The repair of DNA alkylation damage in human cells is poorly understood. We have adapted the alkaline elution technique for use with human peripheral blood lymphocytes in culture. We have also established conditions necessary for short-term culture of human lymphocytes. Lymphocyte growth which can be maintained for up to 30 days is dependent upon irradiated TK6 feeder cells and T-cell growth factor (crude TCGF). The amount of damage induced by a given concentration of methyl methane-sulfonate (MMS) is dependent upon cell number per ml of growth medium. The DNA damage measured, in lymphocytes, by alkaline elution is a composite of single strand breaks and alkali-labile lesions. Repair of this damage after appropriate recovery periods is also detectable. The irradiated feeder TK6 cells do not contribute to the number of strand breaks detected or the amount of recovery after treatment. This method offers a quick and reproducible means of detecting DNA damage and repair in human T-lymphocytes.

Murine cytolytic T lymphocyte (CTL) clones specific for type A influenza virus antigens were generated by in vitro stimulation with syngeneic virus-infected cells in the presence of T cell growth factor (TCGF). All CTL clones recognize viral determinants shared by PR8 and X31 influenza viruses in association with a class I antigen, coded either by the H-2K or H-2D end of the appropriate haplotype. All clones express the Lyt2 antigen marker. Two of five clones also express an antigenic determinant of the V beta chain of the T cell receptor (TCR) identified by F23.1 monoclonal antibody. To effectively generate F23.1+ and antigen-specific CTL clones, heterogenous CTL lines were expanded with F23.1 coated Sepharose beads in the presence of TCGF and then stimulated with PR8 virus-infected cells. Thus, both the proliferative activity to PR8 and the expression of the F23.1 marker was increased significantly. Alternatively, F23.1+ T cells were sorted from in vivo primed mice and expanded with PR8 virus-infected stimulator cells in the presence of TCFG. This F23.1+ T cell line exhibited antigen-specific cytotoxicity for PR8 virus-infected target cells. Additionally, in an 'FcR-focused killing' assay only the F23.1+ CTL line and F23.1+ clones lysed Fc receptor bearing target cells in the presence of F23.1 antibody. These findings indicate that antigen-specific and F23.1+ clones can be selected with high efficiency by alternating stimulation with influenza virus-infected cells and with F23.1-coated Sepharose beads or through the use of a cytofluorograph. The usefulness of antigen-specific and F23.1+ CTL clones and other possible strategies for their selection are discussed.

We have previously demonstrated the ability of soluble factors derived from cultured murine and human bone marrow supernatants to modulate a variety of lymphoid functions, including DNA synthesis. In the present report, we show that human marrow supernatants contain a suppressor factor (BSF) that suppresses T-lymphoid colony formation, and an augmenting factor (BEF) that enhances T-colony growth. BSF suppression exhibits no tissue specificity, affecting marrow-derived and peripheral T colonies similarly. The suppressive activity occurs prior to mitogenic activation by TCGF. In contrast, a preferential augmentation of the size and number of marrow-derived T-cell colonies, as compared to peripheral T-cell colonies, was observed in the presence of BEF. BEF required prior mitogen activation of the colony inocula to effect colony enhancement. In addition, the response to BEF was greater for E- than for E+ colony-forming cells, indicating the target of BEF activity to be an early T cell. The active subfraction of BEF with colony-enhancing activity was found to be between 8000 and 30,000 daltons.

The purposes of the current study were: (1) to investigate the immunoregulatory effects of T-cell growth factor (TCGF) on the activation and differentiation of syngeneic cytotoxic T lymphocyte (CTL) populations generated against a 20-methylcholanthrene-induced ependymoblastoma, 203-glioma, in C57BL/6 mice; and (2) to determine whether the glioma-specific CTL clone (G-CTLL) could be established by TCGF, and whether the in vivo efficacy of the cloned cells could be rendered more effective in adoptive therapy. It was found that TCGF largely allows the CTL populations to proliferate and thus can activate the depressed cytotoxic activity in tumour-bearing mice. Two lines of G-CTLL were successfully obtained by the limiting dilution technique. The G-CTLL retained a TCGF-dependent proliferative growth and a marked cytotoxic activity with target specificity for over 18 months, characterized by a surface phenotype of Lyt-1-.2.3+, Lyt-2 antibody blocking of cytotoxicity and the production of immune interferon in response to mitogen and tumour antigen. In the Winn assay and the adoptive transfer assay, the therapeutic effects were detected in intracranially inoculated tumours in mice. The in vivo efficacy was dependent on the dose of G-CTLL and on the time of the intravenous administration, although the transfer was inversely ineffective in conditions of increased intracranial pressure. The mechanism responsible for the in vivo effect was probably due to the adoptive immunity and/or the tumour-specific interferon production of G-CTLL.

T-lymphocyte colony-enhancing factor (TLCEF), a growth factor for a minute subpopulation of T lymphocytes, is produced, along with other factors, by conditioned media (CM) of mononuclear cells following stimulation with T mitogens, such as phytohaemagglutinin (PHA). A combination of PHA and a co-mitogen, phorbol 12-myristate 13-acetate (PMA), has been shown to have a synergistic effect on the production of TLCEF, yielding levels of activity eight to fifteen times higher than those obtained with either PHA or PMA alone. TLCEF was purified by ammonium sulphate, fractionation hydrophobic interaction chromatography on phenyl-Sepharose and gel filtration. The last step of this purification procedure yielded two peaks of activity purified 13,000- and 29,000-fold, respectively. The first peak eluted from the column with an average molecular weight of 125,000, whereas the second peak was retained on the gel, probably due to non-specific interactions with it. The purified fractions contained none of the following activities: T-cell growth factor (TCGF), colony-stimulating factor-1 (CSF-1), interleukin-3 (IL-3) and interferon. TCGF activity, which is known to be unstable at high degrees of purification, was already lost after the ammonium sulphate fractionation step, most probably because of the low protein and albumin concentrations (0.33 and 0.11 mg/ml, respectively). TLCEF is thus a more stable immunoregulatory factor than TCGF and has a much higher apparent molecular weight.

Six patient with malignant effusions (five with ascites and one with malignant pleural effusion) due to refractory ovarian carcinomatosis, received intracavitary injections of ovarian viral oncolysate (VO). Measurements were made of the intracavitary activities of T cell growth factor (TCGF-IL-2) and B cell growth factor 12-kD (BCGF) and these were correlated with the clinical activity and cytologic changes in the malignant effusions. Both BCGF and IL-2 activities were demonstrated in malignant effusions prior to therapy although these were relatively higher for BCGF. Increases in the activities of both lymphokines were observed in the post VO treatment samples ranging from 4.1% to 45.0% for BCGF and 4.8% - 33.9% for IL-2. One patient who exhibited marked clinical improvement accompanied by mononuclear cell infiltrates and diminution of ascitic fluid malignant cells, also had elevation of lymphokine activities after repeated VO injections. These data provide further support for the role of VO as inducers of regional immunity.

The responses of thymocytes to Concanavalin A (Con A), and interleukin 1 (IL-1), interleukin 2 (IL-2) and phorbol myristate acetate (PMA) were investigated. The enzyme 20-alpha-hydroxysteroid dehydrogenase (20 alpha SDH) was used as a marker to distinguish between various populations of activated thymocytes. Thymocytes that were selected in Con A + pure or crude IL-2 expressed high 20 alpha SDH activity, while those that were selected in Con A + recombinant IL-1 (rIL-1) or crude IL-1, or Con A + PMA expressed low 20 alpha SDH activity. Both groups proliferate in response to Con A and had IL-2 receptors. After selection, the enzymatic phenotype was stable even if the cells were transferred from Con A + IL-2 to Con A + PMA (or IL-1) or vice versa. A third group was selected from thymocytes that were cultured in PMA + T cell growth factor (TCGF). This group expressed low levels of 20 alpha SDH, had IL-2 receptors, but did not respond to Con A. This paper demonstrates that 20 alpha SDH can be used as an enzymatic marker to distinguish between subpopulations of activated T cells, which have not been previously detected by the conventional surface markers.

Six OKT4+ human T-cell lines that require continuing PHA stimulation and TCGF for continuous growth were established. The cells from all six of these T-cell lines became smaller in size and lost the cell surface Ia antigen when they were grown in phytohemagglutinin (PHA)-depleted growth factor. These cells were unable to survive in the absence of PHA even if exogenous factor was present in great abundance. One of the cell lines (FL) was capable of providing helper functions. In the presence of PHA and phorbol myristate acetate, FL cells produced a growth factor, tentatively identified as Interleukin 2 (IL-2) by its ability to promote the proliferation of an IL-2-dependent murine T-cell line. Moreover, when FL cells were cocultured with B cells, pokeweed mitogen-induced immunoglobulin production was enhanced.

Experimental studies were performed in order to establish autologous adoptive immunotherapy against human solid tumor. When peripheral blood lymphocytes (PBL) from a patient with cholangiocarcinoma (Ch-1) were cultured in the medium supplemented with T cell growth factor for two weeks they proliferated markedly, and more than 100-fold increase in cell number was achieved. These two week-cultured lymphoid cells (CLC2w) were revealed to consist mostly of cytotoxic T cells and, in a portion, of NK cells, from the examination of markers such as SRBC-rosette formation, peroxidase, surface Ig, IgG Fc receptors, C3 receptors, OKT3 antigen, OKT8 antigen, and so on. In in vitro 51Cr-release cytotoxicity assay, fresh PBL from the patient with Ch-1 showed no cytotoxicity against autologous Ch-1 cells which had been maintained by serial transplantations to BALB/c nude mice, whereas CLC2w exerted obvious cytotoxicity against the autologous Ch-1 cells. CLC2w, however, did not lyse PHA-stimulated autologous lymphoblasts. CLC2w also prevented growth of the autologous tumor in nude mice. NK cell activity of CLC2w appeared to be increased as compared with that of fresh PBL from the patient.

K-76 COONa is a derivative of a fungal product which blocks complement (C)-mediated lysis by combining with C5 and preventing its activation to C5b. K-76 COONa can also combine with Factor I and inhibit its ability to hydrolyze C3b to iC3b. The inclusion of K-76 COONa at concentrations similar to those which inhibit C lysis blocked both murine cytotoxic-T-lymphocyte (CTL)-mediated lysis (CML) and the lectin-stimulated proliferative response of murine and human T lymphocytes. A modified cation pulse procedure has been used to determine which phases of CML were most sensitive to the drug. K-76 COONa was inhibitory when it was added to CML prior to the early Mg+2-dependent binding phase, but was much less effective when it was added at any time after the formation of CTL-target conjugates. The principal effect of the drug on the proliferative response was also exerted during an early phase of the response. K-76 COONa did not appreciably decrease the production of T-cell growth factor (TCGF), but it did inhibit the induction of TCGF receptor expression by both functional criteria, i.e., induction of responsiveness to TCGF, and by morphological criteria, i.e., the expression of the Tac antigen. Later events, such as the TCGF-dependent proliferation of cycling T cells, were less sensitive to the drug. Evidence is discussed suggesting that molecules similar to Factor I and to C3 may be involved both in the early events of CML and of T-lymphocyte activation.

Rabbit spleen cells (1 X 10(6)/ml) stimulated with Con A (2.5 micrograms/ml) begin producing T cell growth factor (TCGF) activity within 2-3 h at 37 degrees C and reach a plateau after 20-24 h. Lymphocytes from the mesenteric lymph nodes (MLN) also produce TCGF activity but require higher lectin and/or cell concentrations for optimal production. TCGF generation by MLN can be improved by including a small proportion of spleen cells (10-40%). Rabbit lymphocytes also produce TCGF in serum-free, protein-free medium, but the Con A concentration must be reduced to 0.5-1.25 micrograms/ml. Rabbit TCGF activity eluted from gel filtration columns at a volume equivalent to approximately 15,000 molecular weight, similar to human TCGF activity. However, when rabbit, mouse and human TCGFs were compared for their ability to support proliferation by activated cells from the 3 species, rabbit and mouse TCGFs appeared more similar. In all cases, the homologous combinations of cells and TCGF were most efficient. Human TCGF would support some proliferative activity in all 3 cell types but rabbit and mouse TCGFs failed to support the growth of human cells.

Mouse H-Y-specific and I-Ab restricted T-cell clones have been established and compared for their helper effects in the differentiation of both T and B lymphocytes. The results demonstrate that three individual T-cell clones and one subclone could help in the antigen-driven induction of cytotoxic lymphocytes (CTL) from their precursor cells (CTL-P), and were able to activate B cells to develop into antibody-secreting cells (PFC) in the presence of SRBC, provided the cloned T cells were restimulated by H-Y antigen on antigen-presenting cells. In addition, antigen or lectin could induce the same H-Y-specific T-cell clones to secrete factor(s) expressing helper activities similar to that of the cloned T cells. Furthermore, it is shown that the T cell-derived soluble mediator(s) was distinct from T-cell growth factor (TCGF) and from immune interferon (IFN-gamma). The data reveal a new type of T cell with helper potential for the activation of CTL-P and B lymphocytes, and suggest the existence of distinct T helper cells which can provide help for both cytotoxic and antibody responses by virtue of different lymphokine activities.

Calves were infected with 25,000 or 50,000 viable eggs of Taenia saginata. Larval numbers ranged between 2077 and 6005. During infection the animals developed leucocytosis, which was mainly due to lymphocytosis. An apparently positive correlation was observed between the lymphocytosis and the in vitro proliferative response of the lymphocytes to antigen prepared from proglottids. Maximal in vitro blast transformation of the cells stimulated with antigen occurred on Days 12 and 32 post-infection (p.i.). Specific antibodies to T. saginata were demonstrated on Day 14 p.i. At that time, the proliferative response of the cells paralleled the formation of specific antibodies, particularly of the IgG class. The stimulated cells produced a lymphokine showing interleukin 2 (IL 2)-like activity, since the addition of supernatant of such cells to IL 2-dependent concanavalin A (Con A)-blast cells supported the in vitro growth of the cells. In addition, peripheral blood lymphocytes (PBL) specific for T. saginata could be maintained in long-term cultures when they were cultured in medium containing supernatants of MLA-144 cell lines. The data presented in this study indicate that cells specific for T. saginata produced and consumed T cell growth factor (TCGF).

It is known that protein synthesis inhibitors (PSI) do not block production of some immunoregulatory and inflammatory mediators. Here we show that the production of T-cell growth factor (TCGF) by phytohaemagglutinin-stimulated human peripheral blood lymphocytes (PBL) was resistant to inhibition of protein synthesis more than 90-95%. Moreover, TCGF production was significantly accentuated in the presence of low concentrations of PSI in cultures from low- and high-producer donors. The presence of macrophages was non-essential for enhancement of TCGF production by PSI. When high doses of PSI were used, TCGF production was inhibited. In contrast, the synthesis of granulocyte/macrophage colony-stimulating factor (CSF) was blocked in the presence of low concentration of PSI. Hence, the results indicate that human TCGF produced by PBL belongs to a group of soluble factors whose production is relatively resistant to the action of PSI.

Virus particles are frequently able to non-specifically inhibit the capacity of human peripheral blood lymphocytes to respond to mitogenic or antigenic stimuli. In the case of breast cancer patients with advanced disease, the quantity of virus required to abrogate responsiveness to phytohaemagglutinin (PHA) was approximately four-fold less than that found when cells from healthy donors were employed. The results show that such virus co-incubated cultures are deficient with regard to their ability to synthesize detectable quantities of T cell growth factor (TCGF) activity, and that the extent of responsiveness to PHA in each case corresponds roughly to the amount of TCGF activity that is present in the cultures. While the addition of exogenous purified TCGF to cultures containing virus, normal cells and stimulus caused a reversal of the usual inhibitory effect, this finding was generally not obtained in the case of lymphocytes obtained from patients with advanced breast cancer. These data suggest that one mechanism of explaining diminished cellular immune responsiveness in breast cancer patients may be a relative inability of appropriate cells or subsets of cells to respond effectively to TCGF.

Six T-cell growth promoting factor (TCGF)-dependent T-cell cultures and phytohemagglutinin-stimulated peripheral blood lymphocytes from a patient with metatastic malignant melanoma were karyotyped. Three of the TCGF-dependent cultures were noncloned, started after in vitro sensitization against autologous malignant melanoma cells in tissue culture. A fourth was a subculture of one of these. The two remaining T-cell cultures were generated by in vitro sensitization of the same individual's lymphocytes against allogeneic Epstein-Barr virus-transformed B-cell lines. One of the cultures (AV7) and its subculture (AV/LDP39) showed a 17q+ anomaly in 100% of metaphases studied. All other TCGF-dependent T-cell cultures and fresh blood lymphocytes had normal karyotypes. The possible mode of origin of the abnormality and its significance are discussed.

Murine T cell clones, which were retrieved from an A.TH anti-A.TL(lak) T cell line and had been long-term cultured in the medium supplemented with T cell growth factor (TCGF) and mitomycin C(MMC)-treated feeder cells of either Is or Ik haplotype, were found to survive in TCGF-free medium for a long time, quite in contrast to so far reported TCGF-dependent T cell clones. When T cells of these clones at the full growth in the TCGF-medium were transferred to TCGF-free medium, they survived at resting state for a long time, and half-life, i.e., the time when 50% of the transferred cells were still viable, of some clones reached 20 days. The cloned T cells at the resting state retained full responsiveness to the specific lak antigen but lost the responsiveness to TCGF as determined by [3H]thymidine uptake, whereas the same T cells harvested from TCGF-medium did not show the antigen-specific responsiveness. The cloned T cells at the resting state showed marked DNA synthesis in response to the specific antigen but never entered the phase of the cell division. Addition of TCGF to the antigen-activated cloned T cells at their peak DNA synthesis triggered the cell division without time lag. Thus, it was confirmed at a single clone level that two sequential signals, one via the antigen-receptor reacting with specific antigen and another via the TCGF-receptor accepting TCGF, are required for clonal expansion of T cells reacting with antigen. The mitogen-responsiveness among five clones was examined at their resting state; two clones responded to Con A and PHA only in the presence of accessory cells (MMC-treated, T cell-depleted syngeneic spleen cells), and one clone responded well to Con A and PHA in the absence of accessory cells. Thus, most of our clones retained physiologic characteristics of T cells directly collected from mice even after long-term culture in TCGF-medium.

Lymphocytes isolated from the blood of 17 human fetuses, varying in gestational age between 16 and 26 weeks, were tested for their capacity to generate specific cytotoxic cells after mixed lymphocyte culture (MLC) with allogeneic cells in the presence or absence of exogenous T cell growth factor (TCGF). Blood cells from seven fetuses, distributed throughout the age range, failed to generate cytotoxic cells even when TCGF was added in MLC, whereas six others gave positive responses but only when exogenous TCGF was present during the sensitisation phase. The maturation induced in the latter was not caused solely by a direct, non-specific effect of the TCGF, for control responder cells incubated with TCGF in the absence of allogeneic stimulator cells always responded less strongly or not at all. Fetal blood lymphocytes from the remaining four fetuses gave significant cytotoxic responses that were not augmented by TCGF. It is concluded that there can be a clear dichotomy between proliferative and cytotoxic responses to alloantigens and that the inability of human fetal blood lymphocytes to mount cytotoxic responses at this stage of development might be due to deficiencies in helper cells, cytotoxic precursors or both. For three fetuses it was possible to study, additionally, cytotoxic responses of spleen, liver and thymus with and without TCGF. None of them made specific responses even when TCGF was added, though cells from two spleens and one thymus responded directly to TCGF. To ascertain whether the absence of cytotoxic responses might have been caused by a failure of blood, spleen, thymus or liver cells to proliferate, their mixed lymphocyte reactivity, as detected by the uptake of 3H-thymidine, was studied without exogenous TCGF. Whereas thymus cells from only one of five fetuses responded, cells from all other tissues (including blood) responded consistently.

Poly-Si film, due to its favorable piezoresistive properties, has been widely used in piezoresistive sensors. The previous researches have shown that the ultra-thin poly-Si film have better piezoresistive properties than common poly-silicon film, and have promising future of application. A promising method to obtain large grained high quality poly-silicon films by low-temperature crystallization of an amorphous precursor material is the aluminum-induced layer exchange (ALILE). In this paper, ultra-thin poly-Si films were prepared by aluminum induced layer exchange (ALILE). Experimental results of Raman spectroscopy show that a narrow and symmetrical Raman peak at the wave number of about 518 cm(-1) was observed for all samples, indicating that the films were fully crystallized. XRD results show that the crystallites of ultra-thin poly-silicon layer were preferably (111) and (220) oriented. Hall affect measurements show that hole concentration of the films (p-type) were between 9 x 10(18) and 6 x 10(19) cm(-3). Restorative properties show that the piezoresistors exhibit gauge factors (GFs) up to 60, with temperature coefficients of GF (TCGF) between -0.17-0% degree C and temperature coefficients of resistance (TCR) between -0.2 and -0.1% degrees C. The study of the ultra-thin poly-Si films by ALILE is completed, and the study results lay a foundation for application of the film

A large series of mouse monoclonal antibodies was found to inhibit the proliferation of T-cell growth factor (TCGF)-dependent human T-cell blasts as measured by the incorporation of tritiated thymidine. The specificity of the antibody appeared to be irrelevant for inhibition and two T-cell-specific antibodies did not prevent the absorption of TCGF by treated T cells. It is suggested that the antibodies function by the indirect release of suppressor factors by Fc receptor-bearing TCGF-dependent cells.

This study was designed to investigate the effects of allogenic effect factor (AEF), a soluble mediator derived from short-term mixed lymphocyte cultures (MLC) of in vitro alloantigen-primed T cells, on cultures of murine bone marrow cells. Cultures established under suboptimal conditions namely, in the absence of a pre-established adherent cell layer as required in conventional Dexter-type cultures--declined and lost their stem cell activity rapidly. In contrast, supplementation of these cultures, at initiation and thereafter, with AEF, but not with T cell growth factor (TCGF), induced cell growth and proliferation for several weeks. Such AEF-supplemented cultures exhibited cellular heterogeneity and stem cell activity for significantly longer periods than the control cultures. Even in conventional Dexter cultures, established under optimal conditions, AEF had a beneficial effect on cellular growth and proliferation and myeloid progenitor cell (CFU-C) activity. Furthermore, cell capable of synergizing with suboptimal numbers of mature T cells in con A-induced mitogenic responses, shown by others to be pre-T cells, were detected in the AEF-supplemented cultures for several weeks.

The ability of spleen T cells from mice inoculated with influenza virus to transfer delayed hypersensitivity increases after 5 days culture in the presence of TCGF and influenza virus or its hemagglutinin. Enriched T cells have also a greater ability to bind viral hemagglutinin than cells which were not cultured and restimulated with either antigen. The results suggest the selection in vitro of specific antigen-binding T cells, able to transfer delayed hypersensitivity.

Long-term cultures of T lymphocytes from PBL of normal individuals and cancer patients grown in presence of TCGF exhibited cytotoxic activity directed against a variety of targets. In order to separate the subpopulations responsible for the different cytotoxic activities, cultured T lymphocyte cloning was performed. Production of T cell clones were obtained at a high yield by direct isolation and culture of single T cells. Preliminary analysis of cell-mediated cytotoxicity showed that 8 out of 11 clones were cytotoxic against both PHA pretreated L1210 and K562 target cells. In addition, within a clone, only a percentage of cells expressed the cytotoxic function. We suggested that this finding is due to the different stage of maturation of the cloned cells and this hypothesis correlate with their morphology, which is not identical for all the cells within a clone.