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
Cytokine/Rcptr. Flt3L IL15
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
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 ).
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
Viral antigens as ligands for NK cell receptors
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
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
IgV COOH NH 3 Inhibition of lytic function NK Cell membrane I/VxYxxL Cytoplasm IRp60 SHP-1 Target Cell membrane
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
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
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 –
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