RAE1

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Micro 204NK cells

• Lewis L. Lanier
• Lewis.lanier_at_ucsf.edu

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NK cells - What are they?
Human CD3-,CD56 Mouse CD3-,NKR-P1C (NK1.1)

• 3rd lineage of lymphocytes
• Function in innate immunity to protect against
  viruses, bacteria, tumors
• Produce cytokines kill abnormal cells

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NK Cells - Where do they come from?

• NK/T cell progenitor in bone marrow
• Thymus not required
• nude mice have normal NK cells
• Do not rearrange a,b,g or d TcR or Ig
• normal NK cells in scid and RAG-/- mice

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NK cells - Development

• IL-15 is critical!
• Blocked by gene disruption of
• IL-15 pathway (i.e. IL-15, IL-15R-a, IL-2R-g
  -b, IRF-1, JAK3, Id2 or Ikaros (no IL-2Rg))
• Ets-1
• NOT eliminated by gene disruption of
• CD3 z, FceRIg, DAP12
• IL-1, IL-2, IL-7, IL-12, IL-18, IL-21
• lck, fyn, syk, ZAP70, SHP-1, SHIP

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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
• gt90 of lymphocytes in decidual tissue

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NK cells - Pregnancy

• Maternal NK cells are the major lymphocyte
  population (90) at the maternal - fetal
  interface in the decidua
• Why?
• Fetal protection?
• Fetal sustenance?
• Vascular remodeling (IFNg)

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NK Cells - What do they do?

• Cell mediated-cytotoxicity
• Antibody-dependent cellular cytotoxicity (ADCC)
• Early g-interferon production
• Secretion of TNFa, LTa, GM-CSF, IL-5, M-CSF,
  IL-3, IL-10, IL-13, MIP-1a, MIP-1b, RANTES, etc.

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NK cells- How do they kill?

• Predominantly perforin / granzymes
• Kagi et al Nature 36931, 1994
• Secreted or membrane TNFa
• Degliantoni et al J Exp Med 1621512 1985
• Fas ligand
• Arase et al J Exp Med 1811235, 1995
• TRAIL
• Zamai et al J Exp Med 1882375, 1998

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NK cells - Cytokine Activation

• Interferon-a/b
• augments cytolytic activity
• IL-15
• required for development, induces proliferation,
  increases cytotoxicity
• IL-12 IL-18
• augments INFg production
• IL-2
• induces proliferation, increases
  cytotoxicity..physiological relevance?

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NK cells - what are they good for?

• Protection from viruses
• and maybe bacteria, parasites, tumors

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What are NK cells are good for?

• Humans lacking NK cells susceptible to Varicella
  zoster CMV
• Biron et al N Engl J Med 3201731 1989
• Mouse NK cells protect against CMV - requires
  perforin and IFNg
• Bukowski et al J Exp Med 16140 1985 Welsh et al
  J Exp Med 1731053, 1991 Scalzo et al J Exp Med
  1711469, 1990 Orange et al J Exp Med 1821045,
  1995
• Mouse NK cells protect against mousepox -
  requires IFNg
• Chaudhri et al PNAS 1019057, 2004
• Mouse NK cells protect against Ebola virus -
  requires perforin, not IFN-\g
• Warfield et al J Exp July 2004

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NK cells - Parasites Bacteria

• NK cells protect against certain parasites
• Toxoplasma gondii, Leishmania, Plasmodium
  falciparum, Trypanosoma cruzi
• NK cells in bacterial infection
• TLR signaling of myeloid cells induces IL-12
• IL-12 induces NK cells to make INFg
• INFg augments macrophage function
• Evidence for NK cell protective role against
  Shigella flexneri, Legionella pneumophila
• In Listeria monocytogenes infection, NK cell
  depletion causes the bacteria to grow LESS well

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NK cells - Influence on Adaptive Immunity

• Early INFg production by NK cells may skew CD4
  T cells towards Th1
• INFg production by NK cells may cause IgG class
  switching in B cells
• Cross-talk with dendritic cells

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NK cells - Dendritic Cells

• NK cells are activated by co-culture with DC
• Fernandez et al Nat Med 5405, 1999 Ferlazzo
  et al 195343, 2002
• Mouse CMV infection - CD8 dendritic cells
  produce IL-12 IL-18. activate NK cells in
  vivo
• Depletion of mouse CD8 DC prevents NK cell
  activation and control of infection
• In the absence of mouse NK cells, CD8 DC are
  lost after MCMV infection
• Andrews et al Nat Immunol 4175, 2003
• Human NK cell - DC interactions
• TLR-stimulated pDC or myeloid DC secrete INFa -
  enhances NK cytotoxicity
• TLR-stimulated myeloid DC secrete IL-12 -induces
  NK cell IFNg
• Activated NK cells - mature DC
• Gerosa et al. J Immunol 174727, 2005

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NK cells are required for a Th1 response?

• Upon immunization, NK cells enter the lymph nodes
• Secrete IFNg
• Promote generation of CTL and Th1 response
• Martin-Fontecha et al. Nat Immunol 51260, 2004

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Th17 cells develop BETTER in NK cell-depleted
mice!
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NK cells and NKT cells-What is the difference?

• NKT cells rearrange TcR genes and express a
  CD3/TcR receptor- NK cells dont
• NKT cells usually refer to a subset of T cells
  with an invariant ab-TcR that recognize lipid
  antigens presented by CD1
• Some people call a T cell an NKT cell if this T
  cell expresses any receptor in common with NK
  cells - a very confusing and imprecise name that
  is meaningless

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RBC, neural cells
Normal healthy cells
Virus-infected cells, tumor
Virus-infected cells, tumor
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Immune surveillance for Missing Self

• NK cells preferentially kill cells that have lost
  MHC class I
• Provides protection against cells escaping T cell
  recognition
• Predicts existence of inhibitory receptors for
  MHC class I that spare normal cells from NK cell
  attack
• Karre et al Nature 319675, 1986

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Cytomegalovirus (CMV) infection down-regulates
MHC class I on human fibroblasts
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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 UL83 Blocks proteasome
• MCMV m152 Retain in ER
• MCMV m04 Associates with H-2
• MCMV m06 Lysosomal degradation
• HHV8 K3, K5 Endocytosis
• HIV-1 Nef Endocytosis

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Immunoreceptor Tyrosine-based Inhibitory Motif
(ITIM)

• Prototype I/V/L/SxYxxL/V
• pY binds cytoplasmic tyrosine phosphatase (SHP-1
  or SHP-2) - inhibits activation
• Present in receptors on B cells (FcgRII, CD22), T
  cells (CD5, LAIR, PD-1), NK cells (KIR, Ly49,
  NKG2A), myeloid cells (ILT,CD33, SIRPa)
• Prevents autoimmunity
• -Ravetch Lanier, Science 29084, 2000

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Multi-subunit immune receptors with ITAM-bearing
transmembrane adapters
Cytoplasmic ITAM (D/ExYxxL-X6-8-YxxL) Activate
Syk ZAP70 tyrosine kinases
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ITAM-bearing adapters in NK cells

• DAP12, FceRIg? and z
• Charged D residue in transmembrane required for
  association with receptors
• ITAMrecruits tyrosine kinases Syk and ZAP70

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Human Chr 19q13
Killer cell Ig-like Receptors (KIR) Activating
inhibitory NK receptors
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Killer Cell Ig-like Receptors (KIR)

• Ig superfamily
• 7-12 functional genes on human chromosome 19q13
• Extensive allelic polymorphism alternative
  splicing (no rearrangement)
• Mono-allelic expression possible
• Expressed by subsets of NK cells and memory T
  cells (usually CD8 T cells)

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DO
D1
D2
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Inhibitory and activating KIRNK cell receptors
KIR2/3DS
KIR2/3DL
DAP12
-
-
-
-
-

-
-

-
ITAM Recruit syk / ZAP70 kinases - activate
ITIM Recruit phosphatases - inhibit responses
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KIR - Functional Isoforms

• KIR2D - 2 Ig-like domains
• recognize polymorphic HLA-C ligands
• KIR3D - 3 Ig-like domains
• recognize polymorphic HLA-B HLA-A ligands
• KIR2DL and KIR3DL
• Long (L) cytoplasmic domain with ITIM
• KIR2DS and KIR3DS
• Short (S) cytoplasmic domain..no intrinsic
  signaling capacity..use DAP12 adapter to activate
  NK cells

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Human NK Receptors for HLA class I
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Killer Cell Ig-like Receptors (KIR)

• Inhibitory isoforms with ITIM bind HLA-A, -B,
  -C ligands
• Activating isoforms no intrinsic signaling
  capacity
• associate with activating DAP12 adapter protein
• Physiological ligands unknown - weak binding to
  HLA

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Human KIR Haplotypes Differ in Gene Content and
Organization
3DL1
2DL2
2DS2
2DL5B
2DS3
2DL1
3DP1
2DL5A
2DS4
2DS5
2DS1
3DL2
2DL4
2DP1
2DL3
3DL3
3DS1
A haplotype
2DS4
2DL1
3DL2
3DL1
2DL4
2DL3
3DL3
B haplotype
2DL2
2DS2
2DL1
2DL5A
2DS1
3DL2
3DS1
2DL4
3DL3
2DS5
Generated by gene conversion - exon
swapping Difficult to distinguish loci versus
alleles
Uhrberg et al. 1997 Wilson et al. 2000 Vilches
et al. 2000 Hsu et al. 2002
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KIR Genotype Variation in a Panel of Individuals
3DL3
2DL3
2DS2
2DL2
2DL5
2DS3
2DL1
2DL4
3DL1
3DS1
2DS4
2DS5
2DS1
3DL2
Donor ethnicity
Caucasian
Caucasian
Caucasian
African American
East Asian
Asian Indian
Caucasian
East Asian
Caucasian
Caucasian
Caucasian
Caucasian
East Asian
East Asian
from 1000 individuals -- 111 genotypes
described (Courtesy P. Parham)
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Allelic polymorphism distinguishes 22 Group A
haplotypes having identical gene content
Shilling et al. JI, 2002
Group A haplotype
2DS4
2DL1
3DL2
3DL1
2DL4
2DL3
3DL3
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10
17
23
14
7
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alleles

• 800,000 possible combinations in the group A
  haplotypes
• (Courtesy P. Parham)

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Rapid Expansion of KIR Genes in Primates
Human
3DL1
2DL5A
2DS4
2DS5
2DS1
3DL2
2DL2
2DS2
2DL5B
2DS3
2DL1
2DL4
3DP1
2DP1
2DL3
3DL3
3DS1
Chimpanzee
2DL5
2DL4
KIRCI
2DL6
3DL4
3DL6
Haplotype 1
2DL5
3DL1/2
2DL4
KIRCI
Haplotype 2
Orangutan
2DL
3DL
2DL4
DP1
2DS
3DL3
P. Parham
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KIR Diversity

• In a single person, KIRs provide a diverse
  receptor repertoire for NK cell responses
• In the same ethnic group, different people have
  different KIR genes and alleles
• In the human population, different ethnic groups
  have different KIR genes, alleles, haplotypes
• KIR genes in humans markedly different from other
  primates.evidence for rapid evolution
• Activating KIR arose from the inhibitory KIR and
  are the most rapidly expanding and contracting
  genes
• pathogen-driven evolution? Abi-Rached
  Parham JEM 181319, 2005

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Receptor diversity within the NK population
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NK cell repertoire - Ground Rules

• KIR are arrayed on overlapping NK subsets
• Individual NK clones simultaneously express
  multiple activating or inhibitory receptors for
  self and non-self ligands
• Most NK cells have at least one inhibitory
  receptor for one self MHC class I
• prevent autoimmunity?
• A few NK cells lack inhibitory receptors for self
  class I - regulated by other inhibitory
  receptors? Anergy?
• Repertoire is genetically determined, with subtle
  alterations caused by host MHC

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KIR in Viral Immunity
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KIR Involvement in Viral Immunity
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HLA-Bw4-80I and KIR3DS1 together protect against
progression to AIDS
AIDS
AIDS-related malignancies
Carrington PLoS Pathogens, 2006
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KIR3DS1, HLA-Bw4, and progression to AIDS in
HIV-infected people
Protection
KIR3DS1 NK cell
Killing ?
Slower progression to AIDS
KIR3DS1
No Effect
HIV-infected cell
No Killing ?
KIR3DS1 NK cell
Usual progression to AIDS
HLA- Bw6
Martin et al. 2002 Nat Genet 31429-34
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KIR in Autoimmunity
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KIR Involvement in Autoimmunity
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KIR in Pregnancy
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• Inhibitory KIR2DL on maternal NK cells binding
  HLA-CLys80 on fetal cells may suppress the
  beneficial effects of NK cells on placental
  vascularization

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Mice lack KIR genesinstead ..NK cell
recognition of MHC class I recognition in rodents
is provided by Ly49 receptors
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Inhibitory and activating rodent Ly49NK cell
receptors
Ly49D,H
Ly49A,C,G...
DAP12
-
-
-
-
-

-
-

-
ITAM Recruit syk / ZAP70 kinases - activate
ITIM Recruit phosphatases - inhibit responses
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Rodent Ly49 receptors

• C-type lectin-like superfamily
• 14 genes (B6) on mouse chromosome 6
• Extensive allelic polymorphism alternative
  splicing (no rearrangement)
• Mono-allelic expression possible
• Expressed by subsets of NK cells and memory T
  cells (usually CD8 T cells)
• Deleted from the human genome.only a single
  y-gene remains on chr 12

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Ly49 Receptors

• Inhibitory isoforms with ITIM bind polymorphic
  H2-D and H2-K ligands
• Activating isoforms no intrinsic signaling
  capacity
• associate with activating DAP12 adapter protein

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Ly49H is the NK receptor for the m157 CMV
glycoprotein protects B6 mice from MCMV infection

• Ligation of Ly49H by m157
• DAP12 phosphorylation
• Recruitment / activation of syk or ZAP70 tyrosine
  kinases
• Activation of MAP kinase, Ca flux
• Cytotoxicity, cytokine production, proliferation

Ly49H
DAP12
-

-
M
M
A
A
T
T
I
I
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CMV
CMV
IPC
DC
TLR9
TLR3
INF?
IL15R?
IL-12
IL15
IL2R???
IL12R
IFN?
NK cell
Ly49H-DAP12
m157
CMV
Proliferation
Perforin Granzymes
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KIR Ly49 receptorsconvergent evolution at work

• Polygenic polymorphic
• Rapidly evolving!
• Mono-allelic expression on NK subsets
• Inhibitory ITIM receptors bind polymorphic MHC
• Ligands of activating receptors mostly unknown
  (likely pathogens?)
• Activating isoforms no intrinsic signaling
  capacity
• associate with DAP12 ITAM-adapter protein

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Activating NK receptors - ligands

• Human/mouse CD16-FceRIg/z.IgG
• Human CD2..CD58
• Human 2B4 (CD244)-SAPCD48
• Human DNAM-1 (CD226).CD112, CD155
• Mouse PILRb-DAP12.PILR-L
• Human NKG2D-DAP10.MICA/B, ULBP
• Mouse NKG2D-DAP10/12RAE-1,H60, MULT1
• Mouse Ly49H-DAP12...MCMV m157
• Mouse Ly49P-DAP12...MCMV ?
• Human/mouse NKp46-FceRIg/z..?
• Human NKp30-FceRIg/z. ?
• Human NKp44-DAP12..?
• Mouse NKR-P1c-FceRIg.?

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• NKG2D
• C-type lectin-like superfamily
• 1 gene, non-polymorphic, conserved mice - humans
• Homodimer expressed on all NK cells, gd 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

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NKG2D ligands in mice and humans
Many genes Many alleles
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NKG2D ligands

• MHC class I-like
• dont 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
• Elevated in autoimmune diseases

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What is the biological role of the NKG2D ligands?

• Danger signals to alert the immune system to
  infection or genomic damage

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NKG2D on NK cells, ?? T cells and CD8 T
cells detect NKG2D ligands on abnormal cells
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NKG2D-RAE-1 interaction overrides self class
I-inhibition in vivo
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NK Cells - Ontogeny FunctionComprehensive
reviews

• Trinchieri Adv Immunol 47187, 1989
• Smyth et al. Nat Rev Cancer 2850, 2002
• Di Santo Ann Rev Imm 24257, 2006
• Lanier Nat Rev Immunol 2008

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NK Cells - Receptors SignalingComprehensive
Reviews

• Kumar et al, Nat Rev Immunol 5363, 2005
• Parham, Nat Rev Immunol 5201, 2005
• Lanier, Ann Rev Immunol 23225, 2005