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FOCiS Advanced Course in Basic & Clinical Immunology

Innate Lymphocytesand iNKT cells

Lewis L. [email protected]

iNKT cell Innate lymphocytes

2018 Advanced Course in Basic & Clinical Immunology February 19, 2018

1

ILC3

NKR+ ILC, LTi

Common Lymphoid Progenitor

NK cell

ILC2

Id2Ets1Nfli3

Zbtb16Gata3

IL-15

ILCProgenitor

RORα

RORγt

IL-7

IL-7

ILC1 IFNγ TRAIL

IFNγperforin

IL-17IL-22

IL-5IL-13

Eomes

Tbet

Spits & Cupedo Ann Rev Imm 2012

ILC2 –protect against parasites and help wound repair


2

Spits & Cupedo Ann Rev Imm 2012

ILC3 –protect against extracellular bacteria and help wound repair

IL-17

Lim et al. Curr Op Imm 2017

Innate Lymphoid Cells adapt to their environment


3

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, require a thymus for development

NK cells are not T cells!

don’t rearrange TcR genes or express CD3 on the cell surface

do not require a thymus

Primordial MHC

MHC I MHC IICD1

MHC and CD1 Evolution and Function

Tim

e

Present

Peptides Lipids Peptides

Thanks Mike Brenner

CD8+ T cell CD4+ T cellNKT


4

Lipid in CD1d binding groove

Top View

Side View

Thanks Mike Brenner

Defining iNKT cells

T cells expressing an invariant TcRα chainand recognizing lipid antigens presented by CD1d

In humans - invariant Vα24 + Jα18 TcRα

In mice - invariant Vα14 + Jα18 TcRα


5

Frequency and distribution of iNKT cells

In mice - <0.5% in blood & peripheral lymph nodes (LN)- 0.5-1% in thymus, spleen, mesenteric LN

- ~20-30% of T cells in liver- CD4+ and CD4-,CD8- subsets

In humans-Typically 10- to 100-fold less abundant-CD4+, CD8+, and CD4-,CD8- subsets

–not prevalent in liver

iNKT cells may play an important role in control of many infections

Thanks Mike Brenner


6

Cohen, Garg, and Brenner Adv Imm 2009

How do iNKT cells get activated?

Microbial lipid antigens

Jα28-/-

wt

CFU lung day 3 p.i.

Kawakami EJI 33:3322, 2003

iNKT cells protect mice against Streptococcus pneumoniae


7

Few lipid antigens found that activate iNKT cells

Mic

robi

al li

pid

antig

ens

for

iNK

TaGalCer - Synthetic

BbGL-IIc - Borellia burgdorferi

a-glucosyldiacyglyerol- Strept pneumoniae

Brigl et al. Nat. Immunol. 2003

iNKT cells are strongly stimulated by CD1d+ dendritic cells pulsed with many types of bacteria

DCiNKT

Gram +Gram -


8

iNKT Cells w/o DC

Brigl et al. Nat. Immun. 2003

IL-12 and dendritic cells induce iNKT cell activation

(without foreign antigens, but requires CD1d + self-lipids)

IL-12 ng/ml

DC

iNKT

Self-lipids recognized by iNKT- iGB3- plasmalogen lyso PE- aGlcDAG (vaccenic acid)

Direct & indirect activation of iNKT cells

Cohen, Garg, and Brenner Adv Imm 2009

Selected microbesS. pneumoniae – aGlcDAGBorellia – a-GalDAGSphingomonas – a-GluCer

Most microbesTLR ligands induce IL-12+CD1d self-lipids


9

Subsets of iNKT cells specialize in cytokine production- similar to Th and ILC subsets

Prevention of autoimmune diseasesdiabetes, collagen-induced arthritis, EAE

Autoimmune diseasesairway hyper reactivitycontact hypersensitivityasthma

iNKTCells

IL-4IL-13

Inflammatory/autoimmune diseasesNZB/NZW lupusgraft vs. host diseaseatherosclerosis

Fight infections & cancerbacterial, parasitic, viral and tumor rejection

Exacerbates

Ameliorates

IFNγIL-17

IFNγIL-4 IL-13

NKT cells function in a variety of immune responses via secretion of Th1-, Th2-, or Th17-type cytokines

Thanks Mitch Kronenberg


10

Questions about iNKT cells?

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


11

Detecting NK cells in human peripheral bloodC

D56

CD3

CD56dim mature CD56bright immature

New England Journal of Medicine 1989


12

Physiological role of NK cellsis 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 cell-deficient patient – caused by loss of one good GATA2 gene

CD3-CD56+ NK cells

CD3+CD56+ T cells

caused by heterozygous loss of GATA2


13

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

Human 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


14

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-1α, MIP-1β, RANTES, etc.

(but NOT IL-2, IL-4, IL-17, or IL-22)

IL-15IL-12

IFN-γpDC


15

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 (IL-15, IL-12, IL-18)

NK cell Cytokine receptor

Interferon-γChemokines

eron-γ


16

“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-encodedprotein

NK cellVirus-specificNK receptor

Kill!Interferon-γ

Host cell VV

Viral infection


17

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


18

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

Structural differences betweenMHC 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


19

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

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

KIR genes are highly polymorphic!

Chromosome 19q13.4

2DL22DS2 2DL5B 2DS3 2DL1 3DP1 2DL5A 2DS4 2DS5 2DS1 3DL2

3DL1

2DL42DP12DL33DL3

A haplotype

2DL1 2DS4 3DL23DL12DL42DL33DL3

B haplotype

3DS1

Chromosome 19q13.4

907 alleles at 14 KIR loci


20

Killer Cell Ig-like Receptors (KIR)

Ig superfamily

7-12 functional genes on human chromosome 19q

Extensive allelic polymorphism (907 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

“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


21


22

You may have more than 30,000 NK cells subsets in your blood- CyTOF analysis by Catherine Blish (Stanford)

Ljunggren & Malmberg NRI 2007

Factors boosting NK cell lytic activity


23

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)

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


24

H60c H60b6H6H60c0c0c0c 6H66H666H666H6H6H6666H6H6H6H6H66H66H66H66666H666H6H6H66H666H66H66H6H6666H666H6H66H666H666H66H6666666H666H66H6H666H6H6H6H6H6H6HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH 0bbbbbbbbb0bbbbbbbbbbbb0b0b0b0b0b00000

Lanier Cancer Immunology Research 2015

NKG2D has many polymorphic ligands in humans and mice

107alleles

42alleles

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


25

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

Therapeutic Uses of Natural Killer Cells


26

NK cell Cellule normale

+

Lysis

+

+

CD16 Tumor antigenAntibodies

Tumor cell

-

Inhibitory receptor MHC class I

Antibody-dependent cellular cytotoxicity

Rituxan, Herceptin, Erbitux, Daratumumab

NK cell Cellule normale

+

Lysis

+

+

Activating receptors

Tumor antigenBispecific antibodies

Tumor cell-


Bispecific antibodies

– anti-tumor x anti-NK activating receptor


27

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

Therapies that up-regulate stress-induced ligands on tumors

or agents that activate NK cells

NK cell

+

-

Lysis

Stressed or damaged cell


+

+

Activating receptors

Stimulatory ligands

Irradiation

Chemotherapy

Interferon-α/β

IL-15

IL-12

Agonist costimulatory antibodies


28

NK cell

+

Lysis

+

+

CARanti-CD19-cytoplamic CD137-CD3ζ

Tumor antigen

Tumor-

Inhibitory receptor

MHC class I

CAR NK cells

Chimeric antigen receptors

Guerra Immunity 2008

Increased incidence of myc oncogene-induced B cell lymphomas in NKG2D-deficient mice


29

NATURAL KILLER CELLS REMEMBER

Cytomegalovirus-seropositive humans have a unique population of CD57+NKG2C++ NK cells

0 103 104 105<PE-Cy5-A>: CD57

0

103

104

105

<PE-

A>: N

KG2C

0.62

2.77 2.44

6133.8

HCMV-seronegative

NK

G2C

HCMV-seropositive

0 103 104 105<PE-Cy5-A>: CD57

0

103

104

105

<PE-

A>: N

KG2C

43.1

7.27 45.5

13.833.4

CD57

CD57+NKG2C++


30

NK cells expressing the activating CD94-NKG2C receptor are expanded in healthy humans infected with CMV

CMVneg CMV+

0.0

2.5

5.0

**

5

15

25

50p<0.001

Patient 4

0 2 4 6 8 10 12 14 16 181.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

CD57+NKG2Chi NK

HCMV DNA

0

25000

50000

75000

Time (days)

Patient 3

0 25 50 75 100 1250

2

4

6

8

CD57+NKG2Chi NKHCMV DNA

1024204840968192163843276865536131072262144

Time (days)

Patient 1

0 5 10 15 20 250

2

4

6

8

10

12

CD57+NKG2Chi NK

1000

10000

100000

1000000

HCMV DNA

Time (days)

Patient 2

0 5 10 15 20 25 30 350

2

4

6

8

10

CD57+NKG2Chi NK

HCMV DNA

0

10000

20000

30000

Time (days)

Viral load

NKG2C++ NK cells

Antiviral drugtherapy

NKG2C++ NK cells specifically expands after CMV reactivation in immunosuppressed solid organ transplant patients


31

ILC and NK cells

• ILC – family of innate lymphocytes – rapid cytokine production

• NK cells keep you alive during certain viral infections

• NK cells regulated by inhibitory and activating receptors

• NK receptors are evolving rapidly

• NK cells possess immunological memory

Recent Reviews

ILC1 and NK Spits et al. Nature Immunology17:758, 2016

NK Cerwenka & Lanier Nature Review Immunology 16:112, 2016

ILCLim et al. Curr Opin Immunol 44:61, 2017Klose & Artis Nature Immunology 17:765, 2016

iNKT cellsCrosby & Kronenberg Immunogenetics 68:639, 2016


32

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