Electrophoretic mobility of human lymphocytes purified by nylon columns and spontaneous rosettes]

Cell electrophoresis enables the separation of the lymphocytes in normal human blood into two principal groups, as a function of their migration speed in relation to 1 mum.sec -1..v-1.cm. In 42 healthy adults, 19,9 % of the lymphocytes have a slower migration, and 80,1 % a faster migration than the reference speed. Two known methods are used for the selection of the lymphocytic populations : spontaneous rosetting with sheep red blood cells, a property of the T lymphocytes, and the adherence to nylon wool columns, which is dominant in the case of B lymphocytes. The cells which do not form spontaneous rosettes, and the cells adhering to nylon wool columns have above all a slow migration. On the contrary, cells which do not adhere to nylon columns have a fast migration. These arguments are in favour of the T nature of the rapid migrating lymphocytes, and of the B nature of the slow migrating lymphocytes.     

Identification of T and B cell subpopulations in human peripheral blood: electrophoretic mobility distributions associated with surface marker characteristics.

The electrophoretic mobility distributions of human peripheral blood lymphocytes isolated on Ficoll-Hypaque gradients were characterized by laser Doppler spectroscopy. Three major subpopulations were spectrally resolved, due to differences in their mobility, when an electric field was applied to the scattering cuvette. The fastest component (centered at 2.35 mum/sec/V/cm for 25 degrees C, 0.28 M sucrose medium of 0.005 ionic strength) passed through nylon fiber columns and was identified as a T cell subpopulation. The slowest component (1.85 mum/sec/V/cm) which was further enriched by a one-step rosette procedure with sheep erythrocytes, reacted with activated-complement (C3) and antiserum to human immunoglobulins and was therefore identified as a B cell subpopulation. The intermediate component (centered at 2.15 mum/sec/V/cm) appears to be another T cellsubpopulation. Although cells of this component did not pass through nylon fiber columns, they did rosette with sheep erythrocytes. Furthermore, these cells did not appear to have surface immunoglobulins or complement receptors.     

Changes in the electric charge of blood lymphocyte populations after secondary immunization with tetanus antitoxin in man]

The electrophoretic mobility of circulating lymphocytes has been studied in normal human subjects after immunization by tetanus toxoid. The mean migration speed was shown to increase, particularly two and three days after secondary immunization. This increase appeared to be due to the elevation of percentage of T cells migrating at 1.20 and 1.35 micrometer. sec.-1v.-1 cm. (active rosettes-forming cells), with a decrease of the percentage of B cells and T lymphocytes migrating at 1.10 micrometer. sec.-1v.-1 cm. The return to the anterior status was observed between day 4 and 8 after immunization.     

Decreased membrane potassium permeability and transport in human chronic leukemic and tonsillar lymphocytes.

Human blood T-lymphocytes increase their potassium (K+) permeability and active K+ transport following lectin or antigen stimulation. We have studied the permeability and active transport of K+ by lymphocytes in chronic lymphocytic leukemia (CLL) to determine if their membrane K+ transport was similar to resting or lectin-stimulated normal blood lymphocytes. K+ transport was assessed both by the rate of isotopic 42K+ uptake and by the rate of change in cell K+ concentration after inhibition of the K+ transport system with ouabain. CLL lymphocytes had a marked decrease in membrane K+ permeability and active transport of K+ when compared to blood T lymphocytes. K+ transport in five subjects with CLL (10 mmol.1 cell water-1.h-1) was half that in normal blood T-lymphocytes (20 mmol.1 cell water-1 h-1). Phytohemagglutinin (PHA) treatment of CLL lymphocytes did not increase significantly their active K+ transport, whereas K+ transport by normal T-lymphocytes increased by 100%. Since there were 73% T-lymphocytes in normal blood and 14% in CLL blood, the difference in membrane K+ turnover could be related either to neoplasia or to the proposed B-lymphocyte origin of CLL. We studied human tonsillar lymphocytes which contained a mean of 34% T-cells. In five studies of tonsils, K+ transport was 14 mmol.1 cell water-1.h-1 and treatment with PHA increased K+ transport only 30%. The intermediate values of basal K+ transport and K+ transport in response to PHA in tonsillar lymphocytes were consistent with the proportion of T-lymphocytes present. These data suggest that B-lymphocytes have reduced membrane permeability and active transport of K+. Thus the marked decrease in CLL lymphocyte membrane K+ permeability and transport may be a reflection of its presumed B-cell origin, rather than a membrane alteration related to malignant transformation.     

Development of the mucosa and differentiation of the lymphocytes of the vermiform appendix of the human fetus

One hundred and twenty-two appendices have been obtained from 12-30-week-old human fetuses and studied histologically (90) and immune-morphologically (32). Lymphoid follicles in the organ appear on the 17th week. Character of the epithelial and the reticular tissue structure in the area of the cupola including the lymphoid follicle have been studied. T- and B-lymphocytes have been stated to be present in the appendix of a 17-week-old fetus. As the fetus is further developing, the number of lymphocytes in the appendix increases: the amount of T-lymphocytes is practically constant, and that of B-lymphocytes increases. Direction of the lymphocytes migration out of the follicle is demonstrated. The lymphoid formations of the appendix are necessary for certain local protective reactions and already in the fetus they begin to participate in the general system of the organism's immunogenesis.     

Role of B-lymphocytes in inactivating allogenic stem cells]

Antisera to membrane antigens of B lumphocytes eliminated the capacity of lymphocytes to inactivate allogenous stem cells by 60%; however, lymphocytes from the lymph nodes of B mice possessed no inactivating capacity. T-lymphocytes were the main criteria inactivating allogenous stem cells. Cooperating with T-lymphocytes, B-lymphocytes probably contributed to inactivation of precursor cells realized by T-lymphocytes. However, the presence of B-lymphocytes in the killer cells population was not a determinant, since T-lymphocytes were capable of inactivating allogenous stem cells without any participation of B-lymphocytes.     

Comparative study of the T- and B-cell immunity systems in human and animal embryogenesis

Data are presented on the spleen and thymus structure, time of appearance of the lymphocytes and their heterogeneity in the liver, thymus, spleen, lymph nodes and blood of human foetuses with hemochorial placenta (3 to 34 weeks) and of the minipigs foetuses with epitheliochorial placenta (32 to 95 days). In both foetuses the first T- and B-lymphocytes are found in liver, T-lymphocytes are then found in thymus and later in spleen and lymph nodes whereas B-lymphocytes are found, after liver, in spleen. Kinetics of T- and B-lymphocytes during embryogenesis is described. Reaction of the minipig lymphocytes to mitogens was demonstrated.     

Surface proteins of thymus-derived lymphocytes and bone-marrow-derived lymphocytes. Selective isolation of immunoglobulin and the θ-antigen by non-ionic detergents

Accessible surface proteins of thymus-derived lymphocytes (T-cells) of normal CBA mice and bone-marrow-derived lymphocytes (B-cells) of congenitally athymic nu/nu mice were analysed. The surfaces of lymphocytes were radioiodinated by using the enzyme lactoperoxidase (EC 1.11.1.7), then solubilized either in acid-urea or in the non-ionic detergent Nonidet P-40. These lysates were then precipitated with antisera specific to either immunoglobulin or the theta-alloantigen in order to assess the presence of these surface markers. Comparable amounts of radioactivity in proteins specifically precipitable as immunoglobulin were obtained from T-lymphocytes and B-lymphocytes when the cells were disrupted by acid-urea. This immunoglobulin had mol. wt. approx. 180000 and was composed of light chains and mu-type heavy chains. When radioiodinated lymphocytes were solubilized with Nonidet P-40, 3-4% of radioiodinated high-molecular-weight protein of B-cells consisted of immunoglobulin, a result similar to that found with acid-urea extraction. However, with the detergent extraction, only 0.1% of T-cell surface protein was precipitable by anti-globulin reagents. The theta-alloantigen was isolated from CBA T-cells both by acid-urea and by detergent lysis. This protein possessed a mobility on polyacrylamide-gel electrophoresis in sodium dodecyl sulphate which was consistent with a mol. wt. of 60000. An identical component was isolated from the theta-positive thymoma WEHI 105. The theta-antigen was not isolated from B-cells by either of the extraction procedures used. These results provide further evidence that the surface membranes of normal T-cells and B-cells differ in physicochemical properties. In particular, various surface components possess differential solubilities in non-ionic or organic solvents. This observation provides an explanation for discrepant results that have appeared in the literature concerning the isolation of immunoglobulin from T-lymphocytes.     

Epigenome-wide profiling identifies significant differences in DNA methylation between matched-pairs of T- and B-lymphocytes from healthy individuals

Multiple reports now describe changes to the DNA methylome in rheumatoid arthritis and in many cases have analyzed methylation in mixed cell populations from whole blood. However, these approaches may preclude the identification of cell type-specific methylation, which may subsequently bias identification of disease-specific changes. To address this possibility, we conducted genome-wide DNA methylation profiling using HumanMethylation450 BeadChips to identify differences within matched pairs of T-lymphocytes and B-lymphocytes isolated from the peripheral blood of 10 healthy females. Array data were processed and differential methylation identified using NIMBL software. Validation of array data was performed by bisulfite pyrosequencing. Genome-wide DNA methylation was initially determined by analysis of LINE-1 sequences and was higher in B-lymphocytes than matched T-lymphocytes (69.8% vs. 65.2%, P ≤ 0.01). Pairwise analysis identified 679 CpGs, representing 250 genes, which were differentially methylated between T-lymphocytes and B-lymphocytes. The majority of sites (76.6%) were hypermethylated in B-lymphocytes. Pyrosequencing of selected candidates confirmed the array data in all cases. Hierarchical clustering revealed perfect segregation of samples into two distinct clusters based on cell type. Differentially methylated genes showed enrichment for biological functions/pathways associated with leukocytes and T-lymphocytes. Our work for the first time shows that T-lymphocytes and B-lymphocytes possess intrinsic differences in DNA methylation within a restricted set of functionally related genes. These data provide a foundation for investigating DNA methylation in diseases in which these cell types play important and distinct roles.     

The effect of extracorporeal blood radiotherapy on the peripheral blood T and B-lymphocytes in chronic lymphadenitis]

B-lymphocytes and T-lymphocytes were determined before and after therapy in 12 patients with chronic lymphatic leukemia (CLL) treated with extracorporal blood radiation (ECIB). There was a significant decrease of B-lymphocytes (p less than 0.001) from 85.3 +/- 6.55% to 71.55 +/- 10.60% by ECIB, whereas no significant changes could be found in T-lymphocytes. 12 untreated CLL patients, whose B-lymphocytes amounted 83.25 +/- 6.79% with 7.5 +/- 3.42% of T-lymphocytes, were examined as group of comparison. From these findings ECIB is concluded to cause a decrease of accumulated B-cells.     

Fluorescent probe-indicator of differences between T- and B-lymphocytes in the blood]

A new physico-chemical marker for the human peripheral blood lymphocytes was worked out. The lymphocytes were vitally stained with the fluorescent probe 3-methoxybenzanthrone and measured by microfluorometry. The blood lymphocytes population was found to be heterogeneous; this population consists of the two main groups of cells differing by the intensity of their fluorescence. By means of immunological lymphocyte fractionation it was shown that one of these cell groups was represented by T-lymphocytes, and the other one--by B-lymphocytes.     

Mathematical model of the effect of glucocorticoids on the movement and proliferation kinetics of mammalian lymphocytes

Effect of glucocorticoids on proliferation, death and migration of T-lymphocytes was studied by mathematical methods. A model of the interaction between hormone and thymus T1-lymphocytes is proposed which imitates involution of lymphoid tissue under stress. There were constructed models of migrations of free and radioactive chromium labeled lymphocytes in lymphoid and non-lymphoid organs, blood and lymph at arbitrary concentration of glucocorticoids in the blood. Possible mechanisms of immunodepression due to increased hormone concentrations in the organism are discussed.     

Cytochemistry of leukocytes in childhood. III. Changes of cytochemical reactions in lymphocytes in infections and during immunological reactions]

The present paper gives a report on changes of enzyme reactions, of the glycogen content, and the nucleolus picture in lymphocytes which are based on cytochemical investigations of blood smears taken from 110 children with different diseases. In 20 new-born babies the cytochemical responses of lymphocytes after triple immunization with Di-Pe-Te immunization matter were observed. The findings reveal significant changes to be found predominantly in the activity of alpha-naphthylacetate esterase, PAS-reaction and the nucleolus picture of lymphocytes in immunological reactions. No hints for specific immunological functions of lymphocytes could be detected. The changes may refer to B-lymphocytes and to T-lymphocytes.     

Determination of acid α-naphthyl acetate esterase enzyme activity in peripheral blood leukocytes of gazelles (Gazella subgutturosa)

We examined gazelle peripheral blood leucocytes using the α-Naphthyl acetate esterase (ANAE) staining technique (pH 5.8). Our purpose was to determine the percentage of ANAE positive lymphocytes. The proportion of ANAE positive T-lymphocytes was 72%. T-lymphocytes showed an ANAE positive reaction, but eosinophilic granulocytes and monocytes also showed a positive reaction. By contrast, no reaction was detected in B-lymphocytes, neutrophil granulocytes or platelets. The reaction observed in T-lymphocytes was a red-brown coloration, usually 1–2 granules, but enough granules to fill the cytoplasm were detected rarely. As a result of ANAE enzyme staining, we concluded that the staining technique can be used as a cytochemical marker for gazelle T-lymphocytes.     

Characteristics of individual lymphocyte populations in the blood of virtually healthy persons]

The number of individual lymphocyte populations in the blood of apparently healthy persons was determined by spontaneous rosette-formation with SRBC (T cells) and mouse red blood cells (B cells). In the majority of persons examined the percentage of T-lymphocytes constituted 47.3 +/- 1,6, of B--16.8 +/- 1, and of "zero" cells--33.5 +/- 3.4. There was less T- and B-lymphocytes in the blood of elderly persons (over 50 years of age) than in the young ones. It was also shown that the T-lymphocyte population forming "active" rosettes could be assessed by the number of SRBC sorbed on their surface.     

Relationship between the capacity to be stimulated of lymphocyte subpopulations and the RAI staging in patients with chronic lymphocytic leukemia]

By means of the incorporation rate of 3H thymidine into the lymphocytes of patients with chronic lymphatic leukaemia the possibility of stimulating them by using different mitogens was checked and compared with normal persons. The examination covered 11 patients treated with extracorporeal irradiation of the blood (ECIB), 5 patients treated with a chlorambucil therapy, and 10 untreated patients who were classified according to the staging system proposed by RAI. The lymphocytes of the peripheral blood were stimulated as mixed and isolated T and B-lymphocytes in the microculture by using the mitogens PHA, PWM, ConA, and LPS. In all CLL patients there was a diminished stimulation rate of a mixed lymphocyte population. A relation existed between the seriousness of the stage and the diminution of the incorporation rate of 3H thymidine. A corresponding correlation could not be identified in untreated CLL patients. Isolated T-lymphocytes revealed better results of stimulation than the total population. As to their function B-lymphocytes showed a dependance on the kind of therapy. In the mixed lymphocyte culture of normal persons the best findings could be observed after stimulation with PHA, that is also valid for CLL patients. PHA, PWA, ConA, and LPS were suitable as substances stimulating B-lymphocytes with different efficacy in normal persons and CLL patients. Both collectives showed the best results in the T-lymphocyte culture after stimulation with LPS.     

Cytochemical enzyme staining of fish lymphocytes separated on a Percoll gradient

Brown trout ( Satmo trutta L) lymphocytes were shown to separate into two fractions on a Percoll discontinuous gradient, with 53% of the cells in the 1.07gl^-1 fraction. The cells from the two fractions showed equal enzyme activity when stained for acid esterase and acid phosphatase. About 70% of the lymphocytes gave a positive enzyme reaction, which if the reaction is comparable with mammalian lymphocyte cytochemistry would indicate they were T-lymphocytes. There appears to be increasing evidence among fish for the existence of T- and B-lymphocytes, and cytochemical staining could prove a comparatively convenient method for their demonstration.     

Characterization of human T lymphocytes that express the C3b receptor

The presence of the C3b receptor (C3bR) on human peripheral blood T lymphocytes was recognized by the capacity of rabbit F(ab')2 anti-C3bR and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat F(ab')2 anti-rabbit F(ab')2 to stain 14.5 +/- 3.7% (mean +/- SEM; n = 5) of lymphocytes forming rosettes with sheep erythrocytes (E). The F(ab')2 anti-C3bR also blocked the capacity of peripheral blood lymphocytes stained with OKT11 to form rosettes with bovine E bearing C3b and immunoprecipitated a single membrane protein having a m.w. of approximately 250,000 from detergent lysates of 125I-labeled, purified T cells. Measurement by fluorescent flow cytometry of the quantitative expression of the C3bR indicated that T cells had slightly more antigenic sites/cell than did E and approximately 10-fold fewer sites than were present on B cells. The surface constituents of the peripheral blood T cells expressing the C3bR were assessed in an assay that employed simultaneously three markers: rosette formation with sheep E, TRITC staining with anti-C3bR and fluorescein isothiocyanate (FITC)-staining with a panel of monoclonal antibodies or with aggregated IgG. Among lymphocytes forming rosettes with sheep E and expressing the C3bR, 99.6 +/- 0.4%, 65.0 +/- 5.8%, 17.2 +/- 6.2%, and 15.3 +/- 5.0% of the cells expressed antigens detected by OKT3, OKT4, OKT8, and OKM1 monoclonal antibodies, respectively. Ninety-seven per cent of the C3bR-bearing T cells were also capable of specifically binding aggregated IgG, indicating the presence of Fc receptors for IgG (Fc gamma R) on these cells. The T cells expressing the C3bR had large nuclei, thin rims of basophilic cytoplasm and no azurophilic granules. Thus, the C3bR is present on some T cells, all of which have a typical lymphocyte morphology, the T3 antigen and the Fc gamma R.     

Preparative Electrophoresis of Living Mammalian Cells in a Stationary Ficoll Gradient

An apparatus was designed for the vertical density-gradient electrophoresis of viable mammalian cells. Cultured Chinese hamster cells (M3-1F3) were grown is suspension and layered on top of a 0–10% ficoll gradient which was also an inverse 6.8-5.1% sucrose gradient in phosphate buffer, pB 7.2 and 1Z glucose. A 60-ml vertical gradient of this composition covered the density range 1.04–1.05 g/cm³ over a distance of 15 cm in a 2.3 cm diameter glass column. An electric field of approximately 2.3 V/cm was applied for 5 hr. The cells migrated 4.7 cm during this period. The migration rate was consistent with the cells having an electrophoretic mobility of -1.15 ± 0.20 μm/sec per volt/cm, equal to that determined by microelectrophoresis. The gradient was harvested in 50 fractions after electrophoresis, and the viability of the cells was 75% on the basis of colony formation.     

Topography,ultrastructure and phagocytic capacity of avian lymph nodes

Summary The structure of the avian lymph node (ALN) is characterized by a thin capsule, thin lymphoreticular cords, and an absence of trabeculae. It is not possible to subdivide the ALN into cortex, paracortex and medulla, or to subdivide the system of sinuses into marginal, trabecular and medullary divisions. The lymphoreticular cords contain avian germinal centers (AGC) with B-lymphocytes and the area of T-lymphocytes.Postcapillary venules are responsible for the recirculation of lymphocytes. Sinus reticular cells do not exist in the ALN, but free macrophages are present. The phagocytic capacity of the macrophages was determined by injection of vital dyes (India ink, Berlin blue) and inoculation with Candida cells. Macrophages filled with markers migrate from the lymph sinuses into the lymphoreticular cords and further into the AGC. The mobility of the macrophages is remarkably lower after phagocytosis of Candida cells.This study is dedicated to Prof. Dr. Dr. h.c. Hugo Grau (Weilheim) with sincere appreciation and respect