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

1. Swati Dhar Potential of γδ T lymphocytes stimulated with non-peptidic antigens for immunotherapy of cancer

2. γδ T cells properties and function Modlin & Sieling Science 2005

3. Gene usage of  T cells and αβ T cells 42 (29 Families) 11 V γ V δ γδ T cells V α V γ I-V γ 2, V γ 3, V γ 4, V γ 5, V γ 8 (pairs with V δ 1) V γ II- V γ 9 (pairs with V δ 2) V γ III- V γ 10 V γ IV- V γ 11 47 (24 families) V β (V δ 6,7,8,14) 8 (V δ 1,2,3) No of genes αβ T cells Pairing with other family No of genes

6. Antigens recognized by V  9V  2 T cells Bromohydrin Pyrophosphate (BrHPP) Phoshostim TM (Innate Pharma) Intermediate of the bacterial Rohmer pathway and Mammalian Mevalonate pathway

7. Russel RGG and Rogers MJ Bone 1999 Bisphosphonates

8. CLASSES OF BISPHOSPHONATES Non-nitrogen containing bisphosphonates Nitrogen containing bisphosphonates

9. Roelofs et al., Clin Can Res 2006 Mammalian Mevalonate Pathway , 7 Dehydrocholesterol Farnesol HMG-CoA Reductase

10. Caraglia et al., Endocrine-related Can 2006 Pleiotropic role of Nitrogen containing Bisphosphonates in Tumor cells

12. Chapter 1 Understanding the immunomodulatory role of non-peptidic antigens (IPP/PAM/ZOL) on γδ T cells

13. 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 10x10 6 PBMC in 10 ml of cRPMI 1640 with 10% human AB serum and rIL-2 (100IU/ml) Cultured for 5 days Cultures transferred to 75cm 2 flasks with fresh medium and rIL-2 and expanded for 12-16 days Magnetic Cell Sorting (MACS) to purify γδ T cells Figures indicate representative data for Healthy Donors n=75 Oral Cancer patients n=10 Breast cancer patients n=20 20-30 10-25x10 6 50-80x10 6 Breast cancer patients 15-25 5-10x10 6 30-40x10 6 Oral cancer patients 20-30 25-30x10 6 60-100x10 6 Healthy Donors % Yield of γδ T cells Purified γδ T cells Activated PBMC

14. Phenotype of MACS purified γδ T cells CD3 FITC V γ 9 PE V δ 2 PE V δ 1 FITC 100x 200x Morphology of a  T cell line CD3 PE 90 95 10 Negative Fraction Positive Fraction Washed fraction 0.5% 1% 98% γδ FITC

15. 3 HTdR incorporation (cpm) Antigen ( µM) Induction of proliferation by IPP/PAM/ZOL in V γ 9V δ 2 T cells (A) IPP * @ @ @ * @ @ @ (C) Zoledronate * @ @ @ (B) Pamidronate * p<0.02, @ p<0.05 Data is mean of three independent experiments from γδ T cells of healthy individuals

16. (A) IPP * * * * Induction of cytokine release by IPP/PAM/ZOL in V γ 9V δ 2 T cells * * * * * * * * IFN- γ (pg/ml) Antigens ( µM) (B) Pamidronate (C) Zoledronate Antigen ( µM) * p<0.03 Data is mean of three independent experiments from γδ T cells of healthy individuals

17. 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 V γ 9V δ 2 T cells stimulated with IPP, Pamidronate and Zoledronate V γ 9 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

18. Calcium release (in nM) Time in sec γδ (IPP) γ δ (Pamidronate) γ δ (Zoledronate) α β (anti-CD3 mAb ) Intracellular calcium release in V γ 9V δ 2 T cells stimulated with IPP, Pamidronate and Zoledronate 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)

19. p-p38 pAkt p38 55KDa 46KDa pJNK2 pJNK1 pErk 1 pErk 2 44KDa 42KDa JNK1/2 ERK2 60KDa Akt Untreated IPP PAM ZOL Phospho-protein /total protein (AU) A B Signaling intermediates in V γ 9V δ 2 T cells stimulated with IPP, Pamidronate and Zoledronate 38KDa Representative data from experiments performed with γδ T cells from 2 healthy individuals

21. Chapter 2 Dissecting the molecular pathways of activation of γδ T cells

22. Acetyl CoA+ Acetoacetyl CoA HMG CoA Mevalonate Mevalonate pyrophosphate HMG CoA Reductase Geranyl pyrophosphate Farnesyl pyrophosphate Mevalonate pathway Cholesterol , ubiquinone, vitamins Isopentenyl pyrophosphate FPP synthase Bisphosphonates Pamidronate Zoledronate Mevastatin 7 DHC Farnesol

23. Daudi MCF-7 MDA-MB-231 PC-3 MDA-MB-231 IFN- γ (pg/ml) TNF- α (pg/ml) IFN- γ (pg/ml) Mevastatin ( µM) Farnesol (µM) 7 DHC ( µg) TNF- α (pg/ml) IFN- γ (pg/ml) Activation of V γ 9V δ 2 T cells by tumor cells is abrogated by mevastatin ** * * * * * ** * Untreated tumor cells Mevastatin/Farnesol/7-DHC treated tumor cells ** p<0.03, * p<0.05 Data is mean of 4 independent experiments performed with each cell line * ** ** ** **

24. AW8507 MDA-MB-231 MCF-7 Untreated Mevastatin Morphological changes in tumor cell lines post statin treatment 200x Magnification

25. Mevastatin increases expression of HMG-CoA Reductase in tumor cells AW13516 HMG-CoA Reductase FITC MCF-7 AW 8507 HMG-CoAR 747 bp GAPDH 305bp 1 2 3 4 5 6 7 8 (C) (A) (B) HMG-CoAR/GAPDH GAR FITC ISOCONTROL Representative data from 2 independent experiments

26. Cytokine (pg/ml) IFN- γ TNF- α V γ 9V δ 2 T cells in co culture with tumor cells release higher IFN- γ and TNF- α as compared to normal cells * * * # * * A) B) @ @ Data is mean of 4 independent experiments * p<0.003, # p<0.02, p<0.05

27. γ δ T cell TUMOR Perforin, Granzyme NKG2D Cytolytic mechanism of V γ 9V δ 2 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 with PAM/ZOL (16-18 hours) Targets are washed extensively Labeled with 51 Cr, at 37 0 C 1 hour Targets are washed and co-cultured with V γ 9V δ 2 T cells for 4 hours Supernatants are collected and counted on a gamma counter

28. PC-3 MCF-7 SaOS-2 % Specific lysis Effector:Target Tumor cells treated with Pamidronate and Zoledronate are susceptible to lysis mediated by V γ 9V δ 2 T cells A) B) C) * p<0.0001, @ p<0.005, # p<0.002 Data is mean of 3 independent experiments with each cell line * # @ * # Untreated Pamidronate (100 µM) Zoledronate (100µM)

29. % Specific lysis Effector:Target Differential susceptibility of tumor cells treated with Pamidronate and Zoledronate and lysis of fresh breast and normal breast tumor cells by V γ 9V δ 2 T cells (B) (A) % Specific lysis E:T 30:1 * Cytotoxicity of V γ 9V δ 2 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 Fresh breast tumor cells Normal breast cells

30. A) MCF-7 B) MDA-MB-231 Zoledronate Untreated C) PC-3 Cell cycle arrest induced by Pamidronate and Zoledronate in tumor cell lines Pamidronate G0-G1 49.91% S 50.09% G2-M 0% G0-G1 17.61% S 81.12% G2-M 1.18% G0-G1 50.16% S 21.29% G2-M 28.56% G0-G1 51.85% S 22.82% G2-M 25.33% G0-G1 51.15% S 20.24% G2-M 28.61% Representative data of 2 independent experiments with each cell line G0-G1 32% S 53.98% G2-M 13.96% G0-G1 29.92% S 52.98% G2-M 17.1% G0-G1 33.83% S 66.17% G2-M 0% G0-G1 22.13% S 66.23% G2-M 10.64%

31. MCF-7 MICA FITC MHC I FITC 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 V γ 9V δ 2 T cells NKG2D FITC Isotype control NKG2D FITC (E) MICA MHC 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 Isotype control MCF untreated MCF (Pamidronate) MCF (Zoledronate)

32. MCF-7 (Pamidronate) % Specific lysis @ * Involvement of γδ TCR and NKG2D in modulating c ytotoxicity of V γ 9V δ 2 T cells against Pamidronate and Zoledronate treated MCF-7 (A) (B) MCF-7 (Zoledronate) @ * @ @ @ p<0.003 * p<0.002 Data is mean of 2 independent experiments

33. MCF-7 (Zoledronate) MCF-7 (Pamidronate) % Specific lysis % Specific lysis # # Cytotoxicity of Pamidronate and Zoledronate treated MCF-7 mediated by V γ 9V δ 2 T cells involves the perforin granzyme pathway (A) (B) * * * p<0.001, # p<0.05 Data is mean of 2 independent experiments

34. a c b d Time lapse video microscopy of untreated MCF-7 in co culture with V γ 9V δ 2 T cells Yellow arrows - Untreated tumor cells Black arrows - γδ T cells Figures representative of 4 independent experiments

35. a b c d e f g h f Time lapse video microscopy of Pamidronate treated MCF-7 in co culture with V γ 9V δ 2 T cells Yellow arrows - Pamidronate treated tumor cells Black arrows - γδ T cell Figures representative of 4 independent experiments

36. Time lapse video microscopy of Zoledronate treated MCF-7 in co culture with V γ 9V δ 2 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

38. Chapter 3 Comparative analysis of γδ T cell effector functions of oral and breast cancer patients vis-à-vis healthy individuals

39. 3 HTdR incorporation (counts per minute) 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 (A) IPP (B) Pamidronate (C) Zoledronate * * n=10 n=13 n=8 n=10 n=13 n=8 n=10 n=13 n=8 # # @ @ @ ns ns ns @ p<0.002 * p<0.03 # p<0.05 ns not significant

40. Selective expansion of V γ 9V δ 2 T cells from PBMC of healthy individuals stimulated with IPP, Pamidronate and Zoledronate in the presence of rIL-2 Iso control V γ 9 PE V δ 2 PE CD45RO PE CD3 FITC V δ 2 FITC 33 32 12 41 42 53 65 68 52 41 50 52 (A) (B) (C) (D) Representative data of 3 independent experiments

41. Selective expansion of V γ 9V δ 2 T cells from PBMC of breast cancer patients stimulated with IPP, Pamidronate and Zoledronate in the presence of rIL-2 Iso control V γ 9 PE V δ 2 PE CD45RO PE CD3 FITC V δ 2 FITC 13 15 13 28 25 22 22 23 21 25 23 20 (A) (B) (C) (D) Representative data of 2 independent experiments

42. Selective expansion of V γ 9V δ 2 T cells from PBMC of oral cancer patients stimulated with IPP, Pamidronate and Zoledronate in the presence of rIL-2 V γ 9 PE V δ 2 PE CD45RO PE CD3 FITC V δ 2 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

43. 20-30 10-25x10 6 50-80x10 6 10-12x10 6 Breast cancer Patients (n=20) 15-25 5-10x10 6 30-40x10 6 10-12x10 6 Oral cancer Patients (n=20) 20-30 25-30x10 6 60-100x10 6 10-12x10 6 Healthy Donors (n=85) Yield of γδ T cells % (range) MACS Purified γδ T cells Ex-vivo expanded PBMC on Day 12 PBMC seeded on Day 1 Groups

44. Calcium release from V γ 9V δ 2 T cells of breast cancer patients stimulated with IPP, Pamidronate and Zoledronate Calcium release (in nM) Time in sec γδ (IPP) γ δ (Pamidronate) γ δ (Zoledronate) α β (anti-CD3) IPP/PAM/ZOL treated Untreated Point of addition of stimulus (1% Phytohemaglutinin) Representative data of 2 independent experiments

45. Calcium release from V γ 9V δ 2 T cells of oral cancer patients stimulated with IPP, Pamidronate and Zoledronate Calcium release (in nM) Time in sec γδ (IPP) γ δ (Pamidronate) γ δ (Zoledronate) α β (anti-CD3) IPP/PAM/ZOL treated Untreated Point of addition of stimulus (1% Phytohemaglutinin) Representative data of 3 independent experiments

46. Untreated Pamidronate (100 µM) Zoledronate (100µM) Oral cancer patients MCF-7 Breast cancer patients PC-3 A B % Specific lysis Comparative analysis of cytotoxicity of V γ 9V δ 2 T cells from oral and breast cancer patients against aminobisphosphonate treated tumor cells # Effector:Target * # @ Healthy individuals Healthy individuals * p<0.0001 @ p<0.005 # p<0.002 Data is mean of 5 independent experiments for healthy donors, oral and breast cancer patients

47. Chapter 3 Investigating the immunotherapeutic potential of non-peptidic antigen activated  T cells in tumor-bearing Nude/SCID mice

48. Immunotherapy protocol Detection of V γ 9V δ 2 T cells by Flow cytometry Tumor Apoptosis by Annexin V/PI staining Apoptosis by TUNEL staining Expression of Bax Spleen Nude mice with MDA-MB-231 xenografts (A) 2x10 6 MDA-MB-231 injected at mammary fat pad Day 7 Tumor appearance 15 days Day 21 Day 22 (Group 1-4) Animal sacrifice (NIH III female nude mice 6-10 weeks of age, n=12, Treatment schedule used in 2 independent experiments) Group 1 Control Group 2 IL-2 Group 3 γδ T cells + IL-2 Group 4 γδ T cells + IL-2+ Zoledronate 24 hours later (B) Treatment 10x10 6 V γ 9V δ 2 T cells /mouse i.v, 100 µg Zoledronate/mouse i.p and rIL-2 200IU/mouse i.p V γ 9V δ 2 T cells +Zoledronate+ rIL-2 (Group 4) 10x10 6 V γ 9V δ 2 T cells /mouse i.v and rIL-2 200IU/mouse i.p V γ 9V δ 2 T cells+ rIL-2 (Group 3) 200IU/ animal i.p rIL-2 (Group 2) None Control (Group 1) Treatment Schedule Animal Groups and treatment

49. 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

50. Untreated (Group 1) γδ T cells+ZOL+IL-2 (Group 4) γδ PE Vγ 9 PE Vδ 2 PE Homing of Vγ9Vδ2 T cell to spleen of tumor xenograft mice γδ PE Vγ 9 PE Vδ 2 PE Data is mean of 2 independent experiments 12 7 3 2 0.36 0.23

51. Untreated Group 1 γδ T cells +IL-2 Group 3 γ δ T cells + ZOL+IL-2 Group 4 Annexin V-FITC PI Adoptive transfer of V γ 9V δ 2 T cells induces apoptosis in tumors Data is mean of 2 independent experiments 16 34 36

52. 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

53. 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

54. IL-2 (Group 2) γ δ T cells +IL-2 (Group 3) TUNEL staining for apoptosis detection in tumor sections Data representative of 5 independent fields studied Brown staining indicative of apoptotic nuclei * p<0.05 45* Tumor+ γδ +ZOL+IL-2 38* Tumor+ γδ +IL-2 12 Tumor+IL-2 2 Tumor alone APOPTOTIC INDEX Animal groups

55. (B) V γ 9V δ 2 T cells +ZOL+IL-2 (Group 4) (C) Untreated (Group 1) (A) Negative control Immunohistochemical analysis for expression of Bax in tumor sections

57. Bisphosphonates and interplay of  T cells with bone γδ T cell OSTEOCLAST APOPTOSIS ACTIVATION IFN- γ/TNF-α ??? BISPHOSPHONATES

59. T CELLS IN BONE METABOLISM Walsh et al Ann Rev Imm 2006

60. Cytokine (pg/ml) IFN- γ TNF- α Cytokine profile of activated V γ 9V δ 2 T cells # # A) B) Estimation of sRANKL in supernatants of V  9V  2 T cells stimulated with anti CD3, IPP, Pamidronate and Zoledronate # p<0.03 Data is mean of 2 independent experiments 0 0.203 0.203 0.203 Blank 0.036 0.0175 0.2205 0.22 0.221 γδ +ZOL 0.034 0.017 0.22 0.208 0.232 γδ +PAM 0.034 0.017 0.22 0.209 0.231 γδ +IPP 0.019 0.0135 0.2165 0.21 0.223 γδ +anti CD3 0.08 0.025 0.228 0.233 0.223 γδ only sRANKL (pg/ml) Mean-blank Mean OD2 OD1 Samples

61. 1 2 3 4 5 6 7 RANKL mRNA expression in V γ 9V δ 2 T cells (A) (B) RANKL 464 bp GAPDH 305 bp 1 2 3 4 5 6 7 (C) RANKL/GAPDH (Arbitrary Units) RANKL expression in V γ 9V δ 2 T cells stimulated with non-peptide antigens RANKL FITC Unstimulated IPP anti CD3 Pamidronate Zoledronate αβ T cells

62. (A) M-CSF+RANKL (Positive control) (B) Untreated V γ 9V δ 2 T cell supernatant Tartrate Resistant Acid Phosphatase (TRAP) staining of mouse osteoclast precursors treated with supernatants from untreated V γ 9V δ 2 T cells (C) γδ + anti CD3 (D) γδ +IPP (E) γδ +Pamidronate (F) γδ +Zoledronate Black arrow indicates TRAP positive (red) multinucleated osteoclasts

63. Publications: γδ T cells in cancer immunotherapy: current status and future prospects Chiplunkar SV, Dhar S, Wesch,D and Kabelitz, D Immunotherapy (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

64. THANK YOU