Role of the CD22 human B cell antigen in B cell triggering by anti-immunoglobulin

As B cells mature during ontogeny the CD22 human differentiation Ag is exported from the cytoplasm onto the membrane. Surface expression is lost in terminal differentiation and after activation. In tonsils, CD22 is expressed on the surface of 60 to 80% of the dense B cells. Some IgM+ dense cells, however, and buoyant in vivo activated B cells are CD22-. This differential expression of CD22 and the finding that an anti-CD22 mAb augmented anti-Ig induced B cell proliferation suggested that CD22 may play a role in B cell activation. In this study we have found that CD22+ but not CD22- B cells could be triggered by anti-IgM or anti-IgD to have increased free intracellular calcium ([Ca2+]i). The presence of CD22 rather than of IgD seems to determine the ability of B cells to respond to anti-Ig with a [Ca2+]i flux. Also the proliferative response to anti-Ig or anti-Ig + B cell growth factor was restricted to the CD22+ population. Anti-CD22 mAb, although not inducing [Ca2+]i on their own after binding to B cells, did augment [Ca2+]i fluxes by anti-Ig when cross-linked. Together these results suggest that CD22 may regulate triggering of B cells through surface Ig perhaps by acting as a "bridge" to transmit an early signal into the cytoplasm.     

Activation of human B cell proliferation through surface Bp35 (CD20) polypeptides or immunoglobulin receptors

Human B cells can be activated with monoclonal antibodies (mAb) to surface IgM receptors or mAb to a 35-kilodalton B cell differentiation antigen, Bp35 (CD20). We compared anti-Ig-induced B cell activation with B cell triggering by anti-Bp35. Both anti-Ig- and anti-Bp35-dependent proliferation were augmented by the same co-stimulants, including a partially purified BCGF, recombinant IL 1, TPA, or each other. When anti-Bp35 and anti-Ig were used together to induce proliferation of tonsillar B cells, the strongest response was observed when anti-Bp35 was added 12 to 24 hr before anti-Ig. Anti-Bp35 also was found to act most effectively when added before the BCGF. Blood and tonsillar B cells differed in their proliferative response to anti-Ig or anti-Bp35: unlike dense tonsillar B cells, which consistently proliferated in response to either stimulus, blood B cells from many donors proliferated in response to anti-Ig but not to anti-Bp35 even in the presence of other co-stimuli. Dense tonsillar B cells that proliferate in response to anti-Bp35 appeared to be at a more activated stage than unresponsive blood B cells because they expressed higher levels of HLA class II molecules than blood B cells. Pretreatment of blood B cells with anti-Bp35 converted them to an HLA-DR(bri) phenotype and made them more responsive to anti-Ig-induced proliferation. These results suggest that B cells at different stages of differentiation differ in their response to anti-Bp35 and anti-Ig. The Bp35 surface polypeptide may play an early role in the activation of B cells prior to antigen or other signals.     

CD19 monoclonal antibody HD37 inhibits anti-immunoglobulin-induced B cell activation and proliferation

The 95 Kd CD19 antigen is the broadest lineage specific surface marker for B cells: it is present on the surface of virtually all B lymphocytes, including early B progenitor cells. In this study we have evaluated the function of the CD19 antigen by using the CD19 mAb HD37. Binding of HD37 mAb to B cells at low doses (0.5 microgram/ml) induced a strong inhibition of the proliferative response to anti-Ig. This inhibition was not mediated by the Fc portion of the antibody, since F(ab')2 fragments were as effective as the whole antibody. Both dose-response curve analysis and experiments in which a cross-linking second step anti-mouse antibody was added suggested that cross-linking of CD19 antigens was necessary for optimal inhibition. Early phases in B cell activation were affected by the HD37 mAb: it significantly reduced the number of cells that left G0 and entered the G1 phase of the cell cycle upon triggering with anti-mu. The increase in free intracellular ionized calcium [Ca2+]i that is induced by anti-mu was also consistently reduced by CD19 mAb. Cross-linking was also crucial for this effect, suggesting that a causal relationship may exist between the inhibition of anti-Ig-mediated [Ca2+]i fluxes and inhibition of proliferation. A variable but clear increase in [Ca2+]i levels followed cross-linking of CD19 antigens by specific mAb. This evidence suggests that CD19 molecules may function in the downregulation of B cell growth and proliferation.     

Expression of CD9 antigen on normal activated human B cells

The expression of the CD9 pre-B acute lymphoblastic leukemia (ALL)-associated antigen was studied. CD9-positive B cells were enriched in the in vivo-activated buoyant B cell population isolated from tonsils. Small tonsil B cells activated in vitro with either PWM, phorbol 12-myristate 13-acetate (TPA), or anti-Ig plus low Mr B cell growth factor (BCGF) also demonstrated increased CD9 expression. The peak of CD9 expression (30-40% positive cells) occurred after 4-6 days of activation. The kinetics of increased CD9 expression was similar to that of the 4F2 activation antigen. CD9 antigen expression on tonsillar B cells as well as on pre-B leukemic cell lines was associated with protein kinase C activation. Two phorbols that activate protein kinase C (TPA; phorbol 12,13-dibutyrate) induced expression of the CD9 antigen whereas a phorbol analogue that does not activate C kinase (4-alpha-phorbol 12,13-didecanoate) and an analogue that is a very weak agonist (phorbol 12-myristate 13-acetate-4-0-methyl ether) were unable to induce CD9 expression on tonsil B cells or on the cell lines. The effect of the anti-CD9 monoclonal antibody, DU-ALL-1, on B cell mitogenesis was studied. Dense or buoyant tonsillar B cells were cultured in the presence or absence of DU-ALL-1 antibody plus PWM, anti-Ig, and BCGF, DU-ALL-1 antibody did not inhibit or augment the mitogenic response of resting or activated B cells. These results indicate that the CD9 pre-B ALL antigen is present on a population of normal activated tonsillar B cells and that its induction of expression is associated with protein kinase C activation.     

Inhibitory coreceptors activated by antigens but not by anti-Ig heavy chain antibodies install requirement of costimulation through CD40 for survival and proliferation of B cells

Ag-induced B cell proliferation in vivo requires a costimulatory signal through CD40, whereas B cell Ag receptor (BCR) ligation by anti-Ig H chain Abs, such as anti-Ig micro H chain Ab and anti-Ig delta H chain Ab, alone induces proliferation of B cells in vitro, even in the absence of CD40 ligation. In this study, we demonstrate that CD40 signaling is required for survival and proliferation of B cells stimulated by protein Ags in vitro as well as in vivo. This indicates that the in vitro system represents B cell activation in vivo, and that protein Ags generate BCR signaling distinct from that by anti-Ig H chain Abs. Indeed, BCR ligation by Ags, but not by anti-Ig H chain Abs, efficiently phosphorylates the inhibitory coreceptors CD22 and CD72. When these coreceptors are activated, anti-Ig H chain Ab-stimulated B cells can survive and proliferate only in the presence of CD40 signaling. Conversely, treatment of Ag-stimulated B cells with anti-CD72 mAb blocks CD72 phosphorylation and induces proliferation, even in the absence of CD40 signaling. These results strongly suggest that activation of B cells by anti-Ig H chain Abs involves their ability to silence the inhibitory coreceptors, and that the inhibitory coreceptors install requirement of CD40 signaling for survival and proliferation of Ag-stimulated B cells.     

Epitope specificity of the anti-(B cell lymphoma) monoclonal antibody,LL2

LL2 is a murine monoclonal antibody IgG2a reactive with B cells and non-Hodgkin's B-cell lymphoma, which, in a radioiodinated form, induces responses in lymphoma patients [Goldenberg et al. (1991) J Clin Oncol 9:548–564]. In this report we identify LL2 as a member of the CD22 cluster. The molecular size of the antigen, its expression profile, and competitive blocking studies were used to establish this identification. By Western blot analysis and immunoprecipitation studies using the Raji Burkitt's lymphoma cell line metabolically labelled with [³H]leucine, the LL2 antigen was determined to correspond to a molecular mass of 140 kDa. The molecular mass of the LL2 antigen, and the B-cell-restricted reactivity of the LL2 antibody, were consistent with both the CD21 and CD22 clusters. To assess additional similarities and differences between LL2 and anti-CD22 and anti-CD21, the binding of these mAb to cultured cell lines. Nalm-6 and Molt-4, was compared by flow cytometry. The binding profile of LL2 on these cell lines was consistent with anti-CD22, but not anti-CD21. Sequential immunoprecipitation and cross-blocking studies with anti-CD22 monoclonal antibodies recognizing established CD22 epitopes were performed to confirm that LL2 reacts with CD22 and to determine which epitope LL2 recognizes. Binding of¹³¹I-LL2 to Raji cells is inhibited over 90% by prior incubation of the target cells with unlabelled RFB4, indicating that LL2 belongs to the same epitope group as RFB4, i.e., epitope B.This work was supported in part by USPHS grant CA39841 from the NIH     

Complex formation of CD23/surface immunoglobulin and CD23/CD81/MHC class II on an EBV-transformed human B cell line and inferable role of tetraspanin

CD23, a low-affinity IgE receptor, is a type II transmembrane protein having a C-type lectin domain and it associates noncovalently with MHC class II on B cells. The results of our immunoprecipitation analysis suggest that CD23 co-exists with at least two additional molecules, surface immunoglobulin (sIg) and CD81 (and/or CD9), on the cell surface of L-KT9 cells (an Epstein-Barr virus (EBV)-transformed human B cell line). When both CD23 and sIg molecules were stimulated simultaneously by the corresponding antibodies, a large increase in CD81 in the immunoprecipitation was observed as compared with the case of stimulation by only one antibody. Simultaneous stimulation by anti-CD23 and anti-Ig may mimic the situation of B cells stimulated by an antigen/IgE complex. In addition, a large increase in MHC class II in the immunoprecipitation was also observed by cross-linking of CD23 with anti-CD23 and its second antibody as compared with the case of stimulation by anti-CD23 alone. The cross-linking of CD23 with anti-CD23 and its antibody may mimic the situation of B cells stimulated by an IgE/antigen/IgE complex. Therefore, the complex formation among CD23, sIg, MHC class II, and CD81 on the cell surface of L-KT9 cells by the antigen/IgE or IgE/antigen/IgE complex is most likely to be closely related to B cell regulatory events by signaling through sIg or MHC class II. Tetraspanins such as CD81 and CD9 are thought to be involved in the formation and the preservation of various different membrane complexes consisting of several functional proteins.     

Ex vivo stimulation of B cells latently infected with gammaherpesvirus 68 triggers reactivation from latency

Murine gammaherpesvirus 68 (gammaHV68) infection of mice results in the establishment of a chronic infection, which is largely maintained through latent infection of B lymphocytes. Acute virus replication is almost entirely cleared by 2 weeks postinfection. Spontaneous reactivation of gammaHV68 from latently infected splenocytes upon ex vivo culture can readily be detected at the early stages of infection (e.g., day 16). However, by 6 weeks postinfection, very little spontaneous reactivation is detected upon explant into tissue culture. Here we report that stimulation of latently infected splenic B cells harvested at late times postinfection with cross-linking surface immunoglobulin (Ig), in conjunction with anti-CD40 antibody treatment, triggers virus reactivation. As expected, this treatment resulted in B-cell activation, as assessed by upregulation of CD69 on B cells, and ultimately B-cell proliferation. Since anti-Ig/anti-CD40 stimulation resulted in splenic B-cell proliferation, we assessed whether this reactivation stimulus could overcome the previously characterized defect in virus reactivation of a v-cyclin null gammaHV68 mutant. This analysis demonstrated that anti-Ig/anti-CD40 stimulation could drive reactivation of the v-cyclin null mutant virus in latently infected splenocytes, but not to the levels observed with wild-type gammaHV68. Thus, there appears to be a role for the v-cyclin in B cells following anti-Ig/anti-CD40 stimulation independent of the induction of the cell cycle. Finally, to assess signals that are not mediated through the B-cell receptor, we demonstrate that addition of lipopolysaccharide to explanted splenocyte cultures also enhanced virus reactivation. These studies complement and extend previous analyses of Epstein-Barr virus and Kaposi's sarcoma-associated virus reactivation from latently infected cell lines by investigating reactivation of gammaHV68 from latently infected primary B cells recovered from infected hosts.     

Human peripheral blood T helper cell-induced B cell activation results in B cell surface expression of the CD23 (BLAST-2) antigen

We have developed an in vitro system to assess the early stages of B cell activation induced by peripheral blood T helper cells. Peripheral blood mononuclear cells are cultured for 16 hr with anti-CD3 monoclonal antibody (mAb), T lymphocytes are then removed by sheep red blood cell rosette depletion, and expression of the B cell surface activation antigen CD23 (BLAST-2) is assessed by indirect immunofluorescence. Anti-CD3 mAb, but not a control anti-CD5 mAb, stimulates the expression of CD23 on 20-50% of peripheral blood B cells cultured with autologous T cells. T cell subset depletion studies show that the CD4+ T cell subset is responsible for anti-CD3-mediated induction of CD23 on autologous B cells. Anti-CD3-induced, T helper cell-dependent CD23 expression is not MHC-restricted, as allogeneic combinations of T and non-T cells, cultured in the presence of anti-CD3 antibody, also result in the expression of B cell CD23. Individuals whose monocyte Fc receptors bind murine IgG1 mAb poorly fail to trigger T cell proliferation in response to murine IgG1 anti-CD3 mAb and also fail to express B cell CD23 following culture of PBMC with IgG1 anti-CD3 mAb, while the usual expression of CD23 is seen after culture with IgG2a anti-CD3 mAb. The mechanism of anti-CD3-induced B cell activation was addressed in experiments using a two-chamber culture system. While little IL-4 activity was detected in anti-CD3-stimulated culture supernatants, optimal induction of CD23 was observed when T and B cells were cultured together in a single chamber. This suggests that under physiologic conditions, in which quantities of lymphokine may be limiting, close physical contact between the anti-CD3-activated Th cell and B cell may be required for CD23 expression. The anti-CD3-induced BLAST-2 assay will facilitate the analysis of Th cell-mediated B cell activation in any individual and should permit us to separately evaluate the roles of Th cells and B cells in the impaired immunoregulation characteristic of autoimmune disorders.     

Activation of human B cells. Comparison of the signal transduced by IL-4 to four different competence signals

The effects of the cytokine IL-4 on resting and activated human B cells were compared with the effects of known "competence" signals able to drive resting B cells into the cell cycle, including anti-Ig, PMA, anti-CD20, and a recently described competence signal, anti-Bgp95. In proliferation assays, IL-4 was costimulatory with anti-Ig and anti-Bgp95 but not with anti-CD20 or PMA. IL-4 alone triggered increases in expression of class II DR/DQ and CD40, but it did not trigger increases in intracellular free calcium [Ca2+]i in resting B cells or induce resting B cells to leave G0 and enter the G1 phase of the cell cycle. Although IL-4 has some characteristics of competence signals, it was most effective if added to B cells up to 12 h after anti-Ig or anti-Bgp95 rather than before, and thus, in this respect, works more like a progression signal. Like IL-4, all four competence signals for B cells triggered increases in class II and CD40, but only IL-4 consistently induced increases in CD23 surface levels. IL-4 was costimulatory only with anti-Ig and anti-Bgp95, each of which can trigger increases in [Ca2+]i and new protein synthesis of the proto-oncogene c-myc, and can increase attachment of protein kinase C to the plasma membrane. IL-4 was not costimulatory with signals that 1) did not affect [Ca2+]i yet induced c-myc protein synthesis (anti-CD20), 2) only stimulated the translocation of protein kinase C (PMA), or 3) only stimulated increases in [Ca2+]i (calcium ionophore). These results suggest that resting human B cells require at least two intracytoplasmic signals before IL-4 can effectively promote B cell proliferation.     

Augmentation of normal and malignant B cell proliferation by monoclonal antibody to the B cell-specific antigen BP50 (CDW40)

We recently described a 50,000 dalton polypeptide Bp50 (CDw40) that is expressed on human B cells and plays a role in regulating B cell proliferation. Here we additionally characterize the functional signal given by antibody binding to Bp50 on both normal and malignant B cells. A monoclonal anti-Bp50 antibody could augment the proliferation of B cells activated by anti-IgM, anti-CD20, or 12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulation, but was not co-stimulatory with B cell growth factor (BCGF), interleukin 1, or interleukin 2. The signal did not depend on the Fc portion of the antibody, because F(ab')2 fragments of anti-Bp50 were still functionally active. Both anti-Bp50 and a low m.w. BCGF preparation were similar in that both were co-stimulatory with the same agents and both anti-Bp50 and BCGF affected activated B cells but not resting B cells. However, a panel of B cell malignancies differed in their responsiveness to anti-Bp50 vs BCGF: some tumors proliferated in response to anti-Bp50 but not BCGF, whereas other tumors had the opposite pattern. Bp50 was found to have several properties in common with HLA class II molecules: both Bp50 and class II were expressed at lower levels on blood B cells than on tonsillar B cells; the expression of both Bp50 and class II was increased after activation of blood B cells with TPA or anti-IgM; and the expression of both Bp50 and class II was increased after activation of non T, non-B acute leukemias with BCGF. Thus class II and Bp50 expression may be under common regulatory control. The fact that BCGF modulated the expression of Bp50 on leukemic cells suggests that BCGF and Bp50-mediated signals may be coordinately regulated.     

Functional properties of human germinal center B cells.

Germinal centers (GCs) are histologically defined areas where B cells undergo extensive proliferation and maturation, or die of apoptosis. GC B cells isolated from human tonsils can be phenotypically identified by expression of peanut agglutinin (PNA)-binding sites and can be further divided into subpopulations based on their expression of CD77. To assess the functional potential of GC B cells, we studied CD77+ PNA+ B cells isolated from tonsils by examining their differentiation status and their ability to proliferate in vitro to various cytokines and costimulants. We found that CD77+ GC B cells are less differentiated than CD77- GC B cells; GC B cells less frequently express cytoplasmic IgG and IgM, and spontaneously secrete less Ig compared to CD77- GC B cells. To identify conditions capable of inducing GC B cell proliferation, we examined IL-4, IL-2, IFN-gamma, low molecular weight BCGF (LMW-BCGF), and an MLR supernatant along with costimulants such as anti-IgM antibody, Staphylococcus aureus Cowan I (SAC), PMA, and pokeweed mitogen (PWM). While non-GC B cells proliferate strongly in response to these stimuli, GC B cells did not proliferate. However, CD77+ as well as CD77- GC B cells mounted a rapid and strong proliferative response upon stimulation with IL-4, but only in the presence of anti-CD40 antibody. Moreover, although nine additional cytokines were examined, only IL-4 was capable of supporting CD77+ GC B cell proliferation in the presence of anti-CD40 antibody. When cells were stimulated with IL-4 and anti-CD40 antibody, we also found that IFN-gamma consistently decreased the proliferative response of CD77+ GC B cells without affecting the response of non-GC B cells. Taken together, these data indicate that GC B cells have characteristic growth requirements and that IL-4 may be important for GC B cell growth in vivo.     

Anti-Qa-2-induced T cell activation. The parameters of activation, the definition of mitogenic and nonmitogenic antibodies, and the differential effects on CD4+ vs CD8+ T cells

The MHC Ag Qa-2 is a glycolipid anchored class I molecule expressed at high levels on all peripheral T lymphocytes. In this study we found that anti-Qa-2 antibodies could stimulate the proliferation of murine T cells in vitro. Anti-Qa-2-induced proliferation required secondary cross-linking with anti-Ig antibody and the presence of PMA. Only Qa-2+ strains could be induced to proliferate by anti-Qa-2 antibody, but under the conditions employed, anti-CD3 could induce proliferation in Qa-2+ and Qa-2-strains. Interestingly, only anti-Qa-2 reagents directed against the alpha 3 domain of the Qa-2 class I molecule were effective in inducing proliferation. Furthermore, unlike purified CD4+ cells, purified CD8+ cells were unable to be stimulated by the anti-Qa-2 antibodies. These results lead to the inclusion of Qa-2 in a group of physiologically relevant, glycolipid-anchored, cell-surface molecules, mobilization of which can generate signals that initiate the proliferation of T cells. Such molecules may play a secondary role in cellular activation after the primary engagement of the TCR.     

Distribution and functional analysis of a 120- to 130-kDa T-cell surface antigen

A monoclonal antibody (mAb), SPV-L14, was raised that detected a human T-cell surface antigen with a molecular weight (MW) of 120 kDa on resting and phytohemagglutinin-activated peripheral blood T lymphocytes (PBL). An additional band with a MW of 130 kDa could be precipitated with variable intensities from thymocytes, neoplastic T cells, and CD4+- or CD8+ T-cell clones. Based on their reactivity with SPV-L14 and a mAb directed against CD3, four subpopulations of CD2+ lymphocytes could be detected and their existence was confirmed at the clonal level. The majority (95%) of the CD3+ cells were SPV-L14+, whereas 5% were CD3+, SPV-L14-. Among cloned cell lines CD3-,SPV-L14- and CD3-,SPV-L14+ cells were found to exist. The CD3-,SPV-L14- and CD3-,SPV-L14+ clones were shown to have NK cell activity, indicating that the 120- to 130-kDa antigen is expressed heterogeneously on CD3- NK cell clones. In addition, neoplastic T cells representing these four subpopulations were shown to exist. Although the tissue distribution and the MW of the SPV-L14 target antigen strongly suggest that SPV-L14 reacts with an epitope on CD6, the SPV-L14 mAb did not react with resting or activated B cells or with malignant B cells. Blocking studies showed that SPV-L14 inhibited the proliferative response of PBL, induced by anti-CD3 mAb, but that SPV-L14 did not affect the proliferation induced by phytohemagglutinin. These results suggest that the 120- to 130-kDa MW antigen is associated with T-cell proliferation, depending on the mode of activation.     

Novel binding site for Src homology 2-containing protein-tyrosine phosphatase-1 in CD22 activated by B lymphocyte stimulation with antigen

CD22, a B lymphocyte membrane glycoprotein, contains immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic region and recruits Src homology 2-containing protein-tyrosine phosphatase-1 (SHP-1) to the phosphorylated ITIMs upon ligation of B lymphocyte antigen receptor (BCR), thereby negatively regulating BCR signaling. Among the three previously identified ITIMs, both ITIMs containing tyrosine residues at position 843 (Tyr(843)) and 863 (Tyr(863)), respectively, are shown to be required for CD22 to recruit SHP-1 and regulate BCR signaling upon BCR ligation by anti-Ig antibody (Ab), indicating that CD22 has the SHP-1-binding domain at the region containing Tyr(843) and Tyr(863). Here we address the requirement of CD22 for SHP-1 recruitment and BCR regulation upon BCR ligation by antigen, which induces much stronger CD22 phosphorylation than anti-Ig Ab does. We demonstrate that the CD22 mutant in which both Tyr(843) and Tyr(863) are replaced by phenylalanine (CD22F5/6) recruits SHP-1 and regulates BCR signaling upon stimulation with antigen but not anti-Ig Ab. This result strongly suggests that CD22 contains another SHP-1 binding domain that is specifically activated upon stimulation with antigen. Both of the flanking sequences of Tyr(783) and Tyr(817) fit the consensus sequence of ITIM, and the CD22F5/6 mutant requires these tyrosine residues for SHP-1 binding and BCR regulation. Thus, these ITIMs constitute a novel conditional SHP-1-binding site of CD22 that is activated upon BCR ligation by antigen but not by anti-Ig Ab.     

TIM-1 signaling in B cells regulates antibody production

Members of the T cell Ig and mucin (TIM) family have recently been implicated in the control of T cell-mediated immune responses. In this study, we found TIM-1 expression on anti-IgM- or anti-CD40-stimulated splenic B cells, which was further up-regulated by the combination of anti-IgM and anti-CD40 Abs. On the other hand, TIM-1 ligand was constitutively expressed on B cells and inducible on anti-CD3⁺ anti-CD28-stimulated CD4⁺ T cells. In vitro stimulation of activated B cells by anti-TIM-1 mAb enhanced proliferation and expression of a plasma cell marker syndecan-1 (CD138). We further examined the effect of TIM-1 signaling on antibody production in vitro and in vivo. Higher levels of IgG2b and IgG3 secretion were detected in the culture supernatants of the anti-TIM-1-stimulated B cells as compared with the control IgG-stimulated B cells. When immunized with T-independent antigen TNP-Ficoll, TNP-specific IgG1, IgG2b, and IgG3 Abs were slightly increased in the anti-TIM-1-treated mice. When immunized with T-dependent antigen OVA, serum levels of OVA-specific IgG2b, IgG3, and IgE Abs were significantly increased in the anti-TIM-1-treated mice as compared with the control IgG-treated mice. These results suggest that TIM-1 signaling in B cells augments antibody production by enhancing B cell proliferation and differentiation.     

分子佐剂C3d上调Raji细胞协同刺激分子B7-1和B7-2的表达(简报)

我们在以往研究中,引入选择性增强体液免疫效应的新型分子佐剂C3d,成功构建了重组避孕疫苗hCGB-C3d3,通过免疫Th2型优势的 BALB/c小鼠和,Th1型优势的C57BL/6小鼠,显示分子佐剂C3d在不同品系小鼠均使免疫效应从Th1型细胞免疫向Th2型体液免疫偏倚。     

Signals that initiate somatic hypermutation of B cells in vitro

Somatic hypermutation is initiated as B lymphocytes proliferate in germinal centers. The signals that switch on the mutation process are unknown. We have derived an in vitro system to define signals that will initiate mutation in normal, naive splenic B cells. We find that three signals are required to allow detection of somatic mutation in vitro; these are anti-Ig, anti-CD40, and anti-CD38. If any one of these is omitted, mutation remains off. We show that CD40 is obligatory in vivo, as CD40 knockout mice exhibit no Ag-driven mutation. In contrast, CD38 is not, as CD38 knockout mice mutate normally. We believe that, in vitro, CD38, in combination with other stimuli, drives extensive cell division, allowing the detection of mutated sequences. However, in germinal centers in vivo, proliferative activity is instigated by a different molecule. This is the first demonstration of the initiation of hypermutation in vitro with normal splenic B cells using defined stimuli.     

Studies of in vitro activation and differentiation of human B lymphocytes. I. Phenotypic and functional characterization of the B cell population responding to anti-Ig antibody

Investigation of the activation of splenic B cells by anti-immunoglobulin (Ig) antibody has enabled us to characterize the anti-Ig-responsive B cell and to analyze the phenotypic changes which accompany proliferation and differentiation. The anti-Ig antibody-responsive B cell population was characterized by the expression of high levels of the B2 antigen and represented approximately 40% of splenic B cells. Brisk mitogenesis which peaked at 3 to 4 days was induced by anti-Ig antibody. The proliferative phase was characterized phenotypically by a dramatic decline in B2 antigen expression, with most cells showing no detectable B2 by 4 days post-activation. The other hallmark of this phase was de novo expression of a group of "activation antigens." These included the B cell-restricted antigens B-LAST 1, BB1, and B5, and the T cell-associated interleukin 2 receptor and T12 antigens. Concomitantly, B1, B4, and Ia expression increased, the increase being roughly proportional to the increase in cell size. After day 4, the mitogenic response progressively diminished, while Ig synthesis increased. During this differentiation phase, cell surface antigens again displayed a distinct sequence of changes. The five activation antigens and the B1, B4, and Ia antigens began to decrease. However, two markers, T10 and PCA-1, which are found on plasmacytomas, appeared and their level of expression steadily increased. These changes and the appearance of morphologically identifiable plasma cells required the presence of T cells in this system. T cell supernatants alone induced Ig secretion but did not induce expression of PCA-1 or the appearance of cells with plasma cell morphology. The culture system developed in this study has allowed us to analyze the antigenic changes following activation by anti-Ig antibody. This sequence of changes has not only permitted the identification of antigens which, by their appearance at distinct stages may have an important role in proliferation and differentiation of B cells, but also provides us with the means of studying the function of each antigen.     

B cells activated by lipopolysaccharide,but not by anti-Ig and anti-CD40 antibody,induce anergy in CD8+ T cells: role of TGF-beta 1

B cells recognize Ag through their surface IgRs and present it in the context of MHC class II molecules to CD4(+) T cells. Recent evidence indicates that B cells also present exogenous Ags in the context of MHC class I to CD8(+) T cells and thus may play an important role in the modulation of CTL responses. However, in this regard, conflicting reports are available. One group of studies suggests that the interaction between B cells and CD8(+) T cells leads to the activation of the T cells, whereas other studies propose that it induces T cell tolerance. For discerning this dichotomy, we used B cells that were activated with either LPS or anti-Ig plus anti-CD40 Ab, which mimic the T-independent and T-dependent modes of B cell activation, respectively, to provide accessory signals to resting CD8(+) T cells. Our results show that, in comparison with anti-Ig plus anti-CD40 Ab-activated B cells, the LPS-activated B cells (LPS-B) failed to induce significant levels of proliferation, cytokine secretion, and cytotoxic ability of CD8(+) T cells. This hyporesponsiveness of CD8(+) T cells activated with LPS-B was significantly rescued by anti-TGF-beta1 Ab. Moreover, it was found that such hyporesponsive CD8(+) T cells activated with LPS-B had entered a state of anergy. Furthermore, LPS-B expresses a significantly higher level of TGF-beta1 on the surface, which caused the observed hyporesponsiveness of CD8(+) T cells. Therefore, this study, for the first time, provides a novel mechanism of B cell surface TGF-beta1-mediated hyporesponsiveness leading to anergy of CD8(+) T cells.