Micro 204 Innate Lymphocytes & Natural Killer Cells. Lewis L. Lanier

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1 Micro 204 Innate Lymphocytes & Natural Killer Cells Lewis L. Lanier

2 inkt cell Innate lymphoid cell

3 Nature Reviews Immunology April 2009

4 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

5 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

6 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

7 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

8 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)

9 NK-deficient patient #1 Healthy donor <PE-A>: CD <PE-A>: CD CD3-CD56+ NK cells CD3+CD56+ T cells 0 0 CD56 CD <Pacific Blue-A>: CD <Pacific Blue-A>: CD3 <APC-A>: CD <APC-A>: CD CD3-CD19+ B cells 0 0 CD19 CD <Pacific Blue-A>: CD <Pacific Blue-A>: CD3

10 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)

11 NK-deficient patient #2 CD3-CD56+ NK cells CD3+CD56+ T cells

12 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

13 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

14 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 Cytomegalovirus and other herpesviruses Biron, NEJM Papillomavirus (cervical cancer) and Herpes Simplex Virus Ballas, J. Allergy Clinical Immuno Varicela Zoster Virus Etzioni, J. Peditrics 2005

15 NK cells are critical to early, innate host defense against pathogens 100 % Survival WT B6 Littermates NK-Deficient B6 mice Day Mouse Cytomegalovirus 10 5 pfu i.p. Loh JVI 2005

16 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

17 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)

18 pdc" IL-15" IL-12" IFN-γ

19 How do NK cells sense their environment?

20 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

21 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)

22 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

23 How are NK cell responses regulated?

24 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 Nature 319:675 Class I - tumors are rejected Class I - tumors in NK-depleted mice grow in vivo

25 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

26 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

27 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

28 Ly49 Receptors C-type lectin-like superfamily (but don t bind sugars) 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

29 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

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32 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

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34 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...?

35 RBC neural cells Normal healthy cells Infected cells tumor Infected cells tumor

36 Factors boosting NK cell lytic activity Ljunggren & Malmberg NRI 2007

37 Antibody-dependent cellular cytotoxicity

38 Activating CD16 Fc receptor on NK cells and macrophages mediates Herceptin- and Rituxan-induced human tumor elimination Clynes Nat Med 2000

39 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

40 DNAM-1-deficient mice are more susceptible to chemical (methylcholanthrine)-induced sarcomas Iguchi-Manaka JEM 2008

41 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)

42 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

43 H60c H60b

44 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

45 NKG2D on NK cells, γδ T cells and CD8 + T cells detect NKG2D ligands on abnormal cells

46 Increased incidence of myc oncogene-induced B cell lymphomas in NKG2D-deficient mice Guerra Immunity 2008

47 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

48 5 Abbas & Lichtman, Cellular & Molecular Immunology, 5th Ed, 2003

49 Abbas & Lichtman, Cellular & Molecular Immunology, 5th Ed, 2003

50 Natural Killer cells are critical for control of cytomegalovirus Proliferation Lanier Nat Rev Immunol 8:259, 2008

51 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

52 MCMV-specific NK cells expand during infection and are long-lived! Inject wildtype CD45.1 NK cells MCMV Track NK cells Ly49H-deficient mouse (CD45.2) d 0 d 1.5 d 7 d 14 d 28 d Ly49H CD45.1 (donor type)

53 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 days post-infection Abs. no. of Ly49H + NK cells (spleen) fold expansion days post-infection % Ly49H + NK cells in liver days post-infection Abs. no. of Ly49H + NK cells (liver) fold expansion days post-infection

54 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

55 Secondary expansion of memory Ly49H + NK cells requires m157 viral ligand Abs. No. of Ly49H + NK cells MCMV MCMV-Δm157 MCMV 66.5 MCMV-Δm Days post-infection Fold expansion Naïve NK cells (CD ) DAP12 -/- (CD ) MCMV days Memory NK cells DAP12 -/- (CD ) MCMV or MCMV- Δm days 2 Memory NK cells

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57 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

58 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

59 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

60 NK cells Keep you alive during certain viral infections Regulated by inhibitory and activating receptors Receptors are evolving rapidly Possess immunological memory