Constitutive CD27/CD70 interaction induces expansion of effector-type T cells and results in IFNgamma-mediated B cell depletion.

The interaction between the TNF receptor family member CD27 and its ligand CD70 provides a costimulatory signal for T cell expansion. Normally, tightly regulated expression of CD70 ensures the transient availability of this costimulatory signal. Mice expressing constitutive CD70 on B cells had higher peripheral T cell numbers that showed increased differentiation toward effector-type T cells. B cell numbers in CD70 transgenic (TG) mice progressively decreased in primary and secondary lymphoid organs. This B cell depletion was caused by CD27-induced production of IFNgamma in T cells. We conclude that apart from its role in controlling the size of the activated T cell pool, CD27 ligation contributes to immunity by facilitating effector T cell differentiation.

Characterization of murine CD70, the ligand of the TNF receptor family member CD27.

Human CD70 (CD27 ligand) is a type II transmembrane glycoprotein belonging to the TNF family. The protein is not expressed on resting lymphocytes, but is rapidly induced on these cells after cellular activation. Importantly, interaction of CD70 with its receptor CD27 gives a costimulatory signal for lymphocyte activation. Whereas CD27 has been molecularly characterized in the mouse, murine CD70 (mCD70) was undefined until now. Here, we describe the cDNA cloning and initial characterization of mCD70 and the determination of its gene structure. mCD70 is a polypeptide of 195 amino acids that has 62% homology with its human counterpart. In analogy to human CD70, mCD70 transcript levels are strongly but transiently up-regulated during lymphocyte activation, which is in line with a role for the CD27-CD70 receptor pair early in the immune response. In accordance with the comitogenic activity of mCD27-specific mAb, recombinant mCD70 potently costimulates T cell proliferation. Finally, the mCD70 gene consists of three exons spanning approximately 4 kb of DNA and is localized on chromosome 17.

Alteration of B-cell antigen receptor signaling by CD19 co-ligation. A study with bispecific antibodies.

The activation of B-cell antigen receptor-associated protein tyrosine kinases is an early and crucial event in B-cell signaling. Apart from the B-cell antigen receptor (BCR), the B-cell-specific transmembrane glycoprotein CD19 has also been shown to directly activate intracellular signaling cascades. In addition, because CD19 and the BCR are associated on the surface of activated B-cells, it has been proposed that close approximation between these two entities is crucial for optimal B-cell triggering. To test this hypothesis, bispecific antibodies were generated that bind membrane IgM and CD19 simultaneously. Although CD19 bispecific antibodies strongly induced tyrosine phosphorylation, they were, in contrast to muF(ab)2 fragments, unable to induce a proliferative response. Detailed analysis of the early signaling events showed that compared with muF(ab)2 fragments CD19 bispecific antibodies potently raised the intracellular [Ca2+], which was correlated with an efficient tyrosine phosphorylation of syk. Strikingly, the assembly of Grb2 complexes that may couple the BCR to p21(ras) was clearly altered by the CD19 bispecific antibody. In addition to the reported Shc and 145-kDa phosphoproteins, a prominent 90-95-kDa phosphoprotein resembling CD19 was detected in the Grb2 complexes. Thus, studies with CD19 bispecific antibodies show that CD19 co-ligation both quantitatively and qualitatively alters BCR signaling.

The seven-span transmembrane receptor CD97 has a cellular ligand (CD55, DAF).

CD97 is an activation-induced antigen on leukocytes with a seven-span transmembrane (7-TM) region homologous to the secretin receptor superfamily. However, in contrast to this group of peptide hormone receptors, CD97 has an extended extracellular region with three EGF domains at the NH2 terminus, two of them with a calcium binding site. By demonstrating that lymphocytes and erythrocytes specifically adhere to CD97-transfected COS cells we here show that CD97 in parallel with its molecular evolution has acquired the ability to bind cellular ligands. A mAb selected on its capacity to block the adhesion between CD97 transfectants and red cells was found to be directed to the NH2-terminal short consensus repeat (SCR) of decay accelerating factor (DAF, CD55), a regulatory protein of the complement cascade. The specificity of the interaction of CD97 with CD55 was established by the observation that erythrocytes that lack CD55, obtained from patients with paroxysmal nocturnal hemoglobinuria (PNH) or the CD55, phenotype Inab, failed to adhere to CD97 transfectants. This is the first demonstration of a cellular ligand for a 7-TM receptor.

Hematopoietic cell phosphatase is recruited to CD22 following B cell antigen receptor ligation.

Hematopoietic cell phosphatase is a nonreceptor protein tyrosine phosphatase that is preferentially expressed in hematopoietic cell lineages. Motheaten mice, which are devoid of (functional) hematopoietic cell phosphatase, have severe disturbances in the regulation of B cell activation and differentiation. Because signals transduced via the B cell antigen receptor are known to guide these processes, we decided to analyze molecular interactions between the hematopoietic cell phosphatase and the B cell antigen receptor. Ligation of the B cell antigen receptor induces moderate tyrosine phosphorylation of hematopoietic cell phosphatase and the formation of a multi-molecular complex containing additional 68-70- and 135-kDa phosphoproteins. In resting B cells most of the hematopoietic cell phosphatase proteins reside in the cytosolic compartment, whereas after B cell antigen receptor cross-linking, a small fraction translocates toward the membrane where it specifically binds to the 135-kDa phosphoprotein. This 135-kDa glycoprotein was identified as CD22, a transmembrane associate of the B cell antigen receptor complex. Together these findings provide the first direct evidence that this cytoplasmic tyrosine phosphatase is involved in antigen receptor-mediated B cell activation, suggesting that in vivo B cell antigen receptor constituents or associated molecules may serve as substrate for its catalytic activity.

Antigen receptor nonresponsiveness in chronic lymphocytic leukemia B cells.

B chronic lymphocytic leukemia (B-CLL) are clonal populations of mIgM+ or mIgM+/mIgD+ CD5+ B cells that appear to be arrested in the follicular mantle-zone B-cell stage. Functional analyses have shown two groups of B-CLL that can be distinguished based on their capacity to proliferate in response to B-cell antigen receptor complex (BCR) cross-linking. To investigate the molecular basis for this phenomenon, we have analyzed both architecture and functional properties of BCR complexes on these two groups of B-CLL. Both groups were found to express structurally similar BCR. However, protein tyrosine kinase (PTK) activity associated with and specific for BCR constituents was strongly diminished in nonresponsive B-CLL. Moreover, the PTK-dependent assembly of Shc/Grb2 complexes, which may couple the BCR to p21ras, was absent in these B-CLL. Finally, of all PTKs tested, the expression of PTK syk was found to be considerably lower in nonresponsive B-CLL. Thus, absence of mitogenic responses upon BCR cross-linking in particular B-CLL was found to be strictly correlated with diminished induction of BCR-associated PTK activity and lower levels of PTK syk. Because nonresponsive B-CLL closely resembles tolerant autoreactive B cells both functionally and biochemically, distinction between B-CLL with respect to functional properties in vitro may be determined by differences in antigen encounter in vivo.

B cell antigen receptor cross-linking induces tyrosine phosphorylation and membrane translocation of a multimeric Shc complex that is augmented by CD19 co-ligation.

The SH2 domain-containing transforming Shc protein has been implicated in mitogenic signaling via several surface receptors through p21ras. Following tyrosine phosphorylation by either receptor or non-receptor tyrosine kinases, Shc may interact with the adaptor protein Grb2, which is linked to Sos1, a guanine nucleotide exchange factor for human ras. Ligation of the antigen receptor complex on B cells (BCR) is known to activate various intracellular signaling pathways, which may accumulate in mitogenic responses. With respect to the initial steps, the activation of BCR-associated non-receptor tyrosine kinases appears to be indispensible. In this report we show that Shc proteins become tyrosine phosphorylated after BCR ligation on both transformed and normal human B cells. This is accompanied by the association of Shc with Grb2 proteins and a yet unidentified 145-kDa tyrosine phosphorylated protein. Subcellular fractionation revealed that this activation-induced multimeric Shc complex rapidly translocates towards the plasma membrane. Co-ligation of the BCR with the CD19 molecule results in a marked increase of these events, whereas CD19 cross-linking alone does not induce Shc tyrosine phosphorylation or translocation. Thus, in B cells the Shc complex may represent a molecular junction between the BCR and the mitogenic p21ras cascade.

CD5 is associated with the human B cell antigen receptor complex.

On human B cells the antigen receptor complex is composed of the membrane form of the immunoglobulin molecule and the non-covalently associated Ig alpha/beta heterodimer. A small subpopulation of normal B cells and chronic lymphocytic leukemia B cells express (analogous to T cells) the transmembrane molecule CD5, a counterstructure of B cell-specific CD72. Numbers of CD5+ B cells are increased in several physiological and pathological conditions. Moreover, CD5+ B cells are being held responsible for the production of autoreactive antibodies and seem to have signaling characteristics distinct from conventional B cells. On T cells, CD5 associates with the T cell receptor CD3 complex and ligation of CD5 leads to the generation of co-stimulatory signals, that act on T cell activation. We here demonstrate that CD5 is associated with the B cell receptor (BCR) complex and serves as substrate for BCR-induced tyrosine kinase activity. Hence, CD5+ B cells have a unique potential to modulate BCR signals.

Evidence for a direct physical interaction of membrane IgM, IgD, and IgG with the B29 gene product.

The B cell Ag-receptor complex is composed of membrane immunoglobulin (mIg) and the mb-1/B29 heterodimer. In order to obtain insight into the architecture of the B cell receptor complex, we have looked for conditions that disrupt all disulfide bridges in the complex without affecting the noncovalent interaction between the mIg heavy chain and one or both members of the associated heterodimer. We show that in the presence of the reducing agent beta-mercaptoethanol the m mu, m delta, and m gamma heavy chains remain selectively associated with the B29 members. Our findings implied that if isotype-related differences exist between the mIg-associated dimers, they may reside in B29 and not, as initially suggested, in mb-1. However, sequence analyses of B29 gene transcripts from B cells expressing mIgM, mIgD, or mIgG only revealed no differences in their nucleotide composition. Thus, in spite of their close physical interaction with mIg heavy chain classes, which are significantly distinct in the C-terminal regions, no isotype-specific forms of B29 seem to exist.

Surface expression of immunoglobulin isotypes on primary human B cells: no evidence for glycosyl-phosphatidylinositol linkage.

Surface expression of membrane(m) IgM molecules requires the association of two disulphide-linked transmembrane (TM) glycoproteins, encoded by the B-cell-specific genes mb-1 and B29. We have shown that mIgM, mIgD and mIgG are associated with structurally related heterodimers on primary human B cells. Transfection studies in murine plasmacytoma cells, however, have demonstrated mb-1-independent expression of both mIgD and mIgG. The recent finding that mIgD is expressed on these cells through glycosyl-phosphatidylinositol (GPI) linkage may be interesting in view of the function of mIgD on primary B cells. We therefore investigated whether GPI linkage serves as an additional mechanism for expression of mIgD and the other mIg isotypes on primary human B cells. However, we were unable to demonstrate the release of mIg molecules upon treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) in either immunofluorescence analysis or Ig heavy (H) chain-specific enzyme-linked immunosorbent assay (ELISA). We conclude that primary human B cells, which constitutively express the mb-1 and B29 genes, do not express the mIg isotypes in a GPI-linked manner.

The CR2/CD19 complex on human B cells contains the src-family kinase Lyn.

The complement receptor 2 (CR2 or CD21) can be found in non-covalent association with the B lymphocyte specific CD19 complex at the surface of mature human B cells. Upon ligation of the B cell antigen receptor complex (BCR), members of the CR2-CD19 complex may associate with membrane immunoglobulin (mlg). Moreover, CD19 and CD21 ligands, either murine mAb, C3d fragments or Epstein-Barr virus, are known to have profound effects on B cell activation. We here show that CD19 is tightly linked to the non-receptor src kinase Lyn and that the CD19 glycoprotein itself serves as a substrate for a yet undefined serine/threonine kinase present within the complex. In the process of antigen recognition, mlg and the CR2-CD19 complex may bind different sites of a complement-opsonized antigenic particle. We hypothesize that in this process, approximation to the BCR allows CD19-associated Lyn kinase to phosphorylate potential substrates within the antigen-receptor complex, thereby effecting its coupling to the intracellular compartment.

Comparison of human B cell antigen receptor complexes: membrane-expressed forms of immunoglobulin (Ig)M, IgD, and IgG are associated with structurally related heterodimers.

We have recently reported that on human B lymphocytes, membrane IgM (mIgM) associates with a heterodimer of 47- and 37-kD polypeptides, the 47-kD subunit being encoded by the mb-1 gene. We show here that expression of mb-1, both at the mRNA and the protein level, is not restricted to IgM+ B cells but can also be found in IgM- pre-B cells and mIgM-IgG+ B cells. Membrane forms of IgD and IgG, isolated from freshly isolated human B cells and B cell lines, are expressed together with heterodimeric protein structures biochemically similar to the mIgM-associated polypeptides, and these were shown to comprise the products of the mb-1 and B29 genes, or homologous genes. Finally, all three classes of antigen receptors are linked to protein kinases, capable of phosphorylating the Ig-associated heterodimers. Our findings provide insight in the structural organization of the different antigen receptors on human B cells and have implications for their function.

The membrane IgM-associated heterodimer on human B cells is a newly defined B cell antigen that contains the protein product of the mb-1 gene.

7/8embrane IgM (mIgM) on human B lymphocytes is noncovalently associated with a disulfide-linked dimer that contains phosphoproteins of 47 and 37 kDa. In this study, the biochemical properties and the identity of these Ag receptor-associated components have been addressed. Both subunits carry N-linked carbohydrate groups. After deglycosylation, the 47-kDa and 37-kDa proteins have similar molecular masses, of about 23 kDa, and relatively acidic but different isoelectric points. The accumulated data, together with a previously performed comparison of tryptic peptides, suggest that the two components are structurally distinct and possibly encoded by different genes. Indeed, a mAb, raised against a synthetic peptide that was made on the basis of the published carboxyl-terminal amino acid sequence of the human mb-1 gene product, specifically reacted with the 47-kDa but not the 37-kDa subunit. None of the established B cell-specific mAb characterized in the Fourth International Workshop on Leukocyte Antigens, including CD24, CD37, and CD72, detect the mIgM-linked heterodimer, which makes it a newly defined human B cell Ag.

Requirements for induction of activation and proliferation of human B cells analysed with anti-idiotype monoclonal antibodies.

We have produced five monoclonal antibodies (MoAb) directed against idiotypic determinants of surface immunoglobulins (sIg) expressed on malignant B cells from a patient suffering from prolymphatic leukaemia (B-PLL). The anti-idiotype MoAb were characterized by immunofluorescence-binding studies, allotype reactivity, and immunoprecipitation studies and were found to recognize at least two distinct epitopes on the sIg of the neoplastic B cells. Differential effects of the soluble anti-idiotype MoAb on phorbol myristate acetate (PMA)-induced B-PLL-cell proliferation were found; three types of anti-idiotype MoAb could be distinguished: (1) MoAb that enhanced the PMA-induced B-PLL-cell proliferation, (2) MoAb without effect, and (3) MoAb that inhibited the PMA-induced B-PLL-cell proliferation. Addition of the calcium ionophore A23187 could abolish the differential effects of the anti-idiotype MoAb on PMA-induced B-PLL-cell proliferation. The negative effect of the type 3 MoAb on PMA-induced B-PLL-cell proliferation was associated with a more than 10-fold stronger binding to sIg expressed on the B-PLL cells, compared with the other anti-idiotype MoAb. The differential effects of the types 1 and 2 MoAb cannot be explained solely by differences in the Fc portion of the MoAb and binding characteristics. Neither did differences in epitope specificity necessarily lead to differential effects on PMA-induced B-PLL-cell proliferation. In contrast to the clear differences found in the proliferation induction experiments, all MoAb were equally able to induce an early rise in free intracellular Ca2+ concentration. Our data suggest that for B-cell proliferation to occur, apart from early rises of intracellular Ca2+, more prolonged [Ca2+]i elevations or additional intracellular activation signals are required. The differences in proliferative responses of B-PLL cells to anti-idiotype MoAb may be relevant for immunotherapy of B-cell tumours with anti-idiotype MoAb.

Regulatory role of CD19 molecules in B-cell activation and differentiation.

Cluster of differentiation ([CD]) 19 antigens are B-cell-specific molecules expressed on virtually all human cells of the B-lymphocyte lineage except plasma cells. We produced a new anti-CD19 monoclonal antibody (McAb), CLB-CD19, that was used to study the role of CD19 molecules in B-cell activation. Anti-CD19 McAb induced mobilization of free intracellular calcium ([Ca2+]i) in Daudi cells, but not in normal spleen or tonsillar B cells, for which crosslinking with a second anti-mouse Ig antibody was not required. Anti-CD19 McAb inhibited B-cell proliferation induced by anti-IgM coupled to Sepharose beads. This inhibitory effect was overcome by the addition of nonmitogenic concentrations of phorbol myristate acetate. Anti-CD19 McAb did not interfere with Staphylococcus aureus- or B-cell growth factor-induced B-cell proliferation. Anti-CD19 McAb inhibited T-cell-dependent polyclonal B-cell differentiation in pokeweed mitogen-, interleukin 2-, or anti-CD3-driven culture systems. Delayed addition studies showed that once differentiation of B cells was induced, CD19 molecules lost their regulating function. Taken together, our results indicate that CD19 molecules play a regulatory role in B-cell proliferation and differentiation.