Micro 204 Innate Lymphocytes - UCSF ImmunoX...Ljunggren & Malmberg NRI 2007 Factors boosting NK cell...
Transcript of Micro 204 Innate Lymphocytes - UCSF ImmunoX...Ljunggren & Malmberg NRI 2007 Factors boosting NK cell...
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Innate Lymphoid Cells Lineage-negative, Id2-dependent cells that arise from a common lymphoid precursor Some mediate lymph node organogenesis during fetal development Some become tissue-resident effector cells expressing cytokines driven by transcriptional modules associated with CD4 helper T subsets (Th1, Th2, Th17) Roles in homeostasis - establishing commensals, responding to dietary signals, responding to circadian cues, etc. Roles in inflammation and disease
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Vivier et al. Cell 2018
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Vivier et al. Cell 2018
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ColonnaImmunity2018
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Unclear whether ILC1 cells and NK cells are truly distinct lineages or a spectrum of cells within a single lineage that includes ILC1
cells, immature NK cells and mature NK cells
Nature Immunology 16, 306–317 (2015)
ImmGen Transcriptional Analysis
www.immgen.com
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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
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Godfrey,Immunity,2018
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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
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Detecting NK cells in human peripheral blood
CD
56
CD3
CD56dim mature CD56bright immature
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CD
56
CD3
CD56dim mature CD56bright immature
These are NOT NKT cells!
They are just activated T cells
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New England Journal of Medicine 1989
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�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
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NK cell-deficient patient
CD3-CD56+ NK cells
CD3+CD56+ T cells
caused by heterozygous loss of GATA2
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NK cells are critical to early innate host defense against pathogens
0
20
40
60
80
100
% S
urvi
val
0 5 10 15Day
WT B6 Littermates
NK-Deficient B6 mice
Mouse Cytomegalovirus105 pfu i.p.
Loh JVI 2005
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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 – but increase with infection
>70% of lymphocytes in decidual tissue
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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 TNFα, LTα, GM-CSF, IL-3, M-CSF, IL-10, MIP-1a, MIP-1b, RANTES, etc.
(but NOT IL-2, IL-4, IL-17, or IL-22)
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IL-15IL-12
IFN-γpDC
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Immunity, Volume 40, Issue 1, 2014, 117 - 127
In fungal infections, NK cells respond to IL-17 – producing GM-CSF to recruit granulocytes to control infection
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How do NK cells sense their environment?��
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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-γChemokines
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“Stressed” cells – up regulate 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
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Infected cells express virus-encoded ligands �for activating NK receptors
Host cell
Virus-encodedprotein
NK cellVirus-specificNK receptor
Kill!Interferon-γ
Host cell
Viral infection
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How are NK cell responses regulated?
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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
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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 IAdenovirus E3-k19 Retain in ERHSV-1,2 ICP47 Blocks TAPEBV EBNA1 Block peptide generationHCMV US2, US11 ER to cytosolHCMV US3 Retain in ERHCMV US6 Blocks TAPHCMV US10 Degrades HLA-GMCMV m152 Retain in ERMCMV m04 Associates with H-2MCMV m06 Lysosomal degradationHHV8 K3, K5 Endocytosis HIV-1 Nef Endocytosis
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Structural differences between�MHC class I-specific inhibitory receptors
in mice and humans
s-s
N N
MouseLy49
HumanKiller cell Ig-like Receptor (KIR)
C
ITIM
2 (or 3) Immunoglobulin -like domains
Long cytoplasmic domain
Type II Type I
C-typelectin-like
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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
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2DL22DS2 2DL5B 2DS3 2DL1 3DP1 2DL5A 2DS4 2DS5 2DS1 3DL2
3DL1
2DL42DP12DL33DL3
A haplotype
2DL1 2DS4 3DL23DL12DL42DL33DL3
B haplotype
2DL22DS2 2DL1 2DL5A 2DS1 3DL23DS12DL43DL3
3DS1
2DS5
Different people have different KIR genes
Chromosome 19q13.4
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KIR genes are highly polymorphic!
907 alleles at 14 KIR loci
https://www.ebi.ac.uk/ipd/kir/stats.html
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Killer Cell Ig-like Receptors (KIR)
Ig superfamily
7-12 functional genes on human chromosome 19q
Extensive allelic polymorphism 698 alleles!) no rearrangement; mono-allelic expression possible
Expressed by subsets of NK cells and memory T cells (mostly CD8+ T cells, but also CD4+ 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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Rapid expansion of KIR genes in primates
3DL1Human
Chimpanzee
Haplotype 1
Haplotype 2
2DL2
2DS2
2DL5
B
2DS3
2DL1
2DL5 A
2DS4
2DS5
2DS1
3DL2
2DL4
3DP1
2DP1
2DL3
3DL3
3DS1
2DL5
3DL1
/2
2DL4
KIR
CI
2DL5
2DL4
KIR
CI
2DL6
3DL4
3DL6
2DL
3DL
2DL4
DP1
2DS
3DL3
Orangutan
P. Parham
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Ly49 ReceptorsC-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
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Conserved NKG2 genes on mouse chromosome 6 and human chromosome 12p13.1
Klrg1
Nkrp1a
ClrF
Nkrp1c,d Ly49eLy49a-x
ClrG
Nkrp1f
ClrB
Cd69
Clec2
Cd94
Nkg2d
Nkg2e,c,a
Klre1
KLRG1
NKRP1A
LLT1 YLY49LAICL
KLRF1
CLEC1
CD69 CLEC2 CD94
NKG2D
NKG2F,E,C,A
DECTIN1
Humans
Mice
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Inhibitory CD94-NKG2A and �activating CD94-NKG2C-DAP12 receptors
+ +
NKG2C - CD94
DAP12
-- -
ITIM
Recruit phosphatases - SHP-1 -inhibit responses
CD94 - NKG2A
Recruit Syk & Zap70kinases - activate
Ligand = MHC class I HLA-E
ITAM
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“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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C C
D D K
NKp44
C C
D D
NKp30
DAP12FcεRIγorCD3ζ
K
C C
D D K
FcεRIγorCD3ζ
FcεRIγorCD3ζ
C CDD D
ITAM
NKp46CD16
ITAM-based Activating NK receptors
Miller & Lanier Ann Rev Cancer Biology 2019
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DNAM-1 4-1BB2B4
ITSMSAPorEAT2
PKC
Fyn
TRAFs
NKG2D
DAP10 DAP10
Costimulatory NK receptors
Miller & Lanier Ann Rev Cancer Biology 2019
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LILRB1
KIR
ITIMs
CD94 NKG2AKLRG1
TIM3
CEACAM1 LAG3
PD1
ITSM
CTLA-4
YVKM
Inhibitory NK Receptors
Miller & Lanier Ann Rev Cancer Biology 2019
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You may have more than 30,000 NK cells subsets in your blood- CyTOF analysis by Catherine Blish (Stanford)
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Ljunggren & Malmberg NRI 2007
Factors boosting NK cell lytic activity
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Antibody-dependent cellular cytotoxicity
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Activating CD16 Fc receptor on NK cells and macrophages mediates Herceptin- and Rituxan-induced human tumor elimination
Clynes Nat Med 2000
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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 and 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)
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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
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H60c H60b
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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
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NKG2D on NK cells, γδ T cells and CD8+ T cellsdetect NKG2D ligands on abnormal cells
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Guerra Immunity 2008
Increased incidence of myc oncogene-induced B cell lymphomas in NKG2D-deficient mice
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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�
� �
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How do we engage NK cells in new immunotherapies for cancer?
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NK cell Cellule normale
+
Lysis
+
+
CD16 Tumor antigenAntibodies
Tumor cell
-
Inhibitory receptor MHC class I
Antibody-dependent cellular cytotoxicity
Rituxan, Herceptin, Erbitux, Daratumumab
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NK cell Cellule normale
+
Lysis
+
+
Activating receptors
Tumor antigenBispecific antibodies
Tumor cell-
Inhibitory receptor MHC class I
Bispecific antibodies
– anti-tumor x anti-NK activating receptor
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Tumor cell
LysisInhibitory receptor
Activating receptor
Inhibitory ligand
Stimulatory ligand
Checkpoint blockade therapies
NK cell
+
-
anti-KIR
anti-NKG2A
anti-PD1
anti-Tim3
anti-LAG3
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Therapies that up-regulate stress-induced ligands on tumors
or agents that activate NK cells
NK cell
+
-
Lysis
Stressed or damaged cell
Inhibitory receptor MHC class I
+
+
Activating receptors
Stimulatory ligands
Irradiation
Chemotherapy
Interferon-α/β
IL-15
IL-12
Agonist costimulatory antibodies
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NK cell
+
Lysis
+
+
CARanti-CD19-cytoplamic CD137-CD3ζ
Tumor antigen
Tumor cell-
Inhibitory receptor
MHC class I
Chimeric antigen receptors
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MCMV-infected cell
NK cell
Ly49H
DAP12
DAP10
Ly49H drives NK cell responses �to cytomegalovirus in mice
Syk & ZAP70
CytotoxicityIFN-γ
ProliferationMCMV control
p85 PI3 kinase/Vav1
m157
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Expansion and contraction of 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 foldexpansion
1000 foldexpansion
MCMV Ly49H+ NK
cells
Mouse lacking Ly49H+ NK cells
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Long-lived memory NK cells protect neonatal mice against MCMV infection better than naïve NK cells
MCMV
Adult naïve or memory NK cells
Newborn MCMV-susceptible mice
Memory NK cells kill better and make more cytokine on a per cell basis than naïve NK cells in vitro
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O’Leary NI 2006
Hapten-specific contact hypersensitivity responses in RAG-deficient mice
day 0 & 1 paint hapten on back skinday 6 or 30 – challenge ear – measure swelling
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O’Leary NI 2006
Depletion of NK cells eliminates contact hypersensitivity responses in RAG-deficient mice
day 0 & 1 paint hapten on back skinday 6 deplete NK cells day 6 – challenge ear – measure swelling
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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
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NK cells
• Keep you alive during certain viral infections
• Regulated by inhibitory and activating receptors
• Receptors are evolving rapidly
• Possess immunological memory
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Recent Reviews
ILCColonna Immunity 48:1104, 2018
NK Cerwenka & Lanier Nature Review Immunology 16:112, 2016
NK cell ImmunotherapyMiller & Lanier Ann Rev Cancer Biology 2019