Identification of "active" T lymphocytes among effector cells in guinea pigs (original) (raw)
Guinea pig T lymphocytes have receptors of different affinity for rabbit red blood cells (RRBC): those binding RRBC immediately are termed "active" T cells; the remainder, which bind RRBC only after longer incubation times, are "non-active" or "late-rosetting" T cells. We have found that these two subpopulations have different functional characteristics. Active T cells could not be stimulated effectively with phytohemagglutinin (PHA), and stimulation with concanavalin A (ConA) increased their DNA synthesis only at high concentrations. The non-active subpopulation responded better to PHA but poorly to ConA. Unseparated (total) T cells, however, responded well to both mitogens, suggesting a helper effect by the active T cells. The presence of monocytes in T-cell cultures further enhanced mitogen-induced DNA synthesis. Active T cells were not present in guinea pig bone marrow, whereas they constituted 10% of all lymphocytes in the thymus, 13% in the spleen...
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Immunology and Cell Biology, 1977
Snminar>\ The proliferative response of Mirioiis separated populations of mouse spleen and tliymus lyiiiphoe\ tes to Ihe mito^en phytohat'ninuKl"f'nin (PIIA) was not a direct funelion of the level of responsive T ceils, but uas governed by other reEiiIatory ellet-ts. Tht-se inclnded a stimulation by adherent macrophases, an inhibition by a separate population ol adherent cells and an adherent cell independt-nt restriction of proliferation at high eill (.imcentration. In contra>.t the jtroliferative response to C'oneanavidin A (Con A) wius more clcisely related to the level of responsive T eells. ,\i! densitv' and elettrophoretirally isolated snli-sets of splenic T cell.'; appiari'd eiipable of a proliferalive response tt> PMA aud Con A. althongh under soim-conditions the PIIA res[Kmsivfiiess of (ertiiin fractions was .suppressed. In the thymus. the minor low " subjiopnlation appeared capable of resixwise to iKith niitosens. anti ac(onuted for all the activity of the ni frai tiom-d thvinns cells. No response to either niitoK<-Ti could l)e obtained trom the major-hi^li " thvmoevte population.
Potentiation of the T lymphocyte response to mitogens
Cellular Immunology, 1974
The first papers of the present series establish that mouse thymocytes and peripheral thymus-processed (T) 1 lymphocytes are stimulated to mitosis and their response to such agents as phytohemagglutinin greatly potentiated by factor(s) designated "lymphocyte-activating factor" (LAF), which are produced in cultures of syngeneic or xenogeneic lymphoid cells (1, 2). In this paper, we present data showing that adherent cells, probably macrophages, are the principal source of LAF and that its production is increased by agents which stimulate these cells. Materials and Methods Animals.-Male or female CBA/J mice, 6-12 wk of age, were used without treatment or after irradiation (850 R) and reconstitution with 5)< 106 syngeneic bone marrow cells (XBM), 5-8 X l0 T thymocytes (XT), or both bone marrow and thymus cells (XBMT). Young adult New Zealand albino rabbits of both sexes were purchased from a local dealer and used without treatment. Cell Preparation and Culture.-All the materials and techniques employed for cell preparation and culture are fully described in our previous papers (1-3). The separation of adherent from nonadherent cells was carried out both on plastic (4, 5) and by the use of nylon columns (6, 7). While 8% pooled normal human serum was routinely used for lymphocyte culture, the adherence technique required higher concentrations, 10% human serum being used routinely and 10% fetal calf serum (Grand Island Biological Company, Grand Island, N.Y.
A Study of the Proliferative Response of Rabbit T Cells Using the Brdu-Hoechst Method
Cell Proliferation, 1984
Con-A-and PHA-induced proliferation of cells from rabbit thymus, spleen and mesenteric lymph node was studied with the DNA-fluorescent probe 33258 Hoechst. The fluorescence of this probe is quenched when 5-bromo-2'-deoxy-uridine is incorporated into nascent DNA during the S phase. Fluorescence decreased with increasing content of newly formed DNA per cell. Proliferation kinetics and the number of Con-A-and PHA-reactive cells (Ct and Pt cells) were determined cytofluorometrically. Lymphocytes from control and dexamethasone (DX)-treated animals start their proliferation early: after 42 hr about 25% of the control and the majority of the DX-resistant cells finished their second cell division. Small numbers of C+ (12.0%) and Pt (3.5%) cells were found in control thymus, while these percentages were enhanced in DX thymus: 32.5 and 27.0% respectively; 50% of the spleen T cells in control and DX animals are C+ or P+ and 75% of the lymph-node T cells are C+ (after DX 45%) and 50% are P+ (after DX also 50%). It is concluded that in thymus and lymph nodes, a steroid sensitive (Ss) C+P-, and in lymph nodes a Ss C+P+ cell pool is present. A mitogen non-proliferative cell pool (C-P-) is present in control and DX thymus. Stimulation of lymphocytes by mitogens in vitro is widely used in experimental and clinical immunology. Con-A and PHA are mitogenic lectins able to transform T lymphocytes into proliferative blasts (Oppenheim & Rosenstreich, 1974). The DNA synthetic capacity of these cells is usually quantified with radioactive DNA precursors (usually 3H-thymidine) and the amount of label incorporated has been used as a measure of the proliferative capacity. For both Con-A and PHA large differences in the extent of stimulation have been reported, and heterogeneity among T cells in this respect has therefore been considered (Stobo, 1975). Using elimination of mitogen-reactive subsets by photolysis Touraine ef al. (1976) have demonstrated that a large population of T cells in human peripheral blood is proliferative both to Con-A and well as to PHA (C+ and P+ respectively). Other authors have reported that C+ and P+ cells differ in a number of physical (
Journal of Immunological Methods, 1982
In the present communication we present the results of a study undertaken to assess the effectiveness of several methods of depleting human peripheral blood of thymusderived (T) cells, and to assess the purity of the T cell-enriched populations obtained. The methods of T cell depletion employed included rosetting with unmodified sheep red blood cells (SRBC) in the cold at high erythrocyte to lymphocyte ratios; rosetting with S-2-aminoethylisothiouronium bromide hydrobromide (AET)-treated SRBC; and cytotoxic depletion using anti-Leu-1, a monoclonal antibody directed toward a panT cell antigen, and rabbit complement. The methods used to enrich for T cells included rosetting with unmodified or AET-treated SRBC, and passage through a nylon wool column. The purity of the T-enriched and T-depleted populations obtained was assessed by (a) repeat rosetting with unmodified SRBC, (b) staining for cell surface immunoglobulin (sIg), (c) quantitating the number of Ig secreting cells (ISC) in a reverse hemolytic plaque assay, (d) quantitating the proliferative responses to the T cell mitogens phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) and to the B cell mitogen Staphylococcus aureus Cowan (SAC), and (e) quantitating the development of ISC following culture with PWM, with or without the addition of an irradiated T cellenriched population. By all parameters investigated, the depletion of T cells from peripheral blood by rosetting with AET-treated SRBC or by treatment with anti-Leu-1 and complement was far more effective than by rosetting with unmodified SRBC under optimal conditions. Of particular note was the finding that functional assays (ISC at time zero (To), proliferative response to SAC, the development of ISC on culture with PWM) revealed a much higher degree of B cell contamination in all of the T cell-enriched populations than would have been suspected from the extent of the depletion of sIg staining cells.
European Journal of Immunology, 1977
Selection in long-term culture of alloreactive T cells, by successive in vitro restimulation with semi-allogeneic cells, results in primed responder cell populations which maintain full proliferative reactivity t o allogeneic cells as well as to the T cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA) but are depleted of cells which can effect target cell destruction in either a specific or nonspecific manner. Con A-induced T cell blasts (selected by velocity sedimentation) can revert to small resting lymphocytes in the presence of inert "filler" cells. Con A blasts which have reverted, readily proliferate in response to Con A or allogeneic stimulator cells but are largely depleted of effector killer cells and PHA-responsive cells.
Active" T cells in guinea pig peripheral blood lymphocytes
Clinical immunology and immunopathology, 1981
The ability to form rosettes with rabbit red blood cells (RRBC) has been shown to be characteristic of T but not B cells from guinea pigs. In an attempt to devise an assay system to measure the guinea pig equivalent of human "active" T cells, we have investigated the effects of various conditions on RRBC binding by guinea pig lymphocytes, including incubation time, RRBC:lymphocyte ratio, presence or absence of guinea pig serum or fetal calf serum in the incubation medium, and pretreatment of RRBC with neuraminidase or 2-aminoethylisothiouronium bromide (AET). Without incubation and at a 100: 1 ratio of RRBC to guinea pig lymphocytes, about 35% of guinea pig peripheral blood lymphocytes bound RRBC, similar to the number of "active" T cells in human peripheral blood as measured by rosette formation with sheep red blood cells. Cold incubation increased the number of rosetting cells to over SO'%, presumably the total number of T cells. A relative decrease in the number of RRBC diminished rosetting only at ratios of 1O:l or less. Pretreatment of RRBC with neuraminidase slightly increased, whereas AET pretreatment drastically reduced the number of rosettes. Increasing concentrations of guinea pig serum in the medium up to 100% slightly inhibited rosetting. These results suggest the presence of two T-cell populations in guinea pig peripheral blood, equivalent to the human "active" and "total" rosette-forming T-cell subpopulations.
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