Inhibition Of E-rosette Formation Research Articles

Characterization of a new porcine differentiation antigen involved in the proliferative response to mitogens

The cytotoxic monoclonal antibody (Mab) LIL 13 reacts with a widely distributed antigen that is expressed on 80–95% of swine peripheral blood mononuclear cells (PBMC) and a variety of lymphoid cell types. Using indirect immunofluorescence (IIF) the positive cells (75–100%) were divided between the bright, intermediate and dull populations. The remaining negative cell population contained B-cells, T-cells and probably null cells. Mab LIL 13 did not react with swine major histocompatibility complex (MHC) class I antigens (SLA) and did not inhibit E-rosette formation. Reactivity of LIL 13 with leukocyte function antigen 1 (LFA-1) was excluded by competitive IIF and cytotoxicity tests with cross-reacting anti-human CD 18 or anti-swine LFA-1-specific antibodies. Mab LIL 13 and complement treatment severely reduced mitogen-induced proliferative response to phytohaemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) and lipopolysaccharide (LPS) (90–100%). In the absence of complement, LIL 13 partially reduced proliferation of cells by interfering with the capability of mitogens to bind to the corresponding surface receptor (LIL 13 followed by mitogens), and partially inhibited mitogenic proliferative response following post-treatment (mitogens followed by LIL 13). Biochemical analysis of the antigen using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed bands of approximately 180–190 kDa and 46–50 kDa under reducing conditions and 200 kDa and 46–50 kDa under non-reducing conditions.

DEVELOPMENT AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES TO LYMPHOCYTE CELL SURFACE ANTIGENS OF THE RHESUS MONKEY

Three monoclonal antibodies directed against rhesus lymphocyte cell surface antigens are described. A pan-T mAb, T64, and a T suppressor mAb, T35, showed phenotypic and functional specificity for both human and rhesus cells. In contrast, a third mAb, N42, identifying natural killer cells in rhesus peripheral blood leukocytes, was not crossreactive with the corresponding homologous human cells. N42 reacted with the same cells identified by Leu 11a and Leu 11b in rhesus PBL and in a functional assay decreased NK activity by 80%. N42 precipitated a 50KD protein from rhesus PBL lysates and was reactive with the Fc receptor domain of the NK cell. The T-S functional activity of cells reactive with mAb T35 was demonstrated in a pokeweed-mitogen-driven system for Ig synthesis: removal of the T35 positive cells by complement-mediated lysis led to an enhanced production of Ig by rhesus PBL, and the addition of T35 positive cells to a culture of T helper and B cells resulted in a reduction of this response. T35 was determined to be an IgG2a immunoglobulin and precipitated a 34KD protein from rhesus cell lysates. An IgM immunoglobulin, mAb T64 delineated all T lymphocytes, inhibited E-rosette formation, interfered with the proliferation of cells stimulated with mitogens, and precipitated a 52KD membrane protein. The potential utilization of these mAbs in vivo for organ or tissue transplantation in the rhesus monkey is discussed.

Effects of isoferritins on human granulocytes.

Serum ferritin levels are often elevated in patients with certain cancers and these elevations are, in part, derived from the tumors. In such patients, the increased levels of serum ferritin are associated with a poor prognosis. This association may be explained in part by biological effects of ferritin on lymphocytes: inhibition of E-rosette formation, masking of cell surfaces and suppression of lymphocytes' response to mitogens in vitro. The authors hypothesized that ferritins from tumor tissues also exert adverse effects on human granulocytes that are involved in tumoricidal activity. Three granulocyte functions were tested: nitroblue tetrazolium test, phagocytosis, and production of hydrogen peroxide. The results supported the authors' hypothesis: NBT reduction and phagocytosis are decreased in granulocytes exposed to ferritins, more so with tumor ferritins, than normal ferritin, and H2O2 production is less in granulocytes previously exposed to ferritins from tumor and nontumor tissues than cells not exposed to ferritins. However, the inhibitory effects of ferritins on H2O2 production can be reversed if granulocytes are further stimulated by phorbol myristate acetate (a membrane stimulant). If the elevated serum ferritin in cancer patients impairs granulocyte functions, in vivo, then it may increase the risk of infection, decrease tumoricidal host responses, and, thereby, contribute to the poor prognosis of these individuals.

The inhibitory effects of N-ethylmaleimide, colchicine and cytochalasins on human T-cell functions

The thiol-alkylating agent, N-ethylmaleimide (NEM), was found to inhibit the response of human peripheral blood mononuclear cells to the T-cell mitogen, concanavalin A (Con A). NEM (10 μM) blocked Con A-induced agglutination, production of interleukin-2 (IL-2) and expression of the IL-2 receptors (Tac) without toxicity. In order to determine whether the effectsof NEM on lymphokine production were related to inhibition of agglutination, we compared the immunosuppressive effects of NEM with those of cytochalasin A, cytochalasin B and colchicine. NEM did not inhibit E-rosette (ER) formation, suggesting that it does not interfere with actin filaments. Low concentrations of NEM (4 μM) inhibited IL-2 production and Tac expression without inhibiting agglutination, while 6–10 μM NEM blocked agglutination and DNA synthesis as well. In contrast, 5–10 μM cytochalasin B (CB) inhibited ER formation, agglutination, Tac expression and DNA synthesis, but augmented IL-2 production by three- to ten-fold. Colchicine (0.1–10 μM) had no effect on ER formation or agglutination and augmented IL-2 production by as much as 18-fold. However, colchicine blocked Tac expression by >40% and DNA synthesis by >80%. Cytochalasin A (CA), which has the thiol-reactive properties of NEM, the actin filament-disrupting properties of CB, and the microtubule-distrupting properties of colchicine, exhibited the immunosuppressive effects of all three compounds. These studies suggest that the inhibitory effects of NEM on IL-2 production do not appear to be due to reactivity with the cytoskeleton, but are probably due to effects on signal transduction pathways leading to IL-2 production and expression of IL-2 receptors.

Iron exerts a specific inhibitory effect on CD2 expression of human PBL.

The effect of in vitro exposure to ferric citrate (Fe-citrate) on the expression of human lymphocyte surface markers was studied. The following markers were examined: E-rosette formation, CD2, CD3, CD4, CD8, CD1, CD22, CD10 and HLA-DR. Pretreatment of peripheral blood lymphocytes (PBL) with Fe-citrate at concentrations ranging from 10(-2) M to 10(-5) M resulted in the exclusive inhibition of E-rosette formation and CD2 expression. None of the other surface antigens examined appeared to be sensitive to the Fe-citrate treatment. Competition experiments further indicated that iron interacts specifically with CD2 on T lymphocytes.

Effects of Selenium In Vitro on Human T-Lymphocyte Functions and K-562 Tumor Cell Growth

In vitro E-rosette formation, lymphocyte mitogenesis, and natural killer (NK) cell activity of human blood lymphocytes were strongly inhibited by high concentrations (10(-4) M) of sodium selenite, sodium selenate, and selenium dioxide. Lower concentrations (10(-5) or 10(-7) M) also inhibited E-rosette formation and natural killer cell activity against K-562 tumor cells. Lymphocyte transformation induced by concanavalin A (con A) or pokeweed mitogen (PWM) was also inhibited by all selenium compounds tested, but only at the highest concentrations (10(-5) and 10(-4) M). There was depression of the total number of viable lymphocytes following incubation with selenium dioxide only at a high concentration (10(-4) M). Interferon production was enhanced at lower levels (10(-9) to 10(-6)M) of selenium dioxide while a higher concentration (10(-5) and 10(-4)M) appeared to inhibit its production. The mechanism of inhibition by selenium compounds (10(-4) M) is due, in part, to the decrease of viable lymphocytes. It is unclear how other and lower concentrations (10(-7) or 10(-9) M) of selenium compounds inhibit E-rosette formation, NK activity, or K-562 tumor cell growth.

Relationship of anti-β 2-microglobulin antibodies to T11 and T-cell activation in systemic lupus erythematosus

Monoclonal anti-β 2-microglobulin (β 2m) inhibited in a specific, dose-dependent fashion both in vitro tetanus toxoid-induced human-T-cell proliferation and sheep erythrocyte (E)-rosette formation, a function of the 50 kDa T11 molecule. In these respects, anti-β 2m exhibited effects similar to those of sera from patients with SLE. Although 10 of 16 SLE sera contained antibody to β 2m in monoclonal rosette inhibition assays, the presence of antibody of this specificity contributed only partially to the capacity of SLE serum to inhibit E-rosette formation or the T-cell response to tetanus toxoid. Removal of anti-β 2m from SLE serum by solid phase absorption with β 2m-Sepharose 4B reduced inhibition of the tetanus toxoid response and E-rosette formation in certain cases, but to a lesser extent than that observed following absorption with T-cell blasts, which completely eliminated inhibitory activity. Neither SLE antilymphocyte antibodies (including anti-β 2m) nor heterologous anti-β 2m were directed to the E receptor binding site (T11 1 epitope), as indicated by failure to inhibit OKT11 monoclonal antibody rosette formation or to reduce the relative intensity of OKT11 immunofluorescent staining. These data suggest an interesting functional relationship between β 2m, the E receptor, and T-cell activation. While anti-β 2m antibodies in SLE exert some inhibitory effect on antigen-induced T-cell proliferation, other distinct autoantibody systems to T-cell activation antigens appear to play the predominant role in this regard.

Effects of retinoids on human lymphocyte functions in vitro.

E-Rosette formation in vitro, lymphocyte mitogenesis and natural killer (NK) activity of human blood lymphocytes were strongly inhibited by high concentration (10(-4) M) of retinol or retinal. Other retinoids at 10(-4) M (retinoic acid and 13-cis-retinoic acid) and lower concentrations (10(-7) or 10(-9) M) of retinol, retinal and carotenes also inhibited E-rosette formation. Lymphocyte transformation responses induced by concanavalin A (Con A) or pokeweed mitogen (PWM) were also inhibited while NK activity was not affected. There was a remarkable depression of the total number of viable lymphocytes after incubation with retinol or retinal 10(-4) M. However, other retinoids, 10(-7) and 10(-9) M of retinol and retinal and carotenes did not show marked decrease of lymphocyte number or viability even after prolonged incubation (48 h). The mechanism of inhibition by retinol or retinal (10(-4) M) is due in part to the decrease of viable lymphocytes. It is unclear how other retinoids, carotenes and lower concentrations (10(-7) or 10(-9) M) of retinol or retinal inhibit E-rosette formation or lymphocyte transformation.

Induction of cytotoxic cell activity by peptide fragments of β2-microglobulin

A heptapeptide fragment of β2-microglobulin has been described by Abiko in hemodialysates of uremic patients and found to inhibit E-rosette formation by hyman T cells. In order to study the possible immunoregulatory effects of this heptapeptide, we investigated its effect on human cytotoxic cell activity. Low concentrations (10−8 to 10−7 M) of the heptapeptide enhanced cytotoxic activity of human lymphocytes against herpes simplex virus-infected cells by 50–60% whereas higher concentrations (10−4M) depressed cytoxicity. When lymphocytes were incubated with the heptapeptide during an in vitro sensitization assay, an even stronger enhancement was observed. In an attempt to define possible structure-activity correlations, synthetic peptide fragments were also tested for their effect on cytotoxic cell activity. Thus, the corresponding desHis-hexapeptide (2–7) had little enhancing effect on fresh lymphocyte cytotoxicity but significantly enhanced cytotoxic activity following presensitization. In contrast, further amino acid deletions resulting in 3–7, 4–7, 5–7 or 6–7 fragments had no significant activity. Alternatively, C-terminal deletions resulting in 1–6, 1–5 or 1–4 fragments also failed to exert any effect on lymphocyte-mediated cytotoxicity. We suggest that the heptapeptide and its 2–7 hexapeptide fragment may play a role in regulating host cytotoxic responses to viruses in health and disease.

Lymphocyte subpopulations in patients with systemic lupus erythematosus

A discrepancy between E-rosette-forming cells and total T cells identified by monoclonal antibodies (Leu-1 +) was observed in patients with systemic lupus erythematosus (SLE). E-Rosette-forming cell percentages of peripheral blood mononuclear cells (MNC) were significantly lower than those observed in normal individuals, in contrast to the percentage of Leu-1 + cells in SLE patients which were not different from those of normal values. The cells which did not form E rosettes, but stained positively with the monoclonal anti-Leu-1 antibody (E −, Leu-1 +) had certain functional capability evidenced by their ability to mount a proliferative response to T-cell mitogens, phytohemagglutinin and concanavalin A, but failed to provide T-helper-cell activity to support the differentiation of normal B lymphocytes into immunoglobulin-secreting cells following stimulation with pokeweed mitogen. Inhibition of E-rosette formation, but not of the staining for Leu-1, was demonstrated by incubating sera from SLE patients with normal MNC. These studies suggest that the T-cell markers, E + and Leu-1 +, do not necessarily characterize identical subpopulations of T cells. When referring to E − cells in studies with MNC from SLE patients, it should be realized that this population includes, in addition to B cells and macrophages, cells staining positively for Leu-1 antigen and reactive to T-cell mitogens.

Erythrocyte-rosette receptor expression by chronic lymphocytic leukemia B lymphocytes.

Erythrocyte (E) rosette-forming cells have been investigated in three patients with B chronic lymphocytic leukemia whose leukemic lymphocytes were easily identifiable. A small percentage of fresh neoplastic cells formed E rosettes in two patients. In every patient, most unstimulated, cultured leukemic lymphocytes became E+ and this was further enhanced by phytohemagglutinin and pokeweed mitogen stimulation or neuraminidase treatment. These E+ B cells lacked detectable T cell antigens (except for a weak expression of an antigen associated with the helper T cell subpopulation in one case). They were unreactive or weakly reactive by immunofluorescence with a monoclonal antibody to the E receptor. However, this antibody completely inhibited E-rosette formation. The enhanced expression of E-rosette receptors by in vitro cultured cells appeared to be dependent upon the presence of a small number of E-rosetting cells at the beginning of the culture. E-rosette receptor expression by leukemic lymphocytes was most likely in a fourth case (out of 9 patients studied). This finding may account for some of the discrepancies in the study of so-called T cells in B chronic lymphocytic leukemia.

Human T lymphocyte receptor for sheep erythrocytes: Characterization of a specific antiserum and its application in the detection and quantitation of the receptor in a soluble form

An anti-human T lymphocyte serum specific to the receptor for sheep erythrocytes (E) was produced by immunizing sheep with the complex autologous E-soluble E receptor (ER s). The soluble receptor (R s) was obtained by heating human lymphocytes at 45 °C for 1 hr. The anti-R s serum has been shown to inhibit E-rosette formation, to be cytotoxic to T cells, to identify T lymphocytes by indirect immunofluorescence, and to stimulate blastogenesis. The reaction of anti-R s with R s was directly demonstrated by two newly developed methods: agglutination of complexes formed by the treatment of formolized E with R s (EFR s complexes) and adhesion of a protein A producer strain of Staphylococcus aureus to EFR s treated with anti-R s. The anti-R s antibodies could be neutralized by R s present in supernatant of heated peripheral lymphocytes, inhibiting the above reactions and therefore providing methods to quantitate R s in biological preparations. The importance of these assays is that R s plays an immunoregulatory activity, and high levels of R s in serum are associated with depressed cell-mediated immunity.

Phenomenon of human T cells rosetting with sheep erythrocytes analyzed with monoclonal antibodies. "Modulation" of a partially hidden epitope determining the conditions of interaction between T cells and erythrocytes.

Anti-D66 is a monoclonal antibody able to inhibit E-rosette formation of T cells both at 4 degrees C and at 37 degree C but that does not inhibit T cell rosette formation with neuraminidase or 2-amino-ethylisothiouronium bromide (AET)-pretreated E. As demonstrated by capping experiments, it defines an epitope, D66, that is directly involved in E-rosette formation. D66 is distinct from the epitope defined by 9.6 because 9.6, a previously defined "pan-T" monoclonal antibody, inhibits E(AET) rosette formation and because no cross-blocking occurred between both antibodies fixation. However, 9.6 and D66 are carried by the same molecule, as demonstrated by sequential immunoprecipitation assays performed on two different T cell lines. On the thymocyte surface, also, 9.6 and D66 are most probably carried by the same molecule, as indicated by cocapping and colysostripping experiments. D66 is present at higher densities on thymocytes and activated T cells than on peripheral blood T cells. Investigation of numerous T cell populations, both normal and malignant, showed a straightforward correlation between elevated D66 density and ability to form 37 degrees C stable E-rosettes. Neuraminidase treatment of thymocytes and peripheral blood lymphocytes forming E-rosettes unmasked a large fraction of D66 not readily accessible on their surface. These hidden D66 epitopes appear to be responsible for a surprising observation: the ability of anti-D66 to inhibit E-rosette formation could be totally reversed by fixation on anti-D66 of an antibody to mouse immunoglobulin or an Fab fragment anti-mouse immunoglobulin. This would induce microdisplacement with emergence of hidden D66, as documented by fluorometric studies. Finally, malignant T cells with a differentiative status of mature T cells, but forming no (or low numbers of) E-rosettes, could be induced both to display D66 and to form E-rosettes by neuraminidase treatment.

Inhibition of allogeneic lymphocyte E-rosettes induced by sera from newly diagnosed type I diabetics.

Sera from 64 juvenile onset insulin-dependent diabetics (Type I diabetics) and 30 normal subjects were tested for their ability to inhibit sheep red blood cell rosette formation (E-rosette) by normal allogeneic lymphocytes. Inhibition of E-rosette formation by greater than 20% was found with 16 (66%) sera from newly diagnosed patients, 3 (16%) sera from patients with duration of disease between 2 and 12 months and with 4 (18%) sera from diabetics with duration of disease between 1 and 7 years. On the other hand, only 2 (6%) control sera showed inhibitory effect. No relationship between E-rosette inhibition and islet-cell antibodies presence, anti-lymphocyte antibodies occurrence, anti-HLA activity, blood glucose levels, respectively, was found. Investigation of the properties of this inhibitory factor suggests that it is different from other substances that reportedly inhibit E-rosette formation.

The Roles of β-Lymphotoxin in antibody Dependent Cell-mediated Cytotoxicity

We investigated roles of α- and β-lymphotoxins in antibody dependent cell-mediated cytotoxicity(ADCC) utilizing a homologous human system; skin fibroblasts with known HLA type, allo-antiserum and blood lymphocytes. Under assay conditions using optimal amounts of priming anti-HLA antibody, maximal killing of 60-80% was achieved within 6 hours. At this time, only β-LT was present in supernatants of lytic culture; α-LT was not detected. The amounts of β-LT, as well as the kinetics of appearance, paralleled the extent and kinetics of ADCC killing by lymphocytes. Allo-antibody directed to HLA determinants not on the target cells elicited neither lysis nor β-LT release. Antisera were prepared to β-LT-containing supernatants or to partially purified α-LT. Anti-β-LT serum inhibited essentially only β-LT. Anti-α-LT inhibited from 13-38% of the total activity of standard β-LT preparations. We interpret this as either indicating a 13% contamination of the anti-α-LT with anti-β-LT activity or a 13% contamination of the standard β-LT preparation with α-LT. Both immune sera inhibited EA-rosette formation. In addition, anti-α-LT serum inhibited E-rosette formation. Anti-β-LT serum was markedly effective in inhibiting ADCC. Inhibition by anti-α-LT correlated with its anti-β-LT titer rather than with its anti-α-LT titer. Adsorptions to spleen cells essentially did not reduce the ability to block ADCC and totally removed EA-rosette inhibiting activity. These data suggest that β-LT plays a important role in ADCC target destruction.

Inhibition of Human Lymphocyte E-Rosette Formation by Oxygenated Sterols

25-Hydroxycholesterol, 20 alpha-hydroxycholesterol, 7 alpha-hydroxycholesterol, and 5 alpha-hydroxy-6-ketocholestanol, when added to cultures of human lymphocytes in lipoprotein-depleted medium (LPDM) at a concentration of 2.5 x 10(-6) M, inhibit E-rosette formation with sheep red blood cells. 20 alpha-Hydroxycholesterol, 7 alpha-hydroxycholesterol, and 5 alpha-hydroxy-6-ketocholestanol are more potent inhibitors than 25-hydroxycholesterol. The inhibitory effect of 5 alpha-hydroxy-6-ketocholestanol on E-rosette formation appears after 15 min of exposure; with the other three compounds, an exposure time of 18 hr is necessary. The inhibitory effect of E-rosette formation can be abolished by addition of free cholesterol, low-density lipoprotein, or high-density lipoprotein to the LPDM or by incubation of the cells in normal AB serum, but not by the addition of mevalonic acid to the LPDM. These observations suggest that the capacity of oxygenated sterol compounds (OSC) to inhibit E-rosette formation is independent of their inhibitory effect on sterol synthesis. It is possible that OSC inhibit E-rosette formation as a consequence of their insertion into the lymphocyte membrane as cholesterol analogues.

Interference of levamisole with inhibition of E-rosette formation by Hodgkin's disease and systemic lupus erythematosus cytotoxic sera

A new system has been used to test the influence of levamisole on T- cell function. Evidence has been produced that prior exposure to the drug “protects” normal human peripheral blood lymphocytes from the inhibition that cytotoxic sera from patients with Hodgkin's disease and systemic lupus erythematosus exhibit on their E-rosette-forming capacity. Also, damage of this T-cell function already induced by Hodgkin's sera may be partially corrected.

Interference of Levamisole With Inhibition of E-Rosette Formation by Hodgkin's Disease and Systemic Lupus Erythematosus Cytotoxic Sera

AbstractA new system has been used to test the influence of levamisole on T-cell function. Evidence has been produced that prior exposure to the drug “protects” normal human peripheral blood lymphocytes from the inhibition that cytotoxic sera from patients with Hodgkin's disease and systemic lupus erythematosus exhibit on their E-rosette-forming capacity. Also, damage of this T-cell function already induced by Hodgkin's sera may be partially corrected.

Lithium: a Modulator of Cyclic AMP-Dependent Events in Lymphocytes?

Theophylline, salbutamol, isoproterenol, and dibutyryl cyclic AMP inhibited E-rosette formation by human T lymphocytes and immunoglobulin M secretion from human plaque-forming B cells and augmented T-suppressor cell activity in three patients with agammaglobulinemia. Lithium chloride increased mitogen-induced lymphocyte proliferation and inhibited suppressor cell activity. In the presence of lithium, the effects of all the drugs except dibutyryl cyclic AMP could be prevented. The data suggest a role for lithium in the modulation of cyclic AMP-dependent events in lymphocytes. Its potential role as an inhibitor of suppressor cell activity warrants further attention.

The effect of a synthetic thymosin alpha 1 fragment on the inhibition of E-rosette formation by the serum of a patient with nephrotic syndrome.

The decapeptide corresponding to the C-terminal amino acid sequence (residues 19-28) of bovine thymosin α1 was synthesized using HONB-WSCI as a coupling reagent. After incubation of lymphocytes with various amounts of the decapeptide from 10 to 100 μg/ml in the presence of serum from a patient with nephrotic syndrome, recovery of E-rosette formation was observed.