The role of CD11a/CD18-CD54 interactions in human T cell-dependent B cell activation.

The role of leukocyte function-associated Ag-1 (LFA-1, CD11a/CD18) and intercellular adhesion molecule 1 (ICAM-1, CD54) interactions in human T cell and B cell collaboration was examined by studying the effect of mAb to these determinants on B cell proliferation and differentiation stimulated by culturing resting B cells with CD4+ T cells activated with immobilized mAb to the CD3 molecular complex. In this model system, mAb to either the alpha (CD11a) or beta (CD18) chain of LFA-1 or ICAM-1 (CD54) inhibited B cell responses significantly. The mAb did not directly inhibit B cell function, inasmuch as T cell-independent activation induced by formalinized Staphylococcus aureus and IL-2 was not suppressed. Moreover, DNA synthesis and IL-2 production by immobilized anti-CD3-stimulated CD4+ T cells were not suppressed by the mAb to LFA-1 or ICAM-1. Although the mAb to LFA-1 inhibited enhancement of IL-2 production by co-culture of immobilized anti-CD3-stimulated CD4+ T cells with B cells, addition of exogenous IL-2 or supernatants of mitogen-activated T cells could not abrogate the inhibitory effects of the mAb to LFA-1 or ICAM-1 on B cell responses. Inhibition was most marked when the mAb were present during the initial 24 h in culture. Immobilized anti-CD3-stimulated LFA-1-negative CD4+ T cell clones from a child with leukocyte adhesion deficiency could induce B cell responses, which were inhibited by mAb to LFA-1 or ICAM-1. These results indicate that the interactions between LFA-1 and ICAM-1 play an important role in mediating the collaboration between activated CD4+ T cells and B cells necessary for the induction of B cell proliferation and differentiation, and for enhancement of IL-2 production by CD4+ T cells. Moreover, the data are consistent with a model of T cell-B cell collaboration in which interactions between LFA-1 on resting B cells and ICAM-1 on activated CD4+ T cells play a critical role in initial T cell-dependent B cell activation.     

CD28-independent costimulation of T cells by OX40 ligand and CD70 on activated B cells.

OX40 and its ligand (OX40L) have been implicated in T cell-dependent humoral immune responses. To further characterize the role of OX40/OX40L in T-B cell interaction, we newly generated an anti-mouse OX40L mAb (RM134L) that can inhibit the costimulatory activity of OX40L transfectants for anti-CD3-stimulated T cell proliferation. Flow cytometric analyses using RM134L and an anti-mouse OX40 mAb indicated that OX40 was inducible on splenic T cells by stimulation with immobilized anti-CD3 mAb in a CD28-independent manner, while OX40L was not expressed on resting or activated T cells. OX40L was inducible on splenic B cells by stimulation with anti-IgM Ab plus anti-CD40 mAb, but not by either alone. These activated B cells exhibited a potent costimulatory activity for anti-CD3-stimulated T cell proliferation and IL-2 production. Anti-CD80 and anti-CD86 mAbs partially inhibited the costimulatory activity, and further inhibition was obtained by their combination with RM134L and/or anti-CD70 mAb. We also found the anti-IgM Ab- plus anti-CD40 mAb-stimulated B cells exhibited a potent costimulatory activity for proliferation of and IL-2 production by anti-CD3-stimulated CD28- T cells from CD28-deficient mice, which was substantially inhibited by RM134L and/or anti-CD70 mAb. These results indicated that OX40L and CD70 expressed on surface Ig- and CD40-stimulated B cells can provide CD28-independent costimulatory signals to T cells.     

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.     

Murine B1 B cells require IL-5 for optimal T cell-dependent activation

T helper cell-driven activation of murine B cells has been shown to depend upon CD40-CD40 ligand (CD40L) interactions and a defined set of cytokines. These observations are primarily based on the use of conventional B cells obtained from the spleen. Therefore, it is presently unclear whether all mature B cell subsets found in the mouse have an equal dependence upon CD40-CD40L interactions and use the same T cell-derived cytokines. The present study tested the response of splenic follicular and marginal zone as well as peritoneal B2 and B1 B cells to Th cell stimulation. Splenic and peritoneal B cell subsets were sort purified based on CD23 expression, and cultured with rCD40L and cytokines or Th2 cells. The results demonstrate that follicular, marginal zone, and peritoneal B2 B cells require CD40-CD40L interactions and preferentially use IL-4 for optimal proliferation, differentiation, and isotype switching. In contrast, peritoneal B1 B cells use IL-5 in conjunction with CD40-CD40L interactions for maximal Th cell-dependent responses. Furthermore, B1 B cells are capable of proliferating, differentiating, and isotype switching in the absence of CD40-CD40L interactions. B1 B cells are able to respond to Th2 clones in the presence of anti-CD40L mAb as well as to Th2 clones derived from CD40L(-/-) mice. The CD40-CD40L-independent response of B1 B cells is attributable to the presence of both IL-4 and IL-5, and may explain the residual Ab response to T cell-dependent Ags in CD40L- or CD40-deficient mice, and in X-linked hyper-IgM (X-HIM) patients.     

HIV-1 Clade B Tat, but Not Clade C Tat, Increases X4 HIV-1 Entry into Resting but Not Activated CD4+ T Cells

CXCR4-using human immunodeficiency virus, type 1 (HIV-1) variants emerge late in the course of infection in >40% of individuals infected with clade B HIV-1 but are described less commonly with clade C isolates. Tat is secreted by HIV-1-infected cells where it acts on both uninfected bystander cells and infected cells. In this study, we show that clade B Tat, but not clade C Tat, increases CXCR4 surface expression on resting CD4⁺ T cells through a CCR2b-dependent mechanism that does not involve de novo protein synthesis. The expression of plectin, a cytolinker protein that plays an important role as a scaffolding platform for proteins involved in cellular signaling including CXCR4 signaling and trafficking, was found to be significantly increased following B Tat but not C Tat treatment. Knockdown of plectin using RNA interference showed that plectin is essential for the B Tat-induced translocation of CXCR4 to the surface of resting CD4⁺ T cells. The increased surface CXCR4 expression following B Tat treatment led to increased function of CXCR4 including increased chemoattraction toward CXCR4-using-gp120. Moreover, increased CXCR4 surface expression rendered resting CD4⁺ T cells more permissive to X4 but not R5 HIV-1 infection. However, neither B Tat nor C Tat was able to up-regulate surface expression of CXCR4 on activated CD4⁺ T cells, and both proteins inhibited the infection of activated CD4⁺ T cells with X4 but not R5 HIV-1. Thus, B Tat, but not C Tat, has the capacity to render resting, but not activated, CD4⁺ T cells more susceptible to X4 HIV-1 infection.     

CD40 stimulation of human peripheral B lymphocytes: distinct response from naive and memory cells

During secondary immune response, memory B lymphocytes proliferate and differentiate into Ig-secreting cells. In mice, the binding of CD40 by CD154 clearly enhances the activation and differentiation of memory B lymphocytes. In humans, the role of CD40-CD154 in the stimulation of memory B lymphocytes is not as obvious since in vitro studies reported positive and negative effects on their proliferation and differentiation in Ig-secreting cells. In this study, we examine the response of peripheral memory and naive cells in relation to the duration of CD40-CD154 interaction. We measured the proliferation and differentiation of both subsets stimulated with CD154 and IL-4 for short- (4-5 days) and long-term (>7 days) periods. Following short-term stimulation, memory B lymphocytes did not expand but represented the only subset differentiating into IgG- and IgM-secreting cells. A longer stimulation of this population led to cell death, while promoting naive B lymphocyte proliferation, expansion, and differentiation into IgM- or IgG-secreting cells. This prolonged CD40 stimulation also triggered naive B lymphocytes to switch to IgG and to express CD27 even in absence of somatic hypermutation, suggesting that these latter events could be independent. This study suggests that naive and memory B lymphocytes have distinct requirements to engage an immune response, reflecting their different roles in humoral immunity.     

Essential role of IL-21 in B cell activation, expansion, and plasma cell generation during CD4+ T cell-B cell collaboration

During T cell-B cell collaboration, plasma cell (PC) differentiation and Ig production are known to require T cell-derived soluble factors. However, the exact nature of the cytokines produced by activated T cells that costimulate PC differentiation is not clear. Previously, we reported that costimulation of purified human B cells with IL-21 and anti-CD40 resulted in efficient PC differentiation. In this study, we addressed whether de novo production of IL-21 was involved in direct T cell-induced B cell activation, proliferation, and PC differentiation. We found that activated human peripheral blood CD4(+) T cells expressed mRNA for a number of cytokines, including IL-21, which was confirmed at the protein level. Using a panel of reagents that specifically neutralize cytokine activity, we addressed which cytokines are essential for B cell activation and PC differentiation induced by anti-CD3-activated T cells. Strikingly, neutralization of IL-21 with an IL-21R fusion protein (IL-21R-Fc) significantly inhibited T cell-induced B cell activation, proliferation, PC differentiation, and Ig production. Inhibition of PC differentiation was observed even when the addition of IL-21R-Fc was delayed until after initial B cell activation and expansion had occurred. Importantly, IL-21 was found to be involved in PC differentiation from both naive and memory B cells. Finally, IL-21R-Fc did not inhibit anti-CD3-induced CD4(+) T cell activation, but rather directly blocked T cell-induced B cell activation and PC differentiation. These data are the first to document that B cell activation, expansion, and PC differentiation induced by direct interaction of B cells with activated T cells requires IL-21.     

Signaling through CD70 regulates B cell activation and IgG production

CD70, the cellular ligand of the TNF receptor family member CD27, is expressed transiently on activated T and B cells and constitutively on a subset of B cell chronic lymphocytic leukemia and large B cell lymphomas. In the present study, we used B cells constitutively expressing CD70 to study the functional consequences of signaling through CD70. In vitro, CD70 ligation with anti-CD70 mAbs strongly supported proliferation and cell cycle entry of B cells submitogenically stimulated with either anti-CD40 mAb, LPS, or IL-4. In this process, the cell surface receptors CD25, CD44, CD69, CD95, and GL7 were up-regulated, whereas the expression of CD21, CD62L, surface IgM (sIgM), and sIgD was decreased. Addition of CD70 mAb to low dose LPS-stimulated CD70-positive B cells strongly diminished IgG secretion and enhanced production of IgM. Signaling through CD70 on B cells was dependent on the initiation of both PI3K and MEK pathways. In vivo exposure to either CD70 mAb or the CD70 counterreceptor CD27 down-regulated CD62L and sIgM on CD70-positive B cells. CD70 signaling during T cell-dependent immune responses also decreased IgG-specific Ab titers. Together, the in vitro and in vivo data demonstrate that CD70 has potent reverse signaling properties in B cells, initiating a signaling cascade that regulates expansion and differentiation.     

Cultured human follicular dendritic cells. Growth characteristics and interactions with B lymphocytes.

Minced human tonsils were digested with DNase and collagenase, and lymphoid cell-depleted low density cells were cultured and grown in granulocyte-macrophage-CSF. Large, morphologically homogenous adherent cells with elongated extensions grew continuously in culture. These nonphagocytic cells appear to be related to follicular dendritic cell (FDC) as they do not have properties of monocytic lineage cells or dendritic cells and because, like FDC, 1) they express CD11b, CD14, CD29, CD40, CD54, CD73, CD74, and VCAM-1, and do not express CD11c, CD22, T cell markers, CD18, CD25 and CD45; and 2) they bind human B lymphocytes and B cell lines, but not T lymphocytes by an adhesion blocked in part by mAb to VLA-4 (CD49d). The cultured FDC also augmented B cell proliferation stimulated by anti-mu sera and/or CD40 mAb. Cultured FDC spontaneously produced low levels of IL-6, but did not produce IL-1 alpha or TNF-alpha; however, after treatment with either IFN-gamma or LPS, they produced more IL-6. The expression of CD54 (ICAM-1) was elevated by treating the cultured FDC with either TNF-alpha, IL-1 beta, IFN-gamma or granulocyte-macrophage-CSF; in contrast, IL-4 had no effect on CD54 but rather up-regulated expression of VCAM-1. IFN-gamma, unlike the other cytokines tested, increased expression of a set of markers on cultured FDC (CD54, VCAM-1, and CD14) and converted these class II-negative cells into class II+ cells. The fact that various T cell-derived cytokines have different effects on FDC suggests that the T cell products may influence the manner by which FDC stimulate B cell proliferation and maturation.     

Human B cells differentiate into granzyme B-secreting cytotoxic B lymphocytes upon incomplete T-cell help

Recently, CD4(+) T helper cells were shown to induce differentiation of human B cells into plasma cells by expressing interleukin (IL-)21 and CD40 ligand (CD40L). In the present study we show, that in the absence of CD40L, CD4(+) T cell-derived IL-21 induces differentiation of B cells into granzyme B (GzmB)-secreting cytotoxic cells. Using fluorescence-activated cell sorting (FACS) analysis, ELISpot and confocal microscopy, we demonstrate that CD4(+) T cells, activated via their T-cell receptor without co-stimulation, can produce IL-21, but do not express CD40L and rapidly induce GzmB in co-cultured B cells in an IL-21 receptor-dependent manner. Of note, we confirmed these results with recombinant reagents, highlighting that CD40L suppresses IL-21-induced GzmB induction in B cells in a dose-dependent manner. Surprisingly, although GzmB-secreting B cells did not express perforin, they were able to transfer active GzmB to tumor cell lines, thereby effectively inducing apoptosis. In contrast, no cytotoxic effects were found when effector B cells were activated with IL-2 instead of IL-21 or when target cells were cultured with IL-21 alone. Our findings suggest GzmB(+) cytotoxic B cells may have a role in early cellular immune responses including tumor immunosurveillance, before fully activated, antigen-specific cytotoxic T cells are on the spot. CD40 ligand determines whether IL-21 induces differentiation of B cells into plasma cells or into granzyme B-secreting cytotoxic cells.     

Anti-CD180 (RP105) activates B cells to rapidly produce polyclonal Ig via a T cell and MyD88-independent pathway

CD180 is homologous to TLR4 and regulates TLR4 signaling, yet its function is unclear. We report that injection of anti-CD180 mAb into mice induced rapid Ig production of all classes and subclasses, with the exception of IgA and IgG2b, with up to 50-fold increases in serum IgG1 and IgG3. IgG production after anti-CD180 injection was not due to reactivation of memory B cells and was retained in T cell-deficient (TCR knockout [KO]), CD40 KO, IL-4 KO, and MyD88 KO mice. Anti-CD180 rapidly increased both transitional and mature B cells, with especially robust increases in transitional B cell number, marginal zone B cell proliferation, and CD86, but not CD80, expression. In contrast, anti-CD40 induced primarily follicular B cell and myeloid expansion, with increases in expression of CD80 and CD95 but not CD86. The expansion of splenic B cells was due, in part, to proliferation and occurred in wild-type and TCR KO mice, whereas T cell expansion occurred in wild-type, but not in B cell-deficient, mice, indicating a direct role for B cells in CD180 stimulation in vivo. Combination of anti-CD180 with various MyD88-dependent TLR ligands biased B cell fate because coinjection diminished Ig production, but purified B cells exhibited synergistic proliferation. Anti-CD180 had no effect on cytokine production from B cells, but it increased IL-6, IL-10, and TNF-α production in combination with LPS or CpG. Thus, CD180 stimulation induces intrinsic B cell proliferation and differentiation, causing rapid increases in IgG, and integrates MyD88-dependent TLR signals to regulate proliferation, cytokine production, and differentiation.     

Distinct activation signals determine whether IL-21 induces B cell costimulation, growth arrest, or Bim-dependent apoptosis

IL-21 costimulates B cell proliferation and cooperatively with IL-4 promotes T cell-dependent Ab responses. Somewhat paradoxically, IL-21 also induces apoptosis of B cells. The present study was undertaken to more precisely define the expression of the IL-21R, using a novel mAb, and the circumstances by which IL-21 promotes B cell growth vs death. The IL-21R was first detected during T and B cell development, such that this receptor is expressed by all mature lymphocytes. The IL-21R was further up-regulated after B and T activation, with the highest expression by activated B cells. Functional studies demonstrated that IL-21 substantially inhibited proliferation and induced Bim-dependent apoptosis for LPS or CpG DNA-activated B cells. In contrast, IL-21 induced both costimulation and apoptosis for anti-CD40-stimulated B cells, whereas IL-21 primarily costimulated B cells activated by anti-IgM or anti-IgM plus anti-CD40. Upon blocking apoptosis using C57BL/6 Bim-deficient or Bcl-2 transgenic B cells, IL-21 readily costimulated responses to anti-CD40 while proliferation to LPS was still inhibited. Engagement of CD40 or the BCR plus CD40 prevented the inhibitory effect by IL-21 for LPS-activated B cells. Collectively, these data indicate that there are three separable outcomes for IL-21-stimulated B cells: apoptosis, growth arrest, or costimulation. We favor a model in which IL-21 promotes B cell maturation during a productive T cell-dependent B cell response, while favoring growth arrest and apoptosis for nonspecifically or inappropriately activated B cells.     

Human resting B lymphocytes can serve as accessory cells for anti-CD2-induced T cell activation.

Although resting B cells are poor accessory cells for signals transmitted through the TCR/CD3 complex, we report that these B cells can support T cell proliferation when T cell activating signals are delivered through CD2. This was first suggested when leucine methyl ester treatment of PBMC abolished proliferation induced by anti-CD3, but not by the accessory cell-dependent anti-CD2 mAb combination, GT2 and OKT11. Then we demonstrated that unstimulated, resting B cells could support the proliferation of both CD4+ and CD8+ T cells. Aggregated IgG inhibited proliferation, suggesting that anti-CD2 mAb bound to T cells were cross-linked by attachment to B cell FcR. Two lines of evidence suggested that lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 interaction was crucial for anti-CD2-induced proliferation. First, proliferation was blocked by mAb against these adhesion molecules. Second, intercellular adhesion molecule-1 expression rapidly increased on resting B cells after the addition of anti-CD2, but not anti-CD3. This was of interest because fixed monocytes, but not fixed B cells, were able to support the proliferative response. In contrast to lymphocyte function-associated Ag-1/intercellular adhesion molecule-1, CD28/B7 interaction was not required for anti-CD2-induced proliferation, although ligation of these molecules provided important costimulatory signals for stimulation by anti-CD3. Finally, neutralizing antibodies against IL-1 alpha, IL-1 beta, and IL-6 showed only modest inhibitory effects on T cell proliferation. The addition of IL-1 and/or IL-6 to T cells failed to substitute for accessory cells and were only partially effective with fixed B cells. Further evidence of a linkage between CD2 and CD45 isoforms was obtained. Anti-CD45RA, but not anti-CD45RO, potentiated anti-CD2-induced T cell proliferation. These studies have revealed a novel role for resting B cells as accessory cells and have documented costimulatory signals that are important for this effect. Because Ag-presentation by resting B cells to T cells generally leads to T cell nonresponsiveness, it is possible that this tolerogenic signal may be converted to an activation signal if there is concurrent perturbation of CD2 on T cells.     

Neomycin B-arginine conjugate, a novel HIV-1 Tat antagonist: synthesis and anti-HIV activities.

HIV-1 transactivating protein Tat is essential for virus replication and progression of HIV disease. HIV-1 Tat stimulates transactivation by binding to HIV-1 transactivator responsive element (TAR) RNA, and while secreted extracellularly, it acts as an immunosuppressor, an activator of quiescent T-cells for productive HIV-1 infection, and by binding to CXC chemokine receptor type 4 (CXCR4) as a chemokine analogue. Here we present a novel HIV-1 Tat antagonist, a neomycin B-hexaarginine conjugate (NeoR), which inhibits Tat transactivation and antagonizes Tat extracellular activities, such as increased viral production, induction of CXCR4 expression, suppression of CD3-activated proliferation of lymphocytes, and upregulation of the CD8 receptor. Moreover, Tat inhibits binding of fluoresceine isothiocyanate (FITC)-labeled NeoR to human peripheral blood mononuclear cells (PBMC), indicating that Tat and NeoR bind to the same cellular target. This is further substantiated by the finding that NeoR competes with the binding of monoclonal Abs to CXCR4. Furthermore, NeoR suppresses HIV-1 binding to cells. Importantly, NeoR accumulates in the cell nuclei and inhibits the replication of M- and T-tropic HIV-1 laboratory isolates (EC(50) = 0.8-5.3 microM). A putative model structure for the TAR-NeoR complex, which complies with available experimental data, is presented. We conclude that NeoR is a multitarget HIV-1 inhibitor; the structure, and molecular modeling and dynamics, suggest its binding to TAR RNA. NeoR inhibits HIV-1 binding to cells, partially by blocking the CXCR4 HIV-1 coreceptor, and it antagonizes Tat functions. NeoR is therefore an attractive lead compound, capable of interfering with different stages of HIV infection and AIDS pathogenesis.     

IL-21 induces differentiation of human naive and memory B cells into antibody-secreting plasma cells

IL-21 is a type I cytokine that influences the function of T cells, NK cells, and B cells. In this study, we report that IL-21 plays a major role in stimulating the differentiation of human B cells. When human B cells were stimulated through the BCR, IL-21 induced minimal proliferation, IgD down-modulation, and small numbers of plasma cells. In contrast, after CD40 engagement, IL-21 induced extensive proliferation, class switch recombination (CSR), and plasma cell differentiation. Upon cross-linking both BCR and CD40, IL-21 induced the largest numbers of plasma cells. IL-21 drove both postswitch memory cells as well as poorly responsive naive cord blood B cells to differentiate into plasma cells. The effect of IL-21 was more potent than the combination of IL-2 and IL-10, especially when responsiveness of cord blood B cells was examined. IL-21 costimulation potently induced the expression of both B lymphocyte-induced maturation protein-1 (BLIMP-1) and activation-induced cytidine deaminase as well as the production of large amounts of IgG from B cells. Despite the induction of activation-induced cytidine deaminase and CSR, IL-21 did not induce somatic hypermutation. Finally, IL-2 enhanced the effects of IL-21, whereas IL-4 inhibited IL-21-induced plasma cell differentiation. Taken together, our data show that IL-21 plays a central role in CSR and plasma cell differentiation during T cell-dependent B cell responses.     

CD19 regulation of human B cell responses. B cell proliferation and antibody secretion are inhibited or enhanced by ligation of the CD19 surface glycoprotein depending on the stimulating signal used.

The regulation of human B cell proliferation and differentiation by the CD19 surface glycoprotein was investigated. As expected, proliferation induced by costimulation with anti-IgM plus IL-4 or IL-2, or with G28.8 antibody plus IL-4 was inhibited by antibody ligation of CD19. In contrast, proliferation of tonsillar B cells to mitogenic doses of PMA (5 ng/ml) or to EBV were enhanced, and proliferation of B cell lines to BCGF(low) was unaffected. Similarly, specific antibody responses by tonsillar B cells to influenza virus, and Ig secretion by the CESS lymphoblastoid cell line in response to IL-6 were inhibited, whereas polyclonal Ig production in response to EBV was enhanced. These results show that human B cell responses may be inhibited or enhanced by CD19 depending on the stimulating signal used. The difference in response to CD19 ligation did not depend on whether proliferation or differentiation was being measured, or whether stimulation was by surface Ig. In experiments using PMA as a T cell independent mitogen, it was found that ligation of CD19 inhibited proliferation of B cells costimulated with low doses of PMA plus G28.5 (CD40) antibody, but enhanced the response to higher (mitogenic) doses with or without costimulation with G28.5. The change from inhibition to enhancement occurred over a very small increase in PMA dose (0.5-1.0 ng/ml) that corresponded exactly to the lowest dose required for mitogenic activity. Finally, we showed that CD19 ligation inhibited the increase in surface expression of CD23, but not IgM, induced by IL-4, showing that CD19 ligation can have opposed effects on different responses to the same signal. Together our results suggest that CD19 activation of human B cells interacts with other signaling events to enhance or inhibit the subsequent response.     

IgM and IgG but not cytokine secretion is restricted to the CD27+ B lymphocyte subset.

In a recent study we reported that CD27 is expressed on a subpopulation of human B lymphocytes and presented circumstantial phenotypic evidence that CD27 expression may be acquired late during B cell differentiation. Here we present functional data showing that, after in vitro stimulation, CD27+ but not CD27- B cells secrete large amounts of both IgM and IgG. Using double immunofluorescence staining of CD27 and IgD, three functionally different B cell subsets representing distinct stages of B cell differentiation can be isolated: 1) the CD27- IgD+ B cells, which do not secrete appreciable Ig; 2) the CD27+IgD+ B cells, which exclusively secrete IgM; and 3) the CD27+IgD- B cells, which comprise the IgG-producing cells. Furthermore, costimulation of CD27- B cells with low m.w. B cell growth factor, in the presence or in the absence of a CD40 mAb, does not induce these cells to become Ig-secreting cells. Although CD27- B cells hardly secrete Ig of any isotype in response to Staphylococcus aureus+IL-2, these cells proliferate vigorously and express the IL-2R alpha chain (CD25) under these stimulatory conditions. Furthermore, both CD27- and CD27+ B cells are capable of producing similar amounts of IL-6 and TNF-alpha. Taken together, these findings indicate that CD27 is a unique non-Ig surface marker discriminating naive from primed B lymphocytes. Furthermore, the capacity to proliferate and to secrete the B cell differentiation factors IL-6 and TNF-alpha already exists at an early B cell differentiation stage at which the cells lack CD27 expression and are not induced to produce Ig.