With the objective of developing human T-T cell hybrids producing B-cell growth factor, we fused concanavalin A-activated T lymphocytes with cells of the Jurkat T cell line. The hybrids were selected on the basis of their ability to form colonies in soft agar, whereas the parent Jurkat T cell line did not. T-T cell hybrids were HLA-typed, screened by functional tests, and recloned by limiting dilution. In addition to obtaining B-cell growth factor-producing hybrids, we also obtained certain other T-T cell hybrids (as determined by HLA-typing) producing suppressor factors inhibiting proliferative responses and antibody production by human lymphocytes. Subsequently, a suppressor factor with similar inhibitory properties was identified in supernatants of the Jurkat T cell line. However, the Jurkat factor exhibited different biochemical and functional properties than the hybridoma-derived suppressor factors. Using two-parameter cell cycle analysis and the metachromatic fluorochrome acridine orange, we found that the hybridoma-derived 160 and 169 suppressor factors arrested phytohemagglutinin-induced proliferative of peripheral blood mononuclear cells in the G0/G1 phase of the cell cycle, whereas the Jurkat suppressor factor arrested proliferation in the S phase. Incubation of peripheral blood mononuclear cells with the 160, 169, or Jurkat suppressor factors for 24 hr at 37 degrees C, followed by washing, did not alter their cell cycle progression (or RNA content) in response to stimulation with phytohemagglutinin. The hybridoma-derived 160 and 169 suppressor factors and the Jurkat factor inhibited the growth but not the viability of cells from the following human tumor cell lines: A673 sarcoma cell line, SK-LC-6 and SK-LC-14 lung cell lines, SB, Raji, and Daudi lymphoblastoid cell lines, and FARR malignant melanoma cell line. In contrast, it did not affect the growth of murine L1210 cells and FS-4 normal human diploid fibroblasts. The hybridoma-derived 160 suppressor factor was selected to investigate its effect on cell-mediated cytotoxicity. The 160 suppressor factor did not inhibit natural killer cytotoxicity or its augmentation by interferon alpha or interleukin 2 or the generation of lymphokine-activated killer cells. However, this factor partially inhibited the generation of specific T cell-mediated cytotoxicity.

Exposure of exponentially growing L1210 cells to 5 mM and higher concentrations of caffeine perturbs their progression through the cell cycle and results in increased sensitivity of DNA in situ to denaturation. The latter is detected by the increased metachromatic stainability of DNA with acridine orange (AO) and sensitivity to S1 nuclease, measured by flow cytometry. Decreased DNA stability is generally characteristic of chromatin condensation and in untreated cells is observed in mitosis or quiescence (G0). The caffeine-induced decrease in DNA stability affects the interphase cells regardless of their position in the cycle and the changes are stochastic, concentration- and time-dependent. Populations of cells responding to caffeine are very heterogenous with respect to the degree of destabilization of DNA; sensitivity of DNA to denaturation of the maximally affected cells is similar to that of untreated cells in mitosis. The present method allows one to quantitatively express effects of caffeine on nuclear chromatin in individual cells of large cell populations and may be employed in studies correlating chromatin changes induced by this agent with its effects in modulation of cell sensitivity to radiation or antitumour drugs.

We have recently identified a family of suppressor factors produced by certain human T-T cell hybridomas that we developed (references 1 and 2) and by the Jurkat T cell line. These suppressor factors significantly inhibited proliferative responses to mitogens and allogeneic cells in mixed lymphocyte culture and antibody production by human peripheral blood mononuclear cells. We investigated and report here the effect of these suppressor factors on certain in vitro murine immune responses. Suppressor factors produced by certain of these hybrids, such as 153, 160, 170 and by the Jurkat T-cell line were able to inhibit: (1) proliferative responses to mitogens of mouse thymocytes and splenocytes; (2) proliferative responses of mouse splenocytes to allogeneic cells in mixed lymphocyte cultures; (3) primary in vitro antibody responses of mouse spleen lymphocytes to sheep erythrocytes; (4) primary in vitro antibody responses of mouse spleen lymphocytes to a T-cell independent antigen (TNP-Ficoll). Inhibition of murine immune responses in vitro by these suppressor factors was regular and reproducible and it was observed in a large number of experiments. In contrast, suppressor factors produced by the 169 and by the 77(38F3) hybrids did not suppress the murine immune responses. The basis for these differences are not known at the present. The ability of human suppressor factors to inhibit effectively mouse immune responses provides an additional opportunity for the characterization of the properties of these factors in vivo using mouse models of human disease.

We have recently developed a new method (Hybridoma 6:589, 1987) for the generation of human T-T cell hybrids. This method is based on a new selection procedure that involves cloning the hybrids in soft agar, screening by HLA-typing or appropriate functional tests and recloning by limiting dilution. T-T cell hybrids were separated from the parent line on the basis of their ability to form colonies in soft agar, whereas the parent lymphoblastoid T cell lines did not. HAT medium was not used in our selection procedure. Using this method, we have succeeded in developing human T-T cell hybrids (as determined by HLA-typing) constitutively producing B cell growth factor (BCGF) (Hybridoma 6:589, 1987) or suppressor factors. These hybrids were obtained by fusing MLC or Con A T cell blasts with cells from the Molt 4 or Jurkat lymphoblastoid T cell lines. T-T cell hybridomas, derived by fusing Con A-stimulated lymphocytes with cells from the Jurkat T cell line, produced suppressor factors inhibiting: (1) proliferative response in vitro of human peripheral blood mononuclear leukocytes to mitogens and to allogeneic cells in mixed lymphocyte culture; and (2) immunoglobulin synthesis and secretion by mononuclear leukocytes in the PWM-induced differentiation system in vitro. A suppressor factor with these inhibitory properties was also identified in supernatants of the Jurkat T cell line. These suppressor factors were ammonium sulphate precipitable, pH 2 labile, non-dialyzable and they were inactivated by treatment at 56 degrees C for 30 minutes. They exhibited a molecular weight in the range of 50,000-70,000, as determined by gel filtration, and were not gamma or alpha interferon or lymphotoxin/TNF. They did not lyse human lymphoblastoid tumor cell lines nor did they affect the viability and cell numbers of human mononuclear cells even after prolonged incubation (88 hr). They appeared to be cytostatic rather than cytotoxic molecules. The Jurkat suppressor factor is different from those produced by the hybrids on the basis of: (a) different isoelectric points; and (b) the ability of the Jurkat factor to arrest proliferation to PHA of human mononuclear cells in the S phase, whereas the 160 and 169 factors arrest proliferation at the G1 phase of the cell cycle. Certain of these suppressor factors (produced by the hybrids 153, 160, 170, and the Jurkat T cell line) also inhibited proliferative responses of mouse lymphocytes in vitro. In contrast, suppressor factors produced by the 169 and 77 hybrids did not inhibit any murine responses.

The indolizidine alkaloid swainsonine (SW) is an inhibitor of lysosomal alpha-mannosidase reported to have antimetastatic activity in animal models. The cells grown in its presence develop truncated (hybrid) surface oligosaccharides that may alter their functional properties dependent on interactions of various ligands with membrane receptors. In the present study we observe that SW enhances stimulation of human lymphocytes induced by suboptimal concentration of concanavalin A. The enhancement is manifested by an increased proportion of cells undergoing transition from G0 (G1Q) to G1 and progressing through the cell cycle (S + G2 + M). In contrast, SW suppresses stimulation of lymphocytes by phytohemagglutinin, and the degree of suppression is greater when measured by the number of cells progressing through the cell cycle (S + G2 + M) than by the proportion of cells entering G1 phase. The suppression remains evident even when SW is added 12 h after phytohemagglutinin, suggesting that SW modifies membrane receptors that develop in G1 and are necessary for cell entrance to S phase. The modification of receptors by SW thus up-regulates stimulation by concanavalin A and down-regulates stimulation by phytohemagglutinin. SW has no effect on lymphocyte stimulation induced by OKT3 monoclonal antibody or on the progression of cells from three leukemic cell lines, HL-60, L1210, and MOLT-4, through the cell cycle. The present data are compatible with the possibility that the reported suppression of the growth of metastatic mouse tumors by SW may be due to the immunomodulatory properties of this alkaloid.

DNA content and in situ sensitivity to denaturation were analyzed by flow cytometry of individual cell nuclei isolated from 40 breast carcinomas, nine fibroadenomas, and 14 samples of normal breast tissue. The extent of DNA denaturation induced by acid was expressed as alpha t, which represents the fraction of DNA staining metachromatically red with the fluorochrome acridine orange. In all cases of normal breast tissue DNA was very sensitive to denaturation and the frequency distribution of alpha t values was unimodal with over 90% of cells having alpha t above 0.6. All fibroadenomas were diploid; four had unimodal alpha t as in normal tissue and five had a bimodal distribution with an additional peak below 0.6. Twenty-seven adenocarcinomas (67%) had a DNA index above 1.0; of these 24 had bimodal alpha t distributions. Among 13 diploid carcinomas 10 had bimodal alpha t distributions. Statistically significant differences were observed in alpha t distributions of normal versus tumor breast tissue (P less than 0.005). In normal tissue and in all tumors a predominant proportion of cells with S and G2 + M DNA content were characterized by DNA resistant to denaturation (alpha t below 0.6). Of interest, the diploid cells from aneuploid tumors which may represent reactive host cells often displayed bimodal distributions of alpha t. These results may be interpreted in light of earlier studies demonstrating increased resistance of DNA to denaturation in diffuse chromatin of proliferating and/or transcriptionally active cells, and greater sensitivity to denaturation of DNA in condensed chromatin of quiescent cells. Thus, the presence of the second peaks representing cells with low alpha t values in breast tumors may indicate a high proportion of proliferating cells, whereas high alpha t populations may represent quiescent and differentiating (condensed chromatin) or dying (pycnotic nuclei) cells. It is likely that the low alpha t diploid cells detected in aneuploid tumors may represent the reactive (transcriptionally active and/or proliferating) infiltrating host cells (i.e., lymphocytes, monocytes) whose presence may also be of prognostic value. The data suggest that a DNA denaturability assay may be useful to characterize tumor and infiltrating host cell populations.

We investigated helper and suppressor functions to B-cell responses and T-T cell interactions of purified T4 and T8 cells from 20 untreated patients with B-cell chronic lymphocytic leukemia (CLL). Appropriate mixtures of purified T4 or T8 cells from patients with CLL were cultured with purified B cells or T4 and B cells from normal donors for 7 days with pokeweed mitogen (PWM). IgM, IgA, and IgG produced were determined in the supernatants of these cultures by a heavy chain- specific enzyme-linked immunosorbent assay (ELISA) and compared to those obtained by the corresponding mixtures of T4, T8, and B cells from normal donors. Purified T4 cells from 14 of 20 patients with CLL exhibited defective helper function (P less than .001) to immunoglobulin (Ig) production by purified B cells from normal donors. Purified T4 cells from 6 of these 14 patients were able to suppress significantly (P less than .001) and in a concentration-dependent manner Ig production by mixtures of T4 and B cells from normal donors, in the absence of T8 cells. These suppressor effector T4 cells from certain patients were partially radiosensitive. Purified T8 cells from 8 of 20 patients with CLL exhibited excessive suppressor activity. These cells significantly suppressed (P less than .001), Ig production by mixtures of T4 and B cells from normal donors to a degree significantly higher (P less than .005) than that observed by equal numbers of T8 cells from normal donors. This inhibition was dependent on the numbers of the T8 CLL cells added to the cultures. Excessive suppressor activity by T8 CLL cells was at least in part radiosensitive in four of eight patients. These results demonstrate a wide range of immunoregulatory T-cell abnormalities in patients with CLL. Naturally occurring T4 suppressor effector cells, directly inhibiting Ig production by mixtures of T4 and B cells, in the absence of T8 cells, are present in certain patients with CLL.

Monoclonal antibodies (mAb's) recognizing the CD3 T-cell differentiation antigen induced the generation of suppressor cells. These cells inhibited (1) proliferative responses of human peripheral blood mononuclear cells (PBMC) to PHA and allogeneic cells in mixed leukocyte culture; (2) proliferative responses of purified E-rosette-negative cells to Staphylococcus aureus Cowans I; and (3) de novo immunoglobulin synthesis and secretion in the pokeweed mitogen (PWM)-induced differentiation system. Monoclonal antibodies recognizing other T-cell differentiation antigens (anti-Leu 2a, anti-Leu 3a, and anti-Leu 5) did not induce the generation of suppressor cells, even at very high antibody concentrations. Statistically significant differences were not observed in the ability of the OKT3 and anti-Leu 4 mAb's to induce suppressor cells. Monocytes were not required for the generation of anti-CD3-induced suppressor cells. F(ab')2 fragments of the OKT3 mAb's were equally effective when compared with intact antibody molecules in inducing suppressor cells, although they did not induce proliferative responses. Proliferation was not required for the induction of suppressor cells. Irradiation (2500 rad) of PBMC before incubation with the anti-CD3 mAb did not affect the generation of suppressor cells. Furthermore, anti-CD3-induced suppressor cells were radioresistant. Addition of recombinant IL-2 to the cultures of responding cells and suppressor cells did not reverse the suppression. In vitro treatment of anti-CD3-induced suppressor cells with either the OKT4 mAb plus complement or the OKT8 mAb plus complement partially decreased the suppression of proliferative responses of PBMC to PHA or allogeneic cells in mixed lymphocytes culture. However, treatment with both OKT4 and OKT8 mAb's plus complement or the OKT11 mAb plus complement completely abolished the suppression. These results suggest that the suppressor cells are of the T11+T4+T8- and T11+T4-T8+ phenotypes. In other experiments, T4+T8- and T8+T4- cells were isolated from PBMC treated for 48 hr with anti-CD3 mAbs. Both these two populations significantly inhibited proliferative responses of autologous PBMC to PHA and de novo immunoglobulin synthesis and secretion by mixtures of purified T4 and B cells from normal donors, in the PWM-induced differentiation system. These results demonstrate that anti-CD3-induced suppressor cells are of the T4 or T8 phenotype. Treatment of purified T4+T8- and T8+T4- cells with anti-CD3 mAb's resulted in the generation of suppressor cells, suggesting that the precursors of the anti-CD3-induced suppressor cells can belong to either of these two populations.(ABSTRACT TRUNCATED AT 400 WORDS)

Modal DNA (ploidy) and sensitivity of DNA in situ to denaturation by acid have been analyzed by flow cytometry of 10 colorectal adenomas and 35 adenocarcinomas; 39 normal mucosa samples served as controls. A new method was developed to denature DNA in chromatin of the freshly isolated, intact, and unfixed individual cell nuclei from surgically resected material. The sensitivity of DNA denaturation (T alpha) was assayed by metachromatic staining with acridine orange and calculated as a ratio of the alpha t index of the tumor sample to the alpha t index of normal mucosa; the alpha t index is that fraction of DNA, following treatment at pH 1.4, that stains metachromatically with acridine orange at pH 2.6. All adenomas were diploid and in nine of 10 the T alpha value was close to 1.00, indicating no difference from control specimens in DNA sensitivity to denaturation. Forty-nine% of adenocarcinomas were aneuploid. Forty-six% of adenocarcinomas differed from normal in sensitivity of DNA to denaturation; the T alpha value was lower than 0.90 indicating that chromatin of the tumor cells was more resistant to denaturation than control cells. There was no correlation between sensitivity to denaturation of DNA and incidence of aneuploidy. However, there was a correlation between T alpha and the pathologically determined stage of disease. There was increased resistance to denaturation in 58% of tumors classified as Dukes' C/D stage, in 36% of tumors classified as Dukes' B, and in 20% classified as Dukes' A stage of the disease. Statistical analysis of these results revealed significant differences between distributions of T alpha in noninvasive (Adenomas and Dukes' A) versus invasive (Dukes' B and C/D) tumors with level of significance at P = 0.02. The data suggest that acid denaturation of DNA in situ may be a valuable adjunct in assessing the biology of colon cancer. The molecular basis for this phenomenon is discussed.

We investigated the ability of purified E-rosette negative largely leukemic B cells from 15 patients with B-cell chronic lymphocytic leukemia (CLL) to synthesize and secrete IgM, IgA and IgG spontaneously or in the presence of purified autologous or allogeneic T4 cells from normal donors, in PWM-induced differentiation system. We observed moderate but significant IgM synthesis and secretion (19.7 ± 8.9 μg/dl, n = 5) by leukemic B cells alone in 5 of 15 patients examined. These IgM concentrations were significantly higher (p < 0.005) than those produced by purified E-rosette negative cells from normal donors (4.3 ± 4.5 μmg/dl; n = 6) in the absence of T cells. Purified E-rosette negative leukemic B cells alone from patients with CLL did not produce IgA or IgG. Addition of purified autologous or allogeneic T4 cells from normal donors resulted in significant increase of IgM production by leukemic B cells from certain patients or initiated IgM secretion in others. However, these IgM levels (73.9 ± 56.6 μg/dl) were significantly lower (p < 0.003) to those produced by mixtures of T4 cells and B cells form normal donors (211.6 ± 58.0 μg/dl, n = 6). Addition of purified autologous or allogeneic T4 cells from normal donors to purified largely leukemic B cells from patients with CLL resulted in production of very small amounts of IgA in 4 of 15 patients (10.6 ± 6.3 μg/dl vs 154.7 ± 35.8 μg/dl produced by T4 and B cells from normal donors; n = 6), but did not support IgG synthesis and secretion. Purified T4 cells from certain patients with CLL exhibit defective helper function to immunoglobulin production by E-rosette negative cells from normal donors.