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PubMed1982

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.

Animals↗
PubMed1982

Molecular heterogeneity of D-end products detected by anti-H-2.28 sera. II. B10.D2(M504) (H-2dm1) mutant fails to express one of the two H-2.4-, 28 + Dd region molecules.

By the technique of antibody-induced redistribution of cell surface antigens (capping), two serologically distinct molecules that do not react with anti-D-private sera were detected in the products of Dd and Dq regions. One of them is H2L, H-2Ld and H-2Lq. The other molecule is different from H-2L as well as from any known Qa molecule and it is either an H-2-like or Qa-like molecule. We designate it provisionally L2d and L2q, respectively. All these molecules carry the specificity H-2.28. Nevertheless, one antiserum against a H-2.28-like specificity, D29 (s X k anti-m) shows differences between these molecules. This antiserum reacted with H-2Ld and H-2Lq but not with L2d and Leq molecules. The same antiserum reacted, however, only with the H-2.4 positive (H-2D) molecule in mutant B10.D2 (M504) (H-2dml) cells. No antipublic serum was found that was able to distinguish different H-2.4 negative molecules in dml haplotype. The serological characterization of H-2.4 negative molecule in dml mutant indicated that this molecule is similar to the L2d molecule detected in the parental H-2d haplotype. Thus, dml mutant fails to express the H-2Ld molecule but does express H-2.4 negative, H-2.28 positive molecule, analogical to L2d. Together with our recent data demonstrating previously unknown molecules in the products of Dd and Kd regions, six serologically distinct molecules other than Qa-2 controlled by H-2d haplotype could be detected -- H-2K1d, H-2K2d, H-2Dd, H-2Md, H-2Ld, and L2d. BALB/c-H-2dm2 mutant fails to express H-2Ld and L2d molecules, while B10.D2(M504) (H-2dm1) mutant fails to express H-2Md and H-2Ld molecules.

Animals↗
PubMed1988

Comodulation of CD3 and CD4. Evidence for a specific association between CD4 and approximately 5% of the CD3:T cell receptor complexes on helper T lymphocytes.

The aggregation of a specific class of lymphocyte surface molecules results in patching, capping, and surface modulation of the aggregated ligand. Both CD4, an associative recognition structure found on helper T lymphocytes, and CD3, a component of the T cell receptor complex, are members of this functional subgroup. When 125I-labeled monoclonal antibodies reactive with either CD4 (19Thy 5D7) or CD3 (RW24B6) were bound to T lymphocytes, the subsequent addition of goat anti-mouse Ig resulted in their rapid, temperature-dependent internalization. Whereas the binding of 125I-19Thy 5D7 (anti-CD4) was inhibited by greater than 90% in the presence of unlabeled 19Thy 5D7, no inhibition occurred in the presence of unlabeled antibody reactive with CD3 (RW28C8). We took advantage of the fact that these antibodies were of different isotypes (19Thy 5D7:IgG2a; RW28C8:IgGl) to determine whether the internalization of CD3 induced the comodulation of CD4. T lymphocytes preincubated with 125I-19Thy5D7 (anti-CD4) and unlabeled RA28C8 (anti-CD3) were treated with goat anti-mouse IgGl under conditions shown to quantitatively internalize CD3. After 1 h at 37 degrees C, T lymphocytes had internalized 10.5 +/- 2.6% (n = 3) of their antibody-bound cell surface CD4. After similar incubations with media alone or with goat anti-mouse IgGl in the absence of prebound RW28C8 (anti-CD3), no internalization of CD4 could be detected. Control antibodies reactive with CD45R (2H4, IgGl) also failed to induce the internalization of CD4. Similar results were obtained by using a helper T cell clone (T4C1) that internalized 9.6 +/- 2.8% (n = 3) of its antibody-bound cell surface CD4 in response to CD3 modulation. In a reciprocal experiment, 125I-anti-CD3 (RW24B6, IgG2b) was preincubated with T4Cl cells together with unlabeled anti-CD4 (12T4D11, IgG1) prior to the addition of goat anti-mouse IgGl. The quantitative modulation of CD4 induced the co-internalization of 4.6 +/- 0.6% (n = 3) of cell surface CD3. These results suggest that approximately 5% of the CD3:T cell receptor complexes on helper T lymphocytes are specifically associated with CD4. Furthermore, our results suggest that an average of two CD4 molecules associate with each CD3:T cell receptor complex.

Antibodies, Monoclonal↗
PubMed1977

Evolution of the lymphoid system. II. Evidence for immunoglobulin determinants on all rainbow trout lymphocytes and demonstration of mixed leukocyte reaction.

Indirect fluorescent antibody analyses utilizing antiserum specific for rainbow trout serum immunoglobulin (Ig) demonstrated that essentially all rainbow trout lymphocytes from thymus, spleen, anterior kidney and peripheral blood possess either Ig determinants or structures which cross-react with those determinants found on serum Ig. Peripheral blood leukocytes from rainbow trout were found to be caple of participating in a mixed leukocyte reaction. This observation is discussed in terms of the evolution of T cell function.

Animals↗
PubMed1978

The relationship of membrane antigens on 141 (NZB) and EL4 (C57BL) lymphoma cells as demonstrated by antibody-induced resistance to complement-mediated cytotoxicity.

We have studied the ability of various antisera to induce resistance to complement-dependent lysis (CdL) (lysostripping) of surface antigens of B and T lymphoma cells. Alloantisera, xenoantisera, as well as anti-Rauscher-leukemia virus (RLV) antisera, were used. Whereas all antisera could induce resistance to CdL mediated by the same serum that served for lysostripping (autolysostrip), only some of the tested antisera could induce resistance to CdL by other antisera (co-lysostrip). In some combinations co-lysostrip was unidirectional, i.e., in a pair of antisera, one induced autolysostrip as well as co-lysostrip, whereas the other antiserum induced autolysostrip only. In other combinations co-lysostrip was reciprocal. These investigations do not support the possibility that viral components and alloantigenic determinants are associated on mouse lymphoma cells.

Animals↗
PubMed1980

Solubilization of complexes between tumor-associated and H-2 antigens from a tumor cell plasma membrane.

The tumor-associated cell surface antigens (TAA) gp 71 and AgI cocap H-2, but are not associated with H-2 following solubilization of membrane with detergent. We have attempted to solubilize intact TAA/H-2 complexes from surface-labeled L cells using decreasing concentrations of the nonionic detergent Nonidet-P 40 (NP 40). Associations between gp 71 and H-2 and between AgI and H-2 in solution were detected using sequential immunoprecipitation. gp 71/H-2 and AgI/H-2 complexes were solubilized at the lowest detergent concentration used, 0.01% NP 40. No coprecipitation of these antigen pairs was seen at any higher NP 40 concentration tested. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed lthat precipitation of antigens solulbilized in 0.01% NP 40 leads to coprecipitation of a small number of other protein peaks and does not appear to involve wholesale inclusion of labeled membrane proteins. These results, together with previous observations of cocapping between TAA and H-2 antigens, suggest that the molecules are associated with one another in the membrane. Detergent solubilization of membranes with all but the most gentle conditions leads to disruption of these complexes.

Animals↗
PubMed1982

The nylon wool adherence marker of the B cell lineage appears at the resting pre-B cell stage.

The proliferative state of functionally defined populations of size-separated pre-B cells was investigated. The results presented here support the hypothesis that pre-B cells are in the G2 phase when stable membrane IgM is first expressed. Small resting precursor cells significantly adhere to nylon wool columns, unlike larger cycling cells which can be enriched in the nonadherent fraction. These results suggest that the nylon wool "receptor' is a useful early B cell marker. Since the in vitro recruitment of both cycling and resting precursors was shown to be essentially dependent on agar mitogen(s), cumulative data suggest that the receptors for agar mitogen(s) appear shortly before surface IgM on proliferating pre-B cells. Nylon wool adherence potential develops next on their resting progeny, followed by full lipopolysaccharide reactivity as cells further mature.

Animals↗
PubMed1984

Ontogeny of surface markers on functionally distinct T cell subsets in the chicken.

Three subsets of chicken peripheral T cells (T1, T2 and T3) have been identified in peripheral blood of adult chickens on the basis of fluorescence intensity after staining with certain xenogeneic anti-thymus cell sera (from turkeys and rabbits). They differentiate between 3-10 weeks of age in parallel with development of responsiveness to the mitogens concanavalin A (Con A), phytohemagglutinin (PHA) and pokeweed mitogen (PWM). Functional tests on the T subsets, sorted with a fluorescence-activated cell sorter, have shown that T2, 3 cells respond to Con A, PHA and PWM and are capable of eliciting a graft-vs.-host reaction (GvHR). In contrast, although T1 cells respond to Con A, they respond poorly to PHA and not at all to PWM or in GvHR. There was some indication of cooperation between T1 and T2,3 cells for the PHA response. Parallels between these chicken subsets and helper and suppressor/cytotoxic subsets in mammalian systems are discussed.

Aging↗
PubMed1984

Surface carbohydrate epitopes of Thy-1.1 and Thy-1.2 thymocytes are distinguished by a monoclonal antibody with a specificity common to peanut agglutinin.

NIMF4-31.7 is a monoclonal IgM antibody with affinity for terminal galactose residues of membrane glycoproteins, Sepharose and agarose. It is cytotoxic for a subpopulation of murine thymocytes and peripheral lymphocytes, corresponding to the peanut agglutinin-positive population, but reacts with a majority of cells after neuraminidase treatment. It has revealed a genetic linkage between the Thy-1 locus and quantitative expression of carbohydrate determinants on the thymocyte membrane.

Animals↗
PubMed1987

A new epitope of the T200 molecule family defined by the 3A35 monoclonal antibody and expressed by macrophages and activated T lymphocytes.

A monoclonal antibody (mAb), 3A35, produced against mouse macrophages (M phi) was found to react against certain activated T cells. This mAb, a rat IgM, resulted from a cell fusion between a mouse plasmacytoma and rat lymphocytes immunized against mouse M phi. It bound more avidly to activated than to resident M phi. It did not react against B cells and resting T lymphocytes but recognized certain dividing T cells like EL4 lymphoma, concanavalin A-activated and interleukin 2-expanded spleen cells, and helper T cell hybridomas. By contrast, other T lymphocyte-derived cell lines such as YAC-1 and CTLL2 were unreactive. No clear relationship was found between the binding of 3A35 to cells and the expression of L3T4 and Lyt-2 antigens. The specific stimulation of T cell clones with antigen rapidly induced a strong reactivity with 3A35 mAb which declined thereafter to a low (helper clones) or non-reactivity (cytotoxic clones) after 10 days of culture. Immunoprecipitation experiments, performed with M phi derived from bone marrow cell cultures, surface iodinated with 125I or metabolically labeled with [35S]methionine, showed that 3A35 bound to a 200-kDa molecule, shifting to 175 kDa under reducing conditions. In peritoneal M phi activated in vivo, in addition to the 175-kDa band, new bands migrating at 140, 120 and 85 kDa were identified by 3A35 and could be absorbed on a commercial anti-T200 mAb bound to Sepharose beads. After strengthening the cell binding of 3A35 to EL4 lymphoma cells by a cross-linking agent, only a 85-kDa molecule was immunoprecipitated. Thus, 3A35 identifies a new epitope of the T200 molecule family which is expressed on M phi and activated T cells.

Animals↗
PubMed1987

Selective effects of cholera toxin on the activation of mouse B cells by different polyclonal activators.

Murine B cells were stimulated in vitro with anti-immunoglobulin (Ig) antibodies, lipopolysaccharide, or with various combinations of phorbol dibutyrate and ionomycin. Very low concentrations (ca. 10(-14) M) of cholera toxin inhibited anti-Ig-stimulated DNA synthesis, while the response to LPS was only abrogated by 2 X 10(4)-10(5)-fold greater concentrations of the toxin. Earlier responses in anti-Ig-stimulated B cells, such as increases in Ia antigen levels, were not affected by the toxin. Protein kinase C-activating phorbol esters, together with Ca2+ ionophores, are believed to stimulate DNA synthesis in lymphocytes by mimicking the two second messengers resulting from ligation of the antigen receptors. However, concentrations of cholera toxin which totally abolish anti-Ig-induced B cell proliferation significantly enhanced DNA and RNA synthesis induced by phorbol dibutyrate plus ionomycin. The results are discussed in terms of possible effects of cholera toxin on guanine nucleotide-binding (G) proteins controlling receptor coupling to second messenger-generating systems in B cells.

Animals↗
PubMed1993

A role for immunoglobulin D: interference with tolerance induction.

We have studied the induction and maintenance of tolerance in the B lymphocyte compartment. Neonatal and adult transgenic mice which expressed either surface IgM (sIgM) or sIgM and sIgD anti-2,4,6-trinitrophenyl (TNP) were treated with soluble mono- and multivalent forms of TNP-modified carriers. We compared the B cell compartment of mice treated with antigen and of littermates injected with phosphate-buffered saline. Antigen-mediated cross-linking of membrane-bound IgM (sIgM) caused deletion of B cells both in neonatal and adult mice with mu and kappa transgenes. Deletion was the result of apoptosis. In mice that carried an additional delta transgene sIgD interfered with tolerance induction. The stage in which the cells were sensitive to deletion was characterized as a transitional stage between immature (sIgMdull, heat-stable antigenbright, B220dull, sIgD-) and more mature (IgMbright, heat-stable antigendull, B220bright, sIgD-) B cells. Surviving cells were functional as measured by receptor-mediated changes in the intracellular free Ca2+ concentration. We propose that when the immature B cells have reached the final stages of maturation IgM always transmits negative signals in the absence of T cell help. When B cells need to be screened against self reactivity IgM is the only antigen receptor expressed. The presence of sIgD protects resting B cells from deletion and allows them to initiate an effective immune response.

Animals↗
PubMed1995

Internalization of B cell and pre-B cell receptors is regulated by tyrosine kinase and phosphatase activities.

Prior to the expression of the B cell antigen receptor, the mu heavy chain associates with two non-polymorphic polypeptides, lambda like and VpreB, which form a pseudo-light chain complex in pre-B cells and pre-B cell lines. Surface expression of the so-called pre-B cell receptor (pre-BCR) occurs only in the presence of Ig alpha and Ig beta, known to be involved both in B cell antigen receptor (BCR) signaling and trafficking. Although the pre-BCR organization is consistent with an efficient transport to the cell surface, most of the newly synthesized receptor remains within the cells, and so far, no data are available concerning the rate of exit from the endoplasmic reticulum. Using the human pre-B cell line Nalm-6, we found that only a small fraction (2%) of newly synthesized pre-BCR is transported to the cell surface within 4-6 h after synthesis, where it is constitutively re-internalized. Membrane Ig-heavy chain cross-linking induced internalization of surface pre-BCR within a few minutes, and the mechanisms underlying endocytosis were analyzed by immunofluorescence and confocal microscopy. Preincubation of the cells with either genistein or orthovanadate, which inhibit, respectively, tyrosine kinases and tyrosine phosphatases, blocked pre-BCR internalization in a dose-dependent manner, indicating that both activities are required for endocytosis. BCR internalization was also inhibited in a reversible manner by the drugs. In contrast, neither drug affected the size of the steady-state pool of internalized transferrin receptors. Thus, our data show that tyrosine phosphorylation and dephosphorylation are both required for cross-linking-induced pre-BCR and BCR internalization.

B-Lymphocytes↗
PubMed1996

CD66: role in the regulation of neutrophil effector function.

Neutrophils express several heavily glycosylated carcinoembryonic antigen (CEA)-related glycoproteins (CD66 antigens) which have been implicated in adhesion to E-selectin and as receptors for the lectins galectin 3 and bacterial type-1 fimbriae. The role of the CD66 antigens in neutrophil effector function was examined using non-cross-reacting and cross-reacting domain-mapped CD66 monoclonal antibody (mAb), which recognize epitopes on biliary glycoprotein (BGP; CD66a), CEA gene family member 6 (CGM6; CD66b), nonspecific cross-reacting antigen 90 (NCA90; CD66c) or CGM1 (CD66d). We show that BGP-specific mAb which recognize an AB-domain epitope strongly augment adhesion to fibrinogen by an Fc receptor- and beta2 integrin-dependent mechanism. Co-ligation of BGP with the glycophosphatidylinositol (GPI)-anchored CGM6 and NCA90 also caused increased beta2 integrin-mediated adhesion, receptor clustering and priming of formyl-Met-Leu-Phe (fMLP)-induced oxidant production by neutrophils, but only a small change in expression of L-selectin and CR3 compared to the chemotactic peptide fMLP. Ligation of CGM6 or NCA90 alone did not cause activation of the neutrophil in any of the assays used and did not cause priming of fMLP-induced oxidant production even when a secondary cross-linking reagent was used. We propose that specific cross-linking of neutrophil BGP with CGM6 and NCA90 contributes significantly to the regulation of neutrophil function during neutrophil recruitment.

Antibodies, Monoclonal↗
PubMed1989

Antigenic modulation and internalization of monoclonal antibody to human colonic carcinoma cells detected by enzyme-linked immunosorbent assay.

We studied antigenic modulation and internalization of monoclonal antibody (MAb) A7 using the enzyme-linked immunosorbent assay (ELISA) and biotin-labelled antibody-staining techniques. Incubation of the colonic SW1116 cell line with an excess of MAb A7 induced modulation of the cell-surface antigen. When the line was assayed by ELISA, a change in cellular reactivity with MAb A7 was seen after 1 hr. After 24 hr, the cellular reactivity showed a 52% decrease in absorbance. Modulation was inhibited by 0.1% sodium azide and acetone fixation, suggesting that this is an energy-dependent phenomenon. The internalization of biotinylated MAb A7 was examined. Internalized biotinylated MAb A7 was detected in cells fixed before labelling with avidin-biotin peroxidase complex. It was observed that the amount of MAb A7 which remained associated with the cell surface had decreased since A7 was internalized.

Antibodies, Monoclonal↗