Ph D Swati Dhar

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

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Ph D Swati Dhar

  1. 1. Swati Dhar Potential of γδ T lymphocytes stimulated with non-peptidic antigens for immunotherapy of cancer
  2. 2. γδ T cells properties and function Modlin & Sieling Science 2005
  3. 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
  4. 4. V  9V  2 T cells <ul><li>Dominant subset constituting 95% of the total peripheral blood  population </li></ul><ul><li>Antigen recognition independent of MHC I or II involvement </li></ul><ul><li>Recognize small molecular weight phosphorylated compounds, nonpeptide antigens </li></ul><ul><li>Expanded in diseases like tuberculosis, tularemia, listeriosis, malaria and even in HIV infection </li></ul><ul><li>Professional antigen presentation upon activation by non-peptidic antigens </li></ul><ul><li>(Brandes et al Science 2005) </li></ul><ul><li>Anti-tumor activity against lymphomas and solid tumors via perforin, granzyme </li></ul><ul><li>or the Fas-FasL pathway </li></ul>
  5. 5. Casseti et al Cellular and Mol Imm 2008 <ul><li>Human γδ T cells recognize heat shock protein-60 on oral tumor cells </li></ul><ul><li>Laad et al. Int J Can 1999 </li></ul><ul><li>2. Involvement of CD166 in the activation of human γδ T cells by tumor cells sensitized with nonpeptide antigens </li></ul><ul><li>Kato et al. J Immunol 2006 </li></ul>V γ 9V δ 2 T cells: Antigens recognized and role of co stimulatory receptors
  6. 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. 7. Russel RGG and Rogers MJ Bone 1999 Bisphosphonates
  8. 8. CLASSES OF BISPHOSPHONATES Non-nitrogen containing bisphosphonates Nitrogen containing bisphosphonates
  9. 9. Roelofs et al., Clin Can Res 2006 Mammalian Mevalonate Pathway , 7 Dehydrocholesterol Farnesol HMG-CoA Reductase
  10. 10. Caraglia et al., Endocrine-related Can 2006 Pleiotropic role of Nitrogen containing Bisphosphonates in Tumor cells
  11. 11. Aims and Objectives <ul><li>Understanding the immunomodulatory role of non-peptidic antigens </li></ul><ul><li>on  T cells </li></ul><ul><li>Dissecting the molecular pathways of activation of  T cells </li></ul><ul><li>3. Comparative analysis of γδ T cell effector functions from oral and breast </li></ul><ul><li>cancer patients vis-à-vis healthy individuals </li></ul><ul><li>Investigating the immunotherapeutic potential of non-peptidic antigen </li></ul><ul><li>activated  T cells in tumor-bearing Nude/SCID mice </li></ul><ul><li>5. Evaluating the role of activated V γ 9V δ 2 T cells in osteoclastogenesis </li></ul>
  12. 12. Chapter 1 Understanding the immunomodulatory role of non-peptidic antigens (IPP/PAM/ZOL) on γδ T cells
  13. 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. 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. 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. 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. 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. 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. 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
  20. 20. <ul><li>Summary </li></ul><ul><li>IPP/PAM/ZOL induce proliferation and IFN- γ release in γδ T cells which is </li></ul><ul><li>increased in combination with rIL-2 </li></ul><ul><li>Bisphosphonates upregulate activation markers CD25, CD69 and the NK marker CD161 </li></ul><ul><li>Calcium flux shows a biphasic model of response distinct from the αβ T cells </li></ul><ul><li>which could emphasize on the different mode of antigen recognition by V γ 9V δ 2 T cells </li></ul><ul><li>Bisphosphonates recruit MAPK intermediates and the PKB/Akt pathway </li></ul><ul><li>for inducing proliferation, differentiation and survival V γ 9V δ 2 T cells </li></ul>Immunomodulatory effects of aminobisphosphonates on V γ 9V δ 2 T cells (Manuscript communicated)
  21. 21. Chapter 2 Dissecting the molecular pathways of activation of γδ T cells
  22. 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. 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. 24. AW8507 MDA-MB-231 MCF-7 Untreated Mevastatin Morphological changes in tumor cell lines post statin treatment 200x Magnification
  25. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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
  37. 37. Summary <ul><li>Deregulated mevalonate pathway in tumor cells generates elevated phosphorylated </li></ul><ul><li>metabolites like IPP that can act as a danger signal and mediate recognition by V γ 9V δ 2 T cells </li></ul><ul><li>Tumor cells harbor higher levels of phosphorylated metabolites vis-à-vis normal cells and IPP can act as an endogenous tumor-associated antigen </li></ul><ul><li>Aminobisphosphonates (PAM and ZOL) sensitize tumor cells of different origin to lysis by V γ 9V δ 2 T cells efficiently enhancing endogenous IPP accumulation in the mevalonate pathway </li></ul><ul><li>Mechanism of augmented V γ 9V δ 2 T cells lysis of PAM/ZOL treated targets depends on the TCR and partly on the engagement of NKG2D receptor </li></ul><ul><li>V γ 9V δ 2 T cells form strong conjugates with PAM treated tumor cells and lyse targets as against untreated tumor cells </li></ul>Lysis of aminobisphosphonate sensitized breast tumor cells by V γ 9V δ 2 T cells (Manuscript communicated Cancer Immunology Immunotherapy)
  38. 38. Chapter 3 Comparative analysis of γδ T cell effector functions of oral and breast cancer patients vis-à-vis healthy individuals
  39. 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. 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. 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. 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. 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. 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. 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. 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. 47. Chapter 3 Investigating the immunotherapeutic potential of non-peptidic antigen activated  T cells in tumor-bearing Nude/SCID mice
  48. 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. 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. 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. 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. 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. 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. 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. 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
  56. 56. Summary <ul><li>In an in-vivo model of breast tumor, V γ 9V δ 2 T cells are able to recognize and induce apoptosis in </li></ul><ul><li>xenografted tumor </li></ul><ul><li>Treatment modality involving ZOL and IL-2 was more efficient than </li></ul><ul><li>protocol involving only T cells and rIL-2 </li></ul>
  57. 57. Bisphosphonates and interplay of  T cells with bone γδ T cell OSTEOCLAST APOPTOSIS ACTIVATION IFN- γ/TNF-α ??? BISPHOSPHONATES
  58. 58. Common players of the immune system and bone biology RANKL ( Receptor Activator of NF- κ B-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 <ul><li>Reduced bone mineral densities in </li></ul><ul><li>Adult and childhood Leukemia (Oliveri et al 1991) </li></ul><ul><li>Chronic infections like Hepatitis C and HIV (Stellon et al 1989) </li></ul><ul><li>Autoimmune diseases Diabetes Mellitus, Rheumatoid arthritis and SLE (Seitz et al 1985) </li></ul><ul><li>Metastasis related cancers of breast and prostate origin (Coleman et al 1998) </li></ul>
  59. 59. T CELLS IN BONE METABOLISM Walsh et al Ann Rev Imm 2006
  60. 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. 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. 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. 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. 64. THANK YOU

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