Ph D Swati Dhar
-
Upload
swati-dhar -
Category
Technology
-
view
1.450 -
download
2
description
Transcript of Ph D Swati Dhar
Swati Dhar
Potential of γδ T lymphocytes stimulated with non-peptidic antigens for immunotherapy of cancer
γδ T cells properties and function
Modlin & Sieling Science 2005
γδ T cells
No of genes
Pairing with other family
αβ T cells
No of genes
Vδ 8
(Vδ1,2,3)(Vδ 6,7,8,14)
Vβ 47
(24 families)
Vγ 11 VγI-Vγ2, Vγ3, Vγ4, Vγ5, Vγ8
(pairs with Vδ1)
VγII- Vγ9
(pairs with Vδ2)
VγIII- Vγ10
VγIV- Vγ11
Vα 42
(29 Families)
Gene usage of T cells and αβ T cells
V9V2 T cells
Dominant subset constituting 95% of the total peripheral blood population
Antigen recognition independent of MHC I or II involvement
Recognize small molecular weight phosphorylated compounds, nonpeptide antigens
Expanded in diseases like tuberculosis, tularemia, listeriosis, malaria and even in HIV infection
Professional antigen presentation upon activation by non-peptidic antigens
(Brandes et al Science 2005)
Anti-tumor activity against lymphomas and solid tumors via perforin, granzyme or the Fas-FasL pathway
Casseti et al Cellular and Mol Imm 2008
1. Human γδ T cells recognize heat shock protein-60 on oral tumor cells Laad et al. Int J Can 1999
2. Involvement of CD166 in the activation of human γδ T cells by tumor cells sensitized with nonpeptide antigensKato et al. J Immunol 2006
Vγ9Vδ2 T cells: Antigens recognized and role of co stimulatory receptors
Antigens recognized by V9V2 T cells
Bromohydrin Pyrophosphate (BrHPP)
Phoshostim TM (Innate Pharma)
Intermediate of the bacterial Rohmer pathway and Mammalian Mevalonate pathway
Russel RGG and Rogers MJ Bone 1999
Bisphosphonates
CLASSES OF BISPHOSPHONATES
Non-nitrogen containing bisphosphonates
Nitrogen containing bisphosphonates
Roelofs et al., Clin Can Res 2006
Mammalian Mevalonate Pathway, 7 DehydrocholesterolFarnesol
HMG-CoA Reductase
Caraglia et al., Endocrine-related Can 2006
Pleiotropic role of Nitrogen containing Bisphosphonates in Tumor cells
Aims and Objectives
1. Understanding the immunomodulatory role of non-peptidic antigens
on T cells
2. Dissecting the molecular pathways of activation of T cells
3. Comparative analysis of γδ T cell effector functions from oral and breastcancer patients vis-à-vis healthy individuals
4. Investigating the immunotherapeutic potential of non-peptidic antigen activated T cells in tumor-bearing Nude/SCID mice
5. Evaluating the role of activated Vγ9Vδ2 T cells in osteoclastogenesis
Chapter 1
Understanding the immunomodulatory role of non-peptidic antigens (IPP/PAM/ZOL) on γδ T cells
Protocol for purification of γδ T cells
Isolation of Peripheral Blood Mononuclear Cells (PBMC) on Ficoll-Hypaque density gradient
Anti- CD3 (OKT 3) ascites coated flasks are seeded with 10x106 PBMC in 10 ml of cRPMI 1640 with 10% human AB serum
and rIL-2 (100IU/ml)
Cultured for 5 days
Cultures transferred to 75cm2 flaskswith fresh medium and rIL-2 and expanded for
12-16 days
Magnetic Cell Sorting (MACS) to purify γδ T cells
Activated
PBMC
Purified γδ
T cells
% Yield
of γδ
T cells
Healthy
Donors60-100x106 25-30x106 20-30
Oral cancer
patients30-40x106 5-10x106 15-25
Breast cancer
patients
50-80x106 10-25x106 20-30
Figures indicate representative data forHealthy Donors n=75Oral Cancer patients n=10Breast cancer patients n=20
Phenotype of MACS purified γδ T cells
CD3 FITC
Vγ9
PE
Vδ2
PE
Vδ1 FITC
100x 200x
Morphology of a T cell line
CD
3 P
E
90 95 10
Negative Fraction Positive FractionWashed fraction
0.5% 1% 98%
γδ FITC
3 HT
dR
inco
rpor
atio
n (
cpm
)
Antigen (µM)
Induction of proliferation by IPP/PAM/ZOL in Vγ9Vδ2 T cells
(A) IPP
05000
1000015000200002500030000350004000045000
40 20 10 5
Gd only
Gd+IPP
Gd+IL-2
Gd+IPP+IL-2
Gd+IL-15
Gd+IPP+IL-15
* @@ @
0
10000
20000
30000
40000
50000
60000
100 50 25 12.5
Gd only
Gd+PAM
Gd+IL-2
Gd+PAM+IL-2
Gd+IL-15
Gd+PAM+IL-15
* @@
@
(C) Zoledronate
0
10000
20000
30000
40000
50000
60000
100 50 25 12.5
Gd only
Gd+ZOL
Gd+IL-2
Gd+ZOL+IL-2
Gd+IL-15
Gd+ZOL+IL-15
* @@ @
(B) Pamidronate
* p<0.02, @ p<0.05
Data is mean of three independent experiments from γδ T cells of healthy individuals
(A) IPP
0
200
400
600
800
1000
40 20 10 5
Gd only
Gd+IPP
Gd+IL-2
Gd+IPP+IL-2
Gd+IL-15
Gd+IPP+IL-15
***
*
Induction of cytokine release by IPP/PAM/ZOL in Vγ9Vδ2 T cells
0
200
400
600
800
1000
100 50 25 12.5
Gd only
Gd+PAM
Gd+IL-2
Gd+PAM+IL-2
Gd+IL-15
Gd+PAM+IL-15
* ** *
0
200
400
600
800
1000
1200
1400
100 50 25 12.5
Gd only
Gd+ZOL
Gd+IL-2
Gd+ZOL+IL-2
Gd+IL-15
Gd+ZOL+IL-15
*
***
IFN
-γ (
pg/
ml)
(B) Pamidronate
(C) Zoledronate
Antigen (µM)* p<0.03
Data is mean of three independent experiments from γδ T cells of healthy individuals
CD25 PE
CD69-PE
CD161-PE
Unstimulated Pamidronate Zoledronate
HLA DR-PE
CD45RO-PE
45 78 80
39 65 70
36 47 49
62 57
62 58
Immunophenotype of Vγ9Vδ2 T cells stimulated with IPP, Pamidronate and Zoledronate
Vγ9 FITC
IPP
89 77
86 81
59
60
45
Representative data for experiments performed with γδ T cells from 3 independent healthy donors
Figures in the plots represent percent positive cells
0
100
200
300
400
500
0 10 20 30 40 60 90 120 150 180 210 240 270
0
100
200
300
400
500
600
700
0 10 20 30 40 60 90 120 150 180 210 240 270
0
50
100
150
200
0 20 40 90 150 210 270
Cal
ciu
m r
elea
se (
in n
M)
Time in sec
γδ (IPP) γδ (Pamidronate)
γδ (Zoledronate) αβ (anti-CD3 mAb)
Intracellular calcium release in Vγ9Vδ2 T cellsstimulated with IPP,
Pamidronate and Zoledronate
0
100
200
300
400
500
600
700
0 10 20 30 45 60 90 120 150 180 210 240 270
Antigen (IPP/PAM/ZOL) stimulated
Untreated
Representative data of 3 independent experiments from γδ T cells of healthy individuals
Point of addition of stimulus (1% Phytohemaglutinin)
p-p38
pAkt
p38
55KDa
46KDapJNK2
pJNK1
pErk 1
pErk 2
44KDa
42KDa
JNK1/2
ERK2
60KDa
Akt
Untreated IPP PAM ZOL
Ph
osp
ho-
pro
tein
/tot
al p
rote
in (
AU
)
A
B
Signaling intermediates in Vγ9Vδ2 T cells stimulated with IPP, Pamidronate and Zoledronate
0
2
4
6
8
Unst IPP PAM ZOL
0
2
4
6
8
10
12
Unst IPP PAM ZOL
0
1
2
3
4
5
Unst IPP PAM ZOL
00.20.40.60.8
11.21.41.61.8
Unst IPP PAM ZOL
38KDa
Representative data from experiments performed with γδ T cells from 2 healthy individuals
Summary
IPP/PAM/ZOL induce proliferation and IFN-γ release in γδ T cells which is increased in combination with rIL-2
Bisphosphonates upregulate activation markers CD25, CD69 and the NK marker CD161
Calcium flux shows a biphasic model of response distinct from the αβ T cells which could emphasize on the different mode of antigen recognition by Vγ9Vδ2 T cells
Bisphosphonates recruit MAPK intermediates and the PKB/Akt pathway for inducing proliferation, differentiation and survival Vγ9Vδ2 T cells
Immunomodulatory effects of aminobisphosphonates on Vγ9Vδ2 T cells
(Manuscript communicated)
Chapter 2
Dissecting the molecular pathways of activation of γδ T cells
Acetyl CoA+ Acetoacetyl CoA
HMG CoA
Mevalonate
Mevalonate pyrophosphate
HMG CoA Reductase
Geranyl pyrophosphate
Farnesyl pyrophosphate
Mevalonate pathway
Cholesterol , ubiquinone, vitamins
Isopentenyl pyrophosphate
FPP synthaseBisphosphonates Pamidronate Zoledronate
Mevastatin
7 DHC
Farnesol
Daudi
0
50
100
150
200
250
50 25
MCF-7 MDA-MB-231 PC-3
0
100
200
300
400
500
50 25
0
100
200300
400
500
600
700800
900
1000
50 25
MDA-MB-231
IFN
-γ (
pg/
ml)
TN
F-α
(p
g/m
l)
IFN
-γ (
pg/
ml)
Mevastatin (µM)
Farnesol (µM) 7 DHC (µg)
TN
F-α
(p
g/m
l)
IFN
-γ (
pg/
ml)
Activation of Vγ9Vδ2 T cells by tumor cells is abrogated by mevastatin
**
0
200
400
600
800
1000
1200
1400
50 25
*
* *
0
20
40
60
80
100
50 25
****
** *
0
200
400
600
800
1000
200 100
**
0
200
400
600
800
1000
50 25
**
0
20
40
60
80
100
200 100
***
Untreated tumor cells
Mevastatin/Farnesol/7-DHC treated tumor cells
** p<0.03, * p<0.05
Data is mean of 4 independent experiments performedwith each cell line
AW8507
MDA-MB-231
MCF-7
Untreated Mevastatin
Morphological changes in tumor cell lines post statin treatment
200x Magnification
Mevastatin increases expression of HMG-CoA Reductase in tumor cells
AW13516
HMG-CoA Reductase FITC
MCF-7AW 8507
HMG-CoAR
747 bp
GAPDH 305bp
1 2 3 4 5 6 7 8
(C)
(A) (B)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2AW13516
AW13516 (mev 50)
AW8507
AW8507 (mev 50)
MCF-7
MCF-7 (mev 25)
HM
G-C
oAR
/GA
PD
H
GAR FITC
ISOCONTROL
Representative data from 2 independent experiments
Cyt
okin
e (p
g/m
l)
IFN-γ
TNF-α
Vγ9Vδ2 T cells in co culture with tumor cells release higher IFN-γ and TNF-α as compared to normal cells
*
*
*
#
0100200300400500600700800900
0
50
100
150
200
250
**A)
B)
@
@
Data is mean of 4 independent experiments
* p<0.003, # p<0.02, p<0.05
γδ T cell
TUMOR
TUMOR
Perforin, Granzyme
NKG2D
Cytolytic mechanism of Vγ9Vδ2 T cells
LFA-1 ICAM-1
TCR
MICA
NKG2D- Natural Killer Group 2DMICA-MHC Class I A, LFA-1 CD11a Leucocyte Function Antigen
ICAM (CD54)- Intercellular Cell Adhesion Molecule , TCR-T cell Receptor
Tumor cell lysis
51 Cr ASSAY
Tumor cells treated withPAM/ZOL (16-18 hours)
Targets are washed extensively
Labeled with 51Cr, at 370C 1 hour
Targets are washed and co-cultured with Vγ9Vδ2 T cells
for 4 hours
Supernatants are collectedand counted on a gamma counter
PC-3MCF-7
SaOS-2
% S
pec
ific
lysi
s
Effector:Target
0
10
20
30
40
50
60
70
30:1 15:1 7.5 : 1 3.75 : 1 1.8 : 1
*
#
0
20
40
60
80
100
30:1 15:1 7.5 : 1 3.75 : 1 1.8 : 1
@
0
1020
3040
5060
7080
90
30:1 15:1 7.5:1 3.75:1 1.8:1
*
#
Tumor cells treated with Pamidronate and Zoledronate aresusceptible to lysis mediated by Vγ9Vδ2 T cells
A) B)
C)
Untreated
Pamidronate (100µM)
Zoledronate (100µM)
* p<0.0001, @ p<0.005, # p<0.002 Data is mean of 3 independent experiments with each cell line
0
5
10
15
20
25
30
35
30:1 15:1 7.5:1 3.7:1 1.8:1
% S
pec
ific
lysi
s
Effector:Target
Fresh breast tumor cells
Normal breastcells
Differential susceptibility of tumor cells treated with Pamidronate and Zoledronate and lysis of fresh breast and normal breast tumor cells by Vγ9Vδ2 T cells
(B)
(A)
0
10
20
30
40
50
60
70
Untreated Pamidronate Zoledronate
THP-1
AW 8507
MDA-MB-231%
Sp
ecif
ic ly
sis
E:T 30:1
*
Cytotoxicity of Vγ9Vδ2 T cells from healthy donors against breast tumor and normal cells
*p<0.03
Data is mean of 2 independent experiments with each cell line
Data is mean of 5 independent experiments with breast tumor cells and 3 independent experiments with normal breast cells
A) MCF-7
B) MDA-MB-231
ZoledronateUntreated
C) PC-3
Cell cycle arrest induced by Pamidronate and Zoledronate in tumor cell lines
Pamidronate
G0-G1 32%
S53.98%
G2-M 13.96%
G0-G1 49.91%
S 50.09%
G2-M 0%
G0-G1 17.61%
S81.12%
G2-M 1.18%
G0-G1 50.16%
S21.29%
G2-M 28.56%
G0-G1 51.85%
S22.82%
G2-M 25.33%
G0-G1 51.15%
S20.24%
G2-M 28.61%
G0-G129.92%
S52.98%
G2-M17.1%
G0-G133.83%
S66.17%
G2-M0%
G0-G122.13%
S66.23%
G2-M10.64%
Representative data of 2 independent experiments with each cell line
MCF-7
MICA FITCMHC I FITC
Isotype control
MCF untreated
MCF (Pamidronate)
MCF (Zoledronate)
Analysis for expression of molecules involved in γδ T cell tumor cell interactions by flow cytometry
(B)(A)HeLa
ICAM-1 FITC
(C)THP-1 MCF-7
FasL PE
(D)Jurkat MCF-7
MCF-7
Vγ9Vδ2 T cells
NKG2D FITC
Isotype control
NKG2D FITC
(E)
MICAMHC I
ICAM-I FasL
MICA
ICAM-I FasL
Granzyme B PE
Granzyme B PE
Perforin PE
Perforin PE
Data is representative of 3 independent experiments
MCF-7 (Pamidronate)
% S
pec
ific
lysi
s
@
*
Involvement of γδ TCR and NKG2D in modulating cytotoxicity of Vγ9Vδ2 T cells against
Pamidronate and Zoledronate treated MCF-7
(A)
(B) MCF-7 (Zoledronate)
0
5
10
15
20
25
30
35
40
45 (Unt)gd+MCF-7(unt)
(Unt)Gd+MCF-7(PAM)
(Isotype IgG) Gd+MCF-7(PAM)
(anti gd) Gd+MCF-7(PAM)
(anti Vg9)Gd+MCF-7(PAM)
(anti NKG2D)Gd+MCF-7(PAM)
(anti gd+anti NKG2D) Gd+MCF-7(PAM)
(anti Vg9+anti NKG2D) Gd+MCF-7(PAM)
@
0
10
20
30
40
50
60
70(Unt)gd+MCF-7(unt)
(Unt)Gd+MCF-7(ZOL)
(Isotype IgG) Gd+MCF-7(ZOL)
(anti gd) Gd+MCF-7(ZOL)
(anti Vg9)Gd+MCF-7(ZOL)
(anti NKG2D)Gd+MCF-7(ZOL)
(anti gd+anti NKG2D) Gd+MCF-7(ZOL)
(anti Vg9+anti NKG2D) Gd+MCF-7(ZOL)
*
@@@ p<0.003
* p<0.002
Data is mean of 2 independent experiments
MCF-7 (Zoledronate)
MCF-7 (Pamidronate)%
Sp
ecif
ic ly
sis
0
5
10
15
20
25
30
35
40(unt gd)+MCF-7 (unt)
(unt gd)+MCF-7 (PAM)
(Concana A gd)+MCF-7 (PAM)
0
10
20
30
40
50
60
(unt gd)+MCF-7 (unt)
(unt gd)+MCF-7 (ZOL)
(Concana A gd)+MCF-7 (ZOL)
% S
pec
ific
lysi
s
#
#
Cytotoxicity of Pamidronate and Zoledronatetreated MCF-7 mediated by Vγ9Vδ2 T cells
involves the perforin granzyme pathway
(A)
(B)
*
*
* p<0.001, # p<0.05Data is mean of 2 independent experiments
a
c
b
d
Time lapse video microscopy of untreated MCF-7 in co culture with Vγ9Vδ2 T cells
Yellow arrows - Untreated tumor cells
Black arrows - γδ T cells
Figures representative of 4 independent experiments
a b c d
e
f
g hf
Time lapse video microscopy of Pamidronate treated MCF-7 in co culture with Vγ9Vδ2 T cells
Yellow arrows - Pamidronate treated tumor cells
Black arrows - γδ T cell
Figures representative of 4 independent experiments
Time lapse video microscopy of Zoledronate treated MCF-7 in co culture with Vγ9Vδ2 T cells
a b c d
e f g h
Yellow arrows - Zoledronate treated tumor cells
Black arrows - γδ T cells
Figures representative of 4 independent experiments
Summary
1. Deregulated mevalonate pathway in tumor cells generates elevated phosphorylated metabolites like IPP that can act as a danger signal and mediate recognition by Vγ9Vδ2 T cells
2. Tumor cells harbor higher levels of phosphorylated metabolites vis-à-vis normal cells and IPP can act as an endogenous tumor-associated antigen
3. Aminobisphosphonates (PAM and ZOL) sensitize tumor cells of different origin to lysis by Vγ9Vδ2 T cells efficiently enhancing endogenous IPP accumulation in the mevalonate pathway
4. Mechanism of augmented Vγ9Vδ2 T cells lysis of PAM/ZOL treated targets depends on the TCR and partly on the engagement of NKG2D receptor
5. Vγ9Vδ2 T cells form strong conjugates with PAM treated tumor cells and lyse targets as against untreated tumor cells
Lysis of aminobisphosphonate sensitized breast tumor cells by Vγ9Vδ2 T cells
(Manuscript communicated Cancer Immunology Immunotherapy)
Chapter 3
Comparative analysis of γδ T cell effector functionsof oral and breast cancer patients vis-à-vis healthy individuals
3 HT
dR
inco
rpor
atio
n (
cou
nts
per
min
ute
)
Proliferative response of PBMC from Healthy Individuals, Breast cancer and Oral cancer patients in response to IPP, pamidronate and zoledronate in the presence of rIL-2 and rIL-15
0
5000
10000
15000
20000
25000
30000
35000
HealthyIndividuals
Breast cancerpatients
Oral cancerpatients
PBMC
PBMC+IPP
PBMC+IL-2
PBMC+IPP+IL-2
PBMC+IL-15
PBMC+IPP+IL-15
0
5000
10000
15000
20000
25000
HealthyIndividuals
Breast cancerpatients
Oral cancerpatients
PBMC
PBMC+ZOL
PBMC+IL-2
PBMC+ZOL+IL-2
PBMC+IL-15
PBMC+ZOL+IL-15
(A) IPP
(B) Pamidronate
(C) Zoledronate
**n=10 n=13
n=8
n=10
n=13
n=8
n=10n=13
n=8
#
#
0
5000
10000
15000
20000
25000
HealthyIndividuals
Breast cancerpatients
Oral cancerpatients
PBMC
PBMC+PAM
PBMC+IL-2
PBMC+PAM+IL-2
PBMC+IL-15
PBMC+PAM+IL-15
@
@
@
ns
ns
ns
@ p<0.002
* p<0.03
# p<0.05
ns not significant
Selective expansion of Vγ9Vδ2 T cells from PBMC of healthy individuals stimulated with IPP, Pamidronate and Zoledronate in the presence of rIL-2
Iso control
Vγ9
PE
Vδ2
PE
CD
45R
O P
E
CD3 FITC Vδ2 FITC
33 32 12
41 42 53
65 68 52
41 50 52
(A)
(B)
(C)
(D)
Representative data of 3 independent experiments
Selective expansion of Vγ9Vδ2 T cells from PBMC of breast cancer patients stimulated with IPP,
Pamidronate and Zoledronate in the presence of rIL-2
Iso control
Vγ9
PE
Vδ2
PE
CD
45R
O P
E
CD3 FITC Vδ2 FITC
13 15 13
28 25 22
2223 21
25 23 20
(A)
(B)
(C)
(D)
Representative data of 2 independent experiments
Selective expansion of Vγ9Vδ2 T cells from PBMC of oral cancer patients stimulated with IPP, Pamidronate and Zoledronate in the presence of rIL-2
Vγ9
PE
Vδ2
PE
CD
45R
O P
E
CD3 FITC Vδ2 FITC
Iso control
6 10 69
32 34 69
30 37 67
37 32 79
(A)
(B)
(C)
(D)
Representative data of 3 independent experiments
Groups
PBMC seeded on Day 1
Ex-vivo expanded PBMC on Day 12
MACS Purified γδ
T cells
Yield
of γδ
T cells
% (range)
Healthy
Donors (n=85)
10-12x106 60-100x106 25-30x106 20-30
Breast cancer
Patients (n=20)
10-12x106 50-80x106 10-25x106 20-30
Oral cancer
Patients (n=20)
10-12x106 30-40x106 5-10x106 15-25
Calcium release from Vγ9Vδ2 T cells of breast cancer patients stimulated with IPP, Pamidronate and Zoledronate
0
200
400
600
800
1000
1200
0 10 20 30 40 60 90 120 150 180 210 240 270 0
200
400
600
800
1000
1200
1400
0 10 20 30 40 60 90 120 150 180 210 240 270
0
200
400
600
800
1000
1200
1400
0 10 20 30 40 60 90 120 150 180 210 240 270
0
20
40
60
80
100
120
140
0 10 20 30 40 60 90 120 150 180 210 240 270
Cal
ciu
m r
elea
se (
in n
M)
Time in sec
γδ (IPP) γδ (Pamidronate)
γδ (Zoledronate) αβ (anti-CD3)
IPP/PAM/ZOL treated
Untreated
Point of addition of stimulus (1% Phytohemaglutinin)
Representative data of 2 independent experiments
Calcium release from Vγ9Vδ2 T cells of oral cancer patients stimulated with IPP, Pamidronate and Zoledronate
0
200
400
600
800
1000
1200
1400
1600
0 10 20 30 40 60 90 120 150 180 210 240 270
0
50
100
150
200
250
300
350
0 10 20 30 40 60 90 120 150 180 210 240 270
Cal
ciu
m r
elea
se
(in
nM
)
Time in sec
γδ (IPP) γδ (Pamidronate)
γδ (Zoledronate)
αβ (anti-CD3)
IPP/PAM/ZOL treated
Untreated
0
100
200
300
400
500
600
700
800
900
0 10 20 30 40 60 90 120 150 180 210 240 270
0
100
200
300
400
500
600
700
800
900
1000
0 10 20 30 40 60 90 120 150 180 210 240 270
Point of addition of stimulus (1% Phytohemaglutinin)
Representative data of 3 independent experiments
0
10
20
30
40
30:1 15:1 7.5:1 3.7:1 1.8:1
0
20
40
60
80
100
30:1 15:1 7.5:1 3.7:1 1.8:1
Untreated
Pamidronate (100µM)
Zoledronate (100µM)
Oral cancer patients
MCF-7
Breast cancer patientsPC-3
A
B
% S
pec
ific
lysi
s
Comparative analysis of cytotoxicity of Vγ9Vδ2 T cells from oral and breast cancer patients
against aminobisphosphonate treated tumor cells
#
Effector:Target
0
10
20
30
40
50
60
70
30:1 15:1 7.5 : 1 3.75 : 1 1.8 : 1
*
#
0
20
40
60
80
100
30:1 15:1 7.5 : 1 3.75 : 1 1.8 : 1
@
Healthy individuals
Healthy individuals
* p<0.0001
@ p<0.005
# p<0.002Data is mean of 5 independent experiments for healthy donors, oral and breast cancer patients
Chapter 3
Investigating the immunotherapeutic potential of non-peptidic antigen
activated T cells in tumor-bearing Nude/SCID mice
Immunotherapy protocol
Detection of Vγ9Vδ2 T cells byFlow cytometry
TumorApoptosis by Annexin V/PI stainingApoptosis by TUNEL stainingExpression of Bax
Spleen
Nude mice with MDA-MB-231 xenografts
(A)2x106 MDA-MB-231 injected at mammary fat pad
Day 7Tumorappearance
15 days
Day 21
Day 22 (Group 1-4)Animal sacrifice
Animal Groups and treatment Treatment Schedule
Control (Group 1) None
rIL-2 (Group 2) 200IU/ animal i.p
Vγ9Vδ2 T cells+ rIL-2 (Group 3) 10x106 Vγ9Vδ2 T cells /mouse i.v and rIL-2 200IU/mouse i.p
Vγ9Vδ2 T cells +Zoledronate+ rIL-2
(Group 4)
10x106 Vγ9Vδ2 T cells /mouse i.v, 100µg Zoledronate/mouse i.p and rIL-2 200IU/mouse i.p
(NIH III female nude mice 6-10 weeks of age, n=12, Treatment schedule used in 2 independent experiments)
Group 1Control
Group 2 IL-2
Group 3 γδ T cells+ IL-2
Group 4γδ T cells+ IL-2+Zoledronate
24 hours later
(B)
Treatment
Untreated (Group 1)
IL-2(Group 2)
γδ T cells+IL-2(Group 3)
γδ T cells+ZOL+IL-2(Group 4)
Hematoxylin staining of tumor tissue sections
Untreated(Group 1)
γδ T cells+ZOL+IL-2(Group 4)
M1
M2
1212M1
M2
6 7M1
M2
33
γδ PE Vγ9 PE Vδ2 PE
Homing of Vγ9Vδ2 T cell to spleen of tumor xenograft mice
M1
M2
2 M1
M2
0.36 M1
M2
0.23
γδ PE Vγ9 PE Vδ2 PE
Data is mean of 2 independent experiments
16 34 36
UntreatedGroup 1
γδ T cells +IL-2Group 3
γδ T cells + ZOL+IL-2Group 4
Annexin V-FITC
PI
Adoptive transfer of Vγ9Vδ2 T cells induces apoptosis in tumors
Data is mean of 2 independent experiments
Untreated (Group 1)Negative control
TACS nuclease positive control
TUNEL staining for apoptosis detection in tumor sections
Test
Data representative of 5 independent fields studied
Brown staining indicative of apoptotic nuclei
γδ T cells +Zoledronate+IL-2 (Group 4)Negative control
TACS nuclease positive control
TUNEL staining for apoptosis detection in tumor sections
Test
Data representative of 5 independent fields studied
Brown staining indicative of apoptotic nuclei
IL-2 (Group 2)
γδ T cells +IL-2 (Group 3)
TUNEL staining for apoptosis detection in tumor sections
Animal groups APOPTOTIC
INDEX
Tumor alone 2
Tumor+IL-2 12
Tumor+ γδ+IL-2 38*
Tumor+ γδ+ZOL+IL-2 45*
Data representative of 5 independent fields studied
Brown staining indicative of apoptotic nuclei
* p<0.05
(B) Vγ9Vδ2 T cells +ZOL+IL-2 (Group 4)
(C) Untreated (Group 1)
(A) Negative control
Immunohistochemical analysis for expression of Bax in tumor sections
Summary
1. In an in-vivo model of breast tumor, Vγ9Vδ2 T cells are able to recognize and induce apoptosis in xenografted tumor
2. Treatment modality involving ZOL and IL-2 was more efficient than protocol involving only T cells and rIL-2
γδ T cellOSTEOCLAST
APOPTOSISACTIVATION
IFN-γ/TNF-α ???
BISPHOSPHONATES
Bisphosphonates and interplay of T cells with bone
Common players of the immune system and bone biology
RANKL (Receptor Activator of NF-κB-Ligand , TRANCE TNF-related Activation-induced Cytokine)
Expressed on osteoblasts and activated T cells
Induces osteoclastogenesis upon interaction with cognate receptor RANK
IFN- , TNF-α, IL-1, IL-11, IL-17
Bone related maladies
Reduced bone mineral densities in
1. Adult and childhood Leukemia (Oliveri et al 1991)
2. Chronic infections like Hepatitis C and HIV (Stellon et al 1989)
3. Autoimmune diseases Diabetes Mellitus, Rheumatoid arthritis and SLE (Seitz et al 1985)
4. Metastasis related cancers of breast and prostate origin (Coleman et al 1998)
T CELLS IN BONE METABOLISM
Walsh et al Ann Rev Imm 2006
Cyt
okin
e (p
g/m
l)
IFN-γ
TNF-α
Cytokine profile of activated Vγ9Vδ2 T cells
050
100150200250300350400450
Gd only Gd+antiCD 3
Gd+IPP Gd+PAM Gd+ZOL
0102030405060708090
100
Gd only Gd+antiCD 3
Gd+IPP Gd+PAM Gd+ZOL
#
#
A)
B)
Samples OD1 OD2 Mean Mean-blank sRANKL (pg/ml)
γδ only 0.223 0.233 0.228 0.025 0.08
γδ +anti CD3 0.223 0.21 0.2165 0.0135 0.019
γδ +IPP 0.231 0.209 0.22 0.017 0.034
γδ +PAM 0.232 0.208 0.22 0.017 0.034
γδ +ZOL 0.221 0.22 0.2205 0.0175 0.036
Blank 0.203 0.203 0.203 0
Estimation of sRANKL in supernatants of V9V2 T cells stimulated with anti CD3, IPP, Pamidronate and Zoledronate
# p<0.03
Data is mean of 2 independent experiments
1 2 3 4 5 6 7
RANKL mRNA expression in Vγ9Vδ2 T cells
(A) (B)
RANKL 464 bp
GAPDH305 bp
1 2 3 4 5 6 7 (C)
RA
NK
L/G
AP
DH
(A
rbit
rary
Un
its)
0
0.1
0.2
0.3
0.4
0.5
0.6Unstimulated gd
Gd+anti CD3
Gd+IPP
Gd+Pamidronate
Gd+Zoledronate
RANKL expression in Vγ9Vδ2 T cells stimulated with non-peptide antigens
RANKL FITC
Unstimulated IPPanti CD3
Pamidronate Zoledronate
αβ T cells
(A) M-CSF+RANKL (Positive control) (B) Untreated Vγ9Vδ2 T cell supernatant
Tartrate Resistant Acid Phosphatase (TRAP) staining of mouse osteoclast precursors treated with supernatants from untreated
Vγ9Vδ2 T cells
(C) γδ + anti CD3 (D) γδ +IPP
(E) γδ +Pamidronate (F) γδ +Zoledronate
Black arrow indicates TRAP positive (red) multinucleated osteoclasts
Publications:
γδ T cells in cancer immunotherapy: current status and future prospectsChiplunkar SV, Dhar S, Wesch,D and Kabelitz, DImmunotherapy (2009) 1(4):663-678
Published a chapter entitled ‘Potential of γδ T cells in immunotherapy of cancer’ S.V.Chiplunkar*, N Atre, S. Dhar and K.A.Pathak Immunotherapeutics and disease management, Twelfth annual symposium Ranbaxy Science Foundation, New Delhi, November 2005
THANK YOU