B. Axelsson

Induction of CD43 Expression during Activation and Terminal Differentiation of Human B Cells

Only a small population (25–30%) of human peripheral blood B lymphocytes expresses large sialoglycoprotein (LSGP) (CD43). However, in the presence of autologous T cells and pokeweed mitogen (PWM) a majority (50–90%) of the immunoglobulin‐producing cells (cÍg+ cells) that develop from these B cells express CD43 is detected with anti‐CD43 monoclonal antibodies (MoAb) B1B6, and the proportion of CD43+cIg+ cells increases with time of culture. Furthermore, a relatively larger proportion (60–80%) of the IgG‐producing cIg+ cells are CD43+ compared with IgM‐containing cIg+ cells (30–50%). In human tonsils, signficantly more CD43+ cells (35%) are found in the in vivo‐activated fraction of B cells than in the fraction of resting B cells (5%). A majority of the cIg+ cells that develop from the resting or the in vivo‐activated tonsillar B cells in a PWM‐induced B‐cell differentiation system are CD43+ (80–100%). Furthermore, tonsillar B cells depleted of CD43+ cells give rise to cIg+ cells, of which the majority are CD43+, and the proportion of such cells increases with time of culture (60–90%). Taken together, these results indicate that LSGP belongs to a group of B‐cell membrane molecules that are induced and upregulated upon activation and differentiation.

Enhancement of human spontaneous cell-mediated cytotoxicity by a monoclonal antibody against the large sialoglycoprotein (CD 43) on peripheral blood lymphocytes.

A murine monoclonal antibody, MoAb B1B6 (IgG1χ), which recognizes the large sialoglycoprotein (LSGP)on human peripheral blood lymphocytes (PBL) effectively enhanced the spontaneous cytotoxicity of these cells against She natural killer (NK)‐sensitive target cells K562 and Molt‐4. Whereas preincubation of she lymphocytes with MoAb B1B6 resulted in increased cytotoxicity, preincubation of the target cells had no effect, indicating that the MoAb amplified cytotoxicity at the effector cell level. Kinetic analysis of the data revealed no differences between the control and the MoAb‐treated lymphocytes with regard to Vmax, usually considered to reflect the overall lytic potential of the cells. The slopes of the saturation curves, however, differed significantly for She two cell populations, indicating a substantial increment in the activity of the MoAb‐treated cells. When studied lit the single cell level and with K562 as targets, treatment of PBL with the MoAb resulted in the recruitment of new effector lymphocytes from the pool of non‐binding cells. In contrast, when Molt‐4 cells were employed as targets, no additional effector cells were recruited. These results indicate that the enhanced cytotoxicity induced by MoAb B1B6 is the result of either recruitment of new effector lymphocytes or of an increased recycling capacity of preexisting effector cells. Together with previous observations, these findings support the conclusion that LSGP belongs to the set of surface molecules which regulate human lymphocyte activation.

Expression of CD40 and CD43 during Activation of Human B Lymphocytes

CD40 and CD43 arc two cell‐surface glycoproteins that appear to be functionally involved in the growth stimulation of human B cells. Whereas CD40 is structurally similar to the NGF receptor and is present on all resting B cells. CD43 displays no homology to other known proteins and is expressed only on a subpopulation of these cells. To further understand the extra‐ and intracellular signals regulating these molecules and in which stage of activation they may play a role, we used various activation strategies and studied their expression on tonsillar B cells. As expected, activation of protein kinase C by TPA increased both CD40 and CD43. In contrast, a rise in intracellular Ca2+, e.g. by ionomycin, did not influence the expression of these antigens. However, in the presence of TPA, ionomycin further up‐regulated CD43 but not CD40. Anti‐IgM behaved similarly to ionomycin suggesting that the effect of this reagent was due primarily to its ability to increase intracellular Ca2+. Of three interleukins(lL‐2. IL‐4 and IL‐6) only IL‐4 had a significant effect when used alone in that it up‐regulated CD40 but not CD43. However, in Ihe presence of anti‐IgM, both IL‐2 and IL‐4 synergistically up‐regulated the two antigens. Complementation of antigen receptor stimulation with TPA or IL‐4 increased CD4U during the first 24 h. whereas up‐regulation of CD43 did not occur until 24 to 4S h after stimulation. With regard both to up‐regulation in response to different stimuli and to kinetics, CD40 expression paralleled that of the early activation antigen CD23, whereas CD43 was induced in parallel with the transferrin receptor (CD71). Taken together, our results suggests that the expression of CD40 and CD43 is regulated by different intracellular signals and that CD40 may be important during early activation, whereas CD43 may have its major function during later stages of B‐cell differentiation. These assumptions are in line with the observations that CD4U antibodies can directly activate resting B cells and that CD43 are retained on plasma cells.

Transferrin Receptors on Mitogen-Stimulated Human Thymus-Derived Lymphocytes

The appearance of transferrin receptors on mitogen‐stimulated human thymus‐derived (T) lymphocytes was studied. When indirect immunofluorescence with immunoadsorbent‐purified antitransferrin antibodies was used, ∼ 10% of resting T cells were stained. This proportion increased to 50–80% of the cells 3–4 days after stimulation with the mitogenic lectins concanavalin A (Con A) and leucoagglutinin (La) from Phaseolus vulgaris. Almost all blast cells (≥90%) were positive. Cell binding experiments with 125I‐labelled transferrin indicated the piesence of 1–5×105 transferrin receptor molecules/cell with high avidity for transferrin (K=2–12 × 1081/mol). Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography of cell lysates containing 125I‐labelled T‐cell surface components revealed two surface peptides (90 kdaltons and 80 kdaltons, reducing conditions), which selectively bound to insolubilized antitransferrin antibodies. The 90‐kdalton peptide also bound to insolubilized transferrin. The 80‐kdalton peptide is most probably transferrin and the 90‐kdalton peptide the transferrin receptor. Unreduced transferrin receptor had a molecular weight of 180 kdakon. It is probably a glycoprotein, since it reacted with wheat germ agglutinin, La, and probably also Con A. The properties of the lymphocyte transferrin receptor are similar to those described for transferrin receptors on various in‐vitro‐grown transformed cells. This speaks in favour of a common receptor present on all proliferating human cells.

Persistent Superphosphorylation of Leukosialin (CD43) in Activated T Cells and in Tumour Cell Lines

CD43 (leukosialin) is a highly sialylated, single‐chain molecule expressed on most human leucocytes. Regulatory signals appear to be transduced through the molecule as suggested by the ability of anti‐CD43 antibodies to induce aggregation and proliferation of T cells and to enhance B‐cell proliferation and natural killer cell activity. Activation of protein kinases is an essential event in signal transduction. We were therefore interested to study whether CD43 may function as a substrate for protein kinases during mitogenic activation of lymphocytes. We show that CD43 was rapidly superphosphorylated (within minutes) on serine residues following addition of phorbol ester (PMA) to peripheral blood lymphocytes. PMA treatment of the cells was not followed by rapid down‐regulation of CD43. Activation of the lymphocytes by concanavalin A or anti‐CD3 antibodies (OKT 3) also resulted in superphosphorylation of CD43. However, the phosphorylation was delayed as compared to that induced by PMA and was detected 3–4 h after the addition of the reagents. A plateau was reached after 24–48 h of Stimulation. Interestingly, the high level of phosphonrylation of CD43 was maintained in long‐term cultures of T cells activated by various means. Furthermore. CD43 was found to be constitutively superphosphorylated (on serine and tyrosine) in continuously growing cell lines of T, B, and non‐lymphoid origin. Taken together, the results suggest that CD43 has an important role during both early and late phases of T‐cell activation and that modulation of its biochemical properties by protein kinases may be associated with progression through the cell cycle and with cellular growth.

Enhancement of Human B‐Cell Proliferation by a Monoclonal Antibody to CD43

Monoclonal antibodies (MoAb) to human leucocyte sialoglycoprotein, CD43, have been shown to deliver mitogenic signals to human T cells or to enhance T‐cell proliferation induced by concanavalin A, anti‐CD3 antibodies or phorbol ester. In this paper, we studied the effects of anti‐CD43 MoAb B1B6 on the activation of human B cells Anti‐CD43 MoAb B1B6 was not mitogenic by itself for human B cells. However, when added together with TPA, both resting and in vivo activated tonsillar B cells, containing 5–10% and about 35% CD43+ respectively, responded with three‐ to fivefold higher proliferation compared to that obtained with TPA alone. A peak in the proliferative response was reached on day 3. Optimal proliferation was obtained when the antibody was present from the start of culturing. Addition of MoAb B1B6 together with a calcium ionophore, ionomycin, did not induce B‐cell proliferation. Neither did mAb B1B6 sustain the growth of B cells that were already in the cell cycle, i.e. precultured with phorbol ester (PDB) and ionomycin for 3 days. The results are similar to those obtained with antibodies to CD22 and CD23 and show that early progression signals are delivered to resting B cells through CD43 in the presence of primary activators of protein kinase C.

Studies on the Role of CD43 in Human B‐Cell Activation and Differentiation

The monoclonal antibody (MoAb) B1B6 to human leucocyte sialoglycoprotein, CD43, induces aggregation of T cells and drivers progression signals early during activation of both T and B cells in the presence of primary activators of proton kinase C. In this report we further studied the role of CD43 in human B‐cell activation and differentiation. About 5–10% of resting tonsillar B cells are CD43+. In the presence of TPA or antibodies to CDw40, the proportions of CD43+ cells drastically increased. The expression was optimal on day 3 of culture, when up to 80% and 50%, respectively, were CD43+. Whereas MoAb B1B6 together with TPA induced a three‐ to fivefold higher proliferative response as compared to TPA alone, antibodies to CDw40 did not synergize with MoAb B1B6 in B‐cell proliferation Tonsillar populations depleted of CD43+ B cells responded with lower proliferation to TPA alone or to TPA and B1B6 or anti‐CDw40antibodies. MoAb BlB6 did not affect the production of IgM or IgG as induced by pokeweed mitogen in the presence of autologous T cells, from either peripheral blood or tonsillar B cells. Neither did it affect the IgG production from the CD43+ BSF‐2 sensitive Epstein Barr virus‐transformed lymphoblastoid cell line CESS. The results show that CD43 is upregulated on B cells during activation. Furthermore, CD43+ B cells are included in the population which responds to signals delivered by TPA, anti‐CD43 or anti‐CDw40 antibodies, and the proliferation of this population is not merely due to an expansion of the small population of CD43+ cells present among these cells. Moreover, the epitopes recognized by MoAb B1B6 are not involved in the differentiation of and ultimate Ig‐secretion from activated B cells.

Calculation of affinity constants directly from homologous displacement curves

Homologous displacement experiments are described in which the binding of labelled ligand (tracer) was inhibited with the same ligand unlabelled (inhibitor). Data were evaluated with a direct curve-fitting method. The equation used employs the concentration of unlabelled ligand as the independent variable and the fraction of tracer bound (p) as the dependent variable. The other entities of the equation are the dissociation constant (Kd), the maximal fraction of tracer bound (Po) and the tracer concentration (T). Different options exist regarding the insertion of Po and T as parameters or constants in the curve-fitting. When applied to experimental data obtained from the binding of dinitrophenyl-hapten by a monoclonal antibody, the least interexperimental variation in affinity was obtained when both Kd and Po were estimated as parameters, and T was inserted as a constant. This calculation procedure achieves more reproducible results than those obtained with the Scatchard plot and the Langmuir curve, even after exclusion of the most scattered data points.

Functional Characterization of Human B Cells Carrying the Lymphocyte Large Sialoglycoprotein gpl50

Human B cell‐enriched populations were prepared from buffy coats of healthy donors. By means of affinity chromatography, the B cells were separated into two fractions, one enriched in and the other depleted of cells expressing gpl50, the large sialoglycoprotein of lymphocytes. In the presence of autologous T cells, monocytes and pokeweed mitogen B cell populations enriched for gpl50+ cells gave rise to significantty more plasma cells (clg+ cells) and secreted significantly more IgG than gpl50‐depleted populations. In contrast, more or an equal amount of IgM was secreted in cultures containing gpl50‐depleted cells. The differences between the fractions could not be ascribed to uneven distribution of T3+ cells, OKM1+ cells or B1+ (CD20) cells. However, the gpl50‐enriched population contained significantly more B2+ (CD21) cells than the gpl50‐depleted population. These results suggest that the gpl50+ B cells differ from gpl50‐B cells, not only in their responsiveness to T cell differentiation signals but also in their commitment to Ig heavy chain isotype secretion.

Agonistic properties of anti-B cell antibodies purified on staphylococcal protein A may be due to contaminating protein A

Some antibodies directed to cell surface receptors may mimic the physiological ligands by inducing the transmission of activation or growth signals. Such agonistic antibodies have proven very useful when studying functional properties of various receptor molecules on, e.g., lymphoid cells. However, while investigating the agonistic effects on tonsillar B cells of the anti-CD43 monoclonal antibody (mAb) D4B11 and the anti-CD40 mAb S2C6, we made some observations which emphasize the need for caution when using antibodies purified by protein A affinity chromatography. Both antibody preparations were found to elicit changes in the intracellular free calcium concentration ([Ca2+]i) as well as promoting proliferation of phorbol ester activated cells. However, a closer analysis showed that the increase in [Ca2+]i could be attributed to soluble staphylococcal protein A (SpA) desorbed during antibody purification. By using pure soluble SpA, we were able to show that nanogram amounts were sufficient to increase [Ca2+]i by a mechanism that involved both a mobilization from intracellular stores and an influx across the B cell membrane. A similar effect on cytosolic Ca2+ in B cells was also noted for streptococcal protein G (protein G), another bacterial component used for antibody purification. However, in contrast to SpA, protein G had little effect on cell proliferation. These observations suggest that the presence of trace amounts of SpA or protein G in antibodies purified on these bacterial components may lead to incorrect interpretations of the agonistic properties of such antibodies. When the above findings were taken into account, it was found that the CD43 mAb D4B11, like the CD40 mAb S2C6, stimulated growth of B cells without causing any measurable increase in [Ca2+]i. Both CD40 and CD43 may thus be coupled to signalling pathways which do not involve breakdown of membrane phosphoinositides.