Null Cell Population Research Articles

FUNCTIONAL AND BIOCHEMICAL CHARACTERISTICS OF HUMAN “NULL” LYMPHOID CELLS

Human peripheral null cells (non-T and non-B lymphocytes) were isolated by removing B lymphocytes and monocytes on nylon-wool columns and by E rosette-forming cells depletion. The null cell population contained less than 1% of SIg-bearing cells, cells with complement receptors (EAC+), and E mouse rosette-forming cells, but was contaminated by about 5% of T lymphocytes and 10% of monocytes. High affinity Fc receptors were present on 12% of the null cells. No intracytoplasmic immunoglobulins (CIgs) were detected by immunofluorescence. Culture of these null cells for 2 to 6 days did not modify their surface receptors. Null cells were not stimulated to undergo blastogenesis by the following mitogenic agents: concanavalin A (Con A), phytohemagglutinin-L (PHA-L), lentil lectin, wheat germ agglutinin, pokeweed mitogen (PWM), peanut agglutinin, and neuraminidase-galactose oxidase. PWM did not induce the transformation of null cells into B lymphocytes and no SIg, CIg, and production of Ig were detected by immunofluorescence and by measuring the incorporation of 3H-leucine. However the antibody-dependent cellular cytotoxicity of these null cells was very active compared to unpurified lymphocytes. Analysis of the lectin-binding surface glycoproteins, after labeling of the cells, by polyacrylamide gel electrophoresis (PAGE), showed that the 27,000- to 33,000-dalton component (Ia-like antigen) present in large amounts on B lymphocytes and monocytes was absent in null cells. These results are in agreement with the hypothesis that null cells consist of one or several populations different from the other lymphocytes and that they are not direct precursors or immature T and B lymphocytes.

Secretory Cells in the Medulla of the Bursal Follicle: The Small Lymphocyte-Like Cells are Precursors of the Secretory Cells

In our morphological study we have identified Sll which are structurally similar to small lymphocytes. It is suggested that the Sll belong to the null cell population of the bursa of Fabricius are blood-borne cells with proliferative capabilities and are precursors of the secretory cells. The transformation of Sll into Sc may be facilitated by Cy treatment.

Pseudo-lymphocyte monocytes—the memory cells responsible for the development of epithelioid cell granulomata. A new hypothesis

This hypothesis proposes that, in individuals with an appropriate genetic background, monocyte memory cells are formed when histiocytes and macrophages undergo mitosis following first exposure to a granulomagenic agent and circulate as pseudolymphocytes in the lymphocyte null cell population. It is proposed that epithelioid cell granulomata develop from a clone of cells formed from monocyte memory cells on the second or subsequent exposure to the same granulomagenic agent. Epithelioid cell granuloma formation is therefore not dependent on T-cell function, although the cellular nature of the granuloma appears to depend upon the nature of a concomitant but independent classical immune response. The implications of pseudolymphocyte memory cells on the development of granulomata of both exogenous and endogenous origin, and the relationships between lymphocytes and cells of the monocytic phagocyte series are discussed.

Cytotoxicity from cultured cells: Analysis of precursors involved in generation of human cells mediating natural and antibody-dependent cell-mediated cytotoxicity

Natural cell-mediated cytotoxicity of human peripheral blood lymphocytes natural killer (NK) against K-562 and antibody-dependent cellular cytotoxicity (ADCC) against Chang cells, as measured in a 4-hr 51Cr release assay, were both completely removed by depletion of Fc receptor-positive (FcR+) cells. After in vitro culture for 7 days, however, NK- and ADCC-like activities spontaneously regenerated. The nature of precursor cells was studied by examination of lymphocyte subpopulations required for generation of this cytotoxicity. After depletion of FcR+ cells from PBL, the following subpopulations were prepared: sheep erythrocyte rosette-forming cells (E+), surface membrane immunoglobulin-positive cells (SmIg+), and null cells (lacking E+, SmIg+, or FcR+ markers). Separate cell types or mixtures were cultured in vitro in medium containing 10% fetal calf serum for 7 days and then tested for NK and ADCC. Whereas unseparated FcR-depleted cells developed substantial cytotoxic activity, each of the subpopulations cultured alone was negative or had low activity. Addition of SmIg+ cells to other cell types had no effect; however, mixture of 80% E+ and 20% null cells resulted in optimal NK and ADCC. It is not presently clear which population the precursors were in. However, the requirement for proliferation by the null cell population but not by the E+ cells (as indicated by sensitivity to radiation and mitomycin C) suggested that the precursors for NK cells may be null cells.

Differential effects of pharmacologic agents on EAC3 rosette formation by lymphocytes from normal and asthmatic subjects

We studied the effect of isoproterenol hydrochloride (I) and theophylline (Th) on EAC3 rosette formation by lymphocytes from 30 normal adult subjects, 35 asthmatic subjects, 11 subjects who were age and sex matched with the asthma group, and 10 subjects with allergic rhinitis. Baseline EAC3 rosette numbers were similar for all groups (normal adults, 13.2%; normal children, 15.1%; rhinitis, 14.4%; asthma, 14.6%). Incubation with I and Th in the presence of fetal calf serum caused enchancement of EAC3 rosette formation by lymphocytes from normal adults (mean increase, 52% with I and 53% with Th), from normal age- and sex-matched controls (mean increase, 45% with I and 40% with Th), from subjects with allergic rhinitis (mean increase, 50% with I and 52% with Th), but not by lymphocytes from asthmatic subjects (mean increase, 0% with I and 2% with Th). These results were not affected by bronchodilator therapy. Similar results were obtained with cholera toxin, prostaglandin E 1 and adrenaline, suggesting that the defect in lymphocytes from asthmatic patients resides at the level of 3′, 5′-cyclic adenosine monophosphate (cAMP) production or at the level of cAMP-triggered events. Lymphocyte fractionation experiments into T cell-rich, B cell-rich, and null cell populations revealed that the null cell population was a target for the I-mediated enhancement of EAC3 rosette formation. Null cells from normal subjects, but not from asthmatic subjects, exhibited an increase in the number of EAC3 rosettes formed following incubation with I. This enhancement required protein synthesis as it was prevented by the addition of puromycin hydrochloride. The resistance of peripheral blood lymphocytes from asthmatics to the enhancing effect of I and Th on EAC3 rosette formation may be used to study the biochemical defect in asthma and should be assessed for possible use in the detection of the latent asthmatic.

Lymphocyte subpopulations B, T and null—In Oral Submucous Fibrosis

The circulating T, B and null cell populations were quantified in Oral Submucous Fibrosis (O.S. M.F.), a disease found almost exclusively in Indians. The absolute null count was significantly increased (Sem=8.0122, n=32) while the absolute T lymphocytes were significantly decreased (Sem=3.6250, n=32). The B-cell populations were within normal limits.

Lymphocyte subpopulations in minimal change nephrotic syndrome

Minimal change nephrotic syndrome (MCNS) was characterized by a relative lymphocytopenia, a normal T-cell count, and a discordance between B cells enumerated by the presence of surface immunoglobulins (Bsig) and by C3B receptors (Beac). By either technique of B-cell count, the total number of T and B lymphocytes in MCNS exceeded that of controls, suggesting the presence of “double-marked” cells or decreased null cell population. This alteration in surface receptor characteristics of lymphocytes which was observed both in relapse and in remission, documents an abnormality, in MCNS, possibly of cell-mediated immunity, which may be relevant to its pathogenesis.

Increased Active Rosette Formation in Patients with Gastrointestinal Cancer after Enzyme Treatment of Lymphocytes In Vitro*

Summary: Increased active rosette formation in patients with gastrointestinal cancer after enzyme treatment of lymphocytes in vitro. B. A. J. Walters, J. C. Rutherford and J. R. Wall, Aust. N.Z. J. Med., 1978, 8, pp. 610–614.Total rosette forming cells (RFC) and active rosette forming cells (ARFC) were estimated in 33 patients presenting with gastrointestinal symptoms suggestive of cancer. When the 33 patients were grouped according to whether they had cancer or not, there was a distinct difference in the percentage of ARFC (P < 0001 Mann‐Whitney Test); patients with cancer having much lower values (mean 11 6 ±4–5) than patients without cancer (mean 27 2 ± 3–6). Total RFC were generally lower in the cancer group (mean 54‐8 ± 8‐3) than the non‐cancer group (mean 71‐9 ± 4–5) a/though the range was greater. After treatment of the lymphocytes with papain, the percentage of ARFC increased. In the non‐cancer group the levels reached a mean of 74‐8 ± 1–8, indicating that the T lymphocyte population (mean 71‐9 ± 4–5) was converted into cells all having high affinity receptors for sheep red blood cells (SRBC). In the cancer group, after papain treatment of the lymphocytes, the percentage of ARFC increased to levels greater than that obtained for total RFC, suggesting a contribution from the null cell population. Serum taken from these patients was shown to produce a decrease in the percentage ARFC obtained from normal, young individuals. Further, this decrease could be partially reversed by papain digestion indicating that serum from cancer patients may contain a factor that masks T

1050 DOUBLE-MARKED LYMPHOCYTES IN MINIMAL CHANGE NEPHROTIC SYNDROME (MCNS)

Indirect evidence supports a role for cell mediated immunity in the pathogenesis of MCNS. In the present study, the morphology and surface receptor characteristics of lymphocytes from 16 patients with MCNS and 14 controls were examined. MCNS was characterized by a relative lymphocytopenia, a normal T cell count, and a discordance between B cells enumerated by the presence of surface immunoglobulins (Bsig) and by C3B receptors (Beac). By either technique of B cell count, the total number of T and B lymphocytes in MCNS exceeded that of controls, suggesting the presence of “double-marked” cells or decreased null cell population. This alteration in surface receptor characteristics of lymphocytes which was observed both in relapse and in remission, documents an abnormality, in MCNS, possibly of cell mediated immunity, which may be relavent to its pathogenesis.

Two Small Lymphocyte Subpopulations in Human Peripheral Blood

By means of simple rosette sedimentation methods two subsets from human peripheral blood lymphocytes have been isolated: (1) (E, Fc)- and (2) (E, Ig)-. The first subset was obtained by centrifuging suspensions of macrophage-depleted PBL in which E and EA rosettes had been allowed to form simultaneously. The dominant marker of these E- Fc- cells was surface Ig, and during 4 days of culture this population did not alter its surface markers. Subset 2 was obtained in two ways following rosette centrifugation with AET-treated SRBC and rabbit anti-human Ig-coated autologous RBC. This 'Null cell population' was shown to be highly variable as judged by the surface markers applied after 4 days of culture, and it is suggested that Null cells contain a number of immature lymphoid cells that may acquire their surface marker during culture. It is concluded that the methods described for purification of small subsets of human lymphocytes are effective and easy to perform and might be used to purify cells for functional studies.

Identification of membrane-associated lymphotoxin (LT) on mitogen-activated human lymphocytes using heterologous anti-LT antisera in vitro

Surface-associated lymphotoxin (LT) molecules have been identified on mitogen-activated human lymphocytes employing heterologous anti-α-LT serum in vitro. These membrane-associated LT molecules are present on PHA- or Con A-activated lymphocytes but do not appear to be expressed on unstimulated cells. Furthermore, these molecules were detected primarily on activated T lymphocytes, with little detectable on activated B- or null-cell populations. The removal of surface LT-bearing lymphocytes, using anti-α-LT serum + C′, does not dramatically affect the capacity of the remaining cells to release LT after mitogen restimulation. In addition, the presence of toxic molecules on the surface of activated lymphocytes suggests that these materials may be expressed in an inactive, noncytotoxic form.

Null cells in peripheral blood of normals and systemic lupus erythematosus

Enriched null cell populations were obtained by fractionating blood mononuclear cells on discontinuous bovine serum albumin gradients. Null cells from 22 normals were compared to those from 14 patients with systemic lupus erythematosus who had various degrees of disease activity and to 8 patients with other autoimmune phenomena. Null cell layer lacked T cells (E rosette forming cells), cells with surface membrane immunoglobulin, cells capable of latex particles phagocytosis or cells capable of binding to IgG-coated human erythrocytes (EA rosette assay). Cells carrying receptors for complement were present. Cells of the null cell layer failed to respond in vitro to mitogens and allogeneic cells but were capable of Ig synthesis upon incubation with pokeweed mitogen. On transmission electron microscopy, null cells had large nucleus, narrow cytoplasm, and short surface processes. Null cells of SLE patients, when compared to normals, were increased in numbers ( p < 0.01), and had the capacity to bind to native DNA ( p < 0.01). Binding of cells to DNA could not be blocked by prior treatment of cells with anti-Ig antisera. There was a positive correlation between disease activity, null cell numbers, and DNA binding to serum or to the null cells. Defective maturation and/or differentiation of the null cells of SLE into mature cells with expansion of cell clones reactive to native DNA could be important in the pathogenesis of SLE. This could be due to the lack of maturation factors and/or rapid emigration of the precursor null cells from the stem cell compartment to the circulating pool.

Immunologic Functions of Isolated Human Lymphocyte Subpopulations

Abstract Sephadex G-200 anti-human Fab column chromatography and rosette depletion techniques were used to isolate three distinct subpopulations of human lymphocytes: 1) T cells which are surface Ig negative and E rosette positive, 2) B cells which are surface Ig positive and E rosette negative, and 3) a “Null” cell population which is both surface Ig negative and E rosette negative. All populations were analyzed for their capacity to develop surface Ig and synthesize Ig in vitro. Greater than 50% of cells in the Null cell population developed surface Ig by day 3 of cell culture. Furthermore, in vitro, the Ig content of the Null cell population, as well as their capacity to secrete Ig in culture, becomes comparable to that produced by B cells. In contrast, cultured T cells neither develop surface Ig nor secrete Ig in culture. These data strongly support the idea that the Null population contains a subset of Ig-producing B cells.

Human antibody-dependent cellular cytotoxicity. Isolation and identification of a subpopulation of peripheral blood lymphocytes which kill antibody-coated autologous target cells.

Antibody-dependent cellular cytotoxicity (ADCC), has been shown to be independent in vitro of thymus-derived lymphocytes, but the precise nature of the effector lymphocyte has not been fully clarified. To further study the identity of the ADCC effector cell type(s), peripheral blood leukocytes were purified by Ficoll-Hypaque density centrifugation and fractionated into surface immunoglobulin-positive [Ig(+)] and surface immunoglobulin-negative [Ig(-)] populations by chromatographic separation on Sephadex G-200 anti-human immunoglobulin columns. After column fractionations, the ADCC effector activity against antibody-coated autologous lymphocytes was predominantly and consistently found in the Ig(-) fraction. This latter population was then further fractionated, by rosetting techniques, into two subpopulations, The first was depleted by lymphocytes with surface receptors for sheep red blood cells [E(+)]and the second was depleted of lymphocytes with receptors for sheep red blood cell-antibody-complement [EAC-(+)]. Analysis of these populations showed that ADCC effector activity was predominantly a property of the Ig(-) lmyphocytes which are E(-) but EAC(+). These lymphocytes have been referred to as "null lymphocytes" and probably represent a subset of bone marrow-derived (B) cells. In addition, variable and low levels of ADCC activity were observed in some Ig(+) populations (B cells). Further purification of the null cell population by filtration over nylon wool columns to reduce the number of contaminating latex ingesting monocytes did not reduce ADCC effector activity. Isolated null cell ADCC effector activity was inhibited by either rabbit anti-human F(ab)2 or normal pooled rabbit gamma globulin, but not by rabbit F(ab)2 anti-human F)ab)2 or media. This supports the contention previously suggested in studies using unfractionated lymphocyte populations that the ADCC effector cell recognizes the Fc portion of the antibody molecule. The variable and low level of activity noted in the Ig(+) populations is unexplained but possibly due to a variable population of null cell-derived Ig(+) lymphocytes within the whole Ig(+) population. In conclusion, these experiments demonstrate that, in vitro, the major ADCC effector activity of circulating human peripheral blood lymphocytes resides in the Ig(-), E(-), EAC-(+) subpopulation termed "null cells." Since it has been noted that in certain disease states, such as immunodeficiency syndromes, autoimmune disorders, and neoplasms, the percentage of this population of lymphocytes in the peripheral blood is elevated, it is speculated that these cells, perhaps through their ADCC function, may play an important pathophysiologic role in these diseases.

Immunologic functions of isolated human lymphocyte subpopulations: V. Isolation and functional analysis of a surface Ig negative, E rosette negative subset

Utilizing column immunoabsorbent anti-Fab chromatography and rosette depletion techniques three distinct subsets of human lymphocytes were isolated: (1) T cells which are surface Ig− and E rosette positive, (2) B cells which are Ig+, E rosette negative, and (3) a Null cell population which is both Ig− and E rosette negative. All three populations were analyzed with respect to a variety of assays of cell mediated immunity. The Ig−, E rosette negative (Null cell) population exhibited close functional similarities to B lymphocytes. Both responded poorly to plant lectins, did not proliferate in response to soluble or cell surface antigens, and were not active in cell mediated lympholysis. However, both were efficient effector cells in antibody dependent cellular cytotoxicity. T lymphocytes, in contrast, were highly active in proliferative responses to plant lectins and antigens, were efficient in cell mediated lympholysis, but were not active effector cells in antibody dependent cellular cytotoxicity. These studies support the view that the human Ig−, E rosette negative cells represent a subset of B lymphocytes.

Surface characteristics of synovial fluid and peripheral blood lymphocytes in inflammatory arthritis.

AbstractIn order to characterize the T‐ and B‐cell populations of inflammatory arthritides, synovial fluid and peripheral blood lymphocytes from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), juvenile rheumatoid arthritis (JRA), and rheumatoid variant diseases (RV) were studied. Normal peripheral blood lymphocytes were examined as controls. T cells were identified by spontaneous sheep red blood cell (E) rosette formation. B cells were determined by complement receptor lymphocyte (EAC) rosette formation and by the presence of surface immunoglobulins (SIg) utilizing fluoresceinated polyvalent antiglobulin. Synovial fluids were analyzed in terms of duration, mucin quality, white cell count, and differential. Synovial fluid and peripheral blood lymphocytes from patients with RA and RV were distributed in T‐ and B‐cell percentages similar to those found in normal peripheral blood. In contrast, significant T‐cell depression was observed in the percentage of synovial fluid and peripheral blood lymphocytes of SLE and JRA patients. This depression was apparent in comparison with normal peripheral blood and with the synovial fluid and peripheral blood from RA patients. B‐cell percentages were similar in all patient groups and in comparison to normal peripheral blood lymphocytes. No differences were noted in B‐cell percentages when the EAC and SIg techniques of identification were compared. The percentage of cells bearing neither T‐ nor B‐cell markers (null cells) was enumerated for each patient group and found to be significantly elevated in the synovial fluid and peripheral blood of SLE and JRA patients. Though the mean synovial fluid and peripheral blood null cell percentages in RA patients were similar to those in controls, a definite bimodal distribution was found in the synovial fluids. These data suggest that evaluation of T‐, B‐, and null‐cell populations may be clinically useful in differentiating patients with SLE and JRA from those with rheumatoid arthritis and variant diseases.