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iNKT cell Innate lymphoid cell
Nature Reviews Immunology April 2009
ILC3
NKR+ ILC, LTi
Common Lymphoid Progenitor
NK cell
ILC2
Id2
IL-15
Innate lymphocyte progenitor?
RORα
RORγt
IL-7
Innate Lymphoid Cells
IL-7
ILC1
IFNγ
IFNγ perforin
IL-17 IL-22
IL-5 IL-13
ILC3
NKR+ ILC, LTi
NK cell
ILC2
Innate Lymphoid Cells
ILC1
IFNγ
IFNγ perforin
IL-17 IL-22
IL-5 IL-13
Viruses Intracellular Bacteria
Helminths Epithelial tissue repair
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
3rd lineage of lymphocytes Function in innate immunity to protect against viruses,
bacteria, parasites, fungi, & tumors Produce cytokines & kill abnormal cells
Human CD3-,CD56+, (Nkp46+) Mouse CD3-,NKR-P1C+ (aka NK1.1) (NKp46+)
Natural Killer Cells
NK cell-deficient Patient #1 Clinical features - 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)
0 102
103
104
105
<Pacific Blue-A>: CD3
0
103
104
105
<P
E-A
>: C
D56
5.84
11.9
0 102
103
104
105
<Pacific Blue-A>: CD3
0
103
104
105
<P
E-A
>: C
D56
16.6
2.22
0 102
103
104
105
<Pacific Blue-A>: CD3
0
103
104
105
<A
PC
-A>
: C
D19 9.9
0 102
103
104
105
<Pacific Blue-A>: CD3
0
103
104
105
<A
PC
-A>
: C
D19 4.03
CD
56
CD
19
CD3
CD3-CD19+ B cells
CD3-CD56+ NK cells CD3+CD56+ T cells
CD3
NK-deficient patient #1 Healthy donor
• 31 year-old male
• Unusual clinical history of infections – Sinusitis, cellulitis, folliculitis, boils, warts (HPV)
• Leukopenia, hypocellular bone marrow
• Ankylosing spondylitis (HLA-B27)
NK cell-deficient Patient #2
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
0
20
40
60
80
100
% S
urvi
val
0 5 10 15 Day
WT B6 Littermates
NK-Deficient B6 mice
Mouse Cytomegalovirus 105 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)
IL-15"IL-12"
IFN-γ pDC"
How do NK cells sense their environment?������
Cytokines produced by infected epithelial or myeloid cells
Epithelial or Myeloid cell
TLR
Bacteria, virus, parasites, fungus
Cytokines (IL15, IL12, IL18)
NK cell Cytokine receptor
Interferon-γ Chemokines
“Stressed” cells – upregulate host-encoded ligands��� for activating NK receptors
Host cell Host-encoded
“stress-induced” protein
Bacteria, virus, fungus, parasite
NK cell
“Stress-induced ligand” receptor
(e.g. NKG2D)
Kill! Interferon-γ
DNA damage (tumors)
Host cell
Infected cells express virus-encoded ligands ���for activating NK receptors
Host cell
Virus-encoded protein
NK cell Virus-specific NK receptor
Kill! Interferon-γ
Host cell
Viral infection
How are NK cell responses regulated?
Class I+ tumorsgrow in vivo
Class I- tumorsare rejected
Class I- tumorsin NK-depleted
mice grow in vivo
NK Cells Reject Tumors Lacking MHC Class I
NK cells like to kill cells lacking MHC class I – “missing-self”
Karre et al. 1986 Nature 319:675
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
s-s
N N
Mouse Ly49
Human Killer cell Ig-like Receptor (KIR)
C
ITIM
2 (or 3) Immunoglobulin -like domains
Long cytoplasmic domain
Type II Type I
C-type lectin-like
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
HLA specificities of human KIRs
C
KIR2DL
C
KIR3DL
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); mono-
allelic 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
Killer Cell Ig-like Receptors (KIR)
Ig superfamily 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…………………....……?
Normal healthy cells
RBC neural cells
Infected cells tumor
Infected cells tumor
Ljunggren & Malmberg NRI 2007
Factors boosting NK cell lytic activity
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
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)
Y322 S329
V
V
NH2
S S
S S
COOH
DNAM-1 (CD226) - an activating NK receptor -
Fyn à PKC à
DNAM-1-deficient mice are more susceptible to chemical (methylcholanthrine)-induced sarcomas
Iguchi-Manaka JEM 2008
Koch Trends Imm 2013
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 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
Guerra Immunity 2008
Increased incidence of myc oncogene-induced B cell lymphomas in NKG2D-deficient mice
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
Lanier Nat Rev Immunol 8:259, 2008
Proliferation
Natural Killer cells are critical for control of cytomegalovirus
MCMV-infected cell
NK cell
Ly49H
DAP12
DAP10
Ly49H drives NK cell responses ���to cytomegalovirus in B6 mice
Syk & ZAP70
Cytotoxicity IFN-γ
Proliferation MCMV control
p85 PI3 kinase/Vav1
m157
7.92 1.18
0.99 89.9
7.96 1.95
0.87 89.2
7.53 21.7
0.47 70.3
7.3 16.1
0.37 76.2
1.07 6.75
0.041 92.2
0.8 0.96
0.078 98.2
d 0 d 1.5 d 7 d 14 d 28 d 50
CD45.1 (donor type)
Ly49
H
MCMV-specific NK cells expand during infection and are long-lived!
Track NK cells
MCMV Inject wildtype CD45.1 NK cells
Ly49H-deficient mouse (CD45.2)
Expansion and contraction of adoptively transferred Ly49H+ NK
cells during MCMV infection
0
5
10
15
20
25
0 10 20 30 40 50 days post-infection
% L
y49H
+ N
K c
ells
in s
plee
n
105 104
102
103
104
105
106
0 10 20 30 40 50 days post-infection
Abs
. no.
of L
y49H
+ N
K c
ells
(spl
een)
0
5
10
15
20
25
0 10 20 30 40 50 days post-infection
% L
y49H
+ N
K c
ells
in li
ver
105 104
102
103
104
105
106
Abs
. no.
of L
y49H
+ N
K c
ells
(liv
er)
0 10 20 30 40 50 days post-infection
100 fold expansion
1000 fold expansion
MCMV Ly49H+ NK cells
Mouse lacking Ly49H NK cells
Long-lived memory NK cells protect neonatal mice against MCMV infection better than naïve NK cells
MCMV Adult naïve or
memory NK cells
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
102
103
104
105
0 2 4 6 8
Days post-infection
Abs
. No.
of L
y49H
+ N
K c
ells
MCMV
MCMV-Δm157
66.5
3.6
0 25 50 75 100
MCMV-Δm157
MCMV
Fold expansion
MCMV
DAP12-/- (CD45.2+)
Naïve NK cells (CD45.1+)
30-50 days
Memory NK cells
MCMV or MCMV-Δm157
DAP12-/- (CD45.2+)
30-50 days
2° Memory NK cells
O’Leary NI 2006
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
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
