Pr. Daniel Olive, INSERM UMR 1068, Institut Paoli
Calmettes, Aix Marseille Université
Targeting of Novel
Immunomodulatory Pathways in
Lymphomas
Regulating costimulation
Inhibiting inhibitory pathways including Checkpoint
blockade inhibitors and others
Regulating different immune effector populations
Innate effectors and B cells
Classes of Cosignaling Molecules
Presentation
Escape mechanisms in lymphomas: role of BTLA-
HVEM pathway involving innate effectors
Treg and NHL: regulation by the ICOS-ICOSL
pathway
HVEM LTR
LT
LIGHT PD1 CTLA-4 BTLA
PDL-1 PDL-2 B7-1 B7-2
CD160
TNFR-TNF family CD28-B7 family
W/O viral receptors UL144 and
HSV gD
T Cells & BTLA HVEM
BTLA = B and T lymphocyte attenuator
KO BTLA increased:
- T proliferation & cytokine production (Liang & Sha,Curr Opin Immunol,2002)
- severity and duration of EAE (Watanabe et al.,Nat Immunol,2003)
- hypergammagobulinemia and autoantibodies to nuclear antigens, increased numbers
of activated T cells (Oya et al., Arthritis and Rheumatism, 2008)
- duration of infl. in the lung after airway challenge (Deppong et al.,J.Immunol,2006)
- reject of mismatched cardiac allografts (Tao et al., J.Immunol,2005)
- T cell survival (Deppong et al.,J.Immunol,2008)
- survival of mice infected by MHV3 due to decreased apoptosis of macrophages
through TRAIL (Yang et al., Gut, 2013)
BTLA mainly negatively regulates the T immune response in
these settings
BTLA-HVEM Serves as Negative Regulator
of Immune Responses in Cancer
• Melanoma and leukemic specific T cell responses
BTLA highly expressed on melanoma antigen-specific effector CD8 T cells
BTLA activation suppresses cancer-specific CD8 T cells
(melanomas, AML)
– Derre et al., JCI (2010) Fourcade et al., Cancer Res (2012), Hobo et al., J. Immunol., 2012
• γδ T-mediated anti-lymphoma responses
BTLA strongly expressed on resting γδ T cells
BTLA-HVEM ligation suppresses γδ T responses
Lymphoma cells suppress γδ T cells proliferation via BTLA-HVEM ligation
Suppression blocked by BTLA-HVEM mAbs
– Gertner-Dardenne et al., Blood (2013)
• B cell responses
CpG upregulates BTLA in vitro & in vivo in melanoma
BTLA-HVEM ligation suppresses B cell responses
Suppression blocked by BTLA-HVEM mAbs
– Thibult et al., J Mol Med (2013)
HVEM
HVEM Expression in Melanoma
BTLA Clusters with Vg9Vd2 T CR after
Activation
TC
RV
d2
B
TLA
M
ER
GE
D
API
Resting 1h BrHPP
Escape Mechanism Involving BTLA and HVEM in NHL
TNFSFR14/HVEM in Lymphomas
HVEM expressed on human B cell malignancies
HVEM is a possible tumor suppressor gene since HVEM cross linking
induces lymphoma cell death and sensitizes to Fas killing (Costello, Leukemia
2003; Pasero, Eur.J. Immunol., 2009)
TNFRSF14 gene alterations might be correlated to prognosis in FL
(Cheung et al., Cancer Research, 2010; Launay et al., Leukemia 2011)
BTLA is highly expressed in lymphoma microenvironment (Mhidi et al.,
Am. J. Clin. Pathol., 2009, Gertner-Dardenne et al., Blood, 2013).
HV INF FL0
20
40
60
80
100
p = 0.0312
p = 0.0583
%gd
BT
LA
positiv
e c
ells
BTLA Expression on gd T cells in NHL
Microenvironment
BTLA
FL
62 3.4
NK
0.8%
gd
0.3%
CD3
CD3
VIV
ID
CD
56
CD
4
Vg9
Count
BTLA
Characterization of HVEM Antibodies for the Ability
to Modulate BTLA –Ig Binding to Cell Surface HVEM
00.
010.
03 0.1
0.3 1 3 10 30
0
100
200
300
PD1-3.1HVEM-11.8
HVEM18.10
mAb concentration (Log10 µg/ml)
No
rmaliz
ed m
Abs M
FI
BTLA Blockade Restores Autologous gd T Cell
Proliferation in Coculture with its FL NHL
IgG1 Antag. BTLA Fab Antag.BTLA Agon. HVEM Antag.HVEM
49 86 70 19.5 87
CellTrace
CONCLUSIONS
• BTLA is expressed on Vg9Vd2 T cells
• BTLA engagement inhibits selectively Vg9Vd2 T cells proliferation/activation
(via partial S phase arrest) and accelerates their differentiation
• BTLA belongs to the class of molecules regulating cell cycle progression like
CTLA-4 and PD-1
• BTLA regulates homeostatic proliferation of Vg9Vd2 T cell
• BTLA blockade restores Vg9Vd2 T cell proliferation in co-culture with
HVEM+ lymphoma cells
Escape mechanisms in lymphomas: role of
BTLA-HVEM pathway involving innate effectors
Treg and NHL: regulation by the ICOS-ICOSL
pathway
Objectives
V
V
V
C
ICO
S
ICOS-
L
CM
H TC
R
Activated T
cells, TFH,
Tregs DC, B cells,
Monocytes
Differentiation
Cytokine production
IL-10 , IL-4, IL-21, IFN-γ, TNF-α,…
B TF
H
Cooperation
(germinal center formation
and antibody response)
Th1
Th17
Th0
Th2
Survival and proliferation
TFH
Inducible T Cells Costimulator (ICOS)
ICOSL downregulation
Tregs: Prognosis Marker in Tumor
Tregs
Tregs Tregs
Tregs
CCR4 CXCR
4
CCR5 Tregs
Tumor
cell
MDSC
TAM
Migration
CXCL
17
CXCL
22
CCL5
Tumor site
APC
Tumor
cell MDSCs TAM
sTGF-
β
Tregs Tregs TCR
MHC
II
Tregs ICOS
MHC
II
TCR ICOS
ICOS
L MV
IL-2
Expansion
IDO
pDC
ICOS
L
↓ Anti tumoral
responses
• Adverse Prognosis marker on solid
tumors(breast), positive prognosis
marker (H&N, FL) Mougiakakos et al, 2010
Yang et al, 2007, 2009
Martin-Orozco et al, 2010
Faget et al, 2012
Conrad et al, 2012
TCD4
TCD8
Tγδ
NK
Tcon
v Tcon
v
IL-10
APC TAM
MDSC Tumor
cell
CD70
CD27
Tcon
v
TCR TCR
Conversi
on
sTGF-
β PGE2
COX-
2
MHC
II MHC
II
CD28
CD80
CD86
Accumulation of Tregs and ICOS+ Tregs in FL
CXCR5 PD-1 GITR CTLA-4 Ki67
67.5 7.57
15.9 8.98
78.3 5.23
11.5 5
73.5 9.95
8.62 7.89
31.2 54.4
1.99 12.4
8.56 77
1.08 13.3
ICO
S
Gated on Tregs
CD
127
CD25
CD
8
CD4
Tregs
Tconv TCD8
TCD4
ICOSL is absent from B lymphoma cells, pDC, mDC from FL but present on DLBCL
ICOS / ICOSL Interaction
B cells mDC pDC
ICOSL ICOS
FL DLBCL
TCD4
B
cells
0.7 2 1.5
53.0 90.6
22.0
CD86 ICOSL
MFI
331
MFI
69
MFI
35
MFI
280
ICOS /ICOSL interaction induces downregulation of ICOSL on follicular lymphoma B cells
ICOS / ICOSL Interaction
ICOSL
CD
19
B alone B + TCD4 B + TCD4
Ctrl IGG1
B + TCD4
Anti-ICOS
59.4 13.5 14.1 57.3
23.5 25.9 25.1 28
CD
19
CD86
ICOS / ICOSL Interaction
ICOS /ICOSL interaction induces enrichment of Treg in Follicular lymphoma
Foxp3
CD
25
TCD4 + B TCD4
Cell trace
CD25+ Foxp3+ CD25high Foxp3high
ICOS
ICOS / ICOSL Interaction
ICOS /ICOSL induced enrichment of Treg in Follicular lymphoma is prevented by
antagonist ICOS mAbs
Role of Tregs in the Development of FL B Cells
Lymphoma infiltrating- Tregs suppress lymphoma B cells responses
CD80
B alone B / Treg
39.4
%
14.1
%
Role of Tregs in Development
of Lymphoma B Cells
Lymphoma infiltrating- Tregs suppress T conv induced
lymphoma B cells help
B alone B / Tconv /
Treg B / Tconv
39.4
%
49.6
% 31.7
%
CD80
Conclusions
Treg
ICOS ICOSL
Lymphoma
B cells
Lymphoma
B cells
PD-1
Ki67
HLA-DR
CD39
Treg
Treg
Treg
Agonist anti-ICOS mAb would enhance Tregs proliferation and function
Whereas antagonist anti-ICOS mAb would prevent Treg survival and function
Agonist and Antagonist BTN3A mAbs Regulate
Vg9Vd2 T Cell Functions Against Lymphoma
γδ T cells alone
γδ T cells + Control Isotype
+DAUDI
γδ T cells + anti BTN3 20.1
+DAUDI
γδ T cells + anti BTN3 103.2
+DAUDI
1. Cancer : Transcriptome, IHC,
Deep sequencing, RNA Seq
TAA and TSA typing
heterogeneity
HLA typing
Preexisting immune response
4. Combo personalized medicine
Immunosuppressive mechanisms
Cosignaling, galectins, enzymes,
Tregs, MDSC…
2. Exogenous Immunisation
Peptides
Proteins
Viral vectors
DNA
anti CD mAb fused to
TSA or TAA + adjuvant
3. Endogenous Immunisation
Chemo
Rx
Oncolytic viruses
Therapeutic Mabs
5. Monitoring
PFS
Immune parameters (FCM,
transcriptome, RNAseq)
Cytométrie
Transcriptome
ARNseq Olive D. , Cancer Vaccines, 2014
Collaborative approach
– Marseilles
• INSERM UMR 1068
• Aix-Marseilles University
• Marseilles Cancer Research Center
• Paoli Calmettes Institute
• Marseilles University Hospital
– Rennes: INSERM U917, University
Hospital
– Lyon : Lyon Cancer Research Center
Industrial valorization
– Creation of Imcheck
Therapeutics
– Patents licensed to GSK and
Janssen
– Other IP available
– Research partnerships
Multi-source financial support
– French National Agency for
Research
– French National Cancer Intitute
– SIRIC
– Fondation pour la Recherche
Médicale
– Institut Universitaire de France
– ARC Foundation
– GSK
Novel Immunomodulatory Pathways in
Lymphomas Discovered by CALYM
50 29 25
+ PDL1-Fc + PDL2-Fc + IgG1 + HVEM-Fc
27
CFSE
0
20
40
60
80
100
none HVEM PDL1 PDL2
% d
ivis
ion
BTLA-HVEM Interaction Inhibits Vg9Vd2 T
Cell Proliferation
* * *
ITIM(V/IxYxxL/V)
ITSM (TxYxxV/I)
PD-1
Y
Y
P
d γ 2
ZAP70
ERK P
P
P
PKC P
AKT
BTLA
Y
Y
Y
Y
ITIM
ITSM
YxN
YxN
GRB2
Inhibitory function of PD-1 and BTLA
?
CD28
P
Bcl-XL
IIL-2
TCR signals
NF-B
P
P
PI3K p110
P
p85 PI3K p110 p85
P
TNFSFR14 HVEM in Lymphomas
Mutations and deletions of TNFRSF14 gene have been identified in FL
patients (Cheung et al., Cancer Research 2010, Launay et al., Leukemia 2011)
73 non synonymous mutations out of 387 patients
12 were missense mutations affecting CRD1 of which 7 affect BTLA
binding