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Mitogen-transformed human peripheral blood lymphocytes and tonsil blasts were examined by rosette formation to detect the presence of membrane-bound immunoglobulin (Ig) and surface receptors for fixed IgG and fixed C3. In addition, the capacity of these cells to rosette with sheep erythrocytes was evaluated as a reaction characteristic of T lymphocytes. In order for clear morphological recognition of the rosetting transformed lymphocytes and the rosetting tonsil blasts a cytocentrifuge technique was developed and used in conjunction with autoradiography and/or with Romanowsky stains. Using these techniques and the culture methods described in this paper phytohaemagglutinin, pokeweed mitogen, streptococcal filtrates and purified protein derivative stimulated predominantly T cells in the peripheral blood of man. A minority of the transformed cells in these mitogen-stimulated cultures (<24%) did rosette with B lymphocyte markers and presumably represent a B-cell response. No significant differences were found between the T- or B-cell specificity of the mitogens investigated. Lymphoid preparations from tonsils excised from normal donors with recurrent tonsillitis were found to contain 6-15% lymphoblasts and the iarge majority of these cells formed rosettes with the B-cell markers, less than 20% of these lymphoblasts formed spontaneous sheep erythrocyte rosettes. Using a mixed rosetting technique a small proportion (<5%) of PHA-transformed cells and tonsil lymphoblasts were found to have combined sheep Fc or combined sheep C3 receptors. The investigation of B- and T-lymphocyte surface markers on mitogentransformed lymphocytes was extended to neopiastic lymphocyte populations and it was found that the majority of transformed cells (>70%) present in chronic lymphocytic leukaemia cultures stimulated with PHA after 6 days incubation were transformed T lymphocytes. [ABSTRACT FROM AUTHOR]

Human peripheral lymphocyte preparations were tested with a mixed rosette method for the presence of lymphocytes bearing both the complement receptors characteristic of B lymphocytes, anti the capacity of T lymphocytes to spontaneously bind with sheep red blood cells (SR BC). The large and oval shaped pigeon red blood cells (PRBC) which do not form T-cell rosettes were utilized as indicators for rosette formation with the complement receptor-bearing B cell. In every individual (an average of 2.06%) lymphocytes were observed which formed rosettes with both SRBC' and PRBC indicators. These findings show that independent markers for both T and B cells may be present on the surface of the same lymphocytes. [ABSTRACT FROM AUTHOR]

By the use of an indirect immunofluorescent method for the identification of mouse T lymphocytes forming rosettes with antibody-coated erythrocytes, it was found that about 10% of the T cells in normal mouse spleen have receptors for antibody. Inhibition experiments with purified aggregated immunoglobulins showed that IgG2 is the major class involved. The same receptor was found on T cells activated against H-2 isoantigens. The importance of using the appropriate red cells and antiserum for demonstrating receptors of T cells is stressed. [ABSTRACT FROM AUTHOR]

A method is described for indirect immunofluorescent staining of mouse T cells forming rosettes, involving fixation of rosettes by gluturaldehyde and staining with a rabbit anti-mouse brain serum absorbed so as to be specific for T cells. Using this technique, it was shown that about 44%, of spleen cells forming rosettes with sheep erythrocytes both in non-immunized and immunized mice, are T cells. The number of T rosette-forming cells increased about ten-fold after immunization with sheep erythrocytes. but only when serum antibody was detectable. [ABSTRACT FROM AUTHOR]

An in vitro study of the spleen leucocyte migration ability and the phytohaemagglutinin (PHA) stimulated peripheral lymphocyte response was made on cells derived from mice treated by azathioprine, immune stimulation, or thymectomy and azathioprine. These responses were compared with those shown by normal mice and found to be considerably depressed. Thymectomy combined with azathioprine produced the greatest change which persisted at 40 days. These changes suggested that azathioprine in common with thymectomy, antilymphocyte globulin and radiotherapy produced some of its immunosuppression by depressing T-cell function. [ABSTRACT FROM AUTHOR]

The article focuses on the role of antibody in T-cell responses. In the article the evidence is marshaled for an overall hypothesis according to which many of these activities of the T cell can be explained in terms of passively acquired antibody. In this context it must be said at once that T cells do not appear to carry much Ig. The sensitive lacto-peroxidase technique, by which surface proteins can be iodine-labeled in situ, has demonstrated IgM on thymus and thymus-derived cells in some hands.

Phytohaemagglutinin transformation of normal lymphocytes was significantly inhibited by the plasma and by extracts of lymphocytes in six out of eight cases of untreated chronic lymphatic Ieukaemia compared with similar material from matched control patients. Since the concentration of inhibitor in lymphocytes was much greater than that in plasma and both inhibitors were thermolabile, it is possible that the plasma inhibitor may be produced by the leukaemic cells. These findings indicate modifications of normal T-ceIl function by constituents of neoplastic lymphocytes but the effect may be due to exaggeration of a normal mechanism in which the surface properties of T cells are modified by products of B cells. [ABSTRACT FROM AUTHOR]

The ability of mouse lymphoid cells to show non-specific cytotoxicity to 51Crlabelled mastocytoma cells in vitro was assessed by the release of '''Cr. Little cytotoxicity was shown by normal lymphoid cells. However, incubation for several days with mitogens for T cells, such as PHA. Con A, P. graveolens and lentil mitogen, activated spleen cells for cytotoxic killing providing PHA was present during the killing reaction. Pokeweed mitogen, which is an inferior mitogen for T cells, was less active. Three agglutinins, which did not transform T cells, were ineffective. Con A and PWM also activated cortisone-resistant thymus cells. Activated T cells only killed target cells when a plant agent was present during the killing reaction. The T cell mitogens. which are also strong agglutinins, were effective. PWM was less effective and agents which were not T cell mitogens, including leucoagglutinins, had a variable and usually small effect. Cell division was not essential for the activation of cytotoxic cells as colcemid diminished but did not abolish activation. DNA synthesis was not required during the killing reaction as judged by the failure of hydroxyurea, an inhibitor of DNA synthesis, to block cytotoxicity when added 2 hr before the killing reaction. The relation between the number of lymphocytes added and the number of target cells killed was compatible with the 'one hit' hypothesis that an effective interaction between a single attacking cell and a target cell led to target cell death. Non-specific cytotoxicity does not require a strong antigenic difference between attacking and target cells as syngeneic lymphocytes will kill mastocytoma cells. Killing of human cell lines also occurs. The data suggest that the present type of non-specific cytotoxicity is due to activated T cells (presumably blasts) and that plant mitogens are required both for the conversion of the resting T cells to activated cells and to approximate the activated T cells to the target cells and/or initiate a special terminal cytotoxic activation. [ABSTRACT FROM AUTHOR]

Antisera were prepared in rabbits against lymphoid cells from peripheral blood of a patient with Bruton-type agammaglobulinaemia. Such antisera could be shown to display a two plateau level of cytotoxicity against human peripheral lymphocytes in the, presence of complement. This suggested the presence of species-as well as subgroup-specific antibodies in these antisera. The subgroup-activity of the antisera could be shown to be directed against human T lymphocytes on the basis of the following results. When lymphocytes are filtered through columns coated with anti-immunoglobulin antibodies lymphocytes with high surface concentrations of immunoglobulin are retained. The filtered cells are highly enriched in cells sensitive to the subgroup-specific antibodies. A close to complete inactivation of mixed leucocyte reactivity or stimulability with soluble PHA was induced by preincubating with the antisera and complement. Using identical conditions only marginal inhibition of immunoglobulin production of peripheral lymphocytes in vitro was induced. In conclusion, we believe these antisera to selectively kill human T lymphocytes at serum concentrations, which will not kill or inhibit the function of human B lymphocytes. [ABSTRACT FROM AUTHOR]

The presence of binding sites for concanavalin A, phytohaemagglutinin and lentil mitogen on the surface of mouse B and I lymphocytes has been investigated in relation to the previously described mitogenic selectivity of these stimulants for T cells. Purified T or B or mixed T/B suspensions of cells have been analysed both for proportions of cells with affinity for phytomitogens and for the relative density of binding sites. In addition, the specificity of mitogens for simple sac-charides has been investigated in relation to binding of the former to T and B cells. Microscopic agglutination and immunofluorescent studies show that virtually 100% of T and B lymphocytes have binding sites for the three phytomitogens. Quantitative absorption experiments have demonstrated that there is virtually no difference in the relative average density of binding sites on T and B cells. Immunofluorescent studies with Con-A suggested that binding sites are uniformly present over the entire cell surface. However, when lymphocytes are metabolically active an interesting altered localization of bound Con-A molecules to one pole of the cell takes place. His suggested that binding sites for the various phytomitogens are qualitatively and topographically distinct on the cell surface but that with respect to any one mitogen T and B cells have qualitatively and quantitatively similar binding sites. The relevance of these observations to lymphocyte activation is considered. [ABSTRACT FROM AUTHOR]

The mitogenic selectivity of four phytomitogens, phytohaemagglutinin (PHA), concanavalin (ConA), lentil mitogen (LM) and pokeweed mitogen .(PWM), and a series of heterologous antilymphocyte sera (ALS) for mouse T and B lymphocytes has been investigated using thymidine uptake as a measure of proliferative activity. The results show that PHA, ConA and LM stimulate T but not B cells whereas PWM activates both T and B cells. Of six mitogenically active ALS, five were T cell specific and one B cell specific. Cortisone resistant (T) cells within the thymus showed a much greater response to all mitogens than unselected thymocytes. However, ConA and LM activated a considerable response in the latter population. Selective activation of T and B cells by aspecific means may be useful both as a clinical tool and as an approach to gaining an understanding of antigen induction of immune responses. [ABSTRACT FROM AUTHOR]

The proliferative interaction of mixed homologous chicken thymus cells was studied in a serum-free culture system using thymus cell populations from thymus glands that had been experimentally manipulated to yield varying proportions of lymphocytes from the medulla and cortex of the gland. It could be demonstrated that the proliferative responsiveness of thymus cells to immuno-genetically different cells resided with the lymphocyte population located in the medulla of the thymus. The capability of medullary thymus cells to participate in a mixed lymphocyte interaction (MLI) was found to be nearly equal to that of splenic lymphocytes. The in vitro survival of medullary thymus cells was markedly superior to cortical thymus cells. Kinetic studies with medullary thymus cells revealed that the proliferative response in the MLI was initiated during the first 24-40 hr culture period and generally reached its peak 4 days after culture initiation. It could be demonstrated that responding cells were capable of several repeated divisions. In addition, previously nondividing cells entered the reaction for the first time up to the 3rd and possibly 4th day after culture initiation. Calculations revealed that 0.7-l-9% of the original viable thymus cell population participated in the response. It was also calculated that peripheral blood lymphocytes contaminating the thymus cell populations of both cell donors contributed less than 1% to the total number of responding thymus cells. When the response of mixed homologous thymus cells was compared in a chemically defined and serum containing medium, striking differences in the time course and magnitude of the reaction were seen. It could be shown that the viability and proliferation of thymus cells was adversely affected by serum and markedly enhanced by insulin supplementation to a chemically defined medium. [ABSTRACT FROM AUTHOR]