Ph D Swati Dhar

64
Swati Dhar ntial of γδ T lymphocytes stimulated with non-pepti antigens for immunotherapy of cancer

description

Potential of gamma delta T lymphocytes stimulated with non-peptidic antigens for immunotherapy of cancer

Transcript of Ph D Swati Dhar

Page 1: Ph D Swati Dhar

Swati Dhar

Potential of γδ T lymphocytes stimulated with non-peptidic antigens for immunotherapy of cancer

Page 2: Ph D Swati Dhar

γδ T cells properties and function

Modlin & Sieling Science 2005

Page 3: Ph D Swati Dhar

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

Page 4: Ph D Swati Dhar

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

Page 5: Ph D Swati Dhar

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

Page 6: Ph D Swati Dhar

Antigens recognized by V9V2 T cells

Bromohydrin Pyrophosphate (BrHPP)

Phoshostim TM (Innate Pharma)

Intermediate of the bacterial Rohmer pathway and Mammalian Mevalonate pathway

Page 7: Ph D Swati Dhar

Russel RGG and Rogers MJ Bone 1999

Bisphosphonates

Page 8: Ph D Swati Dhar

CLASSES OF BISPHOSPHONATES

Non-nitrogen containing bisphosphonates

Nitrogen containing bisphosphonates

Page 9: Ph D Swati Dhar

Roelofs et al., Clin Can Res 2006

Mammalian Mevalonate Pathway, 7 DehydrocholesterolFarnesol

HMG-CoA Reductase

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Caraglia et al., Endocrine-related Can 2006

Pleiotropic role of Nitrogen containing Bisphosphonates in Tumor cells

Page 11: Ph D Swati Dhar

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

Page 12: Ph D Swati Dhar

Chapter 1

Understanding the immunomodulatory role of non-peptidic antigens (IPP/PAM/ZOL) on γδ T cells

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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

Page 14: Ph D Swati Dhar

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

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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

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(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

Page 17: Ph D Swati Dhar

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

Page 18: Ph D Swati Dhar

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)

Page 19: Ph D Swati Dhar

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

Page 20: Ph D Swati Dhar

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)

Page 21: Ph D Swati Dhar

Chapter 2

Dissecting the molecular pathways of activation of γδ T cells

Page 22: Ph D Swati Dhar

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

Page 23: Ph D Swati Dhar

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

Page 24: Ph D Swati Dhar

AW8507

MDA-MB-231

MCF-7

Untreated Mevastatin

Morphological changes in tumor cell lines post statin treatment

200x Magnification

Page 25: Ph D Swati Dhar

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

Page 26: Ph D Swati Dhar

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

Page 27: Ph D Swati Dhar

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

Page 28: Ph D Swati Dhar

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

Page 29: Ph D Swati Dhar

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

Page 30: Ph D Swati Dhar

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

Page 31: Ph D Swati Dhar

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

Page 32: Ph D Swati Dhar

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

Page 33: Ph D Swati Dhar

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

Page 34: Ph D Swati Dhar

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

Page 35: Ph D Swati Dhar

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

Page 36: Ph D Swati Dhar

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

Page 37: Ph D Swati Dhar

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)

Page 38: Ph D Swati Dhar

Chapter 3

Comparative analysis of γδ T cell effector functionsof oral and breast cancer patients vis-à-vis healthy individuals

Page 39: Ph D Swati Dhar

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

Page 40: Ph D Swati Dhar

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

Page 41: Ph D Swati Dhar

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

Page 42: Ph D Swati Dhar

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

Page 43: Ph D Swati Dhar

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

Page 44: Ph D Swati Dhar

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

Page 45: Ph D Swati Dhar

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

Page 46: Ph D Swati Dhar

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

Page 47: Ph D Swati Dhar

Chapter 3

Investigating the immunotherapeutic potential of non-peptidic antigen

activated T cells in tumor-bearing Nude/SCID mice

Page 48: Ph D Swati Dhar

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

Page 49: Ph D Swati Dhar

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

Page 50: Ph D Swati Dhar

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

Page 51: Ph D Swati Dhar

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

Page 52: Ph D Swati Dhar

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

Page 53: Ph D Swati Dhar

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

Page 54: Ph D Swati Dhar

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

Page 55: Ph D Swati Dhar

(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

Page 56: Ph D Swati Dhar

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

Page 57: Ph D Swati Dhar

γδ T cellOSTEOCLAST

APOPTOSISACTIVATION

IFN-γ/TNF-α ???

BISPHOSPHONATES

Bisphosphonates and interplay of T cells with bone

Page 58: Ph D Swati Dhar

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)

Page 59: Ph D Swati Dhar

T CELLS IN BONE METABOLISM

Walsh et al Ann Rev Imm 2006

Page 60: Ph D Swati Dhar

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

Page 61: Ph D Swati Dhar

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

Page 62: Ph D Swati Dhar

(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

Page 63: Ph D Swati Dhar

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

Page 64: Ph D Swati Dhar

THANK YOU