Upload
others
View
1
Download
0
Embed Size (px)
Citation preview
30/10/2017
1
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
30/10/2017
2
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
30/10/2017
3
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
30/10/2017
4
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
30/10/2017
5
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.