Micro 204 Innate Lymphocytes & Natural Killer Cells Lewis L. Lanier lewis.lanier@ucsf.edu
inkt cell Innate lymphoid cell
Nature Reviews Immunology April 2009
Innate Lymphoid Cells ILC1 Common Lymphoid Progenitor IL-15 NK cell IFNγ perforin IFNγ Id2 Innate lymphocyte progenitor? RORα IL-7 ILC2 IL-5 IL-13 IL-7 RORγt ILC3 NKR+ ILC, LTi IL-17 IL-22
NK cell ILC1 IFNγ perforin IFNγ Viruses Intracellular Bacteria Innate Lymphoid Cells ILC2 IL-5 IL-13 Helminths Epithelial tissue repair ILC3 NKR+ ILC, LTi IL-17 IL-22 Extracellular bacteria Lymphoid tissue modeling Epithelial tissue repair
Natural Killer versus Natural Killer T (NKT) cells What is the difference? NKT cells are T cells! rearrange TcR genes,express an invariant αβ-tcr, and require a thymus for development NK cells are not T cells! don t rearrange TcR genes or express CD3 on the cell surface and do not require a thymus
Natural Killer Cells Human CD3-,CD56+, (Nkp46+) Mouse CD3-,NKR-P1C+ (aka NK1.1) (NKp46+) 3rd lineage of lymphocytes Function in innate immunity to protect against viruses, bacteria, parasites, fungi, & tumors Produce cytokines & kill abnormal cells
Clinical features NK cell-deficient Patient #1-31 yr old female - Severe HPV and HSV infections, EBV well-controlled, HCMV-negative - HPV-induced vaginal carcinoma - Recurrent HSV-induced severe gingivitis - Type I diabetes (diagnosis age 11) Immunological features - Pan leukopenia; hypocellular bone marrow - B subset functionally normal, normal antibody response to tetanus and other antigens, hypergammaglobulinemia - T cell subset normal, high CTL cytotoxicity Lack of NK cells peripheral and tissue (bone marrow & vulvar biopsy)
NK-deficient patient #1 Healthy donor <PE-A>: CD56 10 5 10 4 10 3 2.22 16.6 <PE-A>: CD56 10 5 10 4 10 3 11.9 5.84 CD3-CD56+ NK cells CD3+CD56+ T cells 0 0 CD56 CD3 10 5 0 10 2 10 3 10 4 10 5 <Pacific Blue-A>: CD3 10 5 0 10 2 10 3 10 4 10 5 <Pacific Blue-A>: CD3 <APC-A>: CD19 10 4 10 3 4.03 <APC-A>: CD19 10 4 10 3 9.9 CD3-CD19+ B cells 0 0 CD19 CD3 0 10 2 10 3 10 4 10 5 <Pacific Blue-A>: CD3 0 10 2 10 3 10 4 10 5 <Pacific Blue-A>: CD3
NK cell-deficient Patient #2 31 year-old male Unusual clinical history of infections Sinusitis, cellulitis, folliculitis, boils, warts (HPV) Leukopenia, hypocellular bone marrow Ankylosing spondylitis (HLA-B27)
NK-deficient patient #2 CD3-CD56+ NK cells CD3+CD56+ T cells
Exome sequencing reveals point mutation in zinc-finger of GATA-2 in NK-deficient patients Father of patient #1 had bone marrow transplant early in life buccal swap reveals same GATA-2 mutation - therefore dominant mutation
GATA-2 Zinc-finger domain containing transcription factor family GATA family regulates hematopoietic gene expression GATA-1: erythropoiesis GATA-2: hematopoietic progenitors GATA-3: T cells, NK cells Gata2-/- or Gata1-/- are embryonic lethal (~day 9.5 due to erythropoietic & hematopoietic defects) Zn fingers binding DNA
Physiological role of NK cells is to protect against viral infections and cancer Humans lacking NK cells are particularly susceptible to: - Epstein-Barr Virus Fleisher, J. Pediatrics 1982 - Cytomegalovirus and other herpesviruses Biron, NEJM 1989 - Papillomavirus (cervical cancer) and Herpes Simplex Virus Ballas, J. Allergy Clinical Immuno 1991 - Varicela Zoster Virus Etzioni, J. Peditrics 2005
NK cells are critical to early, innate host defense against pathogens 100 % Survival 80 60 40 WT B6 Littermates NK-Deficient B6 mice 20 0 0 5 10 15 Day Mouse Cytomegalovirus 10 5 pfu i.p. Loh JVI 2005
NK Cells - Where do they live? ~5-20% peripheral blood lymphocytes ~5% lymphocytes in spleen, abundant in liver Low frequency in thymus, bone marrow, uninfected lymph nodes and lymphatics but increase with infection >70% of lymphocytes in decidual tissue
NK Cells - What do they do? Cell mediated-cytotoxicity natural killing Antibody-dependent cellular cytotoxicity (kill antibody-coated cells via activating Fc receptor CD16) Early γ-interferon production Secrete TNFa, LTa, GM-CSF, IL-3, M-CSF, IL-5, IL-10, IL-13, MIP-1a, MIP-1b, RANTES, etc. (but NOT IL-2 or IL-4 or IL-17 or IL-22)
pdc" IL-15" IL-12" IFN-γ
How do NK cells sense their environment?
Cytokines produced by infected epithelial or myeloid cells Epithelial or Myeloid cell TLR Bacteria, virus, parasites, fungus Interferon-γ Chemokines Cytokines (IL15, IL12, IL18) NK cell Cytokine receptor
Stressed cells upregulate host-encoded ligands for activating NK receptors Host cell Bacteria, virus, fungus, parasite DNA damage (tumors) Kill! Interferon-γ NK cell Host cell Host-encoded stress-induced protein Stress-induced ligand receptor (e.g. NKG2D)
Infected cells express virus-encoded ligands for activating NK receptors Host cell Viral infection Host cell Virus-encoded protein Kill! Interferon-γ NK cell Virus-specific NK receptor
How are NK cell responses regulated?
NK cells like to kill cells lacking MHC class I missing-self NK Cells Reject Tumors Lacking MHC Class I Class I + tumors grow in vivo Karre et al. 1986 Nature 319:675 Class I - tumors are rejected Class I - tumors in NK-depleted mice grow in vivo
Physiological role for NK cell inhibitory receptors for MHC class I- Detection of virus-infected cells? Virus Protein Effect on class I Adenovirus E3-k19 Retain in ER HSV-1,2 ICP47 Blocks TAP EBV EBNA1 Block peptide generation HCMV US2, US11 ER to cytosol HCMV US3 Retain in ER HCMV US6 Blocks TAP HCMV US10 Degrades HLA-G MCMV m152 Retain in ER MCMV m04 Associates with H-2 MCMV m06 Lysosomal degradation HHV8 K3, K5 Endocytosis HIV-1 Nef Endocytosis
Structural differences between MHC class I-specific inhibitory receptors in mice and humans Mouse Ly49 Human Killer cell Ig-like Receptor (KIR) C-type lectin-like 2 (or 3) Immunoglobulin - like domains s-s N N Type II ITIM C Type I Long cytoplasmic domain
HLA specificities of human KIRs KIR2DL C KIR3DL C KIR2DL1 : HLA-C2 allotypes (Cw2, 4, 5, 6=Lys80) KIR2DL2 & KIR2DL3 : HLA-C1 allotypes (Cw1, 3, 7, 8=Asn80) KIR3DL1 : HLA-Bw4 KIR3DL2: HLA-A3 Lanier, Ann Rev Imm 2005
Ly49 Receptors C-type lectin-like superfamily (but don t bind sugars) 10-20 functional genes on mouse chromosome 6 Extensive allelic polymorphism (no rearrangement); monoallelic expression possible Expressed by subsets of NK cells and memory T cells (usually CD8 + T cells) Inhibitory Ly49 recognize polymorphic H-2D and H-2K Activating Ly49 receptors - no intrinsic signaling -associate with DAP12 ITAM-adapter protein
Ig superfamily Killer Cell Ig-like Receptors (KIR) 7-12 functional genes on human chromosome 19q Extensive allelic polymorphism (614 alleles!) no rearrangement; mono-allelic expression possible Expressed by subsets of NK cells and memory T cells (mostly CD8 + T cells) Inhibitory KIR2DL recognize HLA-C; KIR3DL recognize HLA-A, -B Activating KIR - no intrinsic signaling -associate with DAP12 ITAM-adapter protein
Missing-self MHC on a cell is not sufficient for an NK cell to attack NK cells require activating receptors to detect ligands on the target cell to initiate a response
Activating NK receptors - ligands Human & mouse CD16-FcεRIγ or ζ..igg Human CD2... CD58 Human 2B4 (CD244)-SAP CD48 Human & mouse DNAM-1 (CD226)...CD112, CD155 Mouse PILRb-DAP12.PILR-L Human NKG2D-DAP10 MICA/B, ULBP Mouse NKG2D-DAP10/12 RAE-1,H60, MULT-1 Mouse Ly49H-DAP12.MCMV m157 Mouse Ly49P-DAP12..MCMV? Human CD94-NKG2C-DAP12..HLA-E, HCMV? Mouse CD94-NKG2E-DAP12 Qa1, poxvirus? Human & mouse NKp46-FcεRIγ or ζ......? Human NKp30-FcεRIγ or ζ..b7-h6 Human NKp44-DAP12..? Human NKp80 (KLRF1).AICL Human NKp65 (KLRF2).KACL Mouse NKR-P1c-FcεRIγ...?
RBC neural cells Normal healthy cells Infected cells tumor Infected cells tumor
Factors boosting NK cell lytic activity Ljunggren & Malmberg NRI 2007
Antibody-dependent cellular cytotoxicity
Activating CD16 Fc receptor on NK cells and macrophages mediates Herceptin- and Rituxan-induced human tumor elimination Clynes Nat Med 2000
DNAM-1 (CD226) - an activating NK receptor - V V NH 2 S S S S Expressed by NK cells, T cells, DC Triggers cytotoxicity in NK cells, CTL Important cancer immmunosurveillence Promotes Th1 differentiation Ligands - CD155 (PVR) CD112 (PRR-2, nectin-2) Fyn à PKC à Y322 S329 COOH
DNAM-1-deficient mice are more susceptible to chemical (methylcholanthrine)-induced sarcomas Iguchi-Manaka JEM 2008
Natural Cytotoxicity Receptors ITAM-coupled activating receptor NKp46 expressed by most NK cells in humans and mice NKp46 also expressed by some ILC, & γδ T cells NKp30 & NKp44 in humans, not mice Involved in recognition and killing of certain tumors Koch Trends Imm 2013 Ligands poorly defined broadly distributed (except B7-H6 for NKp30)
NKG2D C-type lectin-like superfamily 1 gene, non-polymorphic, conserved mice - humans Homodimer expressed on all NK cells, γδ T cells, and CD8 + T cells R in transmembrane associates with D in DAP10 transmembrane DAP10 10 kd homodimer Cytoplasmic YINM recruits Grb2 & p85 PI3-kinase
H60c H60b
NKG2D ligands MHC class I-like don t require peptide or b2-microglobulin Bind with nm affinity to NKG2D Low levels expressed on healthy tissues Induced on virus-infected cells and tumor cells Induced by DNA damage (ATM/ATR pathway) Elevated in autoimmune diseases (rheumatoid arthritis, celiac disease, diabetes, atherosclerosis)* *Disclosure I have licensed patents on blocking NKG2D in autoimmune disease
NKG2D on NK cells, γδ T cells and CD8 + T cells detect NKG2D ligands on abnormal cells
Increased incidence of myc oncogene-induced B cell lymphomas in NKG2D-deficient mice Guerra Immunity 2008
NK cells and Cancer Caveat All NK receptors are expressed by other cell types No mouse models exist that exclusively lack NK cells without effects on other cell lineages
5 Abbas & Lichtman, Cellular & Molecular Immunology, 5th Ed, 2003
Abbas & Lichtman, Cellular & Molecular Immunology, 5th Ed, 2003
Natural Killer cells are critical for control of cytomegalovirus Proliferation Lanier Nat Rev Immunol 8:259, 2008
Ly49H drives NK cell responses to cytomegalovirus in B6 mice NK cell DAP12 Cytotoxicity IFN-γ Proliferation MCMV control Syk & ZAP70 p85 PI3 kinase/vav1 MCMV-infected cell DAP10 Ly49H m157
MCMV-specific NK cells expand during infection and are long-lived! Inject wildtype CD45.1 NK cells MCMV 7.92 1.18 7.96 1.95 7.53 21.7 7.3 16.1 Track NK cells Ly49H-deficient mouse (CD45.2) d 0 d 1.5 d 7 d 14 d 28 d 50 1.07 6.75 0.8 0.96 Ly49H 89.9 0.99 89.2 0.87 70.3 0.47 76.2 0.37 92.2 0.041 98.2 0.078 CD45.1 (donor type)
Ly49H+ NK cells Mouse lacking Ly49H NK cells MCMV Expansion and contraction of adoptively transferred Ly49H + NK cells during MCMV infection % Ly49H + NK cells in spleen 25 20 15 10 5 0 10 5 10 4 0 10 20 30 40 50 days post-infection Abs. no. of Ly49H + NK cells (spleen) 10 6 10 5 10 4 10 3 10 2 100 fold expansion 0 10 20 30 40 50 days post-infection % Ly49H + NK cells in liver 25 20 15 10 5 0 10 5 10 4 0 10 20 30 40 50 days post-infection Abs. no. of Ly49H + NK cells (liver) 10 6 10 5 10 4 10 3 10 2 1000 fold expansion 0 10 20 30 40 50 days post-infection
Long-lived memory NK cells protect neonatal mice against MCMV infection better than naïve NK cells Adult naïve or memory NK cells MCMV Newborn CMV-susceptible mice Memory NK cells kill better and make more cytokine on a per cell basis than naïve NK cells in vitro
Secondary expansion of memory Ly49H + NK cells requires m157 viral ligand Abs. No. of Ly49H + NK cells 10 5 10 4 10 3 10 2 MCMV MCMV-Δm157 MCMV 66.5 MCMV-Δm157 3.6 0 2 4 6 8 0 25 50 75 100 Days post-infection Fold expansion Naïve NK cells (CD45.1 + ) DAP12 -/- (CD45.2 + ) MCMV 30-50 days Memory NK cells DAP12 -/- (CD45.2 + ) MCMV or MCMV- Δm157 30-50 days 2 Memory NK cells
Hapten-specific contact hypersensitivity responses in RAG-deficient mice day 0 & 1 paint hapten on back skin day 6 or 30 challenge ear measure swelling O Leary NI 2006
Depletion of NK cells eliminates contact hypersensitivity responses in RAG-deficient mice day 0 & 1 paint hapten on back skin day 6 deplete NK cells day 6 challenge ear measure swelling O Leary NI 2006
Antigen-specific NK cell memory *induced against haptens, HIV-I, influenza, VSV, HSV-1 *adoptively transfer hapten-specific contact hypersensitivity response to unimmunized recipients *reside exclusively in liver *require CXCR6 to mediate function *receptors responsible for antigen recognition unknown Paust NI 2010
NK cells Keep you alive during certain viral infections Regulated by inhibitory and activating receptors Receptors are evolving rapidly Possess immunological memory