Effect of Neuraminidase on Lymphoid Cells

After neuraminidase treatment the electrophoretic mobility of mouse lymphocytes (B and T cells) and thymocytes was reduced to a similar value, .a diminution of about 40%, 60%, and 40%, respectively. Thus B and T cells and thymocytes may have a common surface charge after removal of sialic acid. The percentage of cells binding anti-thymocyte serum was not changed; thus sialic acid does not seem to be an important part of the specific antigen of lymphoid cells. Similar amounts of sialic acid were released from B and T Ceils, whereas less than half of this was released from the thymocytes. Thus, whereas thymocytes are characterized by their low electrophoretic mobility and low sialic acid release and T cells by high electrophoretic mobility and high sialic acid release, B-cells in contrast show a low electrophoretic mobility and high sialic acid release.     

B lymphocyte subpopulations in the mouse spleen. A study of the differentiation pathway using free flow electrophoretically separated subpopulations of direct PFC progenitor cells.

Free-flow electrophoretic separation of mouse spleen cells provides three distinct progenitor cells of direct PFC, showing high, medium and low electrophoretic mobility. All progenitor cells possess surface immunoglobulin and mouse B-lymphocyte specific antigen. The progenitor cells of high electrophoretic mobility show high cycling turnover, a spleen seeking capacity of 16%, provide PFC with a maximum 8 days after transfer and reveal an isometrical increase of the PFC dose response line as a function of the graft size. The progenitor cells of medium electrophoretic mobility are low cycling, 16% home to the spleen, a maximum of PFC is developed eight days after transfer and the PFC dose response line increases allometrically. The progenitor cells of low EPM show low cycling activity, 20% home to the spleen, a maximum of PFC is attained six days after transfer and the PFC dose response line rises isometrically. These results suggest that the electrokinetically different PFC progenitors represent biologically distinct subsets. In double transfer experiments, some evidence was obtained that progenitor cells of low electrophoretic mobility are derived from progenitors of higher electrophoretic mobility. The same observation accounts also for the formation of B lymphocytes of low EPM. Since it seemed likely that the PFC progenitor cells represent virgin cells of a single lineage, the results were discussed in the terms of differentiation pathways of B lymphocytes. A model is considered in which a progenitor of medium electrophoretic mobility provides those of high electrophoretic mobility which after passing a transient cycling stage finally produce mature resting B lymphocytes of low electrophoretic mobility.     

Electrophoresis of lymphocytes from normal human subjects and patients with chronic lymphocytic leukaemia

The electrophoretic mobility (EM) of peripheral blood lymphocytes from twelve normal subjects and four patients with chronic lymphocytic leukaemia (CLL) was studied in relation to the thymus-derived (T) lymphocytes and bone marrow-derived (B) lymphocytic system. An attempt was made to correlate electrophoretic results with other methods of identifying T and B lymphocytes—T cells by the formation of sheep-cell rosettes and B cells by the formation of erythrocyte–antibody–complement (EAC') rosettes and by staining for surface immunoglobulin.

In the normal subjects the majority of cells migrated quickly with a small `tail' of slower cells. It is suggested that the faster populations are T cells and the slower, B. In CLL the majority of the cells were slow migrators. There was agreement between the percentage of fast cells as assessed by electrophoresis with that of T cells by sheep-cell rosetting; there was also some agreement between the percentage of electrophoretically slow cells to B cells by EAC' rosettes or surface immunoglobulin.

It was possible to remove some of the B cells by density centrifugation after forming EAC' rosettes. This further defined the T cell peak on electrophoresis.

It is concluded that T and B cells carry different surface charge densities which permit them to be separated by electrophoresis and that the malignant B lymphocytes of CLL migrate electrophoretically in a similar fashion to normal B cells.

     

Studies with the macrophage migration inhibition (MMI) test in patients with malignant disease.

Macrophage migration inhibition (MMI) tests have been carried out using lymphocytes from twenty-six patients with malignant disease and from twenty-two other subjects. The antigens used were the same as those employed in the macrophage electrophoretic mobility (MEM) test. The results indicate that the inforation obtained using the MMI test is similar to that found with the MEM test.     

Thymus specific antigen on electrophoretically separated rat lymphocytes. Tracing of the differentiation pathway of bone marrow-derived thymocytes by use of a surface marker

Rat lymphocytes from different lymphoid organs were separated by means of free-flow electrophoresis and the separated cells subdivided into classes of high (H lymphocytes) and low (L lymphocytes) electrophoretic mobility. A rabbit antiserum against thymocytcs of low electrophoretic mobility was prepared and its cytotoxic activity tested with H and L lymphocytes from spleen, lymph node, thoracic duct lymph, bone marrow and thymus. The cytotoxic activity was bound to the IgC immunoglobulins of the antiserum. An agglutination reaction could be observed with all L lymphocytes, but not with H lymphocytes, and was not further investigated. Cytotoxicity was obtained with bone marrow L lymphocytes and H and L thymocytes, but not with lymphocytes of other sources. It was suggested that this antigen resembled the rat “thymus specific” antigen. The finding that this antigen appeared on L lymphocytes of bone marrow and thymus and diminished on thymus H lymphocytes with be treated by following the differentiation of bone marrow derived thymocytes.     

Density gradient electrophoresis of mouse spleen lymphocytes. Differential mobilities of macrophages, responding and stimulating cells in mixed lymphocyte culture

Preparative density gradient electrophoresis has been employed for the separation of BALB/c mouse spleen T and B lymphocytes, on the basis of their surface charge. The high mobility cells were found to be predominantly T lymphocytes, whereas the low mobility cells were B lymphocytes. One way mixed lymphocyte cultures were prepared using electrophoretically separated BALB/c lymphocytes, either as responding or stimulating cells and unfractionated CBA/H/T6j mouse spleen lymphocytes, in the appropriate combination. The responding lymphocytes were found only among the high mobility cells which are primarily T lymphocytes. In contrast, the stimulating cells were found in the intermediate and low mobility fractions which contain the B lymphocytes and macrophages. Minimal stimulation in mixed lymphocyte culture was observed by T cells in this allogeneic system. Ia-positive cells were found only in the intermediate and low mobility fractions, which exhibited the capacity to stimulate allogeneic cells in MLC. Macrophages, identified by nonspecific esterase staining, exhibited intermediate electrophoretic mobility, their peak height coinciding with the maximum observed stimulation.     

Simplified cell microelectrophoretic method applied to the macrophage electrophoretic mobility test for cancer diagnosis.

Simplified agarose-coated capillary microelectrophoresis was adapted to the Macrophage Electrophoretic Mobility (MEM) test for cancer detection. Guinea pig alveolar macrophages gave superior reproducibility to peritoneal macrophages. As good or better reproducibility was obtained with cryopreserved, as with fresh macrophages. The electrophoretic mobilities of patients' lymphocytes themselves, after incubation with Encephalitogenic Factor (EF) showed a significant increase in electrophoretic mobility, while in control lymphocytes a decrease occurred. Thus, for the detection of the results of the interaction of EF on human lymphocytes, a direct lymphocyte electrophoretic mobility test may suffice, and guinea pig macrophages may no longer be required at all.     

Simplified Cell Microelectrophoretic Method Applied to the Macrophage Electrophoretic Mobility Test for Cancer Diagnosis

Simplified agarose-coated capillary microelectrophoresis was adapted to the Macrophage Electrophoretic Mobility (MEM) test for cancer detection. Guinea pig alveolar macrophages gave superior reproducibility to peritoneal macrophages. As good or better reproducibility was obtained with cryopreserved, as with fresh macrophages. The electrophoretic mobilities of patients' lymphocytes themselves, after incubation with Encephalitogenic Factor (EF) showed a significant increase in electrophoretic mobility, while in control lymphocytes a decrease occurred. Thus, for the detection of the results of the interaction of EF on human lymphocytes, a direct lymphocyte electrophoretic mobility test may suffice, and guinea pig macrophages may no longer be required at all.     

Immunological characteristics of lymphoblasts and lymphocytes in acute lymphoblastic leukaemia in children.

By analysing the electrophoretic mobility (EM) of lymphocytes and by comparing their surface charge it is possible to distinguish rapid-moving lymphocytes (T) and slow-moving lymphocytes (B). The authors studied the EM of blood lymphocytes in children suffering from acute lymphoblastic leukaemia, before they underwent treatment. At the same time they carried out a study of classical immunological markers (E rosettes, membrane immunoglobulins, and blastic-transformation ability). The authors were able to distinguish three types of lymphocytes according to their EM i.e., rapid, slow or intermediate. This heterogeneousness could be explained by the fact that the origin of leukaemic proliferation may vary according to different stages in the maturation of the lymphoid cells. It would seem premature to establish any correlation between the initial clinical signs of the disease and its evolution.     

T and B lymphocytes in pituitary dwarf Snell-Bagg mice.

The lymphocyte composition of the thymus and spleen from weaned (4 month old) hypopituitary dwarf Snell-Bagg mice were compared to those of their phenotypically normal littermates and of hormone (somatotropic hormone plus thyroxine)-treated individuals. Detection of cells bearing receptors for peanut agglutinin, physical analysis and measurement of in vitro reactivities to phytohaemagglutinin and concanavae intra-thymic lymphocyte population of dwarf mice. Examination of spleen-cell suspensions demonstrated a slightly higher frequency of T lymphocytes (Thy 1-2+ alpha-Naphthyl esterase+, high electrophoretic mobility) and lower frequency of B lymphocytes (surface immunoglobulin+, low electrophoretic mobility) in dwarf mice than in control mice. The degree of splenocyte responsiveness to T- and B-cell mitogens, however was similar in the two mouse types. High mobility (T) splenic cells were found to exhibit a smaller modal volume in dwarf mice (110 micron3) than in control mice (122 micron3) but this difference was not corrected by hormone administration. More pronounced were the quantitative differences between the spleens of hormone-deficient and normal mice. Thus, when expressed as a function of body weight, the numbers of splenic T and B lymphocytes in untreated dwarf mice were about half the corresponding values in hormone-reconstituted or normal littermates. These data suggested that in adult life, developmental hormones exert little direct effect on the thymus lymphocytes but influence the size of the pool of both peripheral T and B lymphocytes.     

Detection of T and B cell-specific heteroantigens on electrophoretically separated lymphocytes of the mouse

Mouse spleen lymphocytes were electrophoretically separated into pools of T and B lymphocytes. Heteroantisera were raised in rabbits against these cell pools, the IgG fractions were isolated and their lymphocytotoxicity tested. After appropriate absorption, the antisera were specifically directed against lymphocyte antigens. Further cross-absorption with T and B lymphocytes made the antisera specific for antigens which were mutually exclusively present on T and B cells and were related to neither alloantigens nor immuno-globulins. These anti-B and anti-T cell antisera killed antibody-forming cells and graft-versus-host reactive cells, respectively, in vitro. Furthermore, anti-B cell antiserum was cytotoxic to lymphocytes of low electrophoretic mobility in bone marrow and peripheral organs, except in the thymus, whereas anti-T cell antiserum was cytotoxic to lymphocytes of high electrophoretic mobility in all organs and, in addition, killed all thymocytes of low electrophoretic mobility, which had not been affected by anti-B cell antiserum. This distribution of lymphocytes in the electrophoretic distribution profiles, together with the described properties, gives evidence that heteroantigens were found to be exclusively present on T and B cells. They were designated “mouse B cell-specific” and “mouse T cell-specific” antigens and are part of mouse lymphocyte-specific antigen.     

感染约氏疟原虫小鼠淋巴细胞的电泳行为

本文对感染约氏疟原虫小鼠的脾脏及淋巴结淋巴细胞的电泳行为进行了观察比较。正常小鼠的脾脏及淋巴结淋巴细胞的电泳率均呈双峰型。感染约氏疟原虫小鼠的脾脏及淋巴结淋巴细胞表现为电泳率快的T细胞比例增高。感染后自愈的小鼠的脾脏淋巴细胞突出地表现为电泳率慢的B细胞比例增高,淋巴结淋巴细胞以电泳率呈快慢适中的淋巴细胞比例增高更为明显。对感染约氏疟原虫小鼠免疫状况进行了简要的讨论。     

Immunological activity of blood lymphocyte fractions: a study by the macrophage electrophoretic mobility method

Samples of human blood-lymphocytes from patients with cancer, multiple sclerosis or cerebral haemorrhage were separated on a discontinuous Ficoll gradient. Six to eight fractions were obtained in which one or two fractions contained a large number of small lymphocytes. The fractions were incubated with encephalitogenic factor or tumour antigen and guinea-pig macrophages, after which the macrophages' electrophoretic mobility (EMR) was measured. The fractions with a density between 1·075 and 1·090, and containing a high percentage of small lymphocytes, produced a significant EMR in these patients and it was concluded that the cells responsible for cellular immunological activity were also responsible for reductions in the electrophoretic mobility.     

Cell surface analyses of the age-dependent changes in the electrokinetic properties of human peripheral blood lymphocytes

The age-dependent changes in the surface electrical charge of human peripheral blood lymphocytes were analyzed by establishing the differences between the surface density of four types of chemical groups on lymphocytes isolated from the blood of individuals of increasing ages. Similar determinations were carried out on cord blood lymphocytes which were shown previously to exhibit either a low or a high electrical charge density reflected in the experimentally determined parameter, the anodic electrophoretic mobility (EPM), differing by about 30%. The surface density of carboxyl group of N-acetylneuraminic acid (NANA), protein side chain epsilon-amino groups, and phosphate groups were different for the two subpopulations of CBL. Differences were also observed between the surface density of these groups on the two subpopulations of CBL and the lymphocytes of older individuals, with the exception of carboxyl groups. In some experiments on lymphocytes from adults, the carboxyls of NANA were much more numerous on nearly 1% of the cells.     

Antigen-Specific Electrophoretic Cell Separation (ASECS): Isolation of human T and B lymphocyte subpopulations by free-flow electrophoresis after reaction with antibodies

The electrophoretic mobility of human lymphocytes can be reduced by incubation with surface antigen specific antibodies under non-capping conditions. This renders subpopulations of human peripheral blood lymphocytes accessible to separation by free-flow electrophoresis.After reaction of lymphocyte preparations with anti-IgM antibody and a fluorescent second antibody, B lymphocytes showed a considerable shift in position in preparative cell electrophoresis and could be separated with high yield, purity and vitality. Similarly, a T cell subpopulation reactive with the monoclonal antibody T811 could be isolated, even though only small amounts of this antibody were bound, by using a double-sandwich method.Non-specific antibody uptake via Fc-receptors did not contribute to the observed shift of antibody-labelled cells to lower electrophoretic mobility. Flow cytometric analysis showed that cells were separated according to their antigen density.Thus cell electrophoresis can be used to separate antibody-labelled cells. With a flow rate of 100,00 cells/sec this method has a much higher separation capacity than fluorescence-activated cell sorting. The described method should be applicable to the separation of a wide range of cell populations for which specific antibodies are available.     

Electrophoresis of lymphoid cells. Characterization of human B and T cells in peripheral lymphoid tissues

Small lymphocytes from human blood, tonsils, spleen and lymph nodes showed a bimodal distribution on electrophoresis, the electrophoretic mobility (EPM) of one group being slow, the other fast. The difference in mean EPM between the slow and fast groups was about 30%. The percentage of slow cells in each organ was similar to that of cells with surface-bound immunoglobulin as judged by immunofluorescence studies. It was therefore concluded that a major proportion of B cells has a relatively low net surface charge while that of the T cells is higher.     

Electrophoretic behaviour of blood and synovial fluid lymphocytes in rheumatoid arthritis.

Probit analysis of the electrophoretic mobilities of human blood lymphocytes identifies at least three main subpopulations. According to their rate of movement in an electrical field, the subpopulations are referred to as the fast, intermediate and slow cell distributions. Lymphocytes of the fast and intermediate populations appear to be T cells, while the slow cell population includes cells with B cell characteristics. Compared with normal subjects, lymphocytes of intermediate mobility are significantly increased in the blood of rheumatoid patients and comprise a major fraction of the lymphocyte exudate in rheumatoid synovial fluid.     

Inhibition of monocyte spreading. A direct in vitro test for assessing delayed-type hypersensitivity in man

An in vitro expression of delayed-type hypersensitivity to tuberculin was tested in Mantoux positive and Mantoux negative persons. Their lymphocytes and monocytes were isolated from venous blood and incubated in vaseline chambers with or without tuberculin. In the presence of tuberculin, a substantially lower percentage of monocytes from Mantoux positive persons spread, than monocytes from Mantoux negative persons. This antigen-induced inhibition of monocyte spreading seems to be a reliable measure of tuberculin hypersensitivity, since it occurs only in Mantoux positive persons and correlates well with the intensity of the tuberculin skin reaction. Reproducibility of the test and the speed with which it is performed could constitute a basis for the use of monocyte spreading inhibition in clinical studies of cell-mediated immune reactions.     

Analysis of T cell subpopulations by laser Doppler spectroscopy and the association of electrophoretic mobility differences with differences in rosette-forming affinity.

Rosetting procedures were used to identify and isolate human T lymphocytes having different affinities for sheep red blood cells (SRBC). High-affinity E rosette-forming cells (E-RFC) were obtained by rosetting at 29°C with limited numbers of SRBC. Low-affinity and total E-RFC were obtained by rosetting at 4°C with an excess of SRBC. The E-RFC isolated from these fractions on Ficoll-Hypaque gradients exhibited different electrophoretic mobilities as measured by laser Doppler spectroscopy. The high-affinity E-RFC mobility was centered at 2.35 μm/sec/V/cm (for 25°C, 0.28 M sucrose medium of 0.005 ionic strength), whereas the low-affinity E-RFC mobility was centered at 2.15 μm/ sec/V/cm. The total E-RFC fractions consisted of cells of both mobilities. When IgG-sensitized E monolayers were used to remove cells bearing Fc receptors from mononuclear cell preparations, the non-adherent and the adherent fraction were each found to contain a different subpopulation of T cells having mobilities centered at 2.35 and 2.15 μm/sec V/cm, respectively. Rosetting assays and mobility measurements were performed simultaneously with the mononuclear cell preparations and the non-adherent fraction from IgG-sensitized E monolayers. In both cases, there was quantitative agreement between the number of high-affinity E-RFC and the number of high-mobility cells. In contrast, there were consistently more low-mobility cells than could be accounted for by low-affinity E-RFC. The determination of cell electrophoretic mobility is a direct physical measurement and its association with such surface markers as different affinity receptors for SRBC and Fc receptors for IgG shows that it should be useful parameter in the characterization of lymphocytes.     

Time kinetics of the interaction of concanavalin A with splenic lymphocytes of normal AKR mice.

Interaction of concanavalin A (Con A) with splenic lymphocytes of normal AKR mice led to concentration-dependent biphasic changes in electrophoretic mobility. Kinetics of this interaction suggested that at high concentration Con A interacted in two steps. The first step led to redistribution of Con A receptors and increased EPM. In the second step additional receptors to Con A became accessible and their interaction with Con A reduced surface charge density. The kinetic data thus provide additional evidence for the existence of two sets of qualitatively different receptors for Con A on AKR lymphocytes.