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Silvia Pesce

Cytofluorimetric analysis of

NK cell subsets expressing

different immune

checkpoints

ESCCA Conference Thessaloniki, September 24th 2017

Università degli Studi di Genova

Natural Killer (NK) cells are innate lymphocytes

specialized in early defense against virus-infected

and tumor cells. Natural killer (NK) cells are fast-

acting and versatile lymphocytes that are critical

effectors not only in the context of innate immunity,

but are also involved in a series of events able to

shape adaptive immunity

Natural Killer cells

Inhibitory and activating receptors on NK cells

ILT2/LIR-1 (CD85J)

NKG2A (CD159a)

KIR2DL

KIR2DS

KIR3DL

KIR3DS

NKG2C (CD159c)

HLA-E

HLA-C

HLA-Bw4?HLA-F?

HLA-C

HLA-Bw4

DifferentHLA-I alleles

HLA-E

PD-1 (CD279)

Siglec-7 (CD328)

TIGIT

Ganglioside DSGb5

PVR (CD155)

PD-L1 (CD274)PD-L2 (CD273)

IRP60 (CD300a)Alphaherpesvirus pseudorabies virus Phosphatidylserine Phosphatidylethanolamine

NK cell receptorsLigands

NKp46

NKp30

NKG2D

DNAM1

NKp80/KLRF1

2B4 (CD244)

NKp44

B7-H6 -BAG6/BAT3

MICA –MICB -ULPBs

CD48

Nectin-2 (CD112)PVR (CD155)

AICL

?21spe-MLL5

NTB-A (CD352)

CD2

Tactile (CD96)

CD58

PVR (CD155)

NTB-A (CD352)

CRACC/CS1 (CD319)CRACC/CS1 (CD319)

CD16 (FcγRIII)IgG

NK cell receptorsLigands

Inhibitory receptorsActivatory receptors

Ligands

Del Zotto et al

Cytometry 2017

-

NK cell function is the result of a balance between

inhibitory and triggering signals

Under normal conditions the function of inhibitory

NK receptors overrides that of the triggering ones.

Under pathological conditions (tumors or viral-

infection) the triggering NK receptors are allowed to

induce NK cell activation

Moretta A

Annu Review

1996 2001

How to select NK cells

Lymphocytes

NK cells

FSC

SS

C

From…

FSC

SS

C

FSC

SS

C

Cells isolated from

peritoneal fluid of a

ovarian cancer patient

(PF)

Cells isolated from

peripheral blood of a

healthy donor

(PB HD)

Cells isolated from

biopsy of lung cancer

patient

(LC)

lymphocytes

lymphocytes

lymphocytes

Tumor cells

Tumor cells

NK cells

HD1 HD2

Leukocytes

Leukocytes+Tumor cells

Leuckocytes+Tumor cells

CD45

FS

C

CD3+CD19+CD14

CD

56

CD45

FS

C

CD3+CD19+CD14

CD

56

CD

56

CD45

FS

C

CD3+CD19+CD14

CD

56

CD16

CD16

CD16 CD16

CD

56

CD

56

FSC

SS

C

CD45

FS

C

CD3+CD19+CD14

CD

56

From…

FSC

SS

C

CD45

FS

C

CD3+CD19+CD14

CD

56

CD

56

FSC

SS

C

CD45

FS

C

CD3+CD19+CD14

CD

56

Cells isolated from

peritoneal fluid of a

ovarian cancer patient

(PF)

Cells isolated from

peripheral blood of a

healthy donor

(PB HD)

Cells isolated from

biopsy of lung cancer

patient

(LC)

lymphocytes

lymphocytes

lymphocytes

Tumor cells

Tumor cells

NK cells

CD16

CD16

HD1

CD16 CD16

NK cells

LeukocytesLymphocytes HD2

CD

56

CD

56

How to select NK cells

Leukocytes+Tumor cells

Leuckocytes+Tumor cells

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NK CD56 Bright - Dull - Neg

CD16

CD

56

• High Cytotoxicity

• High Cytokine production

• High Proliferation

• Low Cytotoxicity

• High Cytokine production

NK CD56bright

NK CD56dull

• Low Cytotoxicity

• Low Cytokine production

NK CD56neg

HD1 HD2

Moretta A et al.

Ann. Rev Immunol 1996

gruppo C2 gruppo C1

HLA class I-specific inhibitory receptors

KIR:Killer Ig-like

Inhibitory Receptors

CD94/

NKG2A

ITIM

Inhibitory checkpoints on CD56bright and CD56dull NK cells: NKG2A and KIR

HD-NK

NKG2A

CD

56

KIRs

HD1

NK CD56bright

NK CD56dull

NKG2A

KIR

NCR

NK

NKG2A

NCR

NK

HD2

NK

G2A

KIRs

KIR

2D

L2/L

3

KIR2DL1

KIR

2D

L2/L

3

KIR3DL1

NKG2A+ KIR- NKG2A+ KIR+

1 KIR 2 KIRs 3 KIRs 4 KIRs

CD56dull NK cells

Inhibitory receptors on CD56dull NK cells:

coexpression of different immune checkpoints

NK NK NK NK NK

Inhibitory checkpoints KIR and NKG2A and NK cell maturation

NK

G2A

KIRs

NKG2A+ KIR-

NKG2A+ KIR+

NKG2A- KIR+

NKG2A- KIR-

CD57

CD56dull NK cells

LIR-1CD

56

CD57

Inhibitory checkpoint on NK cell: LIR-1

LIR-1

CD

56

LIR-1

KIR

LIR-1

NK

G2A

CD56dull NK cells

HD1

HD2

LIR-1

(ILT-2)

HLA-I,

UL-18

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During development, NK cells are exposed to inhibitory and stimulatory

interactions with normal cells (left column). The balance of signals upon encounter

with neighboring normal cells (middle column) sets the responsiveness state of the

NK cells (right column).

A, Stimulatory signals from neighboring normal cells are opposed when several

inhibitory receptors are coexpressed on the developing NK cell, which allows it to

reach a state of high responsiveness.

B, Expression of one receptor only partially counters stimulatory signals, such that

residual persistent stimulation induces partial hyporesponsiveness.

C, When no inhibitory receptors are expressed, strong persistent stimulation

induces greater hyporesponsiveness. Thus, the responsiveness of NK cells is

tuned to quantitatively different levels by the quantity of inhibitory and stimulatory

interactions to which the cells are exposed during development.

Tuning NK cell responsiveness: an educational “rheostat.”

Joncker et al. J Immunol. 2009 Apr

NKG2A+ KIR- NKG2A+ KIR+

1 KIR 2 KIRs 3 KIRs 4 KIRs

NK NK NK NK NK

Higher citotoxicityMoretta A et al.

Annual Rev Immunol 2001

Activating receptors HLA I specific: aKIR

SIGNALING ADAPTOR MOLECULE

Discrimination of Activating from Inhibitory KIRs by cytofluorimetric analysis

HD-NK

CD

56

aK

IR2D

L1

aKIR2DL1/S1

aK

IR3D

L1

aKIR3DL1/S1

KIR

Inhibitory checkpoints and Cytolitic activity on NK cells

Two possible immunotherapeutic approach against tumor cells

KILLING

NKG2A

KIR

NCR

KILLING

NKG2A

KIR

NCR

HLA-Ineg TUMOR CELLS

HLA-Ipos TUMOR CELLS

KILLING

KIR

NCR

Non-self HLA Ipos TUMOR CELLS

TUMOR/

VIRUS INFECTED CELLS

NCR Ligands

TUMOR/

VIRUS INFECTED CELLS

HLA-A,B,C

HLA-E

NCR Ligands

Alloreactive NK cells

NK

NK

NK TUMOR/

VIRUS INFECTED CELLS

HLA-A,B,C

HLA-E

NCR Ligands

ALLOREACTIVE NK CELL SUBSET: cells expressing only the inhibitory KIR that does not recognize any KIR-ligand of the patient

Definition of the alloreactive NK cell subset

in donor’s NK cell population in GvH direction

MAb to Receptors

permissive to lysis

MAb to Receptors

blocking lysis

Inhibitory KIR specific for patient's HLA-class I alleles +

NKG2A

Inhibitory KIR specific for HLA-class I alleles

absent in patient's haplotype

Pende et al 2005, 2009

Moretta et al Imm Rev, 2008

Del Zotto et al Cytometry 2017

NK cells kill patient’s DC

thus preventing priming of

allogeneic T cells contained

in the BM graft and, thus,

GvHD.

NK cells kill lymphohemopoietic

cells of the patient, including T

lymphocytes. This prevents the T-

cell mediated graft rejection.

NK cells kill leukemic cells,

residual after chemotherapy

and radiotherapy. This may

prevent leukemic relapses.

NK cells in haploidentical haematopoietic stem cells transplantation

Velardi et al. Trends in Immunol, 2002

Moretta et al. Immunol. Rev. 2008

Locatelli et al Clin Immunol 2009

Generation of “Alloreactive” NK cells

NO GvHD

NO graft rejection

NO leukemic relapses

Moretta L. et al Frontiers Immunol 2014

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Nat Rev Cancer 2012: 12(4), 252-64.

INTRODUZIONE PD1

PD-1

Inhibitory checkpoint in NK cells

? PD-1

CD

56

An NK cell subset expressing

a novel inhibitory checkpoint

Pesce et al.,

JACI 2017

1.The choice of the best reagent:

2.Controls needed:

Instructions for a correct analysis of the PD-1 receptor on NK cells

mAb #1 mAb #2 mAb#3 mAb#4

Isotype

control

Negative

control

Positive

Control 1

Positive

Control 2PD-1

Pesce et al.,

JACI 2017

4.Number of cases to be analyzed

3.Number of events to be acquired

5000 2500 500

NK cells derived from PB of healthy donor

CD

56

PD-1

Events

Pesce et al.,

JACI 2017

Instructions for a correct analysis of the PD-1 receptor on NK cells

5%

Pesce et al.,

JACI 2017

Surface phenotype of PD-1+ and PD-1- NK cell subsets

from PB of PD-1+ HD

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Nivolumab

Tumor cell

PDL

NK

PD-1

NK CELL ACTIVATION

NK CELL INHIBITION BY CONSTITUTIVE PD-1

NK cell inhibition by inhibitory receptors and modulation with

immunotherapeutic blocking mAbs.

NK

NK

PD-1

NK

Nivolumab

Lirilumb

PD-1

KIR2D

HLA-C

PDL

HLA-C

PDL

Muntasell et al

Current Opinion in Immunology

2017,

modified

CD94/

NKG2A

KIR

PD-1

NK cell Tumor cell

HLA E

HLA I

PD-L

TIM-3

TIGIT

LAG-3

GALECTIN 9

HLA II

PVR

Classical and New Inhibitory checkpoints on NK cells

MOLECULAR IMMUNOLOGY LABORATORIES

Head: ALESSANDRO MORETTA

Department of Experimental Medicine

University of Genoa

MARCENARO EMANUELA

GREPPI MARCO

PAROLINI SILVIA

(Univ. Brescia)

TABELLINI GIOVANNA

(Univ Brescia)

RAMPINELLI FABIO

(Spedali Civili Brescia)

MORETTA LORENZO

(Osp. Pediatrico Bambino Gesù, Roma)

OLIVE DANIEL

(Univ. Marseille, France)

THANK YOU !!!

“Prospects for the use of NK cells in immunotherapy of human cancer”

Ljunggren & Malmberg, Nature Review Immunology 2007

Size of the alloreactive NK cell subset as a criterion for donor selection

Clinical application of PD-1 agonists and antagonists. PD-1 agonists that suppress adverseimmune responses may be useful in treating autoimmune diseases, allergy and transplantrejection. PD-1 antagonists that augment immune response may be useful in cancer andinfectious diseases.

The immune system is capable of recognizing tumors and eliminates many

early malignant cells. However, tumors evolve to evade immune attack, and

the tumor microenvironment is immunosuppressive. Immune responses are

regulated by a number of immunological checkpoints that promote protective

immunity and maintain tolerance. T cell coinhibitory pathways restrict

the strength and duration of immune responses, thereby limiting

immune-mediated tissue damage, controlling resolution of inflammation,

and maintaining tolerance to prevent autoimmunity. Tumors exploit these

coinhibitory pathways to evade immune eradication.