Office Phone: +91-22-27405032
Research Overview: The focus of my group is on understanding the immune scenario and reasons for immune dysfunctions in patients with cancer. The group is also interested in studying the role of hematopoietic and mesenchymal stem cells for management of hematological neoplasms, tissue regeneration and repair.
Project 1: Activation and death of γδ T lymphocytes in patients with oral cancer
Chiplunkar, Atre (SRF), Pathak, D’Cruz
γδ T cells play an important role as anti-tumor effector cells. However, their expansion is self-limiting. We investigated the activation induced cell death in γδ T cells after stimulation with heat shock proteins (hsp60, hsp70) that act as ligands for γδ T cells. Characterization of the molecular events in the apoptotic pathway revealed that nitric oxide (NO) is released by γδ T cells after hsp stimulation. Purified γδ T cells when incubated with rhsp60 and rhsp70 as well as artificial NO donors - SNAP and arginine, exhibited changes in mitochondrial membrane potential. Addition of caspase-9 inhibiting peptide Z-LEHD-FMK inhibited DNA fragmentation in γδ T cells and mitochondrial membrane potential changes, indicating involvement of caspases-9 in hsp60/70 induced apoptosis of γδ T cells. Our studies demonstrated a unique strategy adopted by tumor cells to evade immune recognition.
Project2: Potential of γδ T lymphocytes stimulated with non-peptidic antigens for immunotherapy of cancer
Chiplunkar, Dhar (SRF), Pathak, Badwe
We are currently interested in analyzing the non-peptidic ligands recognized by γδ T cells on tumor cells. Our studies demonstrate that T lymphocytes expressing the Vγ9/Vδ2 TCR recognize non-peptidic phosphorylated compounds, their synthetic analogs bisphosphonates and endogenous metabolite Isopentenyl pyrophosphate (intermediate metabolite of mevalonate pathway) in tumor cells. γδ T cells release IFN-gamma and TNF-alpha after co-culture with a panel of oral, breast and prostate tumor cell lines as against lowered cytokines in normal, non-transformed cell lines. Chromium release assay and flow cytometry data reveal increased cytotoxicity of γδ T cells upon activation with bisphosphonates. Current studies aim to identify the nexus between γδ T cells and osteoclasts/ osteoblasts vis-à-vis their role in bone metabolism.
Project 3: Toll-like receptors and antitumor immunity: Role of γδ T lymphocytes:
Chiplunkar, Paleja (JRF), Tongaonkar, D’Cruz, Kane, Deodhar
The present project aims at understanding the role of Toll like receptors (TLRs) and regulatory T cells in modulating the anti tumor immunity mediated by γδ T cells in patients with oral and cervical cancer. Preliminary work demonstrated the presence of TLRs (TLR2, TLR3, TLR4 and TLR9) on γδ T cells from freshly isolated peripheral blood leucocytes (PBLs). γδ T cells were immunomagnetically purified from ex-vivo expanded PBLs and stimulated in the presence of a TCR stimulus (IPP and anti-CD3) or a TLR stimulus (Poly I:C) or both in combination. Immunophenotyping of stimulated and unstimulated purified γδ T cells showed the presence of TLR2, TLR3, TLR4 and TLR9 on these cells. Further the culture supernatant from anti-CD3 stimulated γδ T cells alongwith Poly I:C showed increased IFN-gamma production. Immunomagnetically purified CD4+CD25+regulatory T cells showed high expression of TLR2 and TLR4. Studies are in progress to characterize the down stream signaling pathways initiated by TLR agonists and further analyze the crosstalk between TLR agonist stimulated γδ T cells and regulatory T cells.
Project 4:Role of alkylamines in priming γδ T lymphocytes for antitumor effector functions:
Chiplunkar, K Nirmala (JRF), Tongaonkar, D’Cruz, Kane, Deodhar
Alkylamines are the group of non peptidic molecules, which are capable of stimulating Vγ9/Vδ2 T cells. These alkylamines are secreted at millimolar concentrations in bacterial culture supernatants and also are found at high concentrations in tea and at lower concentrations in other edible plant products such as apples and wine. Our studies demonstrate, ethylamine in combination with cytokines IL-15 is able to activate γδ T cells. Activated γδ T cells express activation markers (CD69 and CD25) and chemokines receptors (CCR5 and CXCR4). Stimulation of γδ T cells with ethylamine in the presence of IL-15 resulted in increased production of IFN-gamma. Studies are under way to evaluate the antitumor effector functions of ethylamine stimulated γδ T cells.
Project 5:Analysis of clonal TCR and gene rearrangements in T-ALL: Significance in diagnosis and prognosis:
Chiplunkar, Kode, Dudhal, Banavali
T-cell Acute Lymphoblastic Leukemia (T-ALL) is a clonal lymphoid malignancy. The present project aimed at determining the incidence of clonal TCR and junctional gene rearrangement status in Indian T-ALL patients at diagnosis and analyzing the suitability of clonal TCR and junctional gene rearrangement status at diagnosis as a prognostic marker for T-ALL. Data on increased number of patients with longer follow up demonstrated that out of thirty patients included in MCP 841 protocol and followed up for more than 5 years (average) TCR+ T-ALL (n=9) showed statistically significant increased survival (p=0.0423) compared to TCR+ T-ALL subgroup. Based on this biomarker risk-based treatment assignment may be utilized for childhood T-ALL patients. Studies re in progress to sequence TCR and junctional (CDR3) regions of T-ALL patients for variable, diversity, junctional, N region and P region bases and compare molecular expression profiles of TCR+ T-ALL and TCR+ T-ALL.
Project 6:Down regulation of CD3 zeta chain expression in T cells of oral cancer patients: a potential biomarker for prognosis :
Chiplunkar, Rao (SRF), Pradhan, Pathak, D’Cruz, Kane, Shastri, Srivastava, Chaturvedi, Agarwal
Immunosuppression appears to be more frequent and more profound in patients with head and neck cancer. The immune impairment often observed in these patients is not restricted to the tumor site but presents itself as a systemic defect. The project aims at understanding the molecular mechanism of CD3 ζ chain degradation, both at the mRNA and protein level. Studies demonstrate a marked reduction in lymphocyte proliferative responses, calcium flux and a strong TH2 bias in OC patients. Results indicate a marked reduction in CD3-ζ chain expression in PBL of OC patients as compared to healthy individual (HI). The CD3-ζ defect was found to be T cell V beta subset-specific. The CD3-ζ mRNA and protein expression (flow cytometry and western blotting) decreased as the tumor stage increased. Studies demonstrate that ubiquitination is also responsible for the altered recycling and internalisation of the CD3 ζ chain receptor. The underlying reason for CD3 ζ chain transcriptional defect in tumor compartment is the decreased expression of CD3 ζ transcription factor Elf 1. Preliminary data indicates that the tumor-derived factor involved in CD3 ζ chain degradation is TNF alpha secreted by oral tumor cells. It has also been noted that patients undergoing a combination of surgery and radiotherapy show a marked increase in the expression of CD3 ζ chain in the peripheral blood compartment. An important outcome of the study is that we are now pursuing CD3 ζ as a biomarker for predicting prognosis of oral cancer.
Project 7: Role of cycloxygenases and its inhibitors in lymphocyte activation and in restoring anti tumor immunity :
Chiplunkar, Patel (SRF), Vetale, Mistry
The present study focuses on understanding the reasons for immune dysfunction in lung cancer (LC) patients and understanding role of cyclooxygenases (COX) inhibitors in modulating anti-tumor immunity. Lymphocytes of LC patients showed decreased proliferation when stimulated with mitogens, decrease in calcium flux, low expression of activation markers and low cytotoxic response with high generation of reactive oxygen species. These lymphocytes exhibited a marked decrease in IFN- and high IL-10 with decrease in transcription factor T-bet and increased expression of GATA-3. Dissecting the signaling events demonstrated over-expression of COX-2, P-p38 MAPK and P-ERK with low expression of P-JNK1/2. Incubation of lymphocytes with specific inhibitors for ERK, p38 MAPK and COX-2 resulted in decreased IL-10 production with increase in IFN-. Under the cover of COX inhibitors significant increase in proliferation, cytotoxic response, intracellular Ca++ release and expression of activation markers was observed with decrease in reactive oxygen species. When treated with COX-2 inhibitors, the expression and activity of T-bet was increased but GATA-3 was decreased in lymphocytes. The IL-10 production in these patients, which is responsible for observed immune dysfunction is regulated by PGE2 via p38 MAPK and ERK pathways. COX-2 appears to be the focal point of prostaglandin and IL-10 production, and thus a potential target of intervention in attempts at restoring anti-tumor effector functions.
Project 8: Prognostic value of tissue polypeptide antigen in oral squamous cell carcinoma
Sharada S. Sawant, Milind M. Vaidya, Devendra Chaukar, Shubhada Kane, Anil Dcruz
Human oral cancer has a high risk of locoregional relapse which is difficult to diagnose early due to lack of prognostic markers. We and others have shown aberrant expression of cytokeratin (CK) 8 and 18 in human oral cancer. Aberrant supra-basal expression of CK19 has been shown earlier. In this study, our aim was to develop a non-invasive test for prognostication of human oral cancer. Immunoradiometric assay (IRMA) was used to measure the circulating levels of TPA in the sera of 80 oral cancer patients before surgery and seven days after surgery. We have shown the presence of CK8, 18 and 19 fragments in the sera of oral cancer patients. We could correlate the higher TPA levels with tumour stage, recurrence and poor survival of the patients. We have also shown that the TPA levels remained high even after surgical removal of the tumour, which could be the result of presence of these proteins in the surrounding uninvolved areas.In conclusion, increased post surgery serum TPA levels could prove to be a useful marker of disease progression in human oral cancer and TPA could be used as a useful non-invasive prognostic assay.
Kishor Amin PhD, Jyoti Kode PhD.
Navnanth Dudhal, Shamal Vetale, Trupti Pradhan, Meena Patkar
Technical Assistants :
Suresh Dakave, Baburao Desai, Raju Sawant
Research Fellows :
Nilangi Atre B Ravi Kiran Swati Patel Dakshayini Rao Swati Dhar Bhairav Paleja Nirmala K
Flow cytometry Facility :
ACTREC :Wadia P, Gupta S, Bakshi A, Sawant R, Prabhash K, Teni T
TMH :Dinshaw K, Banavali SD, Mistry RC,Pathak AK, D’Cruz A, Chaturvedi P, Agarwal JP, Tongaonkar HB, Shastri SS, Shrivastva SK, Badwe RA, Deodhar, Agarwal J, Nair R, Gujral S, Agarwal M, Parikh P, Kane S.
Kidwai Memorial Institute of Oncology: Jayashree RS Christian Medical College: Abraham P IIT-Bombay: Bellare J, Mehta H, Sharma S, Soni V Rajiv Gandhi Centre for Biotechnology: Pillai MR Institute of Cytology and Preventive Oncology: Das BC National Centre for Biological Sciences: Krishna S.
1.Atre N, Thomas L, Mistry R, Pathak K, Chiplunkar S.  Role of nitric oxide in heat shock protein induced apoptosis of T cells. Int. J Cancer, 119, (6), 1368-1376. (figure accepted as cover page for Int. J Cancer  120, 12).
2.Kode JA, Dudhal NV, Banavali SD, Chiplunkar S.V. . T-cell receptor and junctional region gene rearrangements as diagnostic and prognostic biomarker for T-cell Acute Lymphoblastic Leukemia. Leukemia and Lymphoma, 47(4),769-70.
3. Wadia P.P., Atre N., Pradhan T., Mistry R. and Chiplunkar S.V.  Heat Shock protein induced TCR gene rearrangements in patients with oral cancer. Oral Oncology. 41, 175-182.
4. Kode J.A., Dudhal N.V., Banavali S.D., Advani S.H. and Chiplunkar S.V.  Clonal T-cell receptor and gene arrangements in T-cell acute lymphoblastic leukemia at diagnosis: Predictor of prognosis and response to chemotherapy. Leukemia & Lymphoma, 45 (1):125 -133.
5. Dadke D., Jaganath P., Krishnamurthy S. and Chiplunkar S.V.  The detection of HBV antigens and HBx-transcripts in an Indian fibrolamellar carcinoma patient: a case study. Liver 22:87-91.
6. Chiplunkar, S.V.  The immune system and cancer. Current Science, Vol.81, No.5, 542-546.
7. Thomas M.T., Badwe,R.A., Deshpande,R.K., Samant,U.C., Chipunkar,S.V.  Role of adhesion molecules in recruitment of Vdelta 1 T cells from the peripheral blood to the tumor tissue of esophageal cancer patients. Cancer Immunol Immunother 50, 218-225
8. Chatterjee, S., Singh, S., Sohoni, R., Kattige, V., Deshpande, C., Chiplunkar, S. Kumar, N., Sharma, S.  Characterization of domains of the phosphoriboprotein PO of Plasmodium falciparum. Molecular Biochemical Parasitology, 107,143-154.
9. Pichan, D., Thomas, M. L., Damodaran, A., Fakih, A. R.and Chiplunkar,S.V  Characterization of tumor associated antigens on human oral squamous cell carcinomas using monoclonal antibody 3F8E3. Tumori, 86,64-69.
10. Thomas,M. L., Samant,U. C.,Deshpande, R. K., Chiplunkar, S. V.  Gamma Delta T cells lyse autplogous and allogenic esophageal tumors: involvement of heat shock proteins in the tumor cell lysis. Cancer Immunol. Immunotherapy , 48, 653-659.
11. Kode, J.A., Advani, S.H. and Chiplunkar, S.V.  T-cell receptor and gene rearrangements in T-cell acute lymphoblastic leukemia in Indian patients. Leukemia and lymphoma, 36,331-338.
Links: Indian Immunology Society