Experimental Basis of Immunology 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 lysis Large granular lymphocytes CD3-, 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

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

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

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

. . . . 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

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

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

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

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

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 IL15Ra IL2/IL15Rb LTa/LTbR (P) Sgnl. Trnsdcr. Jak3 CD3e tg FceR1g 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(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 98:11521-11526, 2001) or Rae1b (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

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

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 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

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

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

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

NK Cell Activating Receptors 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 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 *Rat NKR-P1A binds an undefined determinant on IC-21 tumor cells

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

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

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

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

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

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

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

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.

Signal Transduction Pathway for NK cells* (NKp44) (KIR2DL1) (NKG2D) (DAP12) Modified from Vely and Vivier, 2005, www.stke.org/cgi/content/full/sigtrans;2005/292/cm6

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 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

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