CD40 and B cell antigen receptor dual triggering of resting B lymphocytes turns on a partial germinal center phenotype.
Journal - The Journal of experimental medicine (UNITED STATES )
Phenotypic alterations occur when resting human B lymphocytes become germinal center (GC) cells. These include the induction of surface CD38, CD95 (FAS/APO-1), and carboxy-peptidase-M (CPM), a recently described GC marker. However, the factors that govern the in vivo induction of these surface molecules on B cells remain unknown. Here, we purified resting (CD38-) human B lymphocytes from tonsils in an attempt to establish culture conditions resulting in the induction of these three GC markers. We show that interferon (IFN) alpha or IFN-gamma, as well as antibodies against the B cell antigen receptor (BCR), could induce CD38 on resting B lymphocytes, a phenomenon further enhanced by CD40 stimulation. Concomitantly, CD95 was upregulated by CD40 ligation and, to a lesser extent, by IFN-gamma. By contrast, CPM expression could be upregulated only through BCR triggering. This CPM induction was specifically enhanced by CD19 or CD40 ligation. CD40 + BCR stimulation of resting B cells with CD40 ligand-transfected fibroblastic cells in the presence of cross-linked anti-BCR monoclonal antibodies resulted in the coexpression of CD38, CD95, and CPM. As GC cells, these cells also expressed CD71, CD80 (B7.1), and CD86 (B7.2), but not CD24. However, CD10+ or CD44- B cells could not be detected in these culture conditions, suggesting that yet other signals are required for the induction of these GC markers. Consistent with a GC phenotype, CD40 + BCR-stimulated cells exhibited reduced viability when cultured for 20 h in the absence of stimulus. These results first demonstrate that cotriggering of resting B cells through BCR and CD40 induces both phenotypic and functional GC features. They also show that IFN and CD19 triggering of resting B cells specifically modulate the expression of GC markers.
|ISSN : ||0022-1007|
|Mesh Heading : ||ADP-ribosyl Cyclase Antigens, CD38 Antigens, CD40 Antigens, CD95 Antigens, Differentiation Antigens, Differentiation, B-Lymphocyte B-Lymphocytes Cell Separation Cells, Cultured Flow Cytometry Germinal Center Humans Immunologic Memory Interferon-gamma Membrane Glycoproteins Metalloendopeptidases N-Glycosyl Hydrolases Palatine Tonsil Phenotype Receptors, Antigen, B-Cell biosynthesis biosynthesis drug effects cytology pharmacology biosynthesis biosynthesis cytology|
|Mesh Heading Relevant : ||Antigens, CD metabolism biosynthesis immunology immunology metabolism|
Negative selection of human germinal center B cells by prolonged BCR cross-linking.
Journal - The Journal of experimental medicine (UNITED STATES )
The antigen receptors on T and B lymphocytes can transduce both agonist and antagonist signals leading either to activation/survival or anergy/death. The outcome of B lymphocyte antigen receptor (BCR) triggering depends upon multiple parameters which include (a) antigen concentration and valency, (b) duration of BCR occupancy, (c) receptor affinity, and (d) B cell differentiation stages. Herein, using anti-immunoglobulin kappa and lambda light chain antibodies, we analyzed the response of human naive, germinal center (GC) or memory B cells to BCR cross-linking regardless of heavy chain Ig isotype or intrinsic BCR specificity. We show that after CD40-activation, anti-BCR (kappa + gamma) can elicit an intracellular calcium flux on both GC and non-GC cells. However, prolonged BCR cross-linking induces death of CD40-activated GC B cells but enhances proliferation of naive or memory cells. Anti-kappa antibody only kills kappa + GC B cells without affecting surrounding gamma + GC B cells, thus demonstrating that BCR-mediated killing of GC B lymphocytes is a direct effect that does not involve a paracrine mechanism. BCR-mediated killing of CD40-activated GC B cells could be partially antagonized by the addition of IL-4. Moreover, in the presence of IL-4, prestimulation through CD40 could prevent subsequent anti-Ig-mediated cell death, suggesting a specific role of this combination in selection of GC B cells. This report provides evidence that in human, susceptibility to BCR killing is regulated along peripheral B cell differentiation pathway.
|ISSN : ||0022-1007|
|Mesh Heading : ||Antibodies, Anti-Idiotypic Antibody Specificity Antigens, CD Antigens, CD40 B-Lymphocyte Subsets B-Lymphocytes Cell Separation Cross-Linking Reagents Germinal Center Humans Immunoglobulin Isotypes Immunoglobulin kappa-Chains Immunoglobulin lambda-Chains Immunologic Memory Interferon-gamma, Recombinant Interleukin-2 Interleukin-4 Lymphocyte Activation Lymphocyte Depletion Receptors, Antigen, B-Cell Recombinant Proteins immunology drug effects immunology immunology immunology pharmacology pharmacology pharmacology pharmacology|
|Mesh Heading Relevant : ||immunology immunology immunology immunology|
Anti-CD40 plus interleukin-4-activated human naive B cell lines express unmutated immunoglobulin genes with intraclonal heavy chain isotype variability.
Journal - European journal of immunology (GERMANY )
Combination of anti-CD40 antibody and interleukin-4 (IL-4) induces B cell clonal expansion reminiscent of the T-dependent proliferation following antigenic challenge in vivo. We have analyzed the usage of CH genes and the presence or absence of somatic mutations within the progeny of a single human naive B cell activated with anti-CD40 + IL-4. To address this issue, single-cell cultures of naive (sIgD+) tonsillar B lymphocytes expressing the VH1-restricted G8 idiotype were set up. After culture and RNA extraction, VH1+ Ig mRNA were reverse-transcribed, amplified by polymerase chain reaction and sequenced. A single sIgD+ B cell could generate clones expressing mu, gamma 1, gamma 3, or epsilon, illustrating that the progeny of a single cell can express different isotypes in response to the same stimulus in vitro. The rate of somatic mutations affecting the immunoglobulin variable heavy chain gene was indistinguishable from the background of errors introduced by Taq polymerase.
|ISSN : ||0014-2980|
|Mesh Heading : ||Antibodies, Anti-Idiotypic Antibodies, Monoclonal Antigens, CD Antigens, CD40 Antigens, Differentiation, B-Lymphocyte B-Lymphocytes Base Sequence Cells, Cultured Humans Immunoglobulin Class Switching Immunoglobulin Heavy Chains Interleukin-4 Lymphocyte Activation Molecular Sequence Data Mutation immunology immunology biosynthesis immunology|
|Mesh Heading Relevant : ||Genes, Immunoglobulin immunology immunology immunology immunology genetics immunology|
CD40-activated surface IgD-positive lymphocytes constitute the long term IL-4-dependent proliferating B cell pool.
Journal - Journal of immunology (Baltimore, Md. : 1950) (UNITED STATES )
In vitro, B cells undergo long term proliferation when triggered through their CD40 surface molecule and in the presence of IL-4. Here, we show that cells that proliferate in this culture system lose their germinal center (GC) features and acquire or maintain non-GC markers. When separated by the magnetic cell separation system, both sIgD+ and sIgD- B cells can proliferate in this culture system, sIgD+ B cells exhibiting a higher rate of growth than sIgD- cells. Simultaneous flow cytometric measurement of sIgD and DNA content revealed that B lymphocytes can keep their sIgD after entry into cell cycle. Experiments using G8 Id-positive B lymphocytes allowed us to follow the evolution of sIgD+ and sIgD- cells in a reconstituted B cell population. Long term proliferating cells are sIgD+/sIgM(+)-derived B lymphocytes whereas the initial sIgD-/sIgM- cells are lost. Taken together, these data show that anti-CD40 + IL-4 activated sIgD+ B blasts express non-GC characteristics and that sIgD+ B cells preferentially proliferate in the CD40 system. The possible in vivo role of IL-4 + CD40 signaling is discussed.
|ISSN : ||0022-1767|
|Mesh Heading : ||Antibodies, Monoclonal Antigens, CD Antigens, CD40 Antigens, Differentiation, B-Lymphocyte B-Lymphocytes Cell Cycle Cells, Cultured Humans Immunoglobulin D Interleukin-4 Lymphocyte Activation Palatine Tonsil Receptors, Antigen, B-Cell immunology immunology immunology immunology immunology cytology|
|Mesh Heading Relevant : ||physiology physiology cytology analysis physiology analysis|