Expression cloning of cDNA clones encoding the human cell surface proteins HuLy-m6 and FMC29.
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12E7 Antigen↗
Biomedical subjects
H Zola
Publications and source records attributed to H Zola.
The CD45RO (p180, UCHL1) marker: complexity of expression in peripheral blood.
Analysis of peripheral blood lymphocytes by two- and three-colour high-sensitivity staining with UCHL1 (CD45RO) and other markers shows that the expression of CD45RO on lymphocyte subsets is more complex than is generally supposed. In addition to the populations which express CD45RO and RA in a mutually exclusive manner, up to 30% of cells in adult blood express both markers, at low levels. This "intermediate" population includes CD4-positive cells, and a proportion of these cells express the p55 chain of the IL-2 receptor (CD25), suggesting that they are activated. In cord blood there are few RO-bright cells, but CD45RO is expressed at low intensity on a proportion of cells. Among the CD45RO-bright cells in adult blood at least two subsets can be detected by using MHC Class II and the homing receptor L-selectin as additional markers. This complexity suggests that memory cells are a subset of CD45RO-expressing cells, but that this marker is also found on cells that are activated but not irreversibly "switched" to memory cells.
Adult↗
Sensitivity of flow cytometry.
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Antibodies, Monoclonal↗
Studies of cytokine receptor expression by cells of the immune system--a waste of time?
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Cytokines↗
Detection of cytokine receptors by high-sensitivity immunofluorescence/flow cytometry.
Cytokines have profound effects on cells, and act through receptors which need only be at low concentrations (around 100 copies per cell) to transmit activation signals. The detection of such low concentrations is possible using monoclonal antibodies and fluorescence/flow cytometry, but only by using specialized techniques. The best results so far have been obtained using biotinylated second antibody followed by phycoerythrin-streptavidin, and batches of these reagents have to be carefully selected. Analysis of the fluorescence is best done using 546 nm excitation from a mercury arc lamp, but 512 nm excitation from an argon-ion laser can also be used. With appropriate alignment, instruments with 488 nm fixed-wavelength lasers can give sensitivity almost as good as the 546 nm system. Working at high sensitivity, background levels also increase, particularly for B lymphocytes. Background staining can be reduced to acceptable levels by blocking the two major mechanisms for non-specific binding. Applications of these methods to the detection of cytokine receptors on normal and malignant cells are reviewed.
Animals↗
T cell memory: a role for MHC class II molecules on T cells?
A proportion of human T cells express MHC class II molecules. In this paper, the hypothesis is advanced that these MHC class II molecules present antigenic peptides acquired from interaction with other antigen-presenting cells (e.g. during the primary response) and can therefore interact in a cognitive manner with T cells with the same specificity. Memory is maintained in the physical form as clusters of T cells expressing T cell receptors and the cognate peptide-MHC class II complex; by virtue of their cognate interaction with each other these cells turn over gradually and return to the cluster when they recirculate. When fresh antigen is introduced, it is presented to some of these T cells by non-T antigen-presenting cells that provide a second signal leading to vigorous activation of the T cell. The proximity and cognate interaction of other T cells of the same specificity allows a chain reaction to occur, leading to rapid development of the secondary response. The hypothesis, which is testable, is proposed because of doubts concerning the current concept of memory as applied to T cells, and a need to understand the consequences of the expression of MHC class II molecules by a subset of T cells.
Animals↗
Expression of interleukin-6 receptor on blood lymphocytes without in vitro activation.
A monoclonal antibody against the interleukin-6 receptor (IL-6R) has been used in a high-sensitivity immunofluorescence technique to study receptor expression on unstimulated blood lymphocytes. Most CD4 cells express IL-6 receptor, whilst a small and variable proportion of CD8 and B cells are positive. CD4+ cells express higher levels of receptor than CD8+ T cells, and CD45RO+ cells express higher levels than CD45RA cells.
B-Lymphocytes↗
FMC46, a cell protrusion-associated leukocyte adhesion molecule-1 epitope on human lymphocytes and thymocytes.
In this report, we describe a 76-kDa glycoprotein recognized by mAb FMC46 that, by virtue of its concentration on cell protrusions involved in motility, may be important in lymphoid cell locomotion. FMC46 detects an epitope of the leukocyte adhesion molecule-1 (LAM-1), a member of the selecting family (LAM-1, Endothelial Leukocyte Adhesion Molecular-1 (ELAM-1), and Granule Membrane Protein-140 (GMP-140), that is expressed on LAM-1-transfected cell lines, is a glycosylation epitope based on its loss after culture in tunicamycin, and is closely related to the LAM-1.2 epitope. FMC46 is expressed at high density on the majority of CD45RA+ and CD45RO+ peripheral blood T cells (60 to 70%) and on a subset of thymocytes that includes the multinegative CD3- CD4- CD8- progenitor cells (100% FMC46hi) and the CD45R0- presumptive thymic generative lineage (70% FMC46hi). It appears at reduced density and frequency on CD45RA- thymocytes (50% FMC46lo), comprised mainly of death-committed thymocytes. Among thymic subsets defined by expression of CD4 and/or CD8, FMC46 is expressed at high density predominantly on a subset of single-positive cells and not on double-positive cells. These results suggest a fundamental role for LAM-1 in thymic development, with a high density preferentially expressed on cells involved in thymic generative processes and a low density on cells progressing to intrathymic death. A major subset of peripheral blood B cells and thymic B cells also express FMC46. Immunohistochemistry on frozen sections indicated strong staining in splenic follicles and around blood vessels, staining of the thymic medulla and subcapsular areas, and staining of the mantle zone of germinal centers of the lymph node. FMC46+ lymphocytes accumulated along high endothelial venules in the lymph node. On locomoting multinegative thymocytes, FMC46 is concentrated on the leading tip of extended processes, on pseudopods, and on ruffles, unlike the distribution of either CD44 or TQ1 (LAM 1.2), suggesting a role in locomotion. On dividing multinegative thymocytes, FMC46 was found almost exclusively along the cleavage furrow, implicating it in detachment processes. We conclude that the properties of the LAM-1 molecule recognized by FMC46 are consistent with a role in detachment phases of motility and of cell interactions.
Antibodies, Monoclonal↗
Patients with HIV infection have a reduced proportion of lymphocytes expressing the IL2 receptor p55 chain (TAC, CD25).
Using a high-sensitivity immunofluorescence procedure, a proportion of blood lymphocytes can be shown to express the p55 chain of the IL-2 receptor (CD25, TAC) without in vitro stimulation. In this study, we measured the proportion of peripheral blood lymphocytes expressing CD25 in patients with a diagnosis of HIV infection and compared the results with cells from controls. The mean value for HIV-positive samples was 15%, while controls gave a mean value of 31%. The difference between the groups was highly significant (P less than 0.001, Mann-Whitney U test). When expression of CD25 was examined separately in CD4-positive and CD8-positive T cells and in B cells, there was a wider spread of values in the HIV group compared with controls, but no systematic difference. In the patient group studied (110 samples from 53 patients) there was a weak correlation between CD25 and CD4 expression (correlation coefficient r = 0.21, P less than 0.02), but there were patients with low CD25 and high CD4 as well as patients with high CD25 and low CD4, suggesting that CD25 can vary independently of changes in CD4 lymphocytes. Although the majority of very low values of CD25 (less than 10%) were found in patients with stage IV disease, such low values were also common in Stage II disease.
Adult↗
Preparation and characterization of a chimeric CD19 monoclonal antibody.
FMC63 is an IgG2a mouse monoclonal antibody belonging to the CD19 cluster. CD19 antibodies react with a 95kDa protein expressed by cells of the B lymphocyte lineage, from pre-B cells to mature B lymphocytes. CD19 antibodies have been suggested as candidates for immunological attack on leukaemic and lymphoma cells of the B lineage because the antigen is restricted to the B lineage. With the potential use of FMC63 in immunotherapy in mind, we have produced a mouse-human chimera in which the genes coding for the VDJ region of the heavy chain and the VJ region of the light chain derive from the FMC63 mouse hybridoma, while the C region genes code for human IgG1. The genes have been transfected back into a mouse myeloma line, which secretes low levels of immunoglobulin. (Ig). This Ig was purified and biotinylated in order to determine the specificity of the antibody. The chimeric antibody has a reaction profile concordant with the original FMC63 antibody, but has the properties of a human IgG1, including the ability to fix human complement. However, the antibody is not cytotoxic in vitro in the presence of complement or cells capable of mediating antibody-dependent cellular cytotoxicity. Possible reasons for this and ways of using the antibody are discussed.
Animals↗
A monoclonal antibody exhibiting reactivity with both X-hapten- and lactose-bearing glycolipids.
The tissue reactivity of a monoclonal antibody raised against human granulocytes, FMC 34, was shown to overlap with antibodies including FMC 10, which is directed against the X-hapten, and with T5A7, which is directed against lactose-bearing antigens. Reactivity of FMC 34 on sections of human cornea was more widespread than that of either FMC 10 or T5A7. High sensitivity fluorescence testing revealed that both FMC 34 and T5A7 reacted weakly with the T-lymphocyte cell line JM, unlike the CD15 antibody FMC 10. Competitive binding experiments showed that binding of FMC 34 to granulocytes was inhibited by preincubation with T5A7, while FMC 34 itself was able to inhibit the binding of both FMC 10 and T5A7. The binding of FMC 10 was not inhibited by preincubation of cells with T5A7. Saturation-binding analyses revealed that each human granulocyte could bind 17.5 x 10(5) molecyles of FMC 34, which was more than three times the number of molecules of FMC 10 which could be bound to each cell. The additional reactivity of FMC 34 appears to be due to its capacity to bind fucosylated and non-fucosylated lactose-bearing structures.
Antibodies, Monoclonal↗
Expression of IL-2 receptor p55 and p75 chains by human B lymphocytes: effects of activation and differentiation.
Whilst B cells in human blood can be shown to express interleukin-2 receptor (IL-2R) p55 and p75 chains, using a high-sensitivity immunofluorescence procedure, fresh tonsil B cells did not show detectable levels of expression. Culture of tonsil B cells led to low levels of expression of the p55 chain of the IL-2R, an effect which was dependent on protein synthesis. The level of expression of IL-2R chains could be modulated by culturing in the presence of a number of factors which activate B cells. p55 levels were more readily modulated than p75 levels. IL-4 and combinations of IL-4 with anti-IgM, IL-2 or tumour necrosis factor-beta (TNF-beta) modulated p55 levels, but IL-5 did not. Changes in IL-2R expression were small when compared with other B-cell activation markers such as CD23. When unfractionated tonsil cells were activated with a polyclonal stimulus, the major change was the expression of p55 by T-cell blasts--p75 expression remained low in T and B cells, and p55 expression by B cells remained low.
B-Lymphocytes↗
Detection by immunofluorescence of surface molecules present in low copy numbers. High sensitivity staining and calibration of flow cytometer.
Receptors for lymphokines and growth factors are present on cell surfaces often at concentrations of 100-500 copies per cell. Although conventional immunofluorescence cannot detect such low levels, cell membrane antigens present at these concentrations can be detected using an optimally set up flow cytometer together with a three-layer immunofluorescence technique, consisting of monoclonal antibody reacted with selected batches of biotinylated horse anti-mouse immunoglobulin and phycoerythrin-streptavidin. In this study we purified and radiolabelled a number of monoclonal antibodies, determined the specific radioactivity by self-displacement analysis, and used the radiolabelled antibody in experiments where the number of molecules of antibody bound per cell and the fluorescence intensity were measured on the same sample. This permitted us to determine the sensitivity of the fluorescence procedure in molecules per cell, using several different antibody/target cell combinations. The method was consistently capable of detecting fewer than 100 molecules of antibody bound per cell.
Antibodies, Monoclonal↗
The leukocyte-common antigen (CD45) complex and B-lymphocyte activation.
In view of the changes reported in expression of the different members of the leukocyte-common family of antigens by T cells as they are activated, B cells were examined with monoclonal antibodies against p220-180 (CD45), p220,205 (CD45R), and p180 (UCHL1). Resting tonsil B cells reacted with CD45 and CD45R antibodies, but not with UCHL1. Activation of B cells by anti-IgM and IL-4 and culture of activated B cells with factors which induce proliferation (B-cell growth factor) and differentiation (IL-6) led to only minor changes in the expression of CD45, CD45R, and p180 antigens. Activation with phorbol ester led to minor increases in reactivity with CD45 and CD45R antibodies and led to a weak reactivity with UCHL1. The results suggest that the functional relationship previously described in T cells between p180 and p220,205 is not seen in B cells. p180 is induced, but only at low levels and only with nonphysiologic activation. CD45R is not lost under the same conditions. However, in terminally differentiated B cells, and in some chronic lymphocytic leukemias, p180 expression was seen, often coexpressed with CD45R.
Antibodies, Anti-Idiotypic↗
Expression of the p70 chain of the IL-2 receptor on human lymphoid cells: analysis using a monoclonal antibody and high-sensitivity immunofluorescence.
Using monoclonal antibody (MoAb) against the p70 (beta) chain of the interleukin-2 receptor (IL-2R) and a high-sensitivity immunofluorescence procedure, p70 can be demonstrated on the majority of lymphocytes from normal blood samples, without in vitro stimulation. A subpopulation of cells bears a high concentration of receptor, and these cells have the physical properties of large granular leucocytes (LGL). The smaller lymphocytes, although weaker, are quite clearly positive. T8 cells stain relatively strongly, while T4 cells and B cells stain relatively weakly. Leukaemic cells showed a variety of phenotypes when examined for expression of the p55 and p70 chains of the IL-2R. The demonstration of IL-2R chains directly by immunofluorescence on unstimulated lymphocytes has potential clinical applications, since this technique can be used in a diagnostic setting.
Antibodies, Monoclonal↗
Modulation of expression of the antigen identified by FMC7 upon human B-lymphocyte activation: evidence for differences between activation in vivo and in vitro.
The antigen detected by the monoclonal antibody (mAb) FMC7 is expressed by a proportion of B lymphocytes, and previous information, based on leukaemias, cell lines and some normal cell studies, suggested that FMC7 reacts with relatively differentiated B cells. In this study, it is shown that cells in the high-density 'resting' fraction of tonsil B lymphocytes have a lower expression of FMC7 than cells in the low-density 'activated' fraction. However, activation of 'resting' B cells with anti-immunoglobulin (anti-IgM) together with IL-4 resulted in a decrease in FMC7 reactivity, whilst recognized markers of activation showed up-regulation, as expected. Activated cells which were cultured for an extra 3 days to allow return to a 'resting' stage showed an increase in FMC7 expression. Culture of activated B cells with low molecular weight B-cell growth factor (LMW-BCGF), tumour necrosis factor (TNF) or interleukin-6 (IL-6), did not lead to further changes in FMC7 reactivity, but stimulation with phorbol ester had an effect similar to that of anti-IgM + IL-4. These results suggest that FMC7 is a marker of differentiation rather than activation, and identifies a relatively mature subfraction of the pool of functionally mature B cells. Consistent with this interpretation, FMC7 and surface IgD tended to be expressed by reciprocal B-cell subpopulations.
Antibodies, Anti-Idiotypic↗
Definition of the thymic generative lineage by selective expression of high molecular weight isoforms of CD45 (T200).
Selective expression of CD45 isoforms distinguishes naive and memory T cells in peripheral blood. Paradoxically, although the most recent thymic emigrants are CD45R+ CD45 p180-, the majority of thymocytes are CD45 p180+. Speculating that the small subset of thymocytes selectively expressing only the high molecular weight isoforms of CD45 constitute the thymic generative lineage giving rise to peripheral T cells, we characterized the phenotypic and functional properties of CD45 p180- thymocytes. All cells bearing CD45 p180 were removed by rigorous depletion or all CD45R+ thymocytes were removed in a parallel depletion. CD45R- thymocytes were essentially the same in phenotype and CD4/CD8 subset distribution as unfractionated thymus, and dissimilar to naive peripheral blood lymphocyte (PBL) T cells. In contrast, CD45 p180- thymocytes, mainly CD45R+, were CD1- CD38- pgp 1+, corresponding closely to the phenotype of naive CD45R+ PBL T cells. This subset is enriched in CD4+ or CD8+ single positives, includes a high proportion of CD4-8- thymocytes which are predominantly CD3-, and appears to have a medullary location. Approximately 40%-50% of CD45 p180- thymocytes expressed a high density of CDw29 (4B4), which in the periphery is expressed at high density only on CD45 p180+ memory T cells and at low density on CD45R+ naive T cells. However, the expression of high density CDw29 in the absence of CD45 p180 indicates a close resemblance to fetal lymphocytes and suggests an essential role for CDw29 in both the least and the most mature of T cells. If CD45 p180- thymocytes constitute the generative lineage and CD45 p180+ cells are commited to intrathymic death, then the CD45 p180- subset should have enhanced proliferative potential. By combining depletion methods with a limiting dilution assay for clonogenic potential, we found that 100% of the clonogenic precursors present in unfractionated thymus were CD45R+ CD45 p180- cells. This indicates that the CD45 p180+ majority of thymocytes has a very limited capability for proliferation consistent with a commitment to intrathymic death. The clonogenic potential of CD45 p180- thymocytes indicates a greater functional resemblance to PBL T cells than to CD45 p180+ thymocytes. In so far as clonogenic potential in vitro reflects generative potential in vivo, expression of high molecular weight CD45 isoforms appears to define the generative thymic lineage. Our working hypothesis proposes that expression of CD45 p180 implements the mechanism for eliminating thymocytes with self-reactive receptor specificities.
Antigens, Differentiation↗
Circulating human T and B lymphocytes express the p55 interleukin-2 receptor molecule (TAC, CD25).
Using a highly sensitive immunofluorescence procedure 15-45% of blood lymphocytes from normal human donors can be shown to react with anti-TAC (CD25) monoclonal antibodies. The standard procedure for indirect immunofluorescence does not allow the detection of these CD25-positive cells. Ten healthy adults were tested, and all showed a population of positive cells. Three donors have been tested more than once, and have given consistent results. Three different CD25 antibodies were used, again with consistent results. Double-marker studies showed that the positive cells included B cells, as well as CD4-bearing and CD8-bearing T cells.
Antibodies, Monoclonal↗