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In these experiments, the cytokinesis-block technique was used to measure the spontaneous and radiation-induced micronucleus frequencies in lymphoblastoid cell lines from normal individuals and individuals with radiation-sensitive syndromes. The radiation-induced micronucleus frequencies were measured at different harvest times to investigate the optimum time to measure radiation-induced cytogenetic damage with the cytokinesis-block technique. The frequency of radiation-induced micronuclei was found to be independent of the frequency of binucleate cells in these cell lines. Longer incubation times were complicated by increasing numbers of mononucleate and multinucleate cells, and scoring slides at an early incubation time after the maximum frequency of binucleate cells is reached was recommended. Two of the cell lines (one established from an individual with ataxia telangiectasia, and one from an infant with mitotic instability) had significantly higher radiation-induced micronucleus frequencies than the control cell lines. Finally, the radiation-induced micronucleus frequencies in three of the cell lines were measured by flow cytometry, and the results ranked the cell lines in the same order of radiosensitivity as the cytokinesis block technique.

Monosomy 21 (M21) is a rare aneuploid condition which in certain cases leads to reduced levels of chromosome 21 gene products. We have prepared an Epstein-Barr virus lymphoblastoid cell-line (LCL) from patient with M21 who has immunological abnormalities, and analysed the expression of lymphocyte function-associated antigen-1 (LFA-1). This heterodimeric leucocyte integrin consists of CD11a (alpha) subunits non-covalently associated with CD18 (beta) subunits coded, respectively, by genes on chromosomes 16 and 21. To determine whether monosomy 21 results in decreased expression of LFA-1, monoclonal antibodies were used to compare the expression of CD11a and CD18 on the M21 LCL with LCL from trisomy 21 (Down's syndrome, T21), normal controls and a possible case of leucocyte adhesion deficiency. In addition, phorbol-ester-induced homotypic adhesion, an LFA-1-mediated effect, was compared in these LCLs. The results are consistent with a gene dosage mediated reduction of LFA-1 expression by the M21 LCL.

Thymidine incorporation in vitro by remission lymphocytes from a total of 6 patients with acute myeloid leukaemia (AML) was measured following stimulation by autochthonous and allogeneic AML blasts and cell lines. The early peak response to autochthonous blasts in 2 of these patients (48-72 h) is consistent with the concept of a population of lymphocytes pre-immunized to antigens carried by the blasts. Although stimulation in one patient was increased in the presence of more stimulating (S) blasts than responding (R) lymphocytes, positive responses in other tests were obtained at an S : R ratio of 1 : 1-5. When different methods of treatment of the stimulating autochthonous blasts were compared with untreated cells, mitomycin C gave the highest stimulation indices 2 out of 3 tests. Tissue culture medium in which autochthonous blasts had been incubated for 3-5 days failed to stimulate either remission lymphocytes alone, or combined cultures of lymphocytes with autochthonous or allogeneic blasts, suggesting that mitogenic factors released from autochthonous blasts are not responsible for lymphocyte stimulation. Treatment of autochthonous or allogeneic AML blasts with glycine-HC1(pH 3-0) to remove putative "blocking" factors failed to increase the stimulatory capacity of the leukaemic blasts.

Allogeneic lymphocytes can stimulate cell-mediated cytotoxicity (CMC) in lymphocytes from leukaemia patients against autologous leukaemia target cells. We have compared the capacity of different allogeneic lymphoid cells to stimulate CMC to fresh (i.e., patient) and cultured (MOLT 4, K562) leukaemic target cells in lymphocytes from an acute leukaemic patient and his HLA-identical siblings. Allogeneic lymphoid cells, and particularly a lymphoblastoid cell line, were effective in stimulating CMC to leukaemia targets. In some instances, however, leukaemia cells derived from the patient, mixed with allogeneic lymphoid cells stimulated synergistic CMC to the patient's leukaemia. We also found that the patient's leukaemia cells alone were able to stimulate CMC in HLA-identical sib lymphocytes to fresh and cultured leukaemia targets. Extra specificities on fresh leukaemia cells were revealed when these cells induced unpredicted CMC on normal lymphocyte targets when added to mixed lymphocyte cultures (MLC) between related and unrelated lymphocytes. Cytotoxic lymphocytes generated in MLC against the patient's HLA antigens were absorbed by monolayers of lymphocytes and leukaemia cells of the same HLA type as the patient, leaving residual CMC to fresh (patient) and cultured (K562) leukaemia target cells. In addition, CMC to the patient's leukaemia cells, stimulated in lymphocytes from the patient's HLA-identical sib by allogeneic cells, was absorbed by a monolayer of these allogeneic cells. This suggests cross reactivity between determinants on the leukaemia and allogeneic lymphocytes. The results of this study are consistent with expression of 'leukaemia antigen', which are not restricted to leukaemia cells but may also be expressed on lymphocytes.

To evaluate the potential of using fluorescence flow cytometry (FFC) in the diagnosis of Down's syndrome we analysed a series of Trisomy 21 lymphoid cell-lines (LCL) with a panel of monoclonal antibodies (MoAb) to the CD11a and CD18 subunits of lymphocyte function associated antigen 1 (LFA-1). In pairwise analysis of Trisomy 21 (T21) and normal LCL with 6 CD18 and 10 CD11a MoAb we found that T21 LCL could be distinguished from normal LCL with both types of MoAb, even though only the CD18 gene is duplicated in Down's syndrome. Experiments in which CD18 or CD11a were capped showed that the two subunits co-migrate on T21 and normal LCL, probably as heterodimers. This appears to explain the increased expression of CD11a on Trisomy 21 LCL. There was no evidence that T21 LCL express the other two leucocyte integrin subunits CD11b and CD11c, indicating that Trisomy 21 has little affect on tissue specific control of these molecules. In contrast, there appears to be reduced expression of an unrelated adhesion molecule LFA-3 on Trisomy 21 LCL. We discuss the relevance of these results in the diagnosis of Down's syndrome.

Twenty-two human leukaemias, comprising acute phase leucocytes from 13 acute myeloid and nine lymphoid leukaemias, were tested for susceptibility to spontaneous cell-mediated cytotoxicity (CMC) by untreated lymphocytes and lymphocytes treated for 18 h with 250 IU lymphoblastoid (Namalva) interferon (IFN-alpha). IFN-amplified killing (IAK) by lymphocytes from 24 normal lymphocyte donors was checked on the K562 erythroleukaemia cell line, for comparison with IAK on fresh leukaemias. Nine leukaemias were tested with lymphocytes from three donors, nine with lymphocytes from six donors, three with lymphocytes from nine donors, and one with lymphocytes from 11 donors. Some degree of susceptibility to IAK was found in five acute myeloid and five lymphoid leukaemias, which was markedly dependent upon the source of the effector lymphocytes and did not correlate with the degree of IAK on K562. The 12 other leukaemias were virtually resistant to IAK. The results emphasize the variability in the capacity of IFN-treated lymphocytes to lyse leukaemias that have not been adapted to tissue culture. The basis of effector recognition of cell line and fresh tumour targets is discussed.

Autologous acute myeloid leukaemia (AML), cells caused the suppression of incorporation of 3H-thymidine (3H-Tdr) by remission lymphocytes stimulated with allogeneic cells. In five patients, autologous AML cells suppressed 3H-Tdr uptake by lymphocytes stimulated with up to three different allogeneic cells. Responses to allogeneic AML cells were more strongly suppressed than responses to pooled allogeneic lymphocytes. Suppression was abolished by ultrasonic disintegration of the autologous AML cells, suggesting that a soluble factor was not involved. Suppression was absent from autologous AML cells exposed to ultraviolet light, or when untreated autologous AML cells were present in ratios of less than 1:1 to lymphocytes, or when added 24 or more hours after stimulation of remission lymphocytes with allogeneic cells. It is suggested that suppression is a property of the differentiative level of AML cells, rather than of their malignant properties, although malignant-transformation may bring AML cells into contact with circulating T cells in vivo. Autologous AML cells seem to interfere with the recognition phase of T cell function.

Leucocytes from sixteen untreated acute myeloid leukaemia (AML) patients were tested for their ability to stimulate lymphocytes from each of twelve normal donors. Of 192 tests between the stimulating AML leucocytes, and responding lymphocytes, 42%, resulted in positive lymphocyte stimulation, in contrast to one-way mixed lymphocyte culture (MLC) responses, involving the same lymphocyte donors, which were 100% positive. Lack of stimulation by AML leucocytes was significantly associated, in 11%, of the tests, with two or more HLA-A and/or-B antigens common to the responding lymphocyte and stimulating AML leucocyte. The most stimulatory of the AML leucocytes were obtained from two high-leucocyte-count acute myclomonocytic leukaemia patients. The stimulatory capacity of AML leucocytes did not correlate with the clinical fate of the cell donor. Presence of contaminating lymphocytes from the patient in the AML leucocyte samples did not account for the differences in stimulatory capacity between AML leucocytes. Limited in vitro viability of AML leucocytes was ruled out as a factor causing poor lymphocyte stimulation. Kinetic studies showed that AML leucocytes induce either an MLC-type response, or no response at all. Differences in response kinetics were observed between two normal and two remission AML patients, the latter receiving active immunotherapy. Pronase treatment of AML leucocytes failed to increase their stimulatory capacity, but distilled H2O markedly reduced it. The significance of the results is discussed in relation to lymphocyte stimulation by other types of allogeneic cell, and in the context of active immunotherapy of AML. [ABSTRACT FROM AUTHOR]

We have analysed homotypic adhesion induced by the phorbol ester TPA in EBV-immortalized Down's syndrome (trisomy 21) and normal lymphoblastold cell lines. Our results show that the trisomy 21 cells aggregated more readily than normal cells. The aggregation of the two types of cell was blocked effectively by the addition of monoclonal antibodies to the α and β chains (CD11a, CD18) of lymphocyte function-associated antigen (LFA-), but not by monoclonal antibodies to LFA-3 and the α chain of p150,95 (CD11c). Since TPA did not increase the expression of CD18 on trisoiny 21 cells, we conclude that the increased adhesiveness is the result of over-expression of CD18 as a result of gene dosage. [ABSTRACT FROM AUTHOR]

Human lymphocytes stimulated with phytohaemagglutinin (PHA) were fused with an HGPRT- murine lymphoma, BW5147, and a hybridoma BwFc93-1 was isolated and cloned in agarose. This human × mouse hybrid and nine clones derived from it were characterized by chromosome analysis, phenotypic and functional assays. Karyotyping and isoenzyme studies showed the presence of live human chromosomes in BwFc93-1 with preferential retention of three chromosomes—6, X and 15—in the clones. Membrane immunofluorescence analysis revealed that all the clones expressed human and mouse class I MHC antigens end the mouse T cell antigens Thy-1 and T200, but were devoid of human OKT3, OKT8 and mouse Lyt-2. Human OKT4 and OKM1 phenotypes were transiently expressed by one clone and mouse Lyt 1 by two other clones. Several T4-, Lyt-1- clones produced and bound human interleukin-2 (IL-2) indicating a lack of correlation between human T cell phenotype and function in those hybrids. There was also evidence of dichotomy in the secretion of IL-2 and expression of the IL-2 receptor since clones were identified which either bound or secreted IL-2. One clone expressing IL-2 receptors could be induced to produce human IL-2 by simultaneously stimulating with PHA and phorbol myristate acetate (PMA). [ABSTRACT FROM AUTHOR]

A single nucleotide polymorphism (SNP) is present at position -174 of the human interleukin-6 gene. The risk of developing Hodgkin's lymphoma (HL) in young adults decreases with an increasing number of C alleles at this position. We analysed the effect of this SNP on incidence and outcome in HL. DNA samples from 408 cases and 349 controls were screened and analysed following stratification by age, histological subtype and Epstein–Barr virus status. Although the risk of classical HL in young adults decreased with increasing C alleles, case–control differences were not significant. An excess of G alleles was observed for nodular lymphocyte predominant HL in young adults (n = 21), which was significant. [ABSTRACT FROM AUTHOR]