A surface membrane determinant shared by subpopulations of thymocytes and B lymphocytes.

Utilizing a quantitative fluorescence assay with the fluorescence-activated cell sorter (FACS), we have demonstrated that a rabbit antiserum obtained by immunization with cells of a mouse IgM-producing plasma cell tumor (MOPC104E) is reactive with at least two surface determinants, designated Th-B and ML2, on subpopulations of normal murine lymphocytes. The ML2 determinant is restricted to B lymphocytes. The Th-B determinant is shared by splenic B lymphocytes and a large subpopulation of thymocytes, the latter of which express a 3-fold higher density of Th-B on their surface than do the B lymphocytes. Neither Th-B nor ML2 were found on peripheral T cells or on brain, liver, or kidney cells. The available evidence suggesting that Th-B may be a stem cell determinant that is lost upon maturation is discussed.     

Characterization of an antiserum specific for cell surface antigens of rat mast cells.

Rabbits were immunized with rat peritoneal mast cells (RMC) in complete Freund's adjuvant. The antisera (anti-RMC) were checked for their reactivity with RMC by intradermal skin tests in rats. The best serum was selected and absorbed with rat liver cells and rat immunoglobulins, including IgE. The absorbed serum (anti-RMCabs), as well as the anti-RMC serum, were then tested for their reactivity with RMC. Both sera were cytotoxic to RMC but only anti-RMC was cytotoxic for rat lymph node cells. Both sera gave positive reactions in rat skin, as seen by the permeability to Evan's blue dye. The binding of rat IgE to RMC was also inhibited by both sera. A control rabbit anti-rat sarcoma serum absorbed with liver cells did not show any interaction with RMC. When 125I-labeled RMC surface antigens were precipitated with anti-RMCabs and analyzed by SDS polyacrylamide gel electrophoresis, several components were observed. Among these was one with a mobility identical to that of a mast cell surface component that had previously been identified as the receptor for IgE or at least a component thereof.     

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.     

Identification of cell surface antigens present on murine hematopoietic stem cells

Nine antigens found on murine bone marrow cells were examined to define their pattern of expression in murine hematopoietic differentiation. Lymphocyte function antigen (LFA-1), heat stable antigen (recognized by M1/69), common leukocyte antigen (CLA, T200, Ly-5) and Lgp100a (recognized by 30-C7) were present on early hematopoietic progenitors, BFU-E, CFU-E, CFU-GM, and CFU-M. All antigens found on progenitors were found on some immature precursor cells, myeloblasts, erythroblasts, or monoblasts, but their pattern of expression on identifiable hematopoietic cells varied. Three of these antigens, LFA-1, heat stable antigen recognized by M1/69, and CLA, were expressed on leukocytes of all stages of maturity but were lost from the erythroid lineage during differentiation. MAC-1, Forssman antigen, heat stable antigen (recognized by M1/75), anti-P-95 (recognized by M5/113), and Ia (recognized by M5/114) were found only on differentiated hematopoietic precursors or mature cells. The expression of these antigens was more lineage-specific. MAC-1 and heat stable antigen (recognized by M1/75) were restricted to either mature myeloid or erythroid cells, respectively. The marked differences in distribution of these antigens suggest that they may be useful in negative or positive selection experiments to enrich progenitors, and that some of them may have a functional role in differentiation.     

Serologically defined subpopulations of human T lymphocytes.

Several rabbit antisera to T cells obtained from various sources (thymus, peripheral blood, brain, T-derived leukemias) were studied with the aim to obtain reagents specific for a subset of T cells. Sera were first absorbed on human tissues and B cells; thereafter these T cell-specific sera were additionally absorbed with T cells of different origin and especially with leukemic T cells, which are likely to represent a clonal expnasion of a subset of T cells with potentially unique antigenic markers. Three antigenically distinct subpopulations of T cells were delineated. The relationship of these subsets with previously defined human T cell subpopulations (T subsets with a receptor for the Fc or IgG or IgM or with a receptor for a lectin from wheat germ agglutinin) was investigated.     

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.     

T cell differentiation antigens on lymphocytes in the human intestinal lamina propria.

Recently, T cell subpopulations presumably representing memory T lymphocytes have been described in vitro. Intestinal lamina propria T cells (LP-T) have characteristics resembling those of memory cells. We therefore investigated the expression of surface Ag associated with memory phenotype in vitro on lamina propria lymphocytes (LPL) and PBL and on the T cell subpopulations defined by the bright expression of CD45R0 by flow cytometric analysis of isolated cell populations. LPL had significantly increased percentages of CD45R0 and CD58 positive cells compared with PBL. Whereas PBL showed bimodal expression profiles of CD45R0, CD58, and CD2, the vast majority of LPL was bright for these Ag. Expression of CD45RA was significantly reduced in both frequency and intensity in LPL, and LPL had significantly reduced percentages of CD11a/CD18 and CD29 positive cells compared with PBL. The CD45R0 bright T cell subpopulations of both PBL and LPL were characterized by a lack of CD45RA. CD45R0 bright T cells from the peripheral blood (PB-T) were predominantly bright for CD2, CD58, CD29, and CD11a/CD18 whereas CD45R0 dim PB-T had bimodal expression profiles and CD45R0 negative PB-T were dim or even negative for these Ag. CD45R0 bright LP-T were also bright for CD2 and CD58 but had significantly reduced surface densities of CD11a/CD18 and CD29 compared with CD45R0 bright PB-T. The surface density of CD29 on CD45R0 bright LP-T corresponded to that of CD45R0 negative PB-T, and a significant proportion of CD45R0 bright LP-T was even negative for CD11a/CD18 and CD29. Additionally, CD45R0 bright LP-T in contrast to PB-T were characterized by a lack of 1-selectin and the expression of CDw49a and the mucosa-specific T cell Ag HML-1 on high percentages of cells. Our results show that the phenotype of CD45R0 bright T cells from the lamina propria clearly deviates from that of memory T cells in vitro and of CD45R0 bright T cells in the peripheral blood. We conclude that memory T cell populations in vivo undergo specific differentiation depending on their tissue localization, leading to unique phenotypic and presumably functional features.     

Hybridomas synthesizing monoclonal antibodies to surface antigens of human lymphocytes

Three types of hybridomas were obtained by fusion of murine myeloma cells (NSI-1-Ag4-1) with splenocytes from mice immunized with human lymphoblastoid cells (RPMI-6410t line, acute myeloblastic leukemia). Hybridomas of the first type synthesize monoclonal antibodies Ma-1, which interact with 6410t-cells, but are not bound to the cells of human Burkitt lymphoma-Raji. Raji cells contain HLA-DRw5 and -DRw6 antigens on cell surface but there are no HLA-A2, -B7 and -B12 antigens (specific for 6410t). Thus, Ma-1 are probably derected against some of HLA antigens of loci A or B. Hybridomas of the second type synthesize Ma-2 antibodies which react with 6410t and Raji cells, but are not bound to peripheral blood lymphocytes (PBL). We suppose that Ma-2 antibodies to tumor specific antigens which have common antigen determinants both for Raji and RPMI-6410t cells. The third type of hybridomas synthesizes monoclonal antibodies Ma-3 reacting with all the three types of target cells: 6410t, Raji, and PBL. Ma-3 seems to be directed against human species-specific lymphocyte antigens which remained in 6410t and Raji cells.     

Monoclonal antibodies against rat Leydig cell surface antigens

Monoclonal antibodies (MAbs) directed against the Leydig cell surface may be used to identify this cell in testicular preparations. Collagenase-dispersed adult rat interstitial cells were fractionated on Percoll density gradients, and Leydig cell-enriched fractions were used to prepare MAbs. Hybridomas were screened by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IIF) on isolated testicular cells and immunocytochemical localization on paraffin sections of adult testes. In density gradient fractions, immunoglobulin (Ig) M MAbs (LC-1C6 and LC-6H6) labeled the surface of cells possessing the morphological characteristics of Leydig cells. The density gradient profiles of MAb-binding activity observed by IIF and ELISA were parallel with the Leydig cell distribution as determined by [125I]-human chorionic gonadotropin (hCG) binding, testosterone response to hCG in vitro, 3 beta-hydroxysteroid dehydrogenase histochemistry and electron microscopy. The MAbs prominently labeled most interstitial cells in sections, but there was little or no labeling of connective tissue, endothelial or seminiferous tubule cells. Both MAbs recognized components of Mr 58,000 in Western blots of Leydig cell-enriched extracts. The results indicate that LC-1C6 and LC-6H6 recognize antigens on the Leydig cell surface that are not present on other isolated testicular cells from the adult rat. These MAbs are specific markers of the Leydig cell in situ and in vitro.     

Rabbit antiserum against a purified surface glycoprotein decompacts mouse preimplantation embryos and reacts with specific adult tissues

A rabbit antiserum against a purified embryonal carcinoma (EC) cell surface glycoprotein interferes with cell-cell interaction in mouse preimplantation embryos. The 123 kD glycoprotein seems not to be an integral membrane component. The reactivity pattern of the antiserum was studied by immunofluorescence on cryostat sections of post-implantation embryos and of adult tissues. During embryonic development positive reactions were found on all epithelial cells, irrespective of their germ layer origin. Epithelial cells of adult tissues—tongue, uterus, gut, kidney, trachea and liver—react with the antibodies. The results are compared with cell-adhesive molecules previously described on EC cells and preimplantation embryos.     

Studies of the cell surface of Paramecium. Ciliary membrane proteins and immobilization antigens.

We have developed a procedure to isolate the ciliary membranes of Paramecium and have analysed the membrane proteins by electrophoresis on polyacrylamide gels containing either Triton X-100 or sodium dodecyl sulphate. The electrophoretic pattern on gels containing sodium dodecyl sulphate showed 12-15 minor bands of mol.wt. 25 000-150 000 and on major band of mol.wt. 200 000-300 000 that contained approximately three-quarters of the total membrane protein. 2. We present evidence that the major membrane protein is related to, but not identical with, the immobilization antigen (i-antigen), which is a large (250 000 mol.w.), soluble, surface protein of Paramecium. The similarity of the i-antigen and the major membrane protein was shown by immunodiffusion and by the electrophoretic mobilities in sodium dodecyl sulphate of these two proteins from Paramecium of serotypes A and B. The non-identity of these two proteins was shown by their different electrophoretic mobilities on Triton X-100 containing gels and their different solubilities. 3. We propose that the major membrane protein and the i-antigen have a precursor-product relationship.     

Rabbit B spleen lymphocytes and T helper cells. I. Responsiveness to mitogens of B cell subpopulations of different sedimentation velocities and subpopulations bearing or lacking Fcgamma receptors.

The response to anti-allotype (anti-Ab4), Nocardia Water Soluble Mitogen (NWSM), pneumococcal polysaccharide type III (SSS III), and human Fc fragments of various purified and unfractionated rabbit spleen cell populations was determined in terms of 3H-thymidine up-take. B cells were isolated either from untreated suspensions of spleen cells or from suspensions from which adherent and phagocytic cells were removed. The purification factor was greater than the enhancement of 3H-thymidine uptake by anti-Ab4, NWSM, and SSS III as compared with the response of unfractionated spleen cells. It thus appears that a helper cell was involved: the mitogen response of purified B cells was enhanced by the addition of T cells. B subpopulations were separated by sedimentation or by rosetting, which allowed us to separate Fcgamma receptor-bearing cells from cells that did not possess this receptor. There were differences between cells responding to B mitogens not only in sedimentation velocity but also in the absolute number of cells. B cells bearing the Fcgamma receptor were less responsive to anti-Ab4 and more responsive to SSS III, NWSM, and human Fc than were B cells lacking the Fcgamma receptor.     

Changes in arachidonic acid content of X-irradiated human lymphocytes and plasma membrane fractions

The 14C-arachidonic acid content of pre-labelled human lymphocytes was followed during in vitro incubation in untreated and X-irradiated cells. Loss of about 35-40 per cent of the incorporated radioactivity was found already at 10 minutes after irradiation as detected in whole cells, phospholipid, free fatty acid and phosphatidylcholine fractions of plasma membranes. When pulse labeling with 14C-arachidonic acid was performed at various intervals after in vitro irradiation, the uptake of precursor by irradiated cells did not differ from untreated ones. At the same time the activity of phospholipid, free fatty acid and phosphatidylcholine fractions was approximately 50 per cent lower compared to the control, then it increased gradually reaching the control level.