Alpha-fetoprotein and human lymphocyte subpopulations.

Peripheral blood lymphocytes were incubated with varying concentrations of alpha-fetoprotein and enumerated for total and "active' T lymphocytes, B lymphocytes, and their proliferative responses to phytohemagglutinin. No significant effect was observed on total T or B lymphocyte proportions. However, there was a dose-related increase in proportions of the so called "active" T lymphocytes. The response of human lymphocytes to phytohemagglutinin was markedly depressed. The alteration in the proportion of active T cells and the inhibition of T lymphocyte response to phyto hemagglutinin by alpha-fetoprotein occurred at higher concentrations than are present in amniotic fluid, serum of pregnant women, or serum of adults, but well within the range reached in fetal serum. The immunoregulatory role of alpha-fetoprotein is discussed.     

Rabbit lymphocyte subpopulations. II. Separation and characterization of two Ig+ cell subpopulations.

Rabbit lymph node cells (Ig⁺Ig^?) were separated into Ig⁺ and Ig^? populations by rosette formation with anti-Ig antibody-coated erythrocytes and centrifugation on Ficoll-Hypaque. Subpopulations of Ig⁺ cells were obtained by treating rosetted cells with autologous serum which dissociated approximately half of the rosettes. The stable rosetted cells (Ig⁺ S) were separated from the labile unrosetted cells (Ig⁺L) by centrifugation on Ficoll-Hypaque. The Ig⁺S population contained most of the Ig-secreting cells and responded poorly to mitogens. The Ig⁺L population contained few Ig-secreting cells and responded well to mitogens. Approximately 50% of Ig⁺L cells became Ig⁺S when cultured with Ig^? cells but this transition did not occur if Ig⁺S cells were added to the culture at the start of the incubation period. Purified Ig⁺ L cells lost their ability to form rosettes when cultured by themselves but retained their ability to form rosettes when cultured wih Ig^? cells. The data indicate that the Ig⁺S and Ig⁺L populations are at different stages in the differentiation of Ig⁺ cells (B cells) and that the Ig⁺L cells are subject to the regulatory influences of both Ig^? and Ig⁺S cells.     

Rabbit lymphocyte subpopulations. I. Separation of Ig+ and Ig- cells and their interaction in cultures stimulated by mitogens.

Rabbit lymph node cells (Ig⁺Ig^?) were rosetted with anti-Ig antibody-coated erythrocytes and the rosetted Ig⁺ cells (B cells) were separated from unrosetted Ig^? cells (T cells) by centrifugation through Ficoll-Hypaque medium. The Ig^? cells were recovered from the top and the Ig⁺ cells from the bottom of the Ficoll-Hypaque layer. Some of the purified Ig⁺ cells lost their ability to form rosettes when cultured with the mitogen associated with streptolysin O. This suggested that the Ig⁺ population might contain two distinct subpopulations. The response of Ig⁺Ig^?, Ig⁺, and Ig^? cells to various mitogens was studied. The Ig^? cells incorporated more ³H-TdR when they were incubated by themselves than when they were cultured with Ig⁺ cells in an Ig⁺Ig^? culture. On the other hand, the Ig⁺ cells incorporated less ³H-TdR when they were incubated by themselves than when they were incubated with Ig^? cells in an Ig⁺Ig^? culture. Thus, Ig⁺ cells suppressed the response of Ig^? cells whereas Ig^? cells enhanced the response of Ig⁺ cells. We conclude that rabbit Ig⁺ cells (B cells) and Ig^? cells (T cells) interact with a feedback pattern of regulation.     

Excretion of DNA by purified human lymphocyte subpopulations.

A large proportion of DNA synthesized in vitro by human lymphocytes stimulated with plant mitogens or specific antigens is selectively excreted from the cells. To determine if DNA excretion differs among various types of lymphocytes, we examined purified human lymphocyte subpopulations for DNA synthesis and excretion in response to stimulation by L-PHA. The relative proportion of newly synthesized DNA that is excreted by unseparated mononuclear cells, macrophage-depleted cells, T, and B lymphocytes is identical despite great differences in the magnitude of their responses. Low levels of both DNA synthesis and excretion by macrophage-depleted cells and B cells can be increased by reconstitution with macrophages and T cells, respectively. These data indicate that DNA exretion is a general property of lymphocytes stimulated to undergo DNA synthesis by plant mitogens.     

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.     

Immunologic functions of isolated human lymphocyte subpopulations. IV. Stimulation of MLC and CML by human T cells.

Surface immunoglobulin positive and immunoglobulin negative human lymphocyte populations were obtained by immunoabsorbent column chromatography. Both cell populations were effective as stimulating and target cells in allogeneic MLC and CML reactions. The immunoglobulin negative population was further depleted of both EAC rosette forming cells and nylon wool adherent cells. The resulting highly purified T cell population was also able to stimulate allogeneic cells in MLC, and induce the generation of specifically cytotoxic killer cells in CML.     

Immunologic functions of isolated human lymphocyte subpopulations. III. Specific allogeneic lympholysis mediated by human T cells alone.

The studies presented herein have evaluated both the specificity and cellular basis of cell-mediated lympholysis (CML) in man. An efficient and quantitative 51Cr release assay was utilized to study the role of highly purified human T and B cells in CML. After in vitro sensitization human T cells develop the capacity to kill specifically allogeneic cells to which they were sensitized. In contrast, B cells were neither triggered to proliferate nor activated to kill allogeneic targets. B cells were not activated to kill even when sensitized in the presence of potentially "helper" T cells, nor did they block T cells from killing during the effector phase. Cell-free supernatants taken from active in vitro sensitization cultures were not lympholytic and did not modulate T cell killing. Hence, these studies show that both the afferent and efferent phases of human CML are T cell functions.     

In vitro sensitization of human lymphocytes against histiocytic lymphoma cell lines. I. Primary sensitization of lymphocyte subpopulations.

Peripheral blood lymphocytes from normal donors expressed spontaneous cytotoxic activity against human diffuse histiocytic lymphoma cell lines. In the unfractionated state, they could not be further sensitized in vitro against these cell lines. By applying cell separation techniques before culture, subpopulations of lymphocytes were obtained which could be sensitized in vitro and manifested cytotoxic activity against human histiocytic lymphoma cells. Three methods of separation were found effective: E rosette enrichment; elimination of Fc receptor positive cells; and removal of nylon wool adherent cells. Under these conditions, cross-reactive cytotoxicity was observed against non-neoplastic lymphoblastoid cell lines, but not against normal lymphocytes.     

Separation of rat leukocytes by countercurrent distribution in aqueous two-phase systems. I. Characterization of subpopulations of cells.

Cells from rat spleen, lymph nodes, and thoracic duct were separated by countercurrent distribution in aqueous two-polymer phase systems containing dextran and polyethylene glycol. Lymphoid cells from the different organs gave distinct, highly reproducible distribution patterns. The yield of separated cells and their viability compared well with other methods of physical separation. The majority of the leukocytes was separated from erythrocytes. Cells with surface immunoglobulin were recovered in one side of the distribution, while thymus-derived lymphocytes as determined by indirect immunofluorescence and histochemical staining were found in all fractions. However, cells responding to PHA and Con A were concentrated in a small area of the distribution, indicating a separation of subpopulations of thymus-derived lymphocytes.     

Separation of granule subpopulations in human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes were isolated, disrupted by sonification and the nuclei and unbroken cells removed by centrifugation. The supernatant was applied on top of an optimised discontinuous Percoll gradient. After centrifugation we found nine gradient bands of distinct density. Both the nine bands and the whole fractionated gradient material were assayed for granule marker enzymes. Granule fractions of distinct density, enclosing different enzyme concentrations demonstrated the existence of granule subpopulations. There were three subpopulations of azurophil granules, about four subpopulations of specific granules, one granule fraction perhaps representing the C-particles, and a fraction of plasma membrane vesicles.     

Peanut agglutinin. I. A new tool for studying T lymphocyte subpopulations.

Fluorescein-coupled peanut agglutinin (PNA) has been used at the single-cell level to study mouse lymphocyte subpopulations. PNA not only binds to most thymocytes, as has already been shown by other authors, but also binds to a small fraction of peripheral lymphocytes that are all T cells (theta+Ig-) or null cells (theta-Ig-). Most PNA-positive thymocytes are sensitive to in vivo corticosteroids and irradiation (450 rads) treatments. Conversely, the positive spleen cells (5% of total spleen lymphocytes) are essentially resistant to corticosteroids and irradiation. Study of PNA binding during ontogenesis shows the occurrence of PNA-positive cells in the fetal liver before thymus constitution and in the very beginning of embryonic thymus and spleen development. These data indicate that PNA is a marker of early T cell subpopulations but that there are probably several distinct subsets of PNA-positive T cells.     

Characterization of culture-induced cytotoxicity from human peripheral lymphocyte subpopulations

Cultured human lymphocyte subpopulations can generate cytotoxicity against K562 leukemia target cells under certain conditions. Such cytotoxicity arises during mixed leukocyte culture or in medium containing fetal calf serum (FCS), mitogenic factors, or interleukin 2. We cultured peripheral blood lymphocytes (PBL) in FCS-containing medium after fractionation of these cells on a Percoll discontinuous density gradient. Higher density cell fractions generated culture-induced spontaneous cytotoxicity (CIC) after 2-3 days in culture. CIC was not due to a loss of suppressor cells during fractionation since culture of PBL prior to fractionation yielded the same results. At least some CIC was associated with the differentiation of higher density cells to newly appearing lower density cells during culture. Most CIC required cells with the HNK-1- OKT3- OKM1+ phenotype. Culture-induced cytotoxicity has some similarities to the previously described lymphokine-activated killing but some important differences are also discussed.     

Glycolipid markers of murine lymphocyte subpopulations.

We have shown previously that purified antibodies to ganglioside GM1 react with peripheral T cells and most thymocytes in several strains of mice, independent of Thy-1 phenotype. GM1 and the Thy-1.2 antigen cap independently on C3H thymocytes, which provides additional evidence that GM1 is not the Thy-1.2 antigen. In C3H and nude mice antibodies to GM1 also react with a population of cells, comprising about 25% of lymphocytes from lymph nodes or spleen, that bear surface immunoglobulin. After removal of immunoglobulin from these cells by digestion with proteolytic enzyme, the GM1+ cells regenerate their surface immunoglobulin during 18 hr in culture, which indicates that these double-labeled cells synthesize their surface immunoglobulin. Protease treatment of lymphocytes reveals receptors for antibodies to GM1 on most cells. These data indicate that T and B cells differ in the accessibility of GM1 to antibody, and not necessarily in their content of GM1. Purified antibodies to asialo GM1 react with mature T cells in all strains of mice tested. In contrast to anti-GM1, these antibodies do not react with most thymocytes, with immunoglobulin-bearing lymphocytes of C3H or nude mice, nor with pronase-treated B cells.     

Functional properties of lymphocyte subpopulations in hepatitis B virus infection. I. Suppressor cell control of T lymphocyte responsiveness

Hepatocellular injury in hepatitis B virus infection may be produced by an autoaggressive hepatocytotoxic immune response. To test the hypothesis that acquired suppressor cell defects may participate in such a response, we assessed the functional integrity of 2 suppressor cell populations in patients with type B viral hepatitis. Spontaneous suppression of the 1-way mixed lymphocyte response by radiation-resistant, adherent peripheral blood mononuclear cells decreases during the acute phase of disease, returns towards normal with clinical recovery, but remains depressed in patients with chronic hepatitis. The degree of spontaneous suppressor cell dysfunction correlates inversely with at least 1 biochemical parameter of hepatocellular injury (SGPT). The functional integrity of this suppressor cell fluctuates during chronic hepatitis and may reflect currently undefined biologic variables in this disease. Mitogen-induced suppression on lymphocyte activation by radiation resistant, nonadherent suppressor cells is also depressed in acute and chronic hepatitis, but it does not correlate with biochemical evidence of hepatocellular injury on an individual-patient basis. Documentation of these generalized defects of nonspecific suppressor cell function establishes a basis for the possible existence of specific anomalies of immuno-regulation that may permit the expression of normally suppressed auoaggressive hepatocytotoxic immune mechanisms in viral hepatitis.     

Activation of human lymphocyte subpopulations by protein A from Staphylococcus aureus bacteria.

Cowan I strain Staphylococcus aureus bacteria were found to be mitogenic for human peripheral and cord blood lymphocytes. Experiments with lymphocyte supopulations otained by nylon wool filtration and/or E-rosette separation revealed that T-lymphocytes are the main target cells, whereas isolated B cells did not respond significantly. Further experiments suggested that B cells could be activated in the presence of mitomycin-treated T cells. Null cell-enriched lymphocyte suspensions could be stimulated by Con A but not by the bacteria or by PHA.     

Distribution of surface IgM and IgD on single cells from lymphocyte subpopulations.

The surface immunoglobulin heavy chains on individual spleen cells fractionated by velocity sedimentation were studied using fluorescent antisera. In adult mice, cells bearing both mu and delta chains were found in all fractions. While there was an increase in the proportion of cells bearing mu only in the medium to large cell fractions, the majority of cells bearing mu only were small lymphocytes. Results obtained using 3-week-old mice were basically similar, but showed both a marked decrease in small mu + delta + cells and a marked increase in small mu + delta - cells when compared with adult animals.     

Peripheral blood lymphocyte subpopulations in sarcoidosis.

We have determined the numbers of thymus-derived (T) and bone marrow-derived (B) lymphocytes in the peripheral blood of 20 patients with sarcoidosis and 15 healthy controls. T cells were estimated from the number of lymphocytes forming rosettes in vitro with unsensitized sheep red blood cells, and B cells were enumerated by immunofluorescent assesssment of membrane-bound immunoglobulins. The total lymphocyte count was lower in patients with sarcoidosis owing to a depletion of T lymphocytes from the blood. Nonetheless, the relative and absolute numbers of B lymphocytes were significantly increased. These alterations in lymphocyte subpopulations did not show any consistent correlation with the duration of the disease, clinical stage, activity, or treatment. Changes in the subpopulations may be related to both decreased cellular immunity and increased reactivity of the antibody-forming system as commonly seen in sarcoidosis.     

X-ray induction of micronuclei in human lymphocyte subpopulations differentiated by immunoperoxidase staining.

In this study we sought to confirm the radiosensitivity of human peripheral blood lymphocyte subpopulations using a micronucleus assay. Mononucleated cells isolated from peripheral blood were irradiated with X rays. After being cultured for 3 days, cells were fixed and stained using the immunoperoxidase staining technique. Lymphocyte subpopulations were characterized by means of the monoclonal antibodies Leu4 (CD3), Leu2a (CD8) and Leu19 (CD56). Dose-response curves were obtained by scoring the number of micronuclei in binucleated cells that reacted with a specific antibody and were then stained. The dose response of CD8+ (suppressor/cytotoxic) cells was quite similar to that of CD3+ (pan T) cells. In comparison, CD56+ (natural killer) cells were significantly less sensitive, although scorable binucleated CD56+ cells made up less than 4% of the total number of binucleated cells.