8. NK Progenitors: Adult . . . Bone marrow Thymus NK HSC CLP NKP p-NK NK
stroma 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+
CD122+
CD161c+
CD49b+
Ly49+
Thymus
p-NK NK .
p-T/NK
p-T T T
Modified from Lian and Kumar, 2002

9. . . . . X Bone Marrow Periphery Stimulus HSC pB CLP MY pT NKP NK NK NK
CTX NK .
Enhanced
CTX NK
Stimulus NK
Cytokines:
IFNg
GM-CSF
TNFa NK NK
Modified from Yokoyama, et al, 2004

10. NK Progenitors: Adult . Periphery Stimulus: Hrs IL2 IL12 IL15 IL23
Cytokines:
IFNg
GM-CSF
TNFa NK . NK
Stimulus: Hrs NK
IL2
IL12
IL15
IL23
IL27
IFNa, -b
Enhanced
CTX NK NK

11. NK Progenitors: Adult . Periphery Stimulus: Days IL2 IL12 IL15 IL23
Cytokines:
IFNg
GM-CSF
TNFa NK
IL2
IL12
IL15
IL23
IL27
IFNa, -b NK NK
Proliferation
Enhanced
CTX, with
broader specificity

12. 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

13. 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

14. 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

15. NK Cell Differentiation Pathway: Informative Gene Knockout and Transgenic Mice
HSC >> CLP >> T/NKP >> NKP >> NK
Trnscrptn. Fctr./DBP Ikaros Ets IRF-1
PU.1 (P)* Id IRF-2
STAT5a/b (P)
MEF (P)
Cytokine/Rcptr Flt3L IL15
IL15Ra
IL2/IL15Rb
LTa/LTbR (P)
Sgnl. Trnsdcr Jak CD3e tg
FceR1g tg

16. 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.

17. 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: [1999]).
In vivo…..

18. 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: , 1985)

19. 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: , 1989)
Identification of receptors on NK cells with coordinate tumor cell ligand was lacking

20. 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: , 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

21. 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 98: , 2001) or Rae1b (Diefenbach, A., et al., Nature 413: , 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

22. 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

23. NK-mediated Response to Virus Infection

24. 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

25. Virus-infected Cell NK Cell 1 2 3 4 5 MHC ClassI Homolog MHC Class I
Inhibitory
Receptor 1
Selective
Expression
MHC Class I
Inhibitory
Receptor 2
Activating
Receptor
Antogonist
Activating
Receptor 3
Virus
Down Regulating
Activating Ligand
Cytokine Binding
Protein
Cytokine
Receptor 4
Cytokine
Receptor
Cytokine
Antogonist 5
NK Cell
Infection

26. 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

27. “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, Immunology Today 11:

28. Receptors in Innate and Adaptive Immunity
Characteristics
Innate
Adaptive
Specificity inherited in the genome
Yes No
Expressed by all cells of a particular type
Trigger immediate response
Recognize broad classes of pathogens
Encoded in multiple gene segments
Require gene rearrangement
Clonal distribution
Able to recognize a wide variety of molecular structures

29. 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

30. 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.

31. 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

32. Leukocyte Receptor Cluster (LRC)
LRC is a ~1 mb region located on chromosome 19q13.42

33. NK Cell Inhibitory Receptors: CLRR and KIR
Name Alternative Name[s] Cellular Ligand Viral Ligand
p58.1 KIR2DL HLA-Cw2,4,5,6
p58.2 KIR2DL HLA-Cw1,3,7,8
p70 KIR3DL HLA-Bw4
p140 KIR3DL HLA-A3, -A11
p49 KIR2DL HLA-G
LIR1 ILT2/LILRB1 HLA-G HCMV-UL18
LIR2 ILT4/LILRB2 HLA-F
CD94* KLRD HLA-E**
NKG2A KLRC1/CD159A HLA-E
NKR-P1B, D CD161B, D Clrb
p40 LAIR ?
IRC1 IRp60/CMRF35H ?
p75AIRM1 Siglec Sialylated sugars
*CD94 forms heterodimers with NKG2A, -C and –E
**CD94/CD159A heterodimer is specific for HLA-E

34. Inhibition of lytic function
Target Cell membrane
NH3
IgV
IRp60
NK Cell membrane
Cytoplasm
SHP-1
COOH
Inhibition of lytic function
I/VxYxxL

35. 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

36. NK Cell Activating Receptors
Name Alternative Name[s] Cellular Ligand Viral Ligand
NKp46 Ly94/NCR ? SV-HA, IV-HA
NKp30 IC7/NCR ?
NKp44 Ly95/NCR ? SV-HA, IV-HA
2B4 CD CD48
NTB-A KALI ?
NKp80 KLRF ?
CD16 FcgRIII IgG
CD2 LFA CD58, LFA-3
DNAM-1 CD PVR/CD155, Nectin-2/CD112
NKG2D D12S2489E/CD159D MICA, MICB, MULT ULBP1-4
NKR-P1A CD161A [IC-21]*
NKR-P1C CD161C ?
NKR-P1F CD161F Clrg
P40 LAIR ?
IRC1 IRp60/CMRF35H ?
p75AIRM1 Siglec Sialylated sugars
*Rat NKR-P1A binds an undefined determinant on IC-21 tumor cells

37. 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

38. NKp46:SV-HA or IV-HA * Activation NK Cell membrane Cytoplasm IgC2 IgC2
NH3
NKp46:SV-HA or IV-HA
IgC2
IgC2 D
FceR1g
CD3z I T A M R *
NK Cell membrane
Cytoplasm
ZAP70
SYK
COOH
Activation

39. NKp44:SV-HA or IV-HA * Activation NK Cell membrane Cytoplasm IgV DAP12
NH3
IgV
DAP12 K * D
NK Cell membrane
Cytoplasm I T A M I T A M
ZAP70
SYK
COOH
Activation

40. NKp30:? [iDCs and some tumors]
NH3
IgV
CD3z R * D
NK Cell membrane I T A M I T A M
Cytoplasm I T A M I T A M
COOH
ZAP70
SYK
Activation I T A M I T A M

41. 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

42. 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

43. NKG2D:MICA, MICB, ULBPs * Cytokine secretion Cytotoxicity
COOH
COOH
CTLD
CTLD D
DAP10 Y x M * R
NK Cell membrane
Cytoplasm
PI3K
Grb2
NH3
NH3
ERK1/2
MAPK
Cytokine secretion
Cytotoxicity

44. 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.

45. Signal Transduction Pathway for NK cells*
(NKp44)
(KIR2DL1)
(NKG2D)
(DAP12)
Modified from Vely and Vivier, 2005,

46. 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

47. Interactions with Dendritic Cells to Promote Adaptive Immune Responses

48. VI. Therapeutic Applications of NK Cells
Biological Response Modifiers
IL2, IL12, IL15, IL21, IFNa, IFNg, IFNb, PolyI:C, 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

49. Utilization of Modified NK-92
Modification of
functional activity
Control of in
vivo Expansion
NK-92-CD20
IL2
Targeting specific
tumor types
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