Sudha G. Gangal

Establishment and characterization of four new squamous cell carcinoma cell lines derived from oral tumors

SummaryFour cell lines were established from squamous cell carcinomas (SCC) of the oral cavity. Cell lines AW 13516 and AW 8507 were derived from poorly differentiated SCC and epidermoid carcinoma of the tongue respectively. Cell line AW 10498 was derived from moderately differentiated SCC of the lower alveolus, and AW 9803 grew from a well-differentiated SCC of a retromolar trigone. The cultures showed typical epithelial cell morphology, numerous mitotic figures, occasional multinucleated giant cells, individual cell diskeratosis and nuclear and nucleolar abnormalities. The cell lines AW 13516 and AW 8507 were fast growers with a doubling time of 35.5 h and 31.9 h, respectively, which was independent of the initial seeding density. Cell lines AW 10498 (doubling time 52.2 h) and AW 9803 (doubling time 66 h) showed slower growth and had shorter doubling times at higher seeding densities. The presence of cytokeratins was detected in all the four cell lines by using polyclonal antikeratin antisera in indirect immunofluorescence and in Western blotting. None of the cell lines expressed major histocompatibility complex (MHC) class II antigens. MHC class I antigens were expressed by three cell lines but not by AW 9803. Flow cytometric analysis of DNA content and chromosomal studies suggested the presence of polyploidy and aneuploidy in all the four cell lines. Ultrastructural studies revealed typical epithelial cell features, such as the presence of desmosomes, tonofilaments and keratin bundles.

Modulation of Natural Killer and Antibody-Dependent Cellular Cytotoxicity by Interferon and Interleukin-2 in Chronic Myeloid Leukemia Patients in Remission

Our earlier studies demonstrated that about 55% of chronic myeloid leukemia (CML) patients in remission exhibited impaired natural killer (NK) cytotoxicity (low NK responders) while antibody-dependent cellular cytotoxicity (ADCC) of these patients, on chicken red blood cells as targets, was within normal range. In this paper, we have attempted to modulate the NK cytotoxicity of CML patients in remission with interferon (IFN) and interleukin-2 (IL-2) singly or together. ADCC using K562 target-directed monoclonal antibody (MAb) 4.6E10 was also modulated by treating the effectors with IFN or IL-2. Pretreatment of nonadherent mononuclear cells from peripheral blood (NAPBMNC) with IFN or IL-2 was found to result in 20 and 21% increase in target cell lysis in case of healthy donors, 79 and 98% increase in case of CML normal NK responders, and 164 and 159% increase in case of CML low NK responders. Combined use of IFN and IL-2 potentiated further the lymphocytotoxicity to 25% in healthy donors, 135% in normal NK responder CML patients and 283% in low NK responder CML patients. This treatment resulted in restoration of cytotoxicity of the latter group of patients to a normal level. The augmentation was seen in 80-100% CML patients. Although ADCC with chicken red blood cells as targets was within normal range, ADCC mediated with MAb to K562 cells was significantly lower in CML low NK responders (24.5%) than CML normal NK responders (42.4%) and healthy donors (65.9%).(ABSTRACT TRUNCATED AT 250 WORDS)

Impairment in proliferation, lymphokine production and frequency distribution of mitogen-responsive and interleukin-2-producing cells in Hodgkin's disease

SummaryIn this paper, we have correlated the ability of peripheral blood lymphocytes (PBL) from Hodgkins Disease patients to proliferate in response to a mitogen, phytohaemagglutinin (PHA), with production of lymphokines interleukin-2 (IL-2) and interferon γ (IFNγ), accumulating in the activated lymphocyte culture supernatants. We have also studied the frequency distribution of PHA-responsive and IL-2-producing T cells from PBL using limiting-dilution analysis. We observed that the levels of IL-2 and IFNγ in the supernatants of activated lymphocytes from patients with Hodgkins disease were significantly reduced compared to those of healthy donors. Substage-B patients showed marked reduction in the ability to produce IFNγ. Levels of IL-2 and IFNγ in the culture supernatants of PBL from Hodgkins disease patients correlated positively with proliferative responses, when analysed by linear regressison (r = 0.79 andr = 0.60 respectively). However, production of the two lymphokines by activated lymphocytes from the same patients did not correlate (r = +0.04). Further, the frequencies of PHA-responsive cells and IL-2-producing cells in the PBL of patients with Hodgkins disease (ranges 1/111–1/554 and 1/3009–1/6709 respectively) were also less than those of the healthy donors (ranges 1/80–1/181 and 1/761–1/1828 respectively). Proliferation, IL-2 production in bulk cultures and frequencies of PHA-responsive and IL-2-producing cells correlated well in individual healthy donors. Whereas, one patient (BC 11 214) with a frequency of PHA-responsive cells within normal limits had a very low frequency of IL-2-producing cells. Taken together, the results indicate abnormalities in cytokine production and frequency distribution of cells required for amplification of immune response in patients with Hodgkins disease.

Propagation of cytotoxic effectors from chronic myeloid leukemia patients and cloning of cytotoxic T cells

SummaryCytotoxic cells (CTCs) generated from peripheral blood lymphocytes of 5 chronic myeloid leukemia (CML) patients in remission on stimulation with autologous leukemic cells and allogeneic lymphocytes (3-cell assay), were propagated in vitro in interleukin-2 (IL-2)-containing medium and periodic stimulation with autologous leukemic cells, for a period of 4 to 6 months. During this period, the cells were assessed for phenotype and for cytotoxic responses in a 4-h 51Cr release microcytotoxicity assay. The CTCs continued to show specific lysis of autologous leukemic cells and bone marrow (BM) cells. However, the nonspecific lysis of natural killer (NK) targets and the proportion of cells showing NK phenotype (HNK-1 antigen) increased progressively on cultivation in IL-2-containing medium. Therefore cells showing CD8 phenotype and specific cytotoxic function were segregated by cloning CTCs under the condition of limiting dilution in the presence of allogeneic feeder cells and IL-2-containing medium. Three cytotoxic T cell (CTL) clones expressing CD3+, CD8+, and HLA DR+ phenotypes were obtained from CTCs of 2 CML patients. These clonoid populations, maintained in IL-2-containing medium and periodic antigenic stimulation with autologous leukemic cells, showed specific lysis of autologous leukemic cells and BM cells even at lower (10:1) effector:target ratios. They did not kill K562 (erythroblastoid leukemic NK target cell line) cells and autologous phytohemagglutinin-induced blasts. These clones apparently functioned in an MHC-restricted manner as they did not lyse allogeneic CML cells which would also express a similar set of maturation antigens if sensitization was, as it appeared, against these antigens. Finally, interaction of autologous BM cells with CTL clones reduced the colony forming potential of BM cells only to the extent of 18%–30%. The results therefore indicate that such CTL clones can possibly be used in adoptive immunotherapy as they showed minimal BM toxicity.

Natural and antibody dependent cellular cytotoxicity in chronic myeloid leukemia patients in remission

Non-adherent peripheral blood mononuclear cells (NA-PBMNC) from 67 chronic myeloid leukemia (CML) patients in the first and two subsequent remissions, and 23 normal healthy donors were tested for NK and ADCC activities in short term chromium release assays using K562 and antibody-coated chicken RBCs as respective targets. CML patients in remission exhibited significantly reduced NK cytotoxicity (16. 1-19.7%) compared to normal healthy donors (47.4%). Of the patients tested, 55% exhibited NK levels below the mean percent cytotoxicity--2SD (12.5%) of normal donors (low responders), while 45% exhibited NK cytotoxicity above the 12.5% level (normal responders). On the other hand, CML patients in remission showed ADCC activity comparable to that of normal healthy donors (53.3%) irrespective of whether they belonged to normal NK responder group (55.5-65.0% ADCC) or low NK responder group (39.4-48.3% ADCC). The low or normal NK responder status of CML patients was not found to be related to either progression on the disease, or the type of drug used to bring about remission, or to the period in remission at the time of testing. In-vitro treatment of effector lymphocytes with recombinant human IFN alpha resulted in augmented of NK activity in both low and normal NK responder patients. The IFN-augmented NK activity in low responder patients however remained below the normal levels.

Modulation of tumour associated antigen expressed on human squamous cell carcinoma cell lines by recombinant interferon-α

Our earlier studies have shown that monoclonal antibody (Mab) 3F8E3 generated against a head and neck cancer cell line LICR-LON-HN2 showed reactivity with squamous cell carcinoma (SCC) irrespective of the tissue of origin and identified antigens on SCC cell lines AW 13516 and AW 8507. The affinity constants (Ka) for binding of Mab 3F8E3 to AW 13516 and AW 8507 cell lines were 6.2 x 10(8) and 4.3 x 10(8) mol/l and it identified 6.8 x 10(4) and 3.77 x 10(4) sites/cell, respectively, as determined by Scatchard analysis. Treatment of both the cell lines with recombinant human interferon-alpha (rHu-IFN alpha) increased the binding affinity of the Mab but did not increase the number of binding sites on the SCC cell lines. Shedding of antigen recognised by the Mab in the culture supernatant of the cell lines was reduced after rHu-IFN alpha treatment. The results suggest that rHu-IFN alpha may bring about a firm anchorage of the tumour associated antigen on the SCC cells. Cells modulated with rHu-IFN alpha may serve as better targets for assessing cell mediated as well as Mab mediated cytotoxicity in oral cancer patients.

Effect of administration of BCG, levamisole and irradiated leukemic cells on immune status and remission status in chronic myelogenous leukemia.

The immunomodulating effects of bacillus Calmette-Guérin (BCG) and levamisole with and without irradiated autologous leukemic cells were tested in patients with chronic myeloid leukemia (CML) after control of white cell count with busulfan. The response has been evaluated with respect to remission period and in vivo and in vitro immunological studies comprising delayed cutaneous hypersensitivity response to recall antigens and dinitrochlorobenzene, T cell and its subsets percentages, T-cell response to phytohemagglutinin, and to leukemia cell extracts by blastogenesis and leukocyte migration inhibition. Patients receiving BCG or levamisole did show marginal prolongation of remission, however immune parameters failed to show any improvement. In contrast, improvements in specific and nonspecific immune parameters were observed in patients receiving BCG or levamisole along with irradiated leukemic cells, however, concomitant clinical benefit was not obtained. Development of a better immunotherapeutic approach appears essential for immunomodulation in CML.

Study on ALL-1 gene alterations in Indian childhood acute leukemias: non-isotopic Southern blotting and molecular cytogenetics

We carried out this study to detect ALL-1 gene alterations in Indian childhood leukemias (n-84) using non-radioactive Southern blotting and FISH techniques. 18 (21.4%) patients showed altered ALL-1 gene. All 18 patients with altered ALL-1 gene did not have high WBC count and or typical CD10-/19+ phenotype. 4/18 were infants, while 14 were of 1-12 years of age. 13/18 children were boys. 14/18 expired within 1 year. Karyotyping detected abnormal chromosome 11 only in 4/43 patients and Classical t(4:11) in one AML patient but combination of Painting FISH and LS-FISH confirmed ALL-1 gene alteration in 17/18 cases. In addition, FISH identified nine translocations and multiple copies of ALL-1 gene in three cases which conventional cytogenetics had failed to detect. Our result indicates that a combination of Southern blotting, cytogenetic and FISH techniques are useful to identify ALL-1 gene alterations in childhood leukemias.

Modulatory effect of cytokines on natural killer and antibody dependent cellular cytotoxicity directed to squamous cell carcinoma targets by lymphocytes from oral cancer patients.

Cells from solid tumours are generally poor targets for natural killer (NK) cytotoxicity and antibody dependent cellular cytotoxicity (ADCC). In this paper, we have analysed NK cytotoxicity and ADCC mediated by peripheral blood mononuclear cells from healthy individuals and oral cancer patients before and after modulation with recombinant interleukin-2 (rIL-2), on target cells derived from two squamous cell carcinoma (SCC) cell lines prior to and after treatment with recombinant interferon-alpha (rIFN alpha). Target SCC cell directed monoclonal antibody 3F8E3 was used in ADCC. The results showed that the unmodulated SCC cells were poor targets for NK and ADCC compared to standard targets (K562 cells and chicken red blood cells, respectively). Modulation of targets alone with rIFN alpha showed moderate increase in their susceptibility while rIL-2 treated effectors could significantly lyse even unmodulated targets. Combined treatment of targets with rIFN alpha and effectors with rIL-2 showed additive enhancement in NK and ADCC activity against SCC cells. Lymphocytes from treated patients with recurrent disease could not efficiently lyse SCC targets even after combined modulation.

Functional Activities of Enriched Fc Receptor Bearing T Lymphocytes in Hodgkin’s Disease

Our earlier studies have demonstrated that the peripheral blood mononuclear cells (PBMNC) of patients with Hodgkins disease (HD) have reduced percentage of T mu cells and increased percentage of T gamma cells, while the splenic MNC showed the reversed proportions. In this paper, the functional abilities of subsets with these phenotypes have been investigated. T gamma and T mu cells were enriched from theophylline-sensitive and -resistant populations of lymphocytes obtained from PBMNC and splenic MNC of untreated HD patients and normal healthy donors. The effect of addition of these enriched populations on PHA and mixed leukocyte culture (MLC) responses of corresponding autologous MNC has been studied. The results showed that both PHA and MLC responses of normal as well as of HD lymphocytes could be suppressed by T gamma cells, while T mu cells failed to show an appreciable helper activity. The T gamma population in HD, therefore, appears to maintain suppressor function, thereby influencing the peripheral blood T cell responses.