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Human squamous cell carcinomas (SCC) of the oral cavity were successfully established as xenografts in nude mice. Tumours with higher malignancy scores and involvement of lymph nodes in patients were more readily accepted as xenografts in nude mice. The xenografted tumours were characterised with respect to morphology, histology, DNA index and expression of tumour-associated antigens (TAA). Flow cytometric analysis of cellular DNA content revealed that many of the xenografts retained the parent tumour DNA pattern while some of the xenografts showed progression to aneuploidy. All the xenografted tumours expressed TAA recognised by monoclonal antibody (MAb) 3F8E3. On Western blotting, MAb 3F8E3 recognised proteins of molecular weight 62-64 kDa on parent and xenografted tumours. In general, the xenografts reflect many of the characteristics of the tumours from which they were derived and may provide a useful model for investigating newer approaches of treatment and diagnosis.

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.

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.

Soluble inhibitory factors (SIF) have been demonstrated in the sera of cancer patients, which interfere with the T-cell activation process. We have shown that the major contributory factor to the inhibitory effect of sera from patients with Hodgkin's disease (HD) could be the soluble form of Interleukin-2 receptors (sIL-2R). The parameters studied to show the presence of SIF include (i) inhibiton of mitogen-induced proliferation; (ii) status of high- and low-affinity IL-2R; and (iii) internalization of IL-2-IL-2R complex, by lymphocytes from healthy donors activated with mitogen in presence of HD sera. Parameters studied to show the inhibitory role of sIL2R include (i) quantitation of sIL-2R in HD sera; (ii) effect of high-sIL-2R-containing sera on mitogen-induced proliferation and detection of IL-2 in activated lymphocyte culture supernatants; (iii) effect of exogenous IL-2 supplementation; and (iv) abrogation of inhibitory activity of sIL-2R-containing sera after passing them through IL-2 affinity columns. Our results show that 6/23 HD sera tested had high inhibitory activity (greater than 50% inhibition of mitogen-induced proliferation). The SIF did not affect expression of high- and low-affinity IL-2 receptors, or internalization of the complex by activated lymphocytes. Ten of the 15 sera tested showed significantly high levels of sIL-2R. Pooled sera with high sIL-2R content inhibited mitogen-induced proliferation of normal lymphocytes with a concomitant reduction in IL-2 activity in the lymphocyte culture supernatants. When supplemented with exogenous IL-2, there was a partial recovery of the inhibitory effect. When sIL-2R containing serum pool was passed on IL-2 affinity columns, the inhibitory effect was reduced. The eluted "sIL-2R" adsorbed on the IL-2 column showed anti-proliferative effect.

In this paper, we have correlated the ability of peripheral blood lymphocytes (PBL) from Hodgkin's Disease patients to proliferate in response to a mitogen, phytohaemagglutinin (PHA), with production of lymphokines interleukin-2 (IL-2) and interferon gamma (IFN gamma), 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 IFN gamma in the supernatants of activated lymphocytes from patients with Hodgkin's disease were significantly reduced compared to those of healthy donors. Substage-B patients showed marked reduction in the ability to produce IFN gamma. Levels of IL-2 and IFN gamma in the culture supernatants of PBL from Hodgkin's disease patients correlated positively with proliferative responses, when analysed by linear regression (r = 0.79 and r = 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 Hodgkin's 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 Hodgkin's disease.

Four 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.

The natural killer (NK) and lymphokine-activated killer (LAK) cell activities of peripheral blood lymphocytes from chronic myeloid leukemia (CML) patients in remission and from healthy donors have been studied. Regression analysis to compare both cytotoxic responses in individual donors and the frequency of LAK cell precursors was also carried out. About 42% of CML patients in remission showed low NK activity (less than the mean percentage NK activity of healthy donors — 2 SD) and were categorised as low NK responders. The stage of remission or the drugs used to bring about remission did not influence the NK status. The LAK activity of low NK as well as normal NK responder CML patients was significantly low against the NK-sensitive K562 cell line and the NK-resistant VIP (melanoma) and T-24 (bladder carcinoma) tumor targets, as assessed by linear regression analysis. Allogeneic leukemic cells were more resistant to killing, especially by patients' LAK cells. The frequency analysis of LAK cell precursors revealed a significant reduction in the LAK cell progenitor frequency in CML patients in remission.

We report the natural killer (NK) and lymphokine activated killer (LAK) cell activities in peripheral blood lymphocytes (PBL) from untreated patients with Hodgkin's disease (HD) and from healthy donors. The frequency of LAK cell precursors was also studied using limiting dilution analysis (LDA). About 75% of the HD patients had normal NK activity. There was a higher percentage of low NK responders (mean percent NK activity of healthy donors--2 SD) in patients with lymphocyte depletion histologic grade of the disease and those who were in clinical stage IV, suggesting a correlation of decrease in NK activity with poor prognosis. We found efficient LAK activity against the NK-sensitive K562 cells and NK-resistant VIP (melanoma) and T-24 (bladder carcinoma) tumour targets in both low and normal NK responder HD patients, irrespective of the histopathological grade and clinical stage of the disease. In concordance with their good LAK cell activity, HD patients showed a frequency distribution of LAK cell progenitors in the PBL comparable to that of healthy donors.

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.

Sera from leprosy patients across the clinical spectrum, healthy contacts, tuberculosis patients, and healthy donors were tested for their reactivity with antigens of mycobacterial strain ICRC (a cultivable mycobacterium) and Mycobacterium leprae by immunoprecipitation technique. Using M. leprae antigens, it was not possible to distinguish between reactivities of sera from lepromatous, borderline lepromatous, borderline tuberculoid, and tuberculoid leprosy patients. All these sera identified M. antigens with molecular masses of 47, 36, 21, and 14 kDa. When the same sera were tested for their reactivities with antigens of mycobacterial strain ICRC, several differences were observed. The 21-kDa antigen of mycobacterial strain ICRC was exclusively precipitated by sera from all lepromatous leprosy patients and from those undergoing erythema nodosum leprosum reaction. Sera from all the other donors tested failed to identify the 21-kDa antigen of mycobacterial strain ICRC. The 14-kDa protein of mycobacterial strain ICRC was identified by sera from a few lepromatous leprosy patients (5 of 26) and all their contacts. Our studies indicate that antigens present on cultivable mycobacteria rather than species-specific antigens may prove to be useful in the serodiagnosis of leprosy.

Three monoclonal antibodies (MAb) raised against K562 cells (erythroleukemic cell line) reacting specifically with myeloid maturation related antigens, were characterized. MAb 4.3 (IgG2bk) reacted with promyelocytes, metamyelocytes and myelocytes, MAb 4.6 (IgG3k) also identified the myeloid blasts, while MAb 4.8 (IgG1k) reacted with metamyelocytes. The affinity constant of the MAbs ranged from 0.8 × 107–3.9 × 108 M−1 and the binding sites on K562 cells varied from 8 × 104–4 × 105. In competition RIA MAbs 4.6 and 4.8 competed with each other for binding sites on K562 cells. In Western blot analysis the MAbs reacted with 66,000 mol.wt and 84,000 mol.wt peptides on K562 cells and 97,000 mol.wt peptide on chronic myeloid leukemia (CML) cells. These MAbs may help in immunophenotyping of myeloid leukemias.

doi of original article 10.1016/S0145-2126(03)00030-4. ∗ Corresponding author. Tel.: +91-22-2412-9786/9787x542; fax: +91-22-2413-7000. E-mail address: jaya vyas@hotmail.com (J. Vyas). 1 Present address: Bombay Hospital, Marine lines, Mumbai 400020, India. 2. The photograph placed with figure legend 5 is to be placed with 4. 3. The photograph placed with figure legend 6 is meant to be placed with 5.

In the present paper attempts have been made to investigate suppressor cell activity in CML patients in first and subsequent remissions in order to study the relationship between suppressor cell activity and progression of the disease. For this purpose, the ability of Con A activated suppressor cells from peripheral blood of CML patients in 1st, 2nd and 3rd remission to suppress PHA response of autologous lymphocytes is investigated and compared with that of normal healthy donors. The ability of Con A activated cell population to form rosettes with autologous RBCs (ARFC) is also investigated. The results indicate that lymphocytes from CML patients in 1st (61.8 +/- 6.1%), 2nd (62.6 +/- 3.0%) and 3rd (55.3 +/- 4.8%) remissions show significantly high suppressor cell activity than normal healthy donors (36.5 +/- 1.9%) when activated with Con A. Similarly, generation of spontaneous suppressor cell activity was also higher in 1st (23.3 +/- 4.7%) and 2nd (25.3 +/- 4.2%) remission lymphocytes than controls (10.1 +/- 2.5%). In the 3rd remission however, the spontaneous suppressor cell activity (14.5 +/- 3.2%) was comparable to controls. Thus it appears that a higher suppressor cell precursor population is present in CML patients in remission. However, this could not be correlated with the progression of the disease. CML patients in 1st remission also revealed an increased percentage of ARFC which correlated with the suppressor cell function. The ARFC activity tested in a few patients in subsequent remissions was comparable with controls although functional suppressor activity was increased.

The immunomodulating effects of bacillus Calmette-Guerin (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 immunomodula-tion in CML.

Peripheral blood lymphocytes (PBL) from patients with oral cancer (treated and untreated), oral leukoplakia and healthy donors; lymphocytes from metastatic and non-metastatic lymph nodes (met LNL and non-met LNL); and tumor infiltrating lymphocytes (TIL) were tested for proliferative response to mitogen PHA (phytohemagglutinin) and its augmentation by recombinant Interleukin-2 (rIL-2), for expression of Tac antigen (CD25) and for production of IL-2. Depressed PHA responses were found in PBL of treated and untreated patients, and in TIL. Addition of IL-2 could bring about 16% to 31% augmentation in lymphocyte response to PHA from all the three sources. PBL from 50% of healthy donors, 45% of patients with leukoplakia, 25% untreated oral cancer patients and 35% treated oral cancer patients showed IL-2 mediated augmentation of PHA response. While, 40% non-met LNL samples, 70% met LNL samples and only 23% TIL samples showed increased mitogen induced proliferation by IL-2. The augmented levels of PHA response of PBL from treated and untreated patients, and of TIL, were still below those of normal PBL. PBL from patients with leukoplakia, treated oral cancer patients and TIL showed depressed CD25 antigen expression. Depressed IL-2 production was observed only in PBL of leukoplakia patients. Thus the IL-2 mediated events of T cell activation from different lymphoid sources in patients with oral cancer did not correlate with their proliferative responses.

Peripheral blood monocytes and draining lymph node macrophages from patients with squamous cell carcinoma of the oral cavity, monocytes from patients with oral leukoplakia and those from healthy donors were assessed for FcR. HLA-DR expression and interleukin-1 (IL-1) production after activation with LPS or IFN gamma. Monocyte cytotoxicity was also tested after activation with LPS, IFN gamma, IL-2 singly, or in combinations at suboptimal concentrations. The results showed that the percentage of activated monocytes expressing FcR was significantly low in untreated oral cancer patients, however, the proportion of HLA-DR positive cells was normal. The unstimulated monocytes from oral cancer patients showed spontaneous generation of IL-1. Upon activation, few patients could produce IL-1 to normal levels. The unstimulated monocytes from untreated patients and treated patients with recurrence also exhibit significantly higher tumoricidal activity. Treatment of monocytes with combinations of two modulators (IFN gamma, LPS and IL-2) induced significantly higher cytotoxicity.

Sixteen untreated patients with squamous cell carcinoma of the oral cavity were tested for in vitro immune status in comparison with the normal healthy donors. The parameters investigated were total leukocyte and lymphocyte counts, percentages and absolute counts of T- and B-cells in circulation, subsets of T-cells identified by the Fc receptors, phytohemagglutinin (PHA), and mixed lymphocyte culture (MLC) responses, natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) activities, and circulating immune complexes (CICs). Eight of these patients were retested 3 to 6 months after surgery. The results showed that there was an increase in leukocyte and lymphocyte counts, an increase in the percentage and absolute number of B-lymphocytes, an increase in the percentage of T-gamma cells, suboptimal PHA and MLC responses, normal NK and ADCC activities, and increased levels of CICs in untreated oral cancer patients. In the postoperative stage, except for a reduction in leukocyte and lymphocyte counts, other abnormalities remained unchanged. The CICs in treated patients correlated with the tumor load in that in three patients showing recurrence, the CIC level remained elevated, whereas in patients without evidence of the disease the CIC level was either low or comparable to the upper normal limits.

Lymphocytes from 13 chronic myeloid leukaemia (CML) patients in remission were tested for their ability to differentiate in vitro into a cell population cytotoxic to autochthonous target leukaemic cells. CML remission lymphocytes were cultured in vitro with autochthonous leukaemic cells and allogeneic normal lymphocytes from an unrelated donor, singly or in combination. The cytotoxic lymphocytes obtained after 7 days of culture were tested for their ability to kill autochthonous leukaemic cells in a 3h 51Cr-release assay. It was found that with the allogeneic stimulus alone, cytotoxicity was generated in 5/13 cases, whilst stimulation of lymphocytes with autochthonous leukaemic cells alone induced cytotoxicity in 7/13 cases. In contrast, anti-leukaemic cytotoxicity was shown in 12/13 cases when responder lymphocytes were stimulated with both autochthonous leukaemic and unrelated allogeneic normal lymphocytes. The specificity of cytotoxicity was confirmed using other targets such as autochthonous PHA-transformed lymphoblasts and mouse L1210 cells. In 1/5 cases, CML remission lymphocytes stimulated with autochthonous leukaemic cells showed cytotoxicity to PHA-transformed autochthonous normal lymphoblasts, whilst 1/4 patients showed nonspecific cytotoxicity to L1210 cells when lymphocytes were cultured in MLC or MLTC, as well as in a 3-cell assay.

Sixteen chronic myeloid leukaemia (CML) patients in remission were tested with solubilized membrane antigens from CML leukaemic cells, CML blasts, AML blasts and ALL blasts for cellular immunity in vitro by lymphocyte transformation (LT) and leucocyte migration inhibition (LMI) assays. Twelve CML patients in remission were tested with allogeneic PHA-transformed normal lymphoblasts. As controls, peripheral-blood leucocytes from 9 healthy persons were tested with the same antigen preparations. It was seen that 8/16 (50%) CML patients responded to CML antigens by both LT and LMI assays, while 5/16 (31%) patients reacted to CML blasts and 44% (7/16) patients reacted to AML blast antigens. It was interesting to note that 5/11 (45%) CML patients reacted to ALL blast antigens by both assays. One out of 12 patients reacted to PHA-transformed lymphoblasts. None of the healthy controls reacted to leukaemia-associated antigens. The results suggest the sharing of antigens between myeloid leukaemic cells, myeloid blasts and lymphoid blasts.

Functional evaluation of T-lymphocytes from peripheral blood and spleens in Hodgkin's disease

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.

Lymphokine-activated killer (LAK) cells, generated from peripheral blood lymphocytes (PBL) from patients with oral cancer or oral leukoplakia and from healthy donors showed comparable lysis of 6 target tumor cell lines, including 3 derived from head and neck and oral cancers. The tumor burden of the host did not appear to influence the systemic LAK activity. LAK activity of lymphocytes infiltrating the tumor tissues (TIL) was also comparable to that of the PBL. Both TIL and PBL showed a parallel increase in proportion of HNK-I ++ and CD-25 + CELLS upon activation with IL-2. The lymph-node lymphocytes (LNL) from metastatic (met) and non-metastatic (non-met) draining lymph nodes, however, showed reduced LAK activity and an increase in CD8 CELLS, in addition to CD25 + and HNK-I + cells, when cultured with IL-2. When IL-2-activated LNL were co-cultured with autologous PBL during IL-2 activation of the latter, a strong suppressive effect was exerted by LNL In contrast, IL-2-activated PBL did not suppress autologous LAK generation in spite of an increase in CD8 + cells seen after activation with IL-2. Frequency distribution of LAK precursors was significantly lower in LNL than in PBL from oral cancer patients. LAK precursor frequency in TIL was comparable to that of PBL. The results show that, in oral cancer, regional lymph nodes may not have adequate IL-2-inducible cytotoxic potential, due to a reduced number of LAK progenitors and possible activation of suppressor cells. Alternatively, TIL can be a potential source for LAK cell function.

Mouse monoclonal antibody (MAb) 3F8E3 (IgG3k) was developed against the head and neck cancer cell line LICR-LON-HN2. Subjected to indirect immunofluorescence, the MAb reacted exclusively with SCC cell lines and showed no reactivity with normal or transformed mouse and human non-SCC cell lines and hematopoietic cell lines. The radiolabelled MAb showed an affinity constant of 1.8 x 10(8) M-1 with HN2 cells and identified 2.07 x 10(4) sites/cell by Scatchard analysis. It identified 2 peptides from membrane extracts of HN2 cells by Western blotting. Avidin-biotin-complexed immunoperoxidase staining on cryostat sections of tumors from various tissues revealed that 3F8E3 reacted mainly with the membrane antigens of well differentiated SCC cells of oral cavity, larynx, esophagus, lung, uterine cervix, metastatic nodes of patients with oral cancer, and dysplastic cells in oral leukoplakia. The MAb did not react with poorly differentiated cells of Ca esophagus, adenocarcinoma of breast, stomach and colon, renal-cell carcinoma and soft-tissue sarcoma.

Peripheral blood lymphocytes (PBL) from chronic myeloid leukemia (CML) patients in remission were stimulated in vitro, in a 3-cell assay with autologous leukemic cells or autologous bone marrow (BM) cells alone, or each in combination with allogeneic PBL. The responder cells were used as effectors in a 4-h 51Cr release cytotoxicity assay using autologous targets such as leukemic cells, BM cells, phytohemagglutinin-induced lymphoblasts, and allogeneic K562 (erythroblastoid leukemic cell line) target cells. Sensitization of lymphocytes from CML patients with either autologous leukemic cells or BM cells generated cytotoxic cells (CTCs) capable of killing both the targets. These results suggested that in CML, the PBL may have been sensitized to myeloid maturation-releated antigens in vivo, which, on secondary stimulation in vitro, may result in differentiation of CTCs cytotoxic to immature myeloid cells, either from autologous leukemic cells or autologous BM. The inability of PBL from patients with oral cancers to lyse autologous BM cells upon in vitro stimulation, supported this possibility. Clonogenic assays conducted to assess the colony forming potential of BM cells which had interacted with CTCs indicated that there was about 37% reduction in committed granulocyte stem cell colony formation without an appreciable change in committed granulocyte/monocyte stem cell units and clusters. Therefore, since the BM toxicity of the CTCs is not very high, these cells may have a potential clinical use in CML.

Circulating immune complexes (CICs) in sera from patients suffering from chronic myeloid leukemia (CML) at initial diagnosis, in 'remission', at relapse and in blastic crisis have been quantitated using fluid [125I]Clq binding assay in terms of per cent binding activity and microgram/ml aggregated human globulin (AHG) equivalents. The Clq binding activity (Clq-BA) has been compared within the groups of CML patients in different phases of the disease as well as with sera obtained from normal healthy donors. The results showed that the mean Clq-BA was significantly increased in CML patients at initial diagnosis (25.74 +/- 3.48, p less than 0.001), in relapse (53.36 +/- 6.9, p less than 0.001) and in blastic crisis (60.5 +/- 8.7, p less than 0.001) when compared to control sera. Sera of 'remission' patients showed significant decrease in Clq-BA when compared to sera collected in active phases of the disease, however, the values were still significantly higher (12.87 +/- 1.58, p less than 0.02) than those of normal healthy donors. When the levels of CICs as assessed by Clq-BA were compared with the WBC/blast counts of CML patients in chronic as well as blastic phase, it was noted that the variations in numbers of circulating leukemic cells do not correlate with the CIC levels. The significance of assessment of CIC levels in monitoring the disease in CML patients is discussed.

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%). When the effectors were treated with IL-2 or IFN, much higher ADCC responses were obtained. Combination of treatment of effectors with IL-2 and targets with 4.6E10 augmented the cytotoxicity of low NK responder CML patients to normal levels.

Our earlier studies have demonstrated that the peripheral blood mononuclear cells (PBMNC) of patients with Hodgkin's 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.

In the present investigations attempts have been made to study factor(s) involved in impairment of cellular immunity in Hodgkin's disease (HD). Peripheral blood lymphocytes from 42 untreated HD patients in differnent stages of the disease showed depressed PHA responsiveness as compared to 23 mormal healthy individuals. Peripheral blood lymphocytes of 7 13 HD patients showed partial restoration of ability to respond to PHA after overnight incubation in tissue culture media, supplemented either with human AB serum or fetal calf serum (FCS). The restoration appeared to be more common in cultures incubated in media supplemented with FCS than with AB serum. Supernatants of lymphocyte cultures from HD patients and their sera showed significant inhibition of PHA induced transformation of lymphocytes obtained from healthy donors. In contrast, normal lymphocyte culture supernatants and sera were less inhibitory to PHA induced transformation of normal lymphocytes. Cultures of HD lymphocytes, which failed to restore PHA responsiveness after overnight incubation, also showed inhibitory factors in supernatants.

Cytotoxic 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.