Separation of human lymphocyte subpopulations by density gradient electrophoresis. I. Different mobilities of T (T mu, T alpha) and B lymphocytes from human tonsils

T and B lymphocytes from human tonsils were separated by density gradient electrophoresis on the basis of their surface charge. The high-mobility cell fractions were found to be highly enriched in T lymphocytes with only very small proportions of B cells. In contrast, the low-mobility fractions were predominantly B lymphocytes, and had only 10 to 30% contamination of T cells. The intermediate-mobility fractions contained both T and B lymphocytes in approximately equal proportions. IgM-bearing lymphocytes, as well as cells with receptors for mouse erythrocytes, the Fc portion of IgG, and complement were found in the intermediate- and low-mobility fractions. T lymphocytes, prepared by E rosetting, were also electrophoresed by this method and found to be of higher mobility as compared with peripheral blood T lymphocytes. T cells with Fc receptors for IgM (Tμ) or IgA (Tα) were found to be considerably heterodisperse with regard to surface charge and were present in all fractions. The separated cell fractions were treated in vitro with various concentrations of concanavalin A and thereafter examined for Tμ, Tγ, and Tα phenotypes. Low concentrations of Con A (2.5 μg/ml) had no effect on cell surface phenotypes. However, higher concentrations of Con A (20μg/ml) significantly reduced the numbers of T cells having IgM receptors (Tμ), but failed to alter the expression of the Tγ phenotype. The latter finding contrasts to that observed with T cells from the peripheral blood where high concentrations of Con A increase the proportions of the Tγ cells. This study demonstrates that density gradient electrophoresis can be used for the separation and study of lymphocyte subpopulations from human tonsils.     

Regulation of bone marrow lymphocyte production: III. Increased production of B and non-B lymphocytes after administering systemic antigens

To examine the influence of exogenous stimuli on the genesis of lymphocytes in mouse bone marrow, the production rate and subsets of marrow lymphocytes were examined after a systemic injection of sheep red blood cells (SRBC). Radioautographic analysis after either pulse labeling or infusion of [³H]thymidine revealed a pronounced increase in the number of newly formed small lymphocytes appearing in the marrow, maximal 4–5 days after SRBC injection and dose related. The resulting expansion of the marrow lymphocyte population included both immature B cells and null cells, as shown by cell surface and cytoplasmic markers. Similar stimulation of marrow lymphocyte production followed an injection of either bovine serum albumin or mineral oil. No comparable stimulation occurred in either the thymus or the spleen. The results demonstrate that antigens and nonspecific irritants can exert a central effect in the bone marrow, producing a surge in the production of both primary B and non-B lymphocytes. The possible role of external stimulants in determining the normal rate of bone marrow lymphocyte production is discussed.     

Characterization of canine T-lymphocyte subpopulations: the detection of T mu and T gamma lymphocytes with homologous and heterologous immunoglobulins and the requirements for Fc receptor expression

Erythrocyte antibody (EA) rosette techniques employing sheep red blood cells sensitized with canine (homologous) and rabbit (heterologous) IgM and IgG antibodies were used to determine the number of cells with Fc receptors for IgM (Tμ) and IgG (Tγ) among T lymphocytes isolated from peripheral blood and lymph nodes of dogs. The percentages of Tμ and Tγ lymphocytes detected were found to be independent of the species origin of sensitizing antibody. Among peripheral blood T lymphocytes there were 53.0 ± 2.7% Tμ cells and 18.4 ± 3.6% Tγ cells. T lymphocytes obtained from lymph nodes were 62.1 ± 5.4% Tγ and 15.7 ± 2.6% Tγ. The number of Tμ cells detected increased from 20.0% when freshly isolated to 49.1 ± 4.1% after in vitro culture for 2–16 hr. The expression of the Fc-μ receptor in culture was inhibited by cycloheximide, demonstrating a requirement for active protein synthesis. In contrast, the number of Tγ lymphocytes detected did not vary between freshly isolated cells and those which had been cultured for 16 hr. Expression of the Fc-γ receptor during this time period was not inhibited by cycloheximide.     

Induction of DNA synthesis in isolated nuclei by cytoplasmic factors from spontaneously proliferating and mitogen-activated lymphoid cells

Cytoplasmic extracts, prepared from continuously proliferating lymphoblastoid cells, as well as mitogen-activated normal lymphocytes, contain an extractable factor capable of inducing DNA synthesis in isolated quiescent nuclei. This factor is not detectable in resting cells. It is nondialyzable, precipitable by 30-50% saturated ammonium sulfate, and inactivated by trypsin. It is heat sensitive, but stable to cold and lyophilization. The molecular weight of the factor is greater than 100,000. This cytoplasmic activator of nuclear DNA replication is not released from the cell, and has no effect on intact cells. This suggests that it serves as an intracellular mitogenic signal in replicating cells.     

Locomotion of human lymphoid cells. I. Effect of culture and con A on T and non-T lymphocytes.

Human peripheral lymphocytes were separated from whole blood on a Ficoll-Hypaque gradient. They were then depleted of monocytes, separated into T and non-T fractions, and assayed for locomotor responses toward casein and endotoxin-activated serum in Boyden chambers. Non-T cells showed higher random motility than did T cells. Culture prior to assay was necessary in order to demonstrate locomotor activity of T cells, but this requirement, although desirable, was not essential for non-T lymphocytes. It was not necessary for Con A to be present in the culture medium or for either T or non-T lymphocytes to be in blast form to show locomotion.     

Regulation of bone marrow lymphocyte production: IV. Cells mediating the stimulation of marrow lymphocyte production by sheep red blood cells: Studies in anti-IgM-suppressed mice,athymic mice,and silica-treated mice

Some cellular requirements have been examined for the stimulation of lymphocyte production in mouse bone marrow by injected sheep red blood cells (SRBC). The increased genesis of marrow lymphocytes after a single dose of SRBC assayed radioautographically after [³H]thymidine labeling was unimpaired in the marrow of mice treated with anti-IgM antibodies from birth to eliminate B lymphocytes, and in congenitally athymic mice lacking T lymphocytes. However, pretreatment of mice with silica to depress macrophage function completely abolished the SRBC effect both on the total lymphocyte production and on the number of B and null small lymphocytes in the marrow. Comparative studies were performed on the thymus and spleen. The results demonstrate that the stimulation of marrow lymphocyte production by SRBC is mediated by a silica-sensitive mechanism, does not require B or T lymphocytes, and is independent of the humoral immune response. Thus, extrinsic agents may amplify the production of primary B cells and other lymphocytes in the bone marrow by an antigen-nonspecific mechanism, putatively mediated by macrophages.     

Ontogeny of diversity in the receptor repertoire of murine cytotoxic lymphocytes. I. Comparison of recognition patterns of activated T cells of B10.D2 and B10.BR mice of different ages and analysis of changes in F1 hybrid and bone marrow radiation chimeras

Cytotoxic T lymphocyte precursors (CTLp) from B10.D₂, B10.BR, and (B10.D₂ × B10.BR)F₁ mice of different ages have been activated by irradiated “wild-type” H2K^b antigens (from B10.A(3R) mice) under limiting dilution conditions such that cytotoxic cells in responder wells represent the progeny of a single CTLp. After expansion in the presence of IL₂ and irradiated C57B1/6Kha spleen cells the contents of each well were divided into equal aliquots and tested for lysis with a panel of selected H2K^b mutant targets. As has been observed for the murine B-cell repertoire, there seems to be substantially more homogeneity in the neonatal allo-T-cell repertoire than in the adult mouse. Furthermore, while the adult F₁ repertoire is markedly distinct from that expressed by either parental T-lymphocyte pool, the neonatal repertoire apparently reflects a relatively accurate composite of each parental population, codominantly expressed. These data, combined with studies of adult bone marrow radiation chimeras, suggest that during development of the adult T-lymphocyte repertoire from the initially expressed restricted (germ-line?) recognition specificities, somatic diversification driven by environmental (MHC?) antigenic determinants occurs. In addition to this ontogenetic development, during senescence another “regulation” of the repertoire becomes apparent, and once more the heterogeneity of recognition specificities is diminished. Nevertheless, the homogeneity seen in aged mice does not represent a simple return to the expression of the limited number of allo-specificities encoded in the neonatal repertoire.     

Inhibition of cell-mediated lympholysis by cloned and uncloned lines of natural killer cells and cytotoxic T lymphocytes with sugars and lectins

This study was designed to further understand the nature of the interaction between natural killer (NK) cells and their susceptible targets. To do this, a panel of sugars and two lectins was tested for the ability to inhibit the lysis of NK-sensitive targets by cloned and uncloned lines of human NK cells. Six of these sugars (beta-gentiobiose, sucrose, alpha-lactose, beta-lactose, N-acetylglucosamine, and N-acetylgalactosamine) and one lectin, wheat germ agglutinin (WGA), proved to be potent inhibitors of the lytic activity of NK cells as well as of cytotoxic T lymphocytes activated in mixed lymphocyte cultures. Both beta-gentiobiose and WGA were shown to inhibit lysis at the level of the killer cell. Finally, the inhibitory effect of WGA could be reversed by addition of its sugar ligand, N-acetylglucosamine, which is itself an inhibitor of lytic function. From these findings it is concluded that these inhibitors probably do not act at the recognition stage of lysis since all of the NK and CTL lines tested, regardless of specificity, were inhibited by the same panel of sugars and lectins. Instead, it appears more likely that these inhibitors block some postrecognition stage of the lytic mechanism. The common inhibition profile by these sugars on NK and CTL activity further suggests that these two cell types may share, at least partially, a common lytic mechanism.     

The role of reactive oxygen compounds derived from 6-hydroxydopamine for bone marrow purging from neuroblastoma cells

6-Hydroxydopamine(6-OHDA), a specific neurotoxin against sympathetic nerve cells, is a drug already used for purging of bone marrow from neuroblastoma cells before autologous bone marrow transplantation. However, we could not detect significant differences in the toxicity of 6-OHDA against neuroblastoma and other tumor cells under the purging conditions clinically used. In contrast, bone marrow stem cells were much more resistant. The unspecific toxic effect of 6-OHDA is caused by H2O2 or H2O2-derived products which are generated by auto-oxidation in the incubation medium before a significant amount of 6-OHDA is taken up by the cells. Withdrawal of oxygen during the incubation period and subsequent incubation with an oxygen containing medium led to a more specific destruction of neuroblastoma cells which can take up 6-OHDA selectively.     

Mouse pre-B cells synthesize and secrete mu heavy chains but not light chains

The immunoglobulins produced by the earliest recognizable B cell precursors (pre-B cells) were characterized in the mouse and human. Immunofluorescent analysis revealed no evidence of surface IgM components, and only mu heavy chains could be detected intracytoplasmically in pre-B cells. Surface IgM components could not be isolated from intact fetal liver cells that lacked sIgM+ B lymphocytes but possessed pre-B cells. Pre-B cells were shown to synthesize and secrete mu heavy chains but not light chains by immunochemical analysis. These mu chains constituted less than 0.01% of TCA precipitable protein synthesized and secreted by fetal liver cells during an 8 hr labelling period. Migration of both intracellular and secreted mu chains on SDS-PAGE suggested that they were smaller than mu chains secreted by mouse and human plasmacytomas. These data indicate that mu chain synthesis precedes light chain expression during B cell ontogeny and suggest a new role for pre-B cells in the generation and expression of a diverse immunoglobulin repertoire.     

Characterization of a monoclonal antibody reactive with rabbit T lymphocytes and neutrophils

An IgG1 monoclonal antibody, termed ACM-1, has been shown to react with rabbit T cells, but not Ig+ cells or macrophages. This antibody appears to recognize the same epitope as the previously described 9AE10 antibody and, together with 9AE10, has been used to obtain highly pure and fully functional T- and B-cell populations. However, the relevant epitope does not appear to be homologous to rodent Thy-1 since quantitative absorptions failed to show reactivity with rabbit brain. Furthermore, attempts to obtain in vivo T-cell depletion resulted in larger decreases in white blood cells than would be expected for simple T-cell removal. In vitro assays on enriched neutrophil preparations revealed that 80-95% of these cells were reactive with ACM-1 and 9AE10. Thus, it appears that in the rabbit, T cells and neutrophils share a major epitope.     

Distinctive expression of the T4 antigen in normal and stimulated lymphocytes

Correlated light scatter and fluorescence flow cytometric analysis of human peripheral blood lymphocytes showed that the expression of the T4 antigen was higher in the larger lymphocytes than in the smaller lymphocytes. A similar expression pattern was observed for HLA Class I antigens but not for T3 and T8, whose expression was independent of cell size. Results with lymphocytes from spleen, lymph node, and tonsil were comparable to those of peripheral blood. Thymocytes, however, were smaller and expressed less T4 and T8 than peripheral lymphocytes. In studies of lymphocytes stimulated in vitro with allogeneic cells or pokeweed mitogen, two populations of T4-positive cells were observed: one of large cells expressing high amounts of T4 and one of small cells expressing low amounts of T4. Similar patterns were seen with T8, although less consistently. In contrast, the expression of T3 was the same in both large and small cells. The large cells expressing high amounts of T4 were not restricted to cells engaged in DNA synthesis or mitosis. This was established by selectively analyzing cells in the G0G1 phases of the cell cycle and by studying stimulated lymphocytes no longer undergoing proliferation. Taken together, these results suggest that immature T lymphocytes are small and express low amounts of T4 and T8. We postulate that as they differentiate, cell size and T4 expression increase proportionally, both parameters increasing even further after antigenic or mitogenic stimulation. The quantitative expression of T4, and probably of T8 but not of T3, is therefore intimately related to maturation and activation of lymphocytes, a fact that may conceivably be related to a functional role of these surface molecules.     

Human autologous rosette-forming cells: II. Specificity of the binding between lymphocytes and erythrocytes

The relationship between autorosettes and allorosettes was investigated using a mixed rosette assay in which the origin of the erythrocytes was assessed by the fluorescein isothiocyanate (FITC) labeling of one type of erythrocyte. The data show that auto- and allorosettes belong to the same T-cell subset: (1) in most of the subjects, the percentages of T cells binding autologous red blood cells (auto-RBC) are equivalent to those binding allogeneic RBC (allo-RBC); (2) the percentage of rosettes formed after the simultaneous addition of auto- and allo-RBC is similar to that of autorosettes alone or allorosettes alone; and (3) nearly 80% of the resetting cells bind both types of RBC as directly visualized in the mixed rosette assay. The experiments in which the lymphocytes are resetted first with one type of RBC, and then with the other type support the finding that auto- and allo-RBC may bind to the lymphocytes through a single receptor which exhibits a varying affinity for RBC according to their origin.     

Actin Heterogeneity in primary embryonic culture cells from Drosophila melanogaster.

We have investigated actin heterogeneity in differentiating primary embryonic cell cultures from Drosophila melanogaster. Proteins labeled with [³⁵S]methionine have been separated using O'Farrell two-dimensional gel electrophoresis. Cultures of heterogeneous cell types synthesize at least three major forms of actin, each differing slightly in isoelectric point. We have used a cell separation technique based on differential cell adhesion in the presence of ethylene glycol-bis(β-aminoethyl ether) N,N′-tetraacetate to prepare cultures either highly enriched for, or highly depleted of, myogenic cells. Postfusion myogenic cells synthesize predominantly the most acidic actin form (actin I), while nonmyogenic cells synthesize almost exclusively the two more basic forms (actins II and III). Synthesis of actins at earlier intervals in myogenic cell differentiation in vitro has also been examined. Immediately prior to the onset of myoblast fusion, the synthesis of actin I represents approximately 40% of total actin synthesis. As myogenic cell differentiation progresses this actin form is synthesized at an increasing rate, relative to actins II and III. Drosophila appears to be quite similar to vertebrates with regard to the number of actin species synthesized, as well as to cell and developmental specificity of actin synthesis.     

Studies on the liver sequestration of lymphocytes bearing membrane-associated galactose-terminal glycoconjugates: reversal with agents that effectively compete for the asialoglycoprotein receptor

The removal of "effete" glycoproteins from the circulation represents a proposed physiologic role for the hepatocyte asialoglycoprotein receptor. Our experiments support the hypothesis that this receptor may also be directly involved in the removal from the circulation of cells bearing asialoglycoconjugates. We report that the enhanced liver localization of neuraminidase-treated lymphocytes can be competitively inhibited by the coinjection of asialofetuin (ASF). Fetuin itself was without effect. Competitive inhibition of the liver receptor allowed normal localization to lymphoid tissues of the enzyme-treated lymphocytes, a condition which persisted as long as free ASF was present in the circulation. Our studies support the concept that cell surface carbohydrates play an important role in the tissue distribution of circulating lymphocytes. The process of thymocyte maturation, bone marrow transplantation, and the adoptive immunotherapy with continuous T-cell lines represent conditions where recirculation potential may be influenced by the presence of galactose terminal glycoconjugates.     

Solubilization and characterization of the leukotriene C4 synthetase of rat basophil leukemia cells: A novel,participate glutathione S-transferase

Rat basophil leukemia cell homogenates effectively catalyze the conversion of leukotriene A₄ to a mixture of leukotrienes C₄ and D₄ in the presence of glutathione. These homogenates also catalyze the formation of adducts of halogenated nitrobenzene with glutathione, as determined spectrophotometrically. While all the classical glutathione S-transferase activity resides in the soluble fraction of the homogenates, the thiol ether leukotriene-generating activity is found in the particulate fraction. This “leukotriene C synthetase” activity has been solubilized from a crude high-speed particulate fraction by means of the nonionic detergent, Triton X-100. The solubilized enzyme is incapable of converting 2,4-dinitrochlorobenzene to a colored product in the presence of glutathione. Nor will it react with 3,4-dichloronitrobenzene. On the other hand, under optimal conditions, this enzyme preparation is capable of generating about 0.1 nmol leukotriene C mg protein^?1 min^?1 in a reaction which continues in linear fashion for at least 10 min. This dissociation in substrate specificity, as well as differences in the inhibition profile, distinguish the enzyme activity in the particulate fraction from rat basophil leukemia cell homogenates from the microsomal glutathione S-transferase which has been described in rat liver homogenates, suggesting that this “leukotriene C synthetase” is a new and unique enzyme.     

Suppressor T cells in thymus-reconstituted nude mice: regulation of mitogen-induced transformation

A population of glass-wool-adherent splenic cells which could suppress the response of other spleen cell populations to T-cell mitogens was isolated from thymus-reconstituted nude mice. Such adherent cells were characterized as sensitive to anti-Thy 1.2 and complement treatment. Glass-wool-adherent cells from athymic mice do not have suppressor activity to self or normal littermate NAC; however, these mice possess precursor suppressor cells as demonstrated by isolation of glass-wool-adherent T regulatory cells in thymus-grafted nude mice. Such cells are generated in either freshly obtained or in vitro cultured thymus. Evidence for suppressor T cells of host genotype was supported by their sensitivity to host-specific anti-Thy serum treatment as well as their generation in alymphoid thymus grafts. Prior anti-Thy 1.2 treatment of GAC partially removed the suppressor activity: however, macrophages and B lymphocytes were shown not to be secondary regulatory cells or suppressor mediators, thus mature T lymphocytes with low amounts of Thy 1.2 antigen may be responsible for this residual suppression. Further characterization of GAC indicates that active cell growth is required for their regulatory function, as irradiation removed the suppressor activity. This report provides evidence for the presence of a T-lymphocyte subpopulation which has a regulatory function and requires a thymus in the generation of these cells.     

Synthesis and characterization of a metal-binding peptide fragment of human carbonic anhydrase B

A 36-amino acid residue peptide containing the presumed metal-binding ligands at the active site of human erythrocyte carbonic anhydrase B was synthesized by the standard solid phase method. The synthetic peptide was purified by ion-exchange chromatography and was homogeneous as judged by cellulose acetate gel electrophoresis. Amino acid analysis, dansylation, C-terminal determination, and four cycles of Edman degradation all gave results consistent with the anticipated sequence. The peptide binds Co(II) with an apparent dissociation constant of about 7 × 10^?5M (uncorrected) but has little, if any, of the catalytic activity of carbonic anhydrase. Possible explanations for the weak binding of the metal ion are discussed along with prospects and strategies for designing polypeptide models of enzymatic catalysts.     

Activation of T cells in tumor-bearing mice

Subcutaneous transplantation of the syngeneic P815 mastocytoma in DBA/2J mice induced an activation of splenic T cells which resulted in a hyperresponsiveness of the tumor-bearing animal to the unrelated antigens pneumococcal polysaccharide (Pn) and sheep red blood cells (SRBC). These tumor-activated T cells appeared to increase the plaque-forming cell (PFC) potential of suboptimal numbers of spleen cells, caused normal spleen cells to express increased numbers of PFC, and produced lymphokine(s) which also increased PFC responses of normal splenocytes. The tumor-activated T cells responsible for stimulating normal splenocytes in an in vitro antibody response were shown to be Ly+2- cells. The activity of the tumor-activated T-cell supernatants was not genetically restricted and required additional Ly1 T cells in order to induce rigorously clean B cells to produce antibody. The T cells capable of stimulating non-specific antibody responses were also capable of slowing tumor growth when injected with tumor cells in normal recipient mice. These results suggest that T cells activated by tumor antigens release immunostimulatory lymphokines and, at the same time, are capable of leading to inhibition of tumor growth.     

Analysis of the effects of erythrocytes on mitogen-dependent clonal proliferation of murine B lymphocytes

Addition of intact erythrocytes to semisolid agar cultures of murine B cells dramatically improves cloning efficiency and affects colony morphology. In this study, we investigated possible mechanisms through which this might occur. Specific modification of sheep erythrocyte (SRBC) membranes by treatment with trypsin but not other enzymes improved colony potentiation and erythrocytes from rats, mice, and humans were also effective after trypsin treatment. In addition, autoantibody-coated murine erythrocytes were superior to normal cells in this regard. These observations suggest that erythrocytes enhance lymphocyte survival and/or proliferation by means of particular membrane-mediated processes. The possible importance of erythrocytes as scavengers of toxic hydroxyl radicals was also investigated. Deliberately generated radicals formed by addition of dihydroxyfumaric acid and iron were effectively countered by addition of SRBC. More detailed analyses revealed that of several endogenously produced toxic species, hydrogen peroxide may be the most important under ordinary culture conditions. That is, addition of catalase but not Superoxide dismutase or mannitol improved cloning efficiency in cultures lacking SRBC. These studies suggest that erythrocytes have a beneficial effect on lymphocyte survival and function in culture through at least two mechanisms.