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T lymphocytes isolated from mice infected with Mycobacterium tuberculosis respond vigorously to proteins secreted by the bacilli and these antigens may be of importance in the generation of protective immunity against the disease. In this study, short-term culture filtrate (ST-CF), which constitutes a complex mixture of secreted proteins, was fractionated by a modified preparative SDS PAGE technique. The ability of each fraction to be recognized by T cells isolated from infected mice was evaluated by quantifying proliferation and IFN-γ production in cell cultures. Two molecular mass regions 4-11 and 26-35 kDa were found to possess marked stimulatory properties. Four potent single antigens were mapped within the stimulatory regions. These purified antigens stimulated T cells isolated from mice at the height of a tuberculous infection to produce large amounts of IFN-γ. Two of these stimulatory antigens belonged to the antigen 85 complex. [ABSTRACT FROM AUTHOR]

We describe methods for the development and partial characterization of antigen-specific T-cell lines from the guinea pig, which is the classical experimental animal in tuberculosis research. T cell were obtained from strain 2 guinea pigs immunized with BCG vaccine or with killed Mycobacterium tuberculosis in oil. T-cell lines were obtained from limited dilution cloning of antigen-stimulated, blast-enriched lymphocyte cultures. The lines were grown with weekly reseedings in the alternating absence and presence of mycobacterial antigen. Antigen reactivity of the cell lines was studied with lymphocyte stimulation tests. With these methods we have consistently obtained antigen-reactive cells lines. When injected in small number intradermally in the presence of antigen in synergenic guinea pigs, some of these cell line gave rise to antigen-specific crythematous tuberculin-like skin reactions. The skin reactions, which were usually without induration, were most pronounced after 24 h. Histological examinations of skin undergoing such reaction showed that the erythemas were not accompanied by mononuclear infiltrations. We expect that transfer experiments with guinea pig T-cell lines will prove useful tools in the analysis of the contribution of defined mycobacterial antigen preparations to tuberculosis immunity. [ABSTRACT FROM AUTHOR]

Mixed lymphocyte reaction (MLR) cultures of human lymphocyte subsets with or without the addition of physiological doses of human alpha interferon (IFN-α) were compared with respect to surface marker phenotypes and proliferative capacities of the responder cells. A selective depression on the T4 (inducer) T-cell subset could be demonstrated as a sequence of events: decreased fluorescence intensity of the T4 inducer cells (day 2 of culture), decreased percentages of T4 cells as demonstrated by cell cytofluorometry (days 3-6 of culture), and decreased ³H-thymidine incorporation of purified T4 cells and decreased numbers of T4 cells harvested from IFN MLRs (days 5-6 of culture). In contrast, it was shown that the T8 (cytotoxic/suppressor) subset in MLRs was either not affected or slightly stimulated by the addition of IFN. The depression of the T4 cells by IFN was accompanied by a decrease in the number of activated T cells expressing Ia antigens. On the other hand, IFN MLRs contained greater numbers of cells expressing the T10 differentiation antigen. In experiments with purified T-cell supers the IFN effect was exerted directly on the T4 cells and not mediated by either T8 suppressor cells or monocytes. These findings are discussed in relation to other immunoregulatory effects of IFN-α. [ABSTRACT FROM AUTHOR]

Autologous monocytes are required for an optimal lymphocyte proliferative response to purified protein derivate of tuberculin (PPD) in vitro and for a mixed lymphocyte culture induced by alloantigens. In the proliferative response to PPD we found that autologous monocytes could be replaced with HLA-DR-compatible monocytes and partly with HLADR semi-identical. In spite of a statistically significant difference between autologous and HLA-DR disparate monocytes in their cooperative capacity with PPD-stimulated lymphocytes, replacement in nearly one third of the cases was possible. These findings were supported by more detailed studies in which increasing numbers of allogeneic and autologous monocytes were added to the isolated lymphocytes in the presence of PPD. It is concluded that the serologically defined H L A-DR antigens a lone give insufficient information of the restriction elements controlling the PPD-stimulated lymphocyte-monocyte interactions. [ABSTRACT FROM AUTHOR]

Inoculation of parental strain lymphocytes into the footpads of young F1 hybrid rats induces GVHR, which can be quantitated by estimation of the weight of the popliteal lymph node 7 days later. In a number of strain combinations adult and old F1 animals were also useful for the assay. In some, however, it was observed that aged F1 rats did not support GVHR to the same extent as young did, when one of the parental strains served as donor For the GVHR, but reacted normally if the other parental strain was used. This phenomenon was analysed in serum and cell transfer experiments as well as in vitro in MLC. It was riot possible to transfer suppression in vivo, whereas some degree of specific MLC inhibition was observed when serum from old Ft animals undergoing GVHR was assessed. The old' pattern of response could be reverted partially by splenectomy arid completely by low dose irradiation of the recipient before GVHR. [ABSTRACT FROM AUTHOR]

Alloreactive cytotoxic lymphocytes were generated during mixed lymphocyte cultures (MLC). Human leucocyte interferon added during MLC gave rise to a moderate inhibition of thymidine uptake, yet augmentation of the allospecific cytotoxic product generated was found. Effector cell activities after MLC in the presence and absence of interferon against autologous cells were negligible, whereas NK-sensitive targets and antibody-coated target cells were killed to a higher extent, comparably, by cells generated during MLC in the absence of interferon. Cell separation experiments and cold target inhibition experiments were performed and showed that the effector cells generated with and without interferon had similar characteristics and were distributed among the same lymphocyte subpopulations. The key conclusion was that the augmented cytotoxicity was caused by alloreactive T cells, most of which were H(C)γ receptor-negative, and was not due to enhanced ‘NK-like’ or antibody-dependent cell-mediated cytotoxic activity induced by interferon. [ABSTRACT FROM AUTHOR]

Two human T lymphocyte subpopulations were purified on the basis of their different affinities to sheep erythrocytes and compared with unseparated and non-T lymphocytes as effectors in cell-mediated lympholysis (CML) after mixed lymphocyte culture (MCL), in antibody-dependent cellular cytotoxicity (ADCC) against the mouse mastocytoma line P 815, and in spontaneous cell-mediated cytotoxicity (SCMC) against the human myeloid leukaemia line K 562 and the Burkitt lymphoma line RAJI. The low-avidity T cells(Ti)had developed into more potent effectors than the high-avidity (Te) when assessed in CML. In ADCC and SCMC both subsets exhibited low but consistent lysis with no demonstrable differences. Addition of human leucocyte interferon to the ADCC and SCMC cultures resulted in enhanced SCMC, most notable and to an equal extent with the T cell subsets. In contrast, ADCC of unseparated cells, non-T cells, and the two T subsets was found to be unchanged after interferon addition. These data are discussed in relation to findings with human T-cell subpopulations purified by other methods. [ABSTRACT FROM AUTHOR]

Human leucocyte interferon (HuLeIF) has now for the first time been purified by a series of techniques involving precipitation, gel filtration, and affinity chromatography with Cu-chelate, blue dextran, and antibody. The two major species of HuLeIF were identified as two clearly separable and stainable proteins representing 85% of the biological activity with molecular weights of 18,400 and 20,180 daltrons. Three more subspecies of HuLeIF were demonstrated with molecular weights of 19,500, 20,900 and 23,130 daltons, representing 15% of the biological activity. Specific activity of pure interferon is 2 × 109 interferon units/mg protein. Recovery was about 50%, and the purification factor exceeded 350,000. [ABSTRACT FROM AUTHOR]

A subpopulation of lymphocytes is defined—which under conditions used for isolating mononuclear cells from peripheral blood by the Isopaque—Ficoll gradient—is lost to the erythrocyte pellet because of rosette formation with the autologous erythrocytes in the gradient. Reapplication of the resuspended erythrocyte pellet on a new gradient with higher density could, however, recover the subpopulation. Lymphocyte surface marker analysis on such cells showed that they were enriched in T cells (judged by rosette formation with sheep erythrocytes) and in auto-rosette-forming cells. The importance of these findings is discussed in relation to the wide use of the Isopaque-Ficoll method. [ABSTRACT FROM AUTHOR]

Mixed lymphocyte cultures (MLC) showed decreased thymidine incorporation when interferon (IF) had been added to the culture medium. The cell-mediated lympholysis (CML) potential generated in mixed lymphocyte cultures grown in the presence of IF was substantially augmented at a certain concentration range. Highly purified leucocyte IF also had this effect, whereas mock preparations did not. The CML-augmenting property was found in the antiviral fraction after separation of semipurified leucocyte IF (by the method of Dahl & Degree). The possibility that the effects were due to a shift in kinetics after culturing in the presence of IF has been excluded. The killer-enhancing properties of IF did not seem to be due to an enhanced expression of histocompatibility antigens, the selective inhibition of CML suppressor cells, or a change in the specificity of recognized target cells. The results of secondary in vitro MLC-CML experiments, in which primary MLC had been carried out in the presence or absence of IF, indicated that the compound acts (in vitro) through preferential selection of cells that are less sensitive to inhibition by IF and more likely to kill. [ABSTRACT FROM AUTHOR]

On the basis of their different abilities to form rosettes with unsensitized sheep erythrocytes (SRBC). two subpopulations of human T lymphocytes were separated by the rosette sedimentation technique. In a series of in vitro experiments the T cells carrying high-avidity receptors for SRBC ('active' T cells) were shown to respond significantly better when stimulated with purified protein derivative (PPD) and allogeneic cells than the subpopulation carrying low-avidity receptors ('non-active' T cells). On the other hand, tbe latter responded better to phytohaemagglutinin, concanavalin A and pokeweed mitogen; it was likewise demonstrated that these cells were better stimulators in a mixed lymphocyte reaction. The role of monocytes in mitogen and PPD responses was investigated by adding such cells to Fe-treated suspensions. These were found to be crucial for an optimal response of both subpopulations. Furthermore, different patterns of activation were observed for the active and non-active cells. Especially interesting was the observation that the Con A response of the active fraction was more monocyte- dependent than that of the non-active one. [ABSTRACT FROM AUTHOR]

Antibodies against partially purified human leucocyte interferon (PIF) were bound to Sepharose 4B and crude interferons applied on this affinity column were purified, up to 8 x 105 interferon units (IFU) per mg protein in one step. Antibodies against PIF were absorbed with immobilized crude human leucocyte interferon bound lo Sepharose, whereby antibodies against impurities were predominantly removed. Extensively absorbed antisera were coupled to Sepharose and used for antibody affinity chromatography of crude interferon preparations. Leucocyte and fibroblast interferons were purified in one step with around 100% recovery, up to 1 x 108 IFU per mg protein, and Namalva interferon up to 2 x 107 IFU/mg. SDS electrophoresis of affinity-purified leucocyte interferon revealed that the interferon activity appeared in two bands (19,000 and 23,000 D). [ABSTRACT FROM AUTHOR]