Scientific Officers: S.V. Chiplunkar, Ph.D. (Head), N.N. Joshi, Ph.D., M.K. Amin, Ph.D., JA Kode, Ph.D.

Research Fellows: P.P. Wadia, N.S. Atre, S.S. Patil, R.A.S. Bhairavabhotla, S.R. Patel.

The projects undertaken by the Immunology group focus on the following aspects:
(i) understanding the reasons for immune dysfunction in cancer patients,
(ii) analysis of the role of various effector cells (gdT cells, NK cells, cytotoxic T cells, NKT cells), and
(iii) development of immunodiagnostic and immunotherapeutic approaches for cancer.

Identification of markers of chronic hepatitis B virus infection and their relation to hepatocarcinogenesis (Indian Council of Medical Research / Indo-German collaboration)

Patients chronically infected with the hepatitis B virus (HBV) face a 100 fold increased risk of developing hepatocellular carcinoma (HCC). In chronic hepatitis B virus infection (CHB), integration of HBV into the host DNA leads to the formation of full-length and truncated HBx transcripts (fHBx and tHBx respectively). The present project focuses on understanding the significance of tHBx as a marker of chronic HBV infection and the analysis of immune responses to this protein. High frequency of tHBx transcripts has been noted in liver and tumour tissues of HCC patients. A majority of CHB and HCC patients show presence of tHBx exclusively or co-expressed with fHBx. Preliminary observations indicate high frequency of tHBx in the sera of CHB patients and indicates that tHBx serves as a marker of chronic HBV infection. HBcAg and fHBxAg elicit a mixed cytokine response with increased secretion of IL-10 and moderate IFN-g production. TCR Vb repertoire of peripheral blood lymphocytes (PBL) in CHB and HCC patients is skewed, with clonal populations of several TCR Vb families occurring more frequently. HBV antigen induced clonal expansion of PBL bearing specific TCR Vb families, and apoptosis of specific Vb subsets upon antigenic stimulation provides an explanation for the immune hypo-responsiveness commonly observed in these patients. Future plans include detection of fHBx and tHBx RNA in the sera of CHB and HCC patients before and after lamuvidine or IFN treatment.

Analysis of T cell receptor repertoire and immune dysfunction in oral cancer patients (Indian Council of Medical Research - Task Force)

Deficiencies in both B and T cell responses have been reported in patients with oral cancer. The present study analyzes the T cell receptor (TCR) repertoire and lymphocyte signaling molecules (CD3-zeta, CD3-epsilon, ZAP-70 and P56lck) in patients with oral cancer to understand the strategies adopted by tumour cells to evade immune destruction. A decreased expression of T cell signaling molecules in lymphocytes of oral cancer patients was observed with a marked decrease in the CD3-zeta chain. This finding correlates with the reduced lymphocyte proliferative responses, decreased calcium flux and reduced ratio of IFNg:IL-10. A marked decrease in CD3-zeta chain expression is also seen when PBL of healthy individuals are cultured with supernatants derived from oral tumours. Decreased CD3-zeta chain expression correlates with tumour stage progression. Studies are also being carried out on gdT cells stimulated by heat shock protein (hsp) antigens to understand the molecular mechanism responsible for the loss of expression of the CD3-zeta chain. Caspase inhibitors ZVAD and DEVD inhibit CD3-zeta chain degradation. A concomitant increase in the ability of gdT cells to lyse oral tumour targets is noted. These findings strongly suggest that activation of intracellular caspases (caspase 3 and caspase 7) plays a role in zeta chain degradation. Future plans include sequencing of the junctional regions of clonal TCR Vb, Vg and Vd chains in lymphocytes obtained from tumour, lymph nodes and peripheral blood (TIL/LIL/PBL) of oral cancer patients, determination of tyrosine phosphorylation status of CD3-zeta chain, and characterisation of the factors responsible for degradation of signaling molecules.

Activation and death of gdT cells: Significance in immunosurveillance

The present project involves an examination of: (i) apoptotic pathways utilised by gdT cells, (ii) role of nitric oxide (NO) in hsp-induced apoptosis, and (iii) activation of gdT cells by cytokines (IL-2 / IL-15) and antigens such as hsp / isopentyl pyrophosphate (IPP). Data reveal that gdT cells release NO into the culture supernatants on stimulation with hsp and IPP. gdT cells stimulated with hsp express iNOS and eNOS exhibit mitochondrial membrane potential changes as an early event during apoptosis. DNA fragmentation analysis of hsp-stimulated gdT cells shows the involvement of caspases. gdT cells show strong cytotoxicity against oral tumours, which is augmented in the presence of the cytokines IL-2 and IL-15. In the presence of anti-IL-2Rß and anti-IL-2Rg antibodies, IL-15-induced proliferation of gdT cells is significantly inhibited in healthy individuals [70-80%] and oral cancer patients [30-40%]. Future plans include dissecting the death pathway in gdT lymphocytes and studying the role of cytokines (IL-12 / IL-15) and chemokines (CC / CXC family) in extravasation of gdT cells from peripheral blood to tumour tissue.

Analysis of cytokine profiles and detection of antibodies to E6 and E7 proteins of HPV in patients with cervical cancer and cervical intraepithelial neoplasia

HPV infection is a major risk factor for the development of cervical intraepithelial neoplasia (CIN) and invasive cervical carcinoma. This study involves detection of HPV16 and HPV18 DNA in CIN and cervical cancer biopsies, and correlating it with expression of cytokines IL-1a, IL-6, IL-10 and IFN-g at the mRNA level. Data reveal the presence of HPV16 in 86% and HPV18 in 13% of cervical tumours, with co-expression of HPV16 and HPV18 noted in 11% cases. Positivity using HPV consensus primers indicates the presence of other HPV subtypes in CIN biopsies. Higher expression of inflammatory cytokines (IL-1a, IL-10 and IL-6) is noted in cervical tumours as compared to CIN. In cervical tumours, a significant association is observed between the presence of HPV16 and expression of IL-10 mRNA. Preliminary results indicate the presence of high titre antibodies to HPV16 E7 proteins in cervical cancer patients. Studies have also focused on understanding the anti-tumour role of NKT cells. Future analysis of NKT cells will include a study of their antigen recognition cytokine profiles and TCR clonality in PB, tumour and lymph nodes.

Cytokine imbalance in lung cancer patients – role of cyclooxygenases

Cyclooxygenases (Cox-1 and Cox-2) catalyse a key step in the formation of prostaglandin (PGE2), which is a potent inducer of IL-10 and is known to suppress cell mediated immune responses. The present study aims at understanding the role of Cox-1, Cox-2 and PGE2 in modulating the production of peripheral blood lymphocyte (PBL)-derived cytokines and their effector function in LC patients. Lymphocytes from PB and tumour of LC patients show high expression of Cox-1, Cox-2 and IL-10 mRNA, and incubation of LC patients’ lymphocytes with tumour supernatants up-regulates expression of Cox-2 and IL-10 mRNA, and also IL-10 secretion. A significant decrease in IL-10 production with a concomitant increase in IFN-g, accompanied by up-regulation in the expression of T cell markers CD3, CD4, CD8, CD56 and HLA-DR was observed in the presence of Cox 2 inhibitors. Upon stimulation with IL-2 + IL-12, LC patients’ lymphocytes treated with Cox inhibitors exhibit a marked increase in their ability to lyse tumour targets. Analysis of IL-12p40 in supernatants of Cox-2 inhibitor-treated lymphocytes shows an inverse correlation with IL-10 production. Future plans include analysis of dendritic cell function - cytokine IL-10 and IL-12p40 production under the influence of Cox inhibitors.

Development and characterisation of single chain fv fragments of MAb 3F8E3 recognizing antigens on squamous cell carcinoma: Applications in immunodiagnosis and therapy

MAb 3F8E3 (IgG3K) developed in this laboratory earlier, shows reactivity to squamous cell carcinoma (SCC) cells irrespective of tissue of origin and also to human SCC xenografted in nude mice. Single chain antigen-binding molecules (ScFv) offer several advantages over intact mouse IgG MAb, because of their small size and rapid penetration in tumours and faster clearance. Towards preparation of (Fab)2 fragments of MAb 3F8E3, RNA was isolated from the 3F8E3 hybridoma cells and cDNA prepared by reverse transcription. PCR was performed using IgH and IgL specific primers. Purified PCR products were cloned into pBluescript II SK+ vector. Future plans include preparation of recombinant (Fab)2 fragments and scFv fragments of MAb 3F8E3, checking their integrity, molecular weight and reactivity with fresh tumours, SCC cell lines and tissue sections vis a vis the whole antibody molecule.

Immunogenetic studies in relation to susceptibility to breast cancer (Department of Biotechnology)

The goal of these studies is to examine the relevance of immune function and related genes in determining susceptibility to breast cancer. Earlier studies demonstrating lower T cell and NK cell function in unaffected members from cancer families have therefore been extended to sporadic cancer patients with early (<32 years) or late (>40 years) age of onset. HLA class I (-A, -B and –C loci) and class II (– DRb and -DQb loci) allele frequencies are also being studied in ethnically selected groups of early / late onset patients and age, sex and ethnicity matched healthy women without any family history of cancer. In immune function studies, while the disease phenotype in the two groups of patients is seen to be comparable, older patients display strikingly lower NK cell functions and IFNg production compared to their age-matched healthy controls. On the other hand, defects at the level of NK activity and NK cell response to cytokines or ability to produce IFNg are not enhanced in younger breast cancer patients compared to their age-matched healthy controls. HLA studies revealed increased frequency of HLA-B*40, -Cw*16, -A*11 alleles in breast cancer patients. The strikingly higher frequency of HLA-B*40 (R.R. 2.2; c2 5.8; p=0.02; corrected p=0.6) in both early and late onset patients is not attributable to increased frequency of any haplotype containing this allele. Homozygosity (at two digit typing level) is suggested at HLA-B locus in 17.6% of early onset patients versus only 7% and 10% of late onset patients and healthy controls respectively. At HLA-C locus, this was observed at even higher frequency with 31% in early onset patients versus 18% in late onset patients and 11% in healthy controls.

Molecular HLA typing in Parsi breast cancer patients and study of NK cell function, killer inhibitory receptor genotypes and activating NK cell receptor expression (Lady Tata Memorial Trust)

An important objective of this project is to examine the relevance of ethnic differences to HLA class I and II allele and haplotype frequencies in age- and gender-matched breast cancer patients and healthy controls of the Parsi community (who are ethnically homogeneous and distinct from other Indians). Data will be compared with the findings of earlier studies in Marathi-speaking Maharashtrian Hindus. Since NK cell function is governed by the interplay between HLA and killer inhibitory receptor (KIR) genotypes, and levels of activating receptors, analysis of KIR genotypes and expression of NK cell activating receptors will also be undertaken. It is proposed to generate EBV-transformed cell lines from the study subjects for the future establishment of a DNA bank. KIR typing has been initiated and the methodology for studying expression of activating receptors is being standardized.

In vivo modulation of immune function in breast cancer patients by tamoxifen, progesterone and a combination of oestrogen and progesterone