Mouse B cell activation is inhibited by CD44 cross-linking

Lymphocyte activation and trafficking are indispensable to the immune system. CD44 is an adhesion molecule with known importance in T cell activation, lymphocyte trafficking, and tumor metastasis. Although CD44 has been shown to participate in the activation, rolling and adhesion, and homing of T cells, the role of CD44 on B cells is relatively unknown. The effects of CD44 cross-linking on murine B cell activation via CD40L was explored using the anti-CD44 mAbs RK3G9 and IM7. When immobilized on a plate, both RK3G9 and IM7 were found to strongly inhibit B cell proliferation and Ig production, especially at lower cell input concentrations. IgE inhibition was especially prominent. In contrast, soluble RK3G9 added to the B cell cultures had no effect. The inhibitory effect of anti-CD44 on B cell activation was not influenced by the addition of the anti-FcγRII, indicating that Fc cross-linking did not play a role in this inhibition. As Ig production requires several days for both B cell proliferation and differentiation to occur, the effects of delayed addition of immobilized anti-CD44 mAbs were studied, and the results indicated no inhibition after 96 hrs of culture. Finally, B cells were activated by either LPS or anti-IgM F(ab')₂. While LPS-induced B cell activation was inhibited by immobilized anti-CD44 mAbs, anti-IgM activation was refractory. Interestingly, addition of both anti-IgM and CD40L or LPS resulted in some modulation of the inhibitory activity. These results suggest that CD44 cross-linking could control polyclonal B cell activation by CD40L, but allow sIgM/CD40L activation to continue.     

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-covalentassociation with the Blymphocyte specific CD19 complex at thesurface of mature human B cells. Upon ligation of the B cellantigen receptor complex (BCR), members of the CR2-CD19 complexmay associate with membrane immunoglobulin (mlg). Moreover,CD19 and CD21 ligands, either murine mAb, C3d fragments or Epstein—Barrvirus, are known to have profound effects on B cell activation.We here show that CD19 is tightly linked to the non-receptorsrc kinase Lyn and that the CD19 glycoprotein itself servesas a substrate for a yet undefined serine/threonine kinase presentwithin the complex. In the process of antigen recognition, mlgand the CR2-CD19 complex may bind different sites of a complement-opsonizedantigenic particle. We hypothesize that in this process, approximationto the BCR allows CD19-associated Lyn kinase to phosphorylatepotential substrates within the antigen—receptor complex,thereby effecting its coupling to the intracellular compartment.     

Signaling through murine CD38 is impaired in antigen receptor-unresponsive B cells

CD38 is a 42-kDa membrane associated enzyme which converts NAD into cyclic ADP-ribose (cADPR), a Ca²⁺-mobilizing second messenger, and ADP-ribose (ADPR). Agonistic antibodies to murine CD38 deliver a potent growth co-stimulus to mature splenic B lymphocytes. In this report we demonstrate a striking relationship between CD38-mediated mitogenesis and the ability of surface IgM to promote B cell proliferation. Tolerized B lymphocytes obtained from a double-transgenic mouse model of B cell tolerance do not proliferate in response to antigen stimulation through the Ig receptor or to agonistic anti-CD38 antibodies. Similarly, B-1 cells isolated from the peritoneal cavity of normal mice, and splenic B cells isolated from newborn mice were also unresponsive to both anti-IgM and anti-CD38 stimulation. All of these CD38-unresponsive B cells expressed normal levels of cell surface CD38 and responded to numerous other stimuli. CD38 immunoprecipitated from these B cell populations was normal in size and effectively hydrolyzed NAD, suggesting that the defect in CD38 signaling likely occurs downstream of CD38 itself. Signaling through CD38 and IgM does not always have identical effects on B cells since anti-CD38 cannot deliver inhibitory growth or differentiation signals to normal B cells or immature B cell lines. Nevertheless, the correlative data with these multiple B cell models of unresponsiveness suggests that the signaling pathway utilized by CD38 and IgM intersect, possibly sharing at least one of the crucial components of the Ig receptor signaling cascade.     

IL-10 is predominantly produced by CD19(low)CD5(+) regulatory B cell subpopulation: characterisation of CD19 (high) and CD19(low) subpopulations of CD5(+) B cells

IL-10 production by CD19(+)CD5(+) B cells was investigated, by determining the expression levels of CD19, a classical B cell marker. Peripheral mononuclear cells were stained with fluorescence-conjugated anti-CD5, anti-CD19, anti-IL-10, and Annexin V. Interestingly, IL-10-producing B cells were found to be localised within the CD19(low)CD5(+) B cell subset. Apoptotic changes were also observed mainly in CD19(low) cells among B cells. Thus, CD5(+) B cells should be classified as CD19(high) and CD19(low) cells, and the immunological significance of CD19 for the IL-10 production by CD5(+) B cells requires further studies.     

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 Igα/β 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.     

Association of CD22 with the B cell antigen receptor

The antigen receptor on B lymphocytes is composed of membrane immunoglobulin sheathed by an α/β heterodimer. This structure is in several respects analogous to the antigen receptor on T cells except that, in the case of the T cell but not the B cell receptor, several receptor-associated proteins have been described which may modulate the effects of antigen interaction (e.g. CD4, CD8, CD2 and CD5). To screen for specific associations with the B cell antigen receptor that might be of only low stoichiometry, we have exploited the sensitivity of in vitro kinase assays. We show that the B cell antigen receptor associates with CD22.The association is specific and stable, but Western blotting reveals it to be of low stoichiometry (0.2 to 2% of membrane immunoglobulin is CD22 associated).The CD22/antigen receptor association was demonstrated with multiple isotypes (IgM, IgD and IgG) and was evident both in Burkitt lymphoma lines and in tonsil cells. Whilst the significance of the association is unknown, it is notable that CD22 is a B cell-specific adhesion molecule which we find contains within its cytoplasmic domain a sequence bearing high homology to the “Reth motif implicated in signal transduction. Indeed, CD22 becomes tyrosine phosphorylated less than one minute after antigen-receptor cross-linking. Thus, it is tempting to speculate that interactions involving CD22 assist in the antigen-mediated triggering of B cell activation.     

CD4-dependent and -independent association of protein tyrosine kinases to the T cell receptor/CD3 complex of CD4+ mouse T lymphocytes

Tyrosine phosphorylation of different substrates is the earliest intracellular signal detected after T cell receptor (TcR) ligation. Several tyrosine kinases have been detected associated to the CD3-TcR complex in stimulated or unstimulated cells, including p56^lck, p59^fyn and ZAP-70. We have observed, in one mouse T helper CD4 T cell line, that most TcR- or CD3-associated tyrosine kinase activity comes from CD4:p56^lck (Diez-Orejas, R., Ballester, S., Feito, M. J., Ronda, M., Ojeda, G., Criado, G., Portolées, P. and Rojo, J. M., EMBO J. 1994. 13: 90). To analyze whether this is a major way of tyrosine kinase association to the TcR in normal CD4⁺ T cells, we examined the nature and mode of association of tyrosine kinases to the TcR complex in normal spleen CD4⁺ T lymphocytes. Our results show that, in normal CD4⁺ T lymphocytes, as in CD4⁺ T cell lines, there is a stable and readily detectable association between CD4: p56^lck and the TcR/CD3 complex, as determined by in vitro kinase activity in immunoprecipitates from cell lysates. However, TcR/CD3 complexes from nature CD4⁺ lymphocytes have detectable amounts of p56^lck associated in a CD4-independent manner, as shown by immunodepletion of the lysates with anti-CD4 antibodies. In addition, TcR/CD3 also bind p59^fyn regardless of the presence of CD4. Conversely, we have observed that CD4 co-precipitates small quantities of p56^fyn in a TcR/CD3-independent manner. Overall, our data suggest the existence of different possible molecular complexes between TcR/CD3, CD4 and their attending kinases, as well as some quantitative and qualitative differences between CD4⁺ T cells and CD4⁺ T cell lines in kinase association to the TcR/CD3 complex.     

Regulation of B cell growth and differentiation via CD21 and CD40

Stimulation in vitro of murine splenic B cells by lipopolysaccharide, anti-ϰ Sepharose, anti-CD40 or allo-reactive T helper cells all up-regulated CD21 and CD23 surface expression. Neither anti-CD21 nor anti-CD23 antibodies induced B cell growth or differentiation when added in soluble form or coupled to Sepharose. However, anti-CD40-stimulated B cells showed increased proliferation in the presence of anti-CD21 antibodies coupled to Sepharose; co-stimulation via CD21 also induced differentiation to immunoglobulin secretion in a fraction of anti-CD40-stimulated B cells. Furthermore, anti-CD40 antibodies inhibited differentiation to immunoglobulin secretion induced by lipopolysaccharide and, hence, appears to be a dominant negative signal for B cell differentiation.     

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 μ heavy chain associates with two non-polymorphic polypeptides, λ 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 Igα and Igβ, 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. Prein-cubation 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 steadystate pool of internalized transferrin receptors. Thus, our data show that tyrosine phosphorylation and dephosphorylation are both required for cross-linkinginduced pre-BCR and BCR internalization.     

Apoptosis via the B cell antigen receptor requires Bax translocation and involves mitochondrial depolarization, cytochrome C release, and caspase-9 activation

Various routes to apoptosis can be active during B cell development. In a model system of mature B cells, differences in caspase-3 processing have suggested that antigen receptor (BCR)-mediated apoptosis may involve a zVAD-insensitive initiator protease(s). In search of the events leading to caspase-3 activation, we now establish that both CD95- and BCR-mediated apoptosis depend on Bax activation and cytochrome C (cytC) release. Nevertheless, the timing and caspase-dependence of mitochondrial membrane depolarization differed considerably after CD95- or BCR-triggering. To delineate events subsequent to cytC release, we compared apoptosis induced via BCR triggering and via direct mitochondrial depolarization by CCCP. In both cases, partial processing of caspase-3 was observed in the presence of zVAD. By expression in 293 cells we addressed the potential of candidate initiator caspases to function in the presence of zVAD, and found that caspase-9 efficiently processed caspase-3, while caspase-2 or -8 were inactive. Finally, retroviral expression of dominant-negative caspase-9 inhibited both CD95- and BCR-mediated apoptosis. In conclusion, we obtained no evidence for involvement of a BCR-specific protease. Instead, our data show for the first time that the BCR-signal causes Bax translocation, followed by mitochondrial depolarization, and cytC release. Subsequent caspase-9 activation can solely account for events further downstream.     

Identification of overlapping epitopes in mutant ras oncogene peptides that activate CD4+ and CD8+ T cell responses

Mutant ras p21 proteins contain sequences which distinguish them from normal endogenous ras and, thus, may represent unique epitopes for T cell recognition of antigen bearing tumor cells. Here, we examined the capacity of a mutant K-ras 9-mer peptide to induce in vivo CD8⁺ cytotoxic T lymphocytes (CTL). The peptide chosen reflected positions 4–12 of the point-mutated sequence of the K-ras oncogene encoding the Gly to Val substitution at codon 12. The overall rationale for selecting this particular 9-mer sequence was threefold: the mutant peptide contained a putative major histocompatibility complex (MHC) class I consensus anchor motif for murine H-2K^d; specific binding to MHC class I may then create an immunogenic complex for the induction of anti-ras CD8⁺ CTL; and finally, the mutant sequence overlapped with a newly characterized anti-ras CD4⁺ T helper type 1 epitope, which may have implications for the coordination and activation of both anti-ras immune mechanisms against the same target cell antigenic determinant. A functional interaction with H-2K^d was demonstrated with the mutant ras4–12(V12) peptide, but not the normal ras4–12(G12) peptide, which specifically inhibited an H-2K^d-restricted, anti-nucleoprotein NP147–155 CTL response in a dose-dependent fashion. An anti-ras CD8⁺ T cell line was then established from immune splenocytes of BALB/c (H-2^d) mice injected with ras4–12(V12) in adjuvant, which mediated peptide-specific lysis of syngeneic P815 tumor targets. Cytotoxicity was restricted by H-2K^d and strongly specific for the mutant ras peptide. Importantly, these anti-ras CTL specifically lysed a syngeneic tumor line (i.e. A20 lymphoma) transduced with the corresponding point-mutated ras oncogene, suggesting T cell receptor recognition of endogenously derived antigen. Overall, these data demonstrated that mutant ras p21 at codon 12 (Gly → Val) contained a peptide sequence which exhibited specific functional binding to a murine MHC class I molecule; the ability of the mutant, but not the normal sequence to bind selectively to murine MHC class I likely reflected the generation of a C-terminal anchor residue; and the ras 4–12(V12) peptide was immunogenic for the production of antigen-specific CD8⁺ CTL, which lysed in vitro a syngeneic tumor cell line harboring the mutant K-ras oncogene.     

Engagement of the B lymphocyte antigen receptor induces presentation of intrinsic immunoglobulin peptides on major histocompatibility complex class II molecules

By means of the clonotypic variable region, the immunoglobulin (Ig) is a tumorspecific antigen on B cell neoplasms. We report that engagement of the B cell antigen receptor (BcR) promotes presentation of peptides derived from the B cell's intrinsic Ig to major histocompatibility complex (MHC) class II-restricted T cells. Thus, anti-Ig endowed normal, ex vivo B lymphocytes from H-2^d, Ig constant heavy chain allotype b (IgC_H^b) mice with the capacity to stimulate an I-A^d-restricted T cell clone which recognizes the γ2a^b 435–451 allopeptide. The corresponding self γ2a^a peptide is cryptic and 6000-fold less antigenic than the γ2a^b allopeptide. Even so, the syngeneic B cell lymphoma A20 which expresses surface (s) IgG2a^a, was also recognized by the T cells after BcR ligation. Thus, anti-Ig triggered the disclosure of a cryptic tumor antigen determinant. We propose that autoantigens, by engaging the BcR of self-reactive B cells, induce presentation of intrinsic Ig peptides to which the T helper cell (Th) repertoire is not tolerant. In this way, B cells with anti-self potential may be activated without Th recognition of nominal autoantigen.     

CD19, CD21, and CD22: Multifaceted Response Regulators of B Lymphocyte Signal Transduction

B lymphocyte development and function depend upon the activity of intrinsic and B cell antigen receptor (BCR)-induced signals. These signals are interpreted, amplified, finetuned, or suppressed through the precise actions of specialized cell surface coreceptors, or “response regulators,” that inform B cells of their extracellular environment. Important cell surface response regulators include the CD19/CD21 complex, CD22, and CD72. CD19 establishes a novel Src-family protein tyrosine kinase (PTK) amplification loop that regulates basal signaling thresholds and intensifies Src-family PTK activation following BCR ligation. In turn, CD22 limits the intensity of CD19-dependent, BCR-generated signals through the recruitment of potent phosphotyrosine and phosphoinositide phosphatases. Herein we discuss our current understanding of how CD19/CD21 and CD22 govern the emergence and intensity of BCR-mediated signals, and how alterations in these tightly controlled regulatory activities contribute to autoimmunity in mice and humans.     

Impairment of B cell receptor-mediated Ca2+ influx, activation of mitogen-activated protein kinases and growth inhibition in CD72-deficient BAL-17 cells

CD72 is a 45 kDa B cell-specific type II transmembrane protein of the C-type lectin superfamily. It was originally defined as a receptor-like molecule that regulates B cell activation and differentiation; however, its precise function remains unclear since more recent functional analyses, including a gene targeting study, suggest that CD72 may serve as a negative or a positive regulator of B cell signaling. In the present study, we analyzed the cell-autonomous function of CD72 in B cell receptor (BCR) signaling using CD72-deficient cells generated from mature BAL-17 cells. We found that BCR-mediated phosphorylation of CD19, Btk, Vav and phospholipase Cgamma2 and association of CD19 with phosphatidylinositol-3 kinase were impaired in CD72-deficient cells. Inositol trisphosphate synthesis was normally induced initially but ablated at 1 min of stimulation in CD72-deficient cells. In the event, Ca(2+) release from intracellular stores remained intact, though influx of extracellular Ca(2+) was severely impaired in CD72-deficient cells. Furthermore, BCR-evoked activation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase, and growth inhibition in BAL-17 cells were blocked in the absence of CD72. Significantly, these effects were largely reversed by re-expression of CD72. Thus, CD72 appears to exert a positive effect on BCR signaling pathways leading to Ca(2+) influx and MAPK activation, which in turn may determine the fate of BAL-17 cells.     

Selective enhancement of B cell antigen receptor-mediated antigen presentation by treatment with transforming growth factor-beta

TGF-beta1 was examined for the ability to regulate Ag-presentation by B cells, using A20-HL B lymphoma cells bearing TNP-specific IgM receptors. Treatment of A20-HL cells with TGF-beta1 at 1 ng/ml, a concentration that inhibited proliferation, enhanced presentation of Ag internalized via surface IgM (sIgM), but not via fluid-phase pinocytosis. TGF-beta1-treatment slightly enhanced surface expression of sIgM, but not of MHC class II molecules. The treatment accelerated recovery of sIgM expression after its removal by ligation with TNP-OVA, and induced prolonged intracellular residence of TNP-OVA internalized via sIgM, which co-localized with intracellular MHC class II molecules. TGF-beta1-treatment increased accumulation of newly synthesized intracellular MHC class II molecules that were localized in compartments positive for lysosome-associated membrane protein 1, although cellular protein synthesis was decreased by the treatment. The accumulated intracellular MHC class II molecules were triggered to the cell surface by ligation of sIgM. Finally, TGF-beta1-treatment induced Igalpha-phosphorylation in response to lower concentrations of TNP-OVA. On the basis of these findings, we conclude that TGF-beta1-treatment of A20-HL cells selectively enhances the ability to present Ag internalized via sIgM, not via fluid-phase pinocytosis, through accelerating sIgM recovery, increasing accumulation of intracellular MHC class II molecules and enhancing the ability of sIgM ligation to induce Igalpha-phosphorylation.     

Engagement of the B cell receptor for antigen differentially affects B cell responses to Toll-like receptor-7 agonists and antagonists in BXSB mice

Nucleic acid sensors of the Toll-like receptor (TLR) family play a well-established role in the pathogenesis of lupus. This is particularly true for a single-stranded RNA-sensing TLR-7 receptor, as lupus mice lacking TLR-7 show ameliorated disease. Cytosine-guanosine dinucleotide (CpG)-DNA-sensing TLR-9, conversely, has a complex regulatory role in systemic lupus erythematosus (SLE). Much less is known about whether signals through the B cell receptor for antigen (BCR) may affect the ability of B cells to respond to suboptimal TLR-7 agonists and antagonists. We studied this question in prediseased BXSB male and female B cells. We found that male B cells responded more vigorously to numerous TLR-7 ligands and this responsiveness was enhanced further upon co-engagement of the BCR. This synergy was seen primarily with the interleukin (IL)-6 secretion. A number of 32-mer inhibitory oligonucleotides (INH-ODNs) with a nuclease-resistant phosphorothioate backbone were capable of blocking TLR-7, but not BCR-induced B cell activation, with an inhibitory concentration (IC)(50) of approximately 100 nm. Surprisingly, while the presence of a single TGC motif at the 5' end of an ODN did not increase its inhibitory capacity, INH-ODNs containing multiple TGC motifs had greater inhibitory potency. When BCR and TLR-7 were co-engaged, INH-ODNs showed a differential effect on B cell activation. Whereas apoptosis protection and G1-M entry completely escaped suppression, IL-6 secretion remained sensitive to inhibition, although with a 10-fold lower potency. Our results suggest that while TLR-7 antagonists may be considered as lupus therapeutics, simultaneous co-engagement of the TLR-7 and BCR might favour autoreactive B cell survival. This hypothesis needs further experimental validation.     

Interleukin-6 induces tyrosine phosphorylation of the Ras activating protein Shc,and its complex formation with Grb2 in the human multiple myeloma cell line LP-1

Like many other cytokines and growth factors, interleukin-6 (IL-6) activates p21^ras. However, the precise biochemical mechanisms inducing this activation are unknown. Therefore, we investigated the effects of IL-6 on some recently identified signaling intermediates, Shc (Src homology and collagen) and Grb2 (growth factor receptor bound protein 2), known to activate p21^ras. In the multiple myeloma cell line LP-1, IL-6 stimulated the tyrosine phosphorylation of Shc in a time- and concentration-dependent manner. This led to the complex formation of Shc with Grb2, an adaptor protein known to relocate a p21^ras-GDP exchange factor, Sos1 (Son-of-sevenless), to the cell membrane. Taken together, these findings suggest that IL-6 might activate the Ras signaling pathway via tyrosine phosphorylation of Shc and subsequent recruitment of Grb2. Further studies will elucidate which of the IL-6 receptor associated non-receptor tyrosine kinases of the Src kinase or Janus kinase family, mediate these effects.     

CD72-mediated B cell activation involves recruitment of CD19 and activation of phosphatidylinositol 3-kinase

Occupancy of the B cell glycoprotein, CD72 results in syk-independent activation of phospholipase-C γ and calcium mobilization. The cytoplasmic tail of CD72 does not contain an immunoreceptor tyrosine-based activation motif to directly transduce signals into the B lymphocyte. Hence, we investigated whether other coreceptors such as CD19 and its associated phosphatidylinositol 3-kinase (PI 3-K) were involved in CD72 signaling. Two specific inhibitors of PI 3-K inhibited CD72-stimulated B cell proliferation in a dose-dependent manner. Activation of B lymphocytes via CD72 resulted in recruitment and activation of PI 3-K, which was mediated by CD19. Accordingly, CD72 ligation induced CD19 tyrosine phosphorylation. Thus, lipid products generated as a result of PI 3-K activation may have an important function in CD72-mediated B lymphocyte activation. The kinetics of CD19 tyrosine phosphorylation induced by CD72 ligation were strikingly different from those seen following B cell antigen receptor (BCR) stimulation. A transient increase in the tyrosine phosphorylation of the complement receptors, CD21 and CD35 was observed in BCR- but not CD72-stimulated cells. Co-cross-linking of CD72 and CD19 failed to induce syk tyrosine phosphorylation suggesting that even under these conditions, CD72 signaling was independent of syk activation. A transient and stimulation-dependent physical association between CD19 and CD72 was observed in CD72-ligated cells. These observations suggest a mechanism by which CD72 can recruit CD19 and influence activation of CD19-associated PI 3-K, which appears to be critical for CD72-mediated B cell activation.     

Human lymphocytes shed a soluble form of CD21 (the C3dg/Epstein-Barr virus receptor,CR2) that binds iC3b and CD23

We report on a soluble (s) form of CD21 (the C3dg/Epstein-Barr virus receptor, CR2) that is spontaneously released by B and T lymphocytes. Immunoprecipitation with anti-CD21 mAb of culture supernatants of surface and biosynthetically labeled B and T cell lines revealed a single band with an apparent molecular mass of 135 kDa. The molecule exhibited a molecular mass 10 kDa lower than that of membrane CD21. The release of soluble CD21 (sCD21) was time dependent and correlated with a parallel decrease in the expression of the membrane-associated molecule. The protein was also found in culture supernatants of tonsillar B cells and normal human thymocytes. Epitopic analysis using combinations of anti-CD21 monoclonal antibodies (mAb) indicated that sCD21 and membrane CD21 were similarly recognized by mAb directed against short concensus repeats (SCR) 1–2, SCR 4–5 and SCR 9–11. Affinity-purified sCD21 was capable of binding to purified human iC3b and to human recombinant CD23, as assessed by enzyme-linked immunosorbent assay and by using the BIAcore^TM technology. In addition, normal human serum was found to contain a soluble form of CD21 that exhibited a similar molecular mass to that of the molecule shed by B and T cells in culture. The serum form of CD21 was recognized by all anti-CD21 mAb that we tested and showed a high reactivity with mAb directed against SCR 1–2. Our observations suggest that B and T cells shed the extracellular portion of CD21 and release a soluble molecule that retains the ligand-binding properties of CD21, thus having a potential role in immunoregulation.