Detectable Surface Immunoglobulin Research Articles

Characterization of Immunoglobulin-Bearing and Other Small Lymphocytes in Mouse Bone Marrow by Sedimentation and Electrophoresis

125I-antiglobulin binding and radioautography have been used to define the sedimentation and electrophoretic properties of immunoglobulin-bearing and other small lymphocytes in bone marrow. Bone marrow cell suspensions from CBA mice were exposed to 125I-labeled rabbit anti-mouse immunoglobulin for 30 min at 0 degrees C. Cell fractions were collected after either sedimentation at unit gravity or continuous free-buffer film preparative electrophoresis. The sedimentation profiles of labeled, antiglobulin-binding small lymphocytes and of unlabeled small lymphocytes were identical, peaking at 2.6 mm/hr, except for 5% of the labeled small lymphocytes which sedimented at 3.8 to 4.1 mm/hr. In electrophoretic fractions most labeled small lymphocytes were contained in a peak of low mobility and unlabeled small lymphocytes were of intermediate mobility, but high mobility fractions also contained small numbers of labeled and unlabeled small lymphocytes. Small lymphocytes comprised 80 to 85% of all nucleated bone marrow cells in fractions taken at the peak of the small lymphocyte distribution profile after either sedimentation or electrophoresis, accompanied mainly by either erythroid or granulocytic precursor cells, respectively. The results demonstrate that small lymphocytes with readily detectable surface immunoglobulin in the bone marrow closely resemble those in peripheral lymphoid tissues with respect to their sedimentation and electrophoretic properties. The possibility is raised that the rapidly sedimenting immunoglobulin-bearing small lymphocytes in bone marrow are a functionally distinct group of potential precursor cells. Non-antiglobulin-binding small lymphocytes in bone marrow resemble electrophoretically the double negative (immunoglobulin -and theta -negative) small lymphocytes in other lymphoid tissues. Small lymphocytes of high electrophoretic mobility, prominent in the theta- bearing lymphocyte populations of peripheral lymphoid tissues, are few in bone marrow. Lymphocyte-rich suspensions, containing either high or low proportions of immunoglobulin-bearing small lymphocytes, can be separated from bone marrow by electrophoresis

Acute lymphoblastic leukaemia: a heterogenous disease.

By using several techniques to detect surface markers on T and B lymphocytes, 11 cases of acute lymphoblastic leukaemia (A.L.L.) were studied. In four cases an insignificant number of markers were detected on the lymphoblast populations. In one case a significant number of blasts formed both sheep red blood cell rosettes and Fc rosettes, suggesting a T-cell origin for the neoplastic cells, and in another case the presence of Fc and C3 receptors on the lymphoblast population indicated a B-cell origin. In a further five cases 14-43% of the blasts had detectable surface immunoglobulin. It is concluded that A.L.L. is a heterogeneous disorder, some cases failing to express surface markers and others having either a T-or a B-lymphocyte origin or both.

Appearance of lymphocyte surface markers and functional responses in neonatal and young rabbit spleens

We examined spleen cells from newborn to 1-month-old rabbits for easily detectable surface immunoglobulin, complement receptors, and for in vitro proliferative responsiveness to anti-immunoglobulin antisera and several mitogens. From birth through the first month of life about 15% of the cells from rabbit spleens had easily detectable surface immunoglobulin while about 45% had C3 receptors. In adults as many as 77% of the spleen cells had easily detectable surface Ig but the proportion with C3 receptors remained about 45%. The proliferative response to anti-allotype antisera was present at birth, and was at adult levels by 1 month of age. The proliferative response to pokeweed mitogen was low when cells were obtained during the first week of life but was comparable in magnitude to the response of adult cells by 2 weeks of age. In vitro responsiveness to concanavalin A was present at low levels at birth and increased sharply during the first week. We did not observe significant stimulation of spleen cells from neonatal to 4-week-old rabbits by lipopolysaccharide from Salmonella typhosa. Our data suggest that lymphocyte surface markers and functional responses appear asynchronously in spleen cells of developing rabbits.

Identification of Two Populations of Immunoglobulin-Bearing Lymphocytes in Man

Human peripheral blood lymphocytes bearing easily detectable surface immunoglobulin were quantitated with routine immunofluorescence procedures. The mean value in 17 healthy adults were 18% (range 9 to 30%). When the cells were preincubated at 37 degrees C and washed at the same temperature before staining, the mean value decreased to 9% (range 4.5 to 18%) (p less than 0.001). The mean value increased to 32% (range 15 to 49%) when the cells were prepared and washed at 4 degrees C, and this value was also significantly different from values at room temperature (p less than 0.01). These temperature-related effects were explained by IgG that remained bound to the cell membrane at 4 degrees C, but eluted at 37 degrees C in serum-free medium. The total number of Ig-bearing lymphocytes and T lymphocytes determined by rosette formation with sheep erythrocytes approached 100%. These studies indicate two populations of Ig-bearing lymphocytes in healthy subjects: one with surface-stable Ig determinants, and another that lacks these markers, but has receptors capable of binding IgG.

Ontogeny of B lymphocytes: I. In vitro appearance of Ig-bearing lymphocytes

During the immediate postnatal period, a striking increase in the fraction and number of splenic lymphocytes which bear easily detectable surface immunoglobulin (Ig) occurs in BALB/c and CDF 1 mice. Thus, in mice < 8 hr of age, approximately 4% of splenic lymphoid cells bear surface Ig whereas 17% of splenic lymphocytes of mice 24 hr of age are positive for surface Ig. This increase in Ig-bearing lymphocytes can be obtained in vitro. Thus, cultures of spleen or liver cells from neonatal mice display a substantial increase in the percent and in the absolute number of cells with easily detectable surface Ig. The in vitro increase in Ig-bearing cells is largely inhibited by mitomycin C or puromycin treatment of neonatal cells. Interestingly, pretreatment of these cells with anti-κ antibody, with or without complement, inhibited the increase in Ig-bearing cells. These results indicate that a substantial portion of Ig-bearing lymphoid cells present at 24 hr of age derive from cells already present in the spleens and livers of neonatal mice. Many of these “precursor” cells appear to bear some surface Ig.

Human Lymphocyte Membrane Immunoglobulin as a Reflection of Maturation

Abstract Peripheral blood lymphocytes can be divided into two subpopulations on the basis of surface antigens and membrane receptors. It is generally well recognized that lymphocytes that contain readily detectable surface or membrane-bound immunoglobulin (M-Ig)3 are B lymphocytes (1). The published data on the normal range of B lymphocytes in various studies have not taken into account the possibility of age-related changes in the number and class distribution of human B lymphocytes (2–12). This study was designed to investigate both the number and class distribution of M-Ig on human B lymphocytes obtained from normal subjects in the 1st through the 8th decade of life. The following points are demonstrated: 1) Children (ages 1 to 10) have B lymphocytes bearing IgM (M-IgM) in significantly greater numbers than adults ages 21 to 90 (t = 6.03, p &amp;lt; 0.001). 2) Lymphocytes bearing IgG (M-IgG) in children appear decreased when compared to adults (t = 2.37, p &amp;lt; 0.02).

Differentiation of lymphocytes in mouse bone marrow: I. Quantitative radioautogrophic studies of antiglobulin binding by lymphocytes in bone marrow and lymphoid tissues

The density of surface immunoglobulin on small lymphocytes in the bone marrow and other lymphoid tissues has been compared by radioautographic measurements of antiglobulin binding. Cell suspensions from CBA mice were exposed to 125I-labeled rabbit anti-mouse globulin in a wide range of concentrations for 30 min at 0 °C. With increasing concentration of antiglobulin- 125I the percentage of labeled antiglobulin-binding small lymphocytes in spleen and lymph node suspensions reached well-defined plateau levels. Very few normal or cortisone-resistant thymus cells were labeled under identical conditions. Bone marrow small lymphocytes showed a linear increment in labeled cells throughout the antiglobulin- 125I dose range, their labeling intensity varied widely, and approximately one half remained unlabeled at high antiglobulin- 125I concentrations. In 6 wk-old congenitally athymic mice the bone marrow small lymphocyte labeling pattern resembled that in CBA mice, while nearly all (91–97%) small lymphocytes in lymph nodes, thoracic duct lymph and blood, and 75% of those in the spleen, became labeled under plateau conditions. Treatment of cells from 10 wk-old CBA mice with AKR anti- θ C3H serum and complement resulted in almost complete (93%) antiglobulin-labeling of residual small lymphocytes from the spleen but had little effect on bone marrow lymphocyte labeling. Under germfree conditions the proportion of antiglobulin-binding small lymphocytes was slightly elevated in all lymphoid tissues of CBA mice. The results demonstrate that many of the small lymphocytes in mouse bone marrow have readily detectable surface immunoglobulin molecules which vary considerably in density from cell to cell, while others neither have detectable surface immunoglobulin, nor are they θ-bearing, thymus-dependent or recirculating cells. The concept of bone marrow small lymphocytes as a maturing cell population is discussed.

Rat B cells: Identity of immunoglobulin-bearing cells with a population of lightly uridine-labeled cells

The mixed antiglobulin reaction, to detect surface immunoglobulin (Ig), was combined with uridine labeling and autoradiography to establish that the rat lymphocytes which take up very little uridine are identical with those cells bearing surface immunoglobulin. Thus, low uridine uptake and membrane Ig are both markers for the same B cells.

Membrane antigenic changes associated with PHA transformation of mouse spleen cells in vitro.

The changes in lymphocyte surface antigens associated with blast transformation have been studied by membrane immunofluorescence. When mouse spleen cells were stimulated in vitro with phytohaemagglutinin for 4 days virtually all of the resulting blast cells were shown to have histocompatibility (H-2d), theta(θ)and mouse-specific T lymphocyte (MTLA) antigens; approximately 50% had detectable surface immunoglobulin and 70% mouse-specific B lymphocyte antigen (MBLA). The intensity of the staining reactions on blast cells was increased for the anti-MTLA serum and reduced for the anti-immunoglobulin and anti-MBLA sera when compared with the staining of fresh spleen cells. No difference was detected for the anti-θ and anti-H-2d sera. Cap formation was detected on fresh spleen cells labelled with any of the antisera, but after stimulation only anti-immunoglobulin-treated blasts showed the cap pattern.The simultaneous expression of B-and T-cell markers on PHA transformed cells suggests a convergence of the lympho...

Membrane Receptors and in Vitro Responsiveness of Lymphocytes in Human Immunodeficiency

Abstract Peripheral blood lymphocytes from 24 patients with well-defined immunodeficiency were studied for membrane characteristics and in vitro responsiveness to various stimuli. The mean percentages of lymphocytes bearing easily detectable surface immunoglobulin (Ig) were similar to those of normals except in patients with infantile x-linked agammaglobulinemia (X Ag) who had a significantly lower percentage of only 1.9% Ig bearing cells (p &amp;lt; 0.001). Despite their marked deficiency in surface Ig bearing cells, patients with X Ag had normal percentages of complement receptor rosettes (EAC, HEAC, or B cell rosettes); percentages of these cells were also normal or elevated in the other patient groups. Percentages of spontaneous sheep erythrocyte rosettes (E or T cell rosettes) were significantly increased in X Ag patients and reduced in patients with ataxia telangiectasia and isolated IgA deficiency. Lymphocyte 3H-thymidine incorporation in response to each of the three mitogens, phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM), was usually normal except in X Ag patients where, in several evaluations, the responses of peripheral blood lymphocytes to PHA were significantly lower than those of normal control cells (p &amp;lt; 0.001). Responses to Con A and PWM were normal or elevated in those same experiments. The selective hyporesponsiveness of X Ag lymphocytes to PHA suggests that these patients may be deficient in a restricted population of T cells. It is possible that this population may be necessary for the full maturation of B cell function. Lymphocytes from all patients, including agammaglobulinemic patients, responded normally to stimulation with one or more antigens.

Functional heterogeneity of murine lymphoid cells. V. Lymphocytes lacking detectable surface theta or immunoglobulin determinants.

An appreciable percent (3-14%) of the lymphocyte-like cells of the mouse spleen lack both the theta-isoantigen and sufficient surface immunoglobulin to be detected by conventional immunofluorescence or autoradiographic procedures. These theta(-),Ig(-) cells are increased in frequency after treatment of mice with antithymocyte serum or in mice that have been thymectomized, irradiated (850 R), and reconstituted with bone marrow cells. Moreover, in chimeric C57BL/6 mice in which the T cells are derived from (BALB/c x C57BL/6)F(1) donors, theta(-),Ig(-) cells also lack BALB/c histocompatibility antigens. These experiments indicate that theta(-),Ig(-) cells are not theta(-) T lymphocytes. Removal of complement receptor lymphocytes from spleen cell populations increases the frequency of theta(-),Ig(-) cells, indicating that such cells lack the complement receptor. Partially purified populations of theta(-),Ig(-) cells have been obtained by cytolysis by anti-theta- and anti-kappa-antibody and complement and by density gradient ultracentrifugation. These cells closely resemble lymphocytes in morphology. The only exceptional feature is the existence of prominent nucleoli. The theta(-),Ig(-) cells lack hemoglobin and endogenous peroxidases, are not actively phagocytic, and do not adhere to glass. This suggests they are not of the erythroid, myeloid, or monocytoid lines. [(3)H]Thymidine labeling studies indicate that theta(-),Ig(-) cells are members of a relatively slowly dividing cell pool. Whether theta(-),Ig(-) cells are members of the "classical" B lymphocyte line or belong to another, as yet undescribed, lineage is not yet certain.

Immunoglobulin molecules on the surface of activated T lymphocytes in the rat.

Lewis strain rat lymphocytes were exposed in vitro to a variety of specific and nonspecific blastogenic stimuli. The surfaces of the transformed lymphocytes were examined by indirect immunofluorescence for the presence of T cell antigens and immunoglobulin molecules. More than 90% of lymphocytes that underwent blast transformation after exposure to foreign histocompatibility antigens (mixed lymphocyte reaction; in vitro allograft reaction), purified tuberculin, phytohemagglutinin (PHA), and concanavalin A (Con-A) had T cell antigenic markers on their surfaces. 70-92% of the antigen-stimulated blast cells also had readily detectable surface immunoglobulin molecules, whereas less than 3% of the PHA- and Con-A-activated cells were Ig(+). Pokeweed mitogen (PWM) appeared to activate both B and T cells, but the T cells did not have detectable surface immunoglobulin molecules. Nonactivated control cultures contained T(+)Ig(-) lymphocytes almost exclusively. The results suggest that thymus-dependent rat lymphocytes express increased amounts of detectable immunoglobulin on their surface in response to specific stimulation with antigen. It is postulated that the acquisition of immunological competence by activated T cells may be related to this expression of surface immunoglobulin.

Immunoglobulins on the Surface of Lymphocytes

Abstract The presence of immunoglobulin (Ig) on the surface of lymphocytes that were negative for Ig by immunofluorescence was investigated by combining a quantitative inhibition assay with immunofluorescence. Thymus cells; thymus, spleen and lymph node cells that had been passed through anti-Ig-coated plastic bead columns; and ϑ-positive mouse lymphomas were evaluated by using these two systems. These cell preparations had 0 to 1% of cells with detectable surface immunoglobulin by immunofluorescence. If the assumption is made that the fluorescent-positive cells were relatively homogeneous with respect to the amount of surface Ig, then it could be calculated that there was more Ig present in the various cell populations studied than was accountable by the small percentage of fluorescent-positive cells. Based on these calculations, the amount of Ig present in the different populations was found to be: fluorescent-positive cells: 5 to 14 ng N/106 cells; fluorescent-negative thymus cells: 0.01 ng N/106 cells; fluorescent-negative spleen and lymph node cells 0.07 to 0.1 ng N/106 cells. Three of four ϑ-positive lymphomas also had detectable Ig in amounts 3 to 10 times that found in normal thymus. Routinely there was more IgM found in both fluorescent-positive and -negative normal cells than IgG2.

Removal of immunoglobulin-bearing lymphocytes by anti-immunoglobulin-coated columns

As measured by several immunochemical techniques, lymphocytes can be divided into two categories: those with easily detectable surface immunoglobulin and those with undetectable immunoglobulin. This paper describes a technique by which mouse spleen cell populations can be depleted of these immunoglobulin-bearing cells. By utilizing anti-immunoglobulin-coated plastic bead columns, we have been able to deplete cell populations of immunofluorescent-positive cells. This depletion was about 90% effective after a single passage through an anti-immunoglobulin-coated column. while serial passage of the cells through several columns resulted in a 99.8% depletion. In addition, two sets of experiments demonstrated that these columns deplete cell populations of certain immunocompetent cells. First, preexisting direct and indirect plaque-forming cells are specifically retained by these columns; and second, a population of cells which is necessary for reconstitution of lethally irradiated mice is removed from normal mouse spleens by these columns.

Receptors for complement and immunoglobulin on human leukemic cells and human lymphoblastoid cell lines.

The bone marrow-derived (B) lymphocyte can be identified by the presence of easily detectable surface immunoglobulin and a receptor for antigen-antibody-complement complexes (EAC'). Monocytes and macrophages also bear a receptor for EAC' and in addition possess a receptor for red cell-IgG complexes (EA). Thymus-derived (T) lymphocytes bear neither of these receptors. The cells of 15 patients with leukemia and 19 human lymphoblastoid cell lines were examined for the presence of the EAC' and EA receptors. Of the human leukemias studied, only the cells from the patients with chronic lymphatic leukemia (CLL) possess the EAC' receptor. The EA receptor could not be demonstrated on CLL cells; hence, CLL cells bear the lymphocyte EAC' receptor and by this criteria represent B lymphocytes. 12/19 of the cell lines studied could be classified as B lymphocytes by the presence of the EAC' receptor and absence of the EA receptor. 2/19 cell lines possessed both the EAC' and EA receptors and thus resemble the monocyte. 5/19 cell lines had no detectable receptor for EAC' or EA. The approach presented in this study for the classification of leukemias and cell lines as to their B lymphocyte, T lymphocyte, or monocyte origin may have useful diagnostic and therapeutic implications.

Immunoglobulin and ϑ-Bearing Murine Leukemias and Lymphomas

Abstract Murine lymphocytes can be separated into two distinct populations. The thymusderived T lymphocytes are responsible for cell mediated immunity and are identified by the ϑ isoantigen. The bone marrow derived B lymphocytes are the precursors of the antibody-forming cells and are identified by the presence of easily detectable surface immunoglobulin. In this study, 24 murine leukemias and lymphomas and eight plasma cell tumors were investigated for the presence of the ϑ isoantigen and determinants of the kappa light chain. Of the leukemias and lymphomas studied, 12 have the ϑ isoantigen and are of T cell origin; one tumor bore kappa light chain and is presumably of B cell origin. Eleven of the tumors studied had no detectable ϑ or kappa determinants. Four of the eight plasma cell tumors have surface kappa determinants. No ϑ positive tumors bore kappa chain determinants and no kappa positive tumors bore ϑ determinants. The characterization of leukemias and lymphomas as to their B or T cell origin will provide large numbers of pure B and T lymphoid cells useful for a variety of studies.

Depletion of Antibody-Forming Cells and Their Precursors from Complex Lymphoid Cell Populations

Abstract Lymphocytes are now known to be a heterogeneous cell population which contains at least two functional subtypes. One subtype of cell (B-lymphocyte) bears easily detectable amounts of membrane-associated immunoglobulin; studies in mice have established that these lymphocytes are of bone marrow origin and include the precursors of antibody-forming cells (1, 2, 3). A small number of cells within this population of surface immunoglobulin bearing lymphocytes from non-immunized animals specifically bind antigen and the frequency of such cells increases subsequent to immunization (4, 5). The other major subtype lacks easily detectable surface immunoglobulin and mediates a variety of cellular immune phenomena (6, 7). These lymphocytes in the mouse are derived from the thymus and possess surface differentiation antigens, such as θ, which allow their identification (1). Further delineation of the structure and function of lymphocyte subpopulation and of the interactions between them would be aided by development of techniques to obtain relatively pure populations.

IDENTIFICATION OF HUMAN T AND B LYMPHOCYTES IN NORMAL PERIPHERAL BLOOD AND IN CHRONIC LYMPHOCYTIC LEUKÆMIA

A sandwich radioimmunolabelling technique has been used to demonstrate two populations of human peripheral blood lymphocytes: one, comprising an average of 34% of lymphocytes, carried a high density of surface immunoglobulin, while the other 66% had a much lower density. Human thymocytes had little detectable surface immunoglobulins. By extension from studies in mice, it is suggested that these two groups represent B and T lymphocytes respectively. Lymphocytes from 3 patients with chronic lymphocytic leukæmia had labelling characteristics of B cells but labelled less heavily than the majority of normal B cells. Almost no immunoglobulin was detected on the surface of blast cells from a patient with acute lymphoblastic leukæmia.