Characterization of rat bone marrow cells. II. Analysis of surface antigens in small lymphocytes with particular reference to thymus antigen-carrying cells.

Rat bone marrow (BM) small cells were enriched by velocity sedimentation, further separated by means of free-flow electrophoresis, and characterized using T- and B-cell-specific surface markers. More than 80% of these cells were small lymphocytes by morphological criteria and reacted with lymphocyte-specific antisera. A minority of cells had high electrophoretic mobility (EPM), carried surface antigens characteristic of mature T cells, and lacked B-cell markers. These cells may represent recirculating T cells. A small number of cells were found with rat B lymphocyte-specific antigen (RBLA) and surface immunoglobulin (sIg) and had medium EPM. These cell fractions also contained “null” cells which were devoid of T- and B-cell-specific antigens. More than 80% of the BM small cells had low EPM and carried the three subspecificities of the Thy-1 antigen complex and the Thy-A antigens. These antigens were found at several-fold higher concentration on the surface of all thymocytes, but are lacking in most other lymphocytes. The thymus antigen-carrying BM cells of low EPM do not carry other T- and B-cell-specific markers found in thymocytes and peripheral T and B lymphocytes. These markers comprise the T-cell antigens RTLA (rat T-lymphocyte-specific antigen) and RHLA (antigens specific for rat T cells of high EPM) and the B-cell markers RBLA and sIg. Thus the majority of rat BM lymphocytes differ from all other lymphocytes of the T- and B-cell series which makes any classification on this basis purely speculative.     

Influence of several experimental conditions on the quantity of E- and EAC-rosette forming cells in peripheral human blood]

The authors analysed the influence of some conditions of studying the rosette-formation between the human peripheral blood lymphocytes and sheep red blood cells, not loaded (a) and loaded with antibodies and a complement (b). It was shown that rosette formation depended on the temperature at which the experiment was conducted. Thus, after the incubation of a mixture of cells at 37 degrees C, and then at 4 degrees C the number of rosette-forming cells was greater than after the incubation at 4 degrees C alone. Substances used for their fixation also influenced the formation of rosettes: in the treatment of erythrocytes with formalin, tannin and glutaraldehyde, as well as introduction of the latter into the mixture of erythrocytes and leukocytes considerably increased the number of a and b (the sum of T- and B-lymphocytes exceeded 100%). Among a and b in such experimental conditions there was seen a great number of monocytes and of segmented leukocytes. Fixation of smears prepared from a mixture of cells after the incubation in formalin vapour did not lead to such increase in the number of rosette-forming cells. Apparently in case of using glutardehyde, formalin and tannin cells for the treatment of cells additional investigations are necessary with the purpose of identification of a and b as T- and B-lymphocytes.     

Characterization of rat bone marrow lymphoid cells. I. A study of the distribution parameters of sedimentation velocity, volume and electrophoretic mobility

Various cell populations in rat bone marrow were characterized by means of a two dimensional separation using velocity sedimentation and free flow electrophoresis and by electrical sizing of the separated cells. Up to 4.5 mm/hr five different populations with discrete distributions in volume (coefficient of variation 10% to 13%) and sedimentation velocity (coefficient of variation 6% to 10%) were observed. Three of the small sized populations represented lymphocytes and small normoblasts and two of the larger sized populations represented myeloid cells. Almost all of these cells were in the G0/G1 cycle phase. In the faster sedimenting fractions which contained immature myeloid, erythroid and undefined blast cells and two S phase populations, discrete volume distributions were not evaluated. The cell populations with homogeneous volume (particularly the small lymphocytes) showed high density variations which condiserably impair the separation resolution. The cells sedimenting slower than 3.5 mm/hr were further separated by means of free flow electrophoresis into three peaks differing in electrophoretic mobility (EPM). The peaks of low and high EPM contained two populations and the peak of medium EPM contained three populations all characterized by normal volume distributions of uniform coefficient of variation between 11% and 14%. The small cells in the peaks of high and medium EPM were normolblasts and the other cells were lymphocytes. The biological significance of these results is discussed.     

Quantitation of T- and B-lymphocytes in peripheral blood of patients with solid tumours. I. Relation to other parameters of in vivo and in vitro immune competence.

The absolute number of T- and B-lymphocytes in the peripheral blood was measured in 22 patients with disseminated nonlymphoid solid malignant tumours. Patients with normal absolute lymphocyte counts had normal absolute numbers of T- and B-lymphocytes; patients with low counts had low numbers. The percentage of T- and B-lymphocytes was similar, in both groups of patients, to that of healthy control subjects. Patients with a normal absolute lymphocyte count had better in vitro lymphocyte responses to mitogenic agents than did patients with low absolute lymphocyte counts. Improvement of depressed in vitro lymphocyte responses to normal on culture of cells in AB plasma was most likely in patients with normal absolute lymphocyte counts. The absolute lymphocyte count by itself, then, provides useful information about the immune status of patients with solid malignant tumours.     

Mechanisms of action of synthetic polyelectrolytes and polyampholytes on the immune response]

We investigated the effect of polyacrilic acid (PAA) on the immune response in mice of various strains on sheep red blood cells and also the influence of poly-2-methyl-5-vinyl-pyridine (PMVY), PAA and their statistical copolymers on antibody-forming cells (AFC) production in cultures of T- and B-lymphocytes in vivo. PAA was seen to increase accumulation of AFC in the spleen of mice depending on their genotypes. PMVP and PAA were found to intensify the cooperating interaction of T- and B-lymphocytes, whereas their copolymers exert quite an opposite effect. The injection of copolymers to the recipients of cooperating T- and B-lymphocytes practically results in the complete elimination of the cooperation effect between T- and B-lymphocytes in the immune response to sheep erythrocytes without cytostatic action of cell proliferation.     

Phytohemagglutinin-induced change in the distribution of acidic sugars in surface membrane of lymphoid cells and blocking of the radiation effect.

Cell electrophoretic mobilities (EPM) of cultured lymphoblastoid cells were measured after removal of acidic sugars to investigate whether the localization of these acidic sugars was altered by the action of phytohaemagglutinin (PHA). After treatment with neuraminidase or hyaluronidase, the EPM of control cells decreased 50.1 and 0.3%, while that of PHA-treated cells decreased 25.2 and 39.0%, respectively. These results suggest that hyaluronic acid appeared at the periphery of the cell surface in place of some sialic acid after incubation with PHA. The change became evident after 10 min incubation with PHA and reached its maximum after 20 min at 37 °C, but no change was observed at 4 °C. The EPM decreased with time after X-irradiation, and reached a minimum value after 4 h. The addition of PHA to culture before irradiation completely blocked the X-ray-mediated reduction in EPM. PHA administration after irradiation stopped further EPM reduction. These results seem to suggest a rapid rearrangement of membrane molecules linking with the receptors and acidic sugars induced by PHA, and blocking of further conformation change by X-irradiation.     

Differential analysis of human leukocytes by dielectrophoretic field-flow-fractionation

The differential analysis of human leukocytes has many important biological and medical applications. In this work, dielectrophoretic field-flow-fractionation (DEP-FFF), a cell-separation technique that exploits the differences in the density and dielectric properties of cells, was used to separate the mixtures of the major human leukocyte subpopulations (T- and B-lymphocytes, monocytes, and granulocytes). The separation was conducted in a thin chamber equipped with an array of microfabricated interdigitated electrodes on the bottom surface, and the separation performance was characterized by on-line flow cytometry. To investigate optimal separation conditions for different leukocyte mixtures, elution fractograms at various DEP field frequencies were obtained for each leukocyte subtype. With appropriately chosen conditions, high separation performance was achieved in separating T- (or B-) lymphocytes from monocytes, T- (or B-) lymphocytes from granulocytes, and monocytes from granulocytes. DEP-FFF does not involve cell-labeling or cell-modification step, and provides a new approach to hematological analysis.     

Role of immune RNA in the interaction of T- AND B-lymphocytes]

"Immune" RNA preparations were obtained from the total population and also from the T- and B-lymphocytes of the spleens of the QBA line. Intact bone marrow cells or splenic cells activated with antigen served as target cells for the "immune" RNA. Investigations were carried out in the system of syngenic transfer. To study the target cells in the activated population of the spleen elimination of T-or B-lymphocytes was realized immediately after the incubation of the suspension of the splenic cells with the RNA preparations with the aid of anti-theta-or anti-B-antilymphocytic sera. T-lymphocytes served as the source of the biologically active RNA in the total preparation. B-lymphocytes of the spleen and the bone marrow served as target cells for the RNA of the cells of thymus origin. However, to detect the inducing action of the RNA simultaneous presence in the population of T- and B-lymphocytes is necessary.     

Increased trypsin sensitivity of cell surface macromolecules after malignant transformation.

The effect of treatment with 0.04% (w/v) trypsin (EC 3.4.4.4) for 3 h on the electrophoretic mobility (EPM) of polyoma-virus malignantly transformed BHK21 cells (Py6) and their normal counterparts has been investigated. These particular conditions were chosen because an earlier study had shown that such treatment released material from the Py6 cells which was not obtained from the BHK21 cells. The negative EPM of the Py6 cells at pH 7.5 was greatly increased by this treatment; whereas the EPM of the BHK21 cells remained unchanged. Active enzyme was required to produce the change. No evidence was obtained for cytolysis, cytotoxicity or uptake of the enzyme by the treated Py6 cells. Measurement of the EPM of the Py6 cells at different pH levels before and after trypsin treatment suggested that the enzyme was removing cationic groupings from the cell surface.     

Characteristics of the lymphoid cells of rabbits]

By means of scanning electron microscopy it has been stated that the thymus gland, spleen, paratracheal lymph node, Peyer's patches and appendix in an intact rabbit of "Shinshilla" strain have T- and B-lymphocytes in different proportion. T-lymphocytes are seen as spherical cells with rough surface, with a few microprocesses, have 3.0 +/- 0.1 mcm--4.7 +/- 0.2 mcm in diameter depending on their location. B-lymphocytes are seen as spherical cells with a considerable amount of microvilli on their surface, have 2.9 +/- 0.1 mcm--4.4 +/- 0.1 mcm in diameter depending also on their location. In the thymus gland, cells with rough surface, a few microprocesses and crater-like hollows prevail. In the spleen and lymph node, most of the cells are of similar diameter and have microvilli on the surface. Lymphoid population in the Peyer's patch and appendix is represented by large cells with microprocesses.     

Cellular bases of immunological recognition. The precursors of antigen-binding T- and B-lymphocytes and the patterns of their maturation]

The peculiarities of carefully studied immunoglobulin receptors of the specialized clones of B-lymphocytes are described. These latter are precursors of antibody-forming cells but play only a limited role in the recognition of foreign antigens which is realized by T-lymphocytes. The differentiation of T- and B-lymphocytes from the single precursor, a bone marrow stem hemopoietic cell, proceeds in the bone marrow itself, thymus and peripheral lymphoid organs in several discrete stages. At each of these stages the cells have definite properties and, in some cases, may be separated one from another. The modern data are given concerning the properties of cells at every stage of antigen-independent differentiation of T- and B-lymphocytes which proceeds during embryonic and postnatal development, its alternative pathways, factors of regulation, role of local microenvironment and possibilities of modelling in vitro.     

Effect of radiation on cell interactions in the phenomenon of non-syngeneic stem cell inactivation. Changes in B-lymphocyte function after irradiation

A study was made of the changes in the mode of interaction between T- and B-lymphocytes of mouse lymph nodes with respect to the phenomenon of inactivation of non-syngeneic haemopoietic stem cells. It was shown that irradiation of B-lymphocytes with doses of 77.4--232.2 mC/kg changes their helper activity into a suppressor activity with regard to T-cell-killers having a low electrophoretic mobility.     

Differential effect of pH on the density and volume of rat reticulocytes and erythrocytes. Relevance to their fractionation by centrifugation.

Rats were injected with 59Fe-ferrous citrate and bled thereafter at different times (16 h to 49 d). This gave rise to red cell populations in which cells corresponding in age to the time elapsed between injection and bleeding were labeled. The anticoagulant used was either acid-citrate-dextrose (ACD) with a pH adjusted to 7.3 or ACD (pH 5.1). Final pH of the collected blood was about 7.2-7.4 in the former case and 6.4-6.7 in the latter. Red cells were then centrifuged (5) and approximately 7-10% of the packed cells from the top and 7-10% from the bottom of the cell column collected. When reticulocytes are the predominant labeled red cell population, as in blood obtained for about 24 h after isotope injection, a fractionation of these cells and mature erythrocytes is in evidence only when blood is collected at the higher pH. Thus, at pH 7.2-7.4 ratios of specific radioactivities of cells in top fraction/cells in an unfractionated sample are about 3, whereas at pH 6.4-6.7, the analogous ratios are 1 or less. These differences in specific activity ratios, as a function of pH at collection, virtually disappear after about 4 d following isotope injection. The lower pH is known to increase the volume and decrease the density of mature red blood cells. The marked effect of pH on cellular fractionation could be correlated with the smaller change in rat reticulocyte density and volume in acid medium. At pH 6.4-6.7, the densities of mature erythrocytes and reticulocytes are so close that their physical separation by centrifugation is not feasible.     

The effect of medium pH on rate of growth, neurite formation and acetylcholinesterase activity in mouse neuroblastoma cells in culture.

Cell division, neurite formation and acetylcholinesterase activity were examined in a clone (NBA2) of mouse neuroblastoma cells maintained for up to 120 hours in medium with pH values between 6.6 and 8.0. Growth rate decreased as pH was reduced from 7.8 to 6.6. Generation time at pH 7.4 was 25 hours, while the rate of cell division was negligible at pH 6.6. The total number of cells at stationary phase was less at the lower pH values. Neurite formation was enhanced markedly as the pH was reduced from 7.4 to 6.6. Acetylcholinesterase activity was 5- to 8-fold greater in cells exposed to medium at pH 6.6 than in cells maintained in medium at pH 7.4. The reduction in the rate of cell division and increases in neurite formation and acetylcholinesterase activity at pH 6.6 were reversible upon exposure of the cells to pH 7.4 medium. Cell viability was greater than 90% at all medium pH values over a period of 120 hours. Uncloned T-59 mouse neuroblastoma cells were affected similarly by changes in pH. These results show that manipulation of the environmental pH can reversibly alter growth, neurite formation, and acetylcholinesterase activity of mouse neuroblastoma cells in culture.     

Some electrical properties of the surfaces of murine B- and T-lymphocytes and thymocytes. II. Effects of neuraminidase and ribonuclease.

The electrical properties of the peripheries of murine thymocytes, B-lymphocytes and T-lymphocytes were studied by measurement of electrophoretic mobilities and electron microscopic quantitation of adsorbed, positively charged CIH particles. The effects of neuraminidase and/or ribonuclease treatment upon these parameters were examined. Neuraminidase-susceptible groups accounted for 17%, 13% and 21% of the net surface negativity of T-lymphocytes, B-lymphocytes and thymocytes, respectively, and 28%, 63% and 78% respectively of particle binding. The calculated numbers of charges at the cellular electrokinetic surface per observed CIH particle were similar in control T-cells and thymocytes and higher than in B-lymphocytes. In neuraminidase-treated T- and B-lymphocytes the calculated charges per CIH particle were much lower than in thymocytes. These results may well indicate heterogeneities in the distribution of groups susceptible to neuraminidase, and also in the distribution and/or chemical nature of anionic groups susceptible to neither neuraminidase nor ribonuclease at the peripheries of different murine lymphocyte populations; however, at present we cannot discriminate between these and other possibilities.     

The pool of free purine and pyrimidine nucleosides and bases in the thymocytes, and splenic T- and B-lymphocytes of C3HA mice during the growth of solid hepatoma 22a

Using reverse phase ion pair high performance liquid chromatography, the levels of free adenosine, inosine, adenine, xanthine, hypoxanthine, guanine and deoxycytidine in thymocytes and splenic T- and B-lymphocytes of C3HA mice, were studied under normal conditions and at different times (5 hrs, 1, 2, 3, 4, 5, 8 and 20 days) after transplantation of solid hepatoma 22a. The adenosine and inosine levels in thymus and spleen lymphocytes were 5 to 10 times as low as that of purine bases. Inosine was totally absent in T-and B-lymphocytes. The absolute content of adenine and guanine in thymus and spleen lymphocytes was higher compared to purine bases. It was shown that in all cases studied the decrease in hypoxanthine, xanthine and guanine levels in T- and B-lymphocytes during maximal tumour growth, i.e., on the 5th and 8th post-inoculation days as well as at the terminal period (20th day), was correlated with the decrease in the adenosine deaminase and functional activities of these cells. The level of free adenine in thymocytes and spleen T-lymphocytes during tumour growth showed a 2-4-fold increase in comparison with normal values. A dramatic decrease of intracellular concentration of deoxycytidine was observed in thymocytes and spleen T- and B-lymphocytes beginning with the 5th hour and over the whole subsequent period. The key role of the deoxycytidine decline during tumour growth as a possible cause of simultaneous impairment of DNA synthesis and purine deoxyribonucleoside phosphorylation in lymphocytes is discussed.     

Functional analysis of mononuclear cells infiltrating into tumors. II. Differential ability of mononuclear cells obtained from various tissues to produce helper factors that are involved in the generation of cytotoxic cells

The requirement of CGF in the generation of cytotoxic cells against syngeneic tumor cells (T-9) and in the rejection of transplanted T-9 cells has been investigated. Spleen cells obtained from sensitized rats showed strong cytotoxicity against 51Cr-labeled T-9 cells upon incubation with CGF for 48 hr. Human recombinant IL 2 and rat IFN failed to generate cytotoxic cells from spleen cells of sensitized rats. CGF are produced by spleen cells upon inoculation of T-9 cells into sensitized rats as a host in vivo immune response. Production of CGF preceded the appearance of cytotoxic cells in regional lymph node and tumor tissues. In those rats, inoculated tumor cells were eventually rejected. In contrast, spleen cells failed to produce CGF upon inoculation of T-9 cells in unsensitized rats. Cytotoxic cells were not detected in unsensitized rats, and inoculated tumor grew in those rats. Thus, CGF is likely to be involved in the generation of cytotoxic cells and in the rejection of inoculated syngeneic tumor cells. A Mono Q anion-exchange column with an FPLC system allowed the chromatographic separation of CGF from IL 1, IL 2, IL 3, and CSF.     

Change in the strength of DNA-protein binding in T- and B-lymphocytes of the spleen of C3HA mice during chemical hepatocarcinogenesis

The tightness of DNA-protein binding in the nuclei of mouse spleen T- and B-lymphocytes was assessed, using nucleoprotein celite chromatography, and changes in the number of T- and B-suppressors in the course of o-AAT-induced chemical hepatocarcinogenesis were studied. Attenuation of DNA-protein bonds in T-lymphocytes at the early stages (up to 3 months) was observed, and by the time of hepatoma formation (8 months) about 50% of T-lymphocyte DNA was loosely bound to proteins, which is a typical feature of quiescent cells. In B-lymphocytes attenuation of DNA-protein interaction was only observed by the 8th month of carcinogenesis. By the time of hepatoma formation the number of T-suppressors in mouse spleen increased 2.8-fold, while the number of B-suppressors in lymph nodes remained unchanged.     

Effect of the antineoplastic antibiotic bruneomycin on lymphocyte population ratio in mice

Data on the quantitative ratio of the populations of T- and B-cells in the lymphoid organs of mice at various periods after oral administration of bruneomycin are presented. It was shown that in contrast to the total number of T- and B-cells in the spleen and mesenteric lymph nodes amounting to the minimal values at the early periods (days 1-3) after the antibiotic injection, their relative content remained rather high. Complete recovery of the number of T- and B-lymphocytes in the above organs was observed only by the end of a month period. Study on the kinetics of the immune response to sheep red blood cells showed that reactivity of the antibody producers in the experiments with bruneomycin increased 1.5-2 fold as compared to the control.