Up-regulated recombination-activating gene expression in slg− variants of a human mature B cell line undergoing secondary Igλ, rearrangements in cell culture

To study the role of surface immunoglobulin (sIg) expression in the control of rearrangement activity at the Ig light chain loci, we established rare sIg^? clones (λ^?) from a human sIg⁺ B cell line (μl⁺). Upon expansion of these sIg^? clones, surface immunofluorescence analysis revealed a gradual emergence of sIg⁺ subpopulations, differing from the original tumor cell line both in their idiotypes and Cλ isotypes. DNA analysis revealed that this sIg heterogeneity resulted from a process of ongoing Igλ, rearrangements. That is, one of the Igλ rearrangements in the parental cell line was replaced by novel Igλ rearrangements in the sIg^? clones, which in turn were replaced by yet additional Igλ rearrangements in the sIg⁺ variants. Northern analysis demonstrated that while the expression of the recombination-activating genes RAG1 and RAG2 was relatively low in the parental cell line, their expression was significantly increased in both the sλ g⁺ variants and their sIg⁺ progenies. We thus describe a human mature B cell line, in which differential RAG expression allows sIg heterogeneity to be generated through secondary Igλ gene rearrangements. Our results indicate that the induction of RAG expression may be inversely associated with sIg expression, but that sIg expression, alone, is not sufficient to down-regulate this expression.     

In rat bone marrow Thy-1 antigen is present on cells with membrane immunoglobulin and on precursors of peripheral B lymphocytes.

Rat bone marrow cells carrying Thy-1 antigen were studied morphologically, and tested for their independence of the thymus and their relationship to the B lymphocyte lineage. Using a fluorescence-activated cell sorter to separate Thy-1⁺ and Thy-1^- fractions, it has been confirmed that up to 50 % of all nucleated bone marrow cells are Thy-1⁺, most of which have the morphology of small lymphocytes. Thy-1^-cells were mainly neutrophils and erythroid. Thy-1⁺ cells were found also in the marrow of B rats (rats thymectomized as adults, irradiated and reconstituted with syngeneic bone marrow from thymectomized donors drained of recirculating lymphocytes), though at a lower frequency (roughly half) than of normal rats. In both normal and B rats about 1/4 of the Thy-1⁺ cells also bore lymphocyte surface immunoglobulin (sIg), and these doubly labeled cells accounted for the majority (～ 2/3) of marrow cells carrying large amounts of sIg. Therefore, unlike mice, Thy-1 is not a marker of thymus-dependent lymphocytes in rats. The B precursor activity of marrow fractions was measured in a long-term re-constitution assay counting sIg⁺ cells in the thoracic duct of lethally irradiated recipients. Virtually all the precursors were in the Thy-1⁺ or sIg^- fractions, and were barely detectable among Thy-1^- or sIg⁺ cells. Thus, in the rat peripheral B lymphocytes descend from precursors bearing Thy-1 antigen but lacking sIg.     

Immature B cells from human and mouse bone marrow can change their surface light chain expression

The capacity of bone marrow-derived surface immunoglobulin-positive (sIg⁺) human and mouse immature B cells, generated either in vitro or in vivo, to change their light (L) chain expression, has been assayed by the number of cells which change in vitro from one type of L chain to the other type, or to no sIg at all. Immature sIg⁺ B cells were generated in vitro from sIg^? precursor cells from human or mouse bone marrow. The immature sIg⁺ cells expressed RAG-1. Human sIg⁺ cells expressed xfr; and λ L chains in ratios between 1:1 and 3:1, whereas in mouse cells, this ratio ranged from 10:1 to 20:1. Upon reculture of the human and mouse xfr;⁺sIg⁺ cells, about half of them remained xfr;⁺, a quarter became λ⁺, and another quarter became sIg^?. Between 1 and 3% expressed both xfr; and λ chains. Of the human λ⁺ cells, about two-thirds remained λ⁺, only 1 to 2% became xfr;⁺, while the other third became sIg^?. Again, between 1 and 3% expressed both xfr; and λ L chains. These results indicate that expression of sIgM in the B cell membrane does not terminate L chain gene rearrangement, and that some order exists in xfr; versus λ gene rearrangements. Hence, human and mouse xfr;⁺ immature B cells can become λ⁺, but very few of the λ⁺ cells can become xfr;⁺, and both can become sIg^?. Further, human CD10⁺/sIg⁺ xfr;⁺ and λ⁺ cells and mouse B220^low/sIg^low xfr;⁺ cells enriched from bone marrow, i.e. immature B cells differentiated in vivo, changed their Ig phenotype upon in vitro culture, but in lower frequencies. By contrast, human and mouse mature B cells did not change their L chain or Ig phenotype. Hence, at least a part of the sIg⁺ immature B cells in bone marrow retain the capacity to change their L chain and Ig phenotype, and this capacity is lost when they become mature, peripheral B cells.     

Localization in situ of the co-stimulatory molecules B7.1, B7.2, CD40 and their ligands in normal human lymphoid tissue

Functional interactions between B and T lymphocytes are known to depend on the expression of co-stimulatory molecules B7.1/CD80, B7.2/CD86 and their counter-receptors CD28 and CTLA4, as well as CD40 and its ligand CD40L. To study the role of these molecules in situ, an immunohistochemical analysis was carried out on normal human lymphoid tissue. In the germinal centers (GC), B7.1 and B7.2 were differentially expressed. In the dark zone, centroblasts were predominantly B7.1⁺, while centrocytes in the light zone were B7-2⁺, resulting in reversed gradients of both markers in GC. Follicle mantle cells were negative for B7.1 and B7.2. Macrophages and interdigitating dendritic cells (IDC) in T cell zones both expressed B7.1 and B7.2. Moreover, clusters of B7.2⁺ T cells were demonstrated in interfollicular areas. Intrafollicular CD4⁺ T cells in GC, predominantly in the apical light zone, expressed CD28 and CTLA4, as did the majority of interfollicular T cells. CTLA4 showed a striking excentric cytoplasmic staining, which was also seen on T cells activated in vitro. CD40 was expressed on all B cells and more strongly on macrophages and IDC. Moreover, small clusters of T cells in a rim outside the GC showed CD40 expression. CD40L was expressed both on intrafollicular CD4⁺ T cells as well as on T cells in T cell zones. The differential distribution of co-stimulatory molecules in different compartments of normal human lymphoid tissue in situ indicates that these interactions play a distinctive role in different stages of B cell differentiation and in the immune response.     

Mechanisms of clonal abortion tolerogenesis. III. Antigen abrogates functional maturation of surface immunoglobulin-negative adult bone marrow lymphocytes.

The fluorescence-activated cell sorter (FACS) was used to fractionate adult CBAmouse bone marrow cells on the basis of their size and the amount of surfaceimmunoglobulin (sIg) expressed. It was noted that a population of viable, small sIg^?cells could generate up to 28% slg⁺ cells within 24 h in tissue culture. The capacity of various cell fractions to respond to T-independent stimulation by antibody productionwas studied. Fractionated cells were placed into microculture at limiting dilution inthe presence of thymus “filler” cells, stimulated with E. coli lipopolysaccharide (LPS)and after 3 to 5 days, assayed for the presence of antibody-forming cell clones to thehapten (4-hydroxy-3-iodo-5-nitrophenyl) acetyl (NIP). Through appropriate statistical means, the number of anti-NIP precursors originally present was calculated. Whilethe sIg⁺ fraction contained approximately 70 cells in 10⁶ capable of immediate development into anti-NIP clones, the slg^? fraction contained very few reactive cells. However, the cloning capacity of this fraction increased markedly following 24 h of maturation in tissue culture. When bone marrow cells were exposed to NIP-coupled human gamma-globulin (NIP-HGG) acting as a B cell tolerogen, some tolerance was induced in vitro reflecting therelative immaturity of these cells. When the FACS-sorted sIg^? cells were similarlytreated, there was virtually complete abrogation of their functional maturation intoclonable anti-NIP precursors. The effect of LPS on cells converting from the sIg^? tosIg⁺ status was investigated. When LPS and NIP-HGG were concomitantly present, about one-third of the cells escaped tolerance induction. LPS thus “rescued” somecells from clonal abortion, but certainly did not totally abolish tolerance induction. The significance of these results for the clonal abortion theory of B lymphocytetolerance is briefly discussed. The conclusion is reached that cells converting from thesIg^? to sIg⁺ status in the normal course of differentiation represent a prime target fortolerance induction.     

Terminal B cell development as seen in different human myelomas and related disorders.

Immunological surface marker analysis of the mature lymphoid and plasma cells of a range of human lymphoproliferative diseases is used to show that the surface immunoglobulin (sIg) phenotype of plasma cells depends upon the isotype being secreted. Using a sensitive rosette assay, the plasma cells of all IgG (eight), IgA (six) and IgE (one) myelomas studied and of one of two cases of IgD myeloma were negative for all classes of sIg. The plasma cells from the other case of IgD myeloma expressed only sIgD, while a proportion of plasma cells from two cases of IgM myeloma and one of BJ myeloma had both sIgM and sIgD at the cell surface. The mature lymphoid and plasma cells from the two patients with mixed lymphoid/plasmacytoid proliferations of IgA type (2) carried sIgA alone, while the mature bone marrow cells from a case of Waldenström''s macroglobulinaemia had only sIgM. In all the various types of immunoproliferative disease studied, a greater proportion of lymphoid cells than plasma cells possessed γFc receptors, and plasma cells were negative for receptors for EAC and μFc, and lacked the Ia-like p29, 34 antigen. These results are used to provide a scheme illustrating the terminal stages of B cell development.Examination of the peripheral blood from nineteen cases without morphological evidence of blood involvement demonstrated a monoclonal B cell proliferation in 40% of these cases. Immunological analysis of lymphocyte subpopulations showed that both E-rosette-forming cells and sIgM⁺ and sIgD⁺ normal B cells were depressed in most cases and that there was a corresponding increase in Fc⁺sIg⁻ and/or null (Fc⁻sIg⁻) cells.     

Ligand-independent signaling during early avian B cell development

Surface immunoglobulin (sIg) expression has been conserved as a critical checkpoint in B lymphocyte development. In the chicken embryo, only sIg⁺ B cells are selectively expanded in the bursa of Fabricius, a primary lymphoid organ unique to the avian species. We have previously demonstrated that an interaction between the antigen-binding sites of sIg and a specific bursal ligand(s) is not required to regulate this developmental checkpoint. Rather, the requirement for sIg expression can be attributed to the surface expression of the Igα/β heterodimer associated with sIg. More specifically, ligand-independent signaling downstream of the Igα cytoplasmic domain drives all bursal stages of B cell development during embryogenesis. We discuss here a site-directed mutagenesis approach to identify the critical membrane proximal events involved in ligand-independent signaling during B cell development.     

Immunization with immune complex alters the repertoire of antigen-reactive B cells in the germinal centers

The differentiation of memory B cells in germinal centers (GC) is selectively enhanced upon administration of antigen-antibody complexes. To characterize the repertoire of this response, we examined the rearranged immunoglobulin heavy chain variable (V_H) genes from mouse splenic GC after a single immunization with either antigen, nitrophenyl (NP) hapten coupled to keyhole limpet hemocyanin, or with a preformed complex of antigen with a monoclonal anti-NP antibody of γ1 isotype. Among antigen-immunized mice, NP-reactive GC B cell populations in the antigen-induced GC consisted mostly of cells expressing the canonical V186.2 gene which contained, on average, 0.8 point mutations/V_H gene by day 8 after immunization. These results are indicative of the beginning of somatic hypermutation and consistent with previously published analyses of NP antigen-driven GC. In contrast, the NP-specific B cells in GC that were elicited by administration of immune complex represented a heterogeneous cell population expressing nine different germ-line segments of the V186.2/V3 (J558) gene family, i.e. V23, V24.8, C1H4, V3, CH10, V165.1, V102, V671.5 and V186.2. Moreover, the average frequency of mutations in these genes was 1.7, reaching up to 4 mutations/V_H in some GC. Administration of the antigen NP in complex with specific antibody apparently alters the process of interclonal competition in the GC and results in loss of dominance by V186.2⁺ cells and nearly stochastic representation of diverse clono-types. These results suggest an important feedback regulation of the B cell repertoire by antibody and indicate a role for immune complexes in the activation of somatic hypermutation.     

Normal human B cells express endogenous sheep erythrocyte (E) receptor after appropriate in vitro culture

When normal human B cells from blood, tonsil or spleen are cultured with 20–30% autologous or allogeneic T cells and mitogen, the majority of cells rosette with sheep erythrocytes (E) after 3–5 days in culture. These E⁺ cells are of transformed appearance and several points indicate that many are derived from B cells. Some simultaneously possess surface immunoglobulin (sIg) or a B cell antigen [detected with BA1 monoclonal antibody (mAb)] and E receptor. Many do not stain with anti-T cell mAb (UCHT1, OKT4, OKT8), while cultured T cells continue to express these antigens. Furthermore, many E⁺ cells appear when the original T cells have been irradiated. All the cultured E⁺ cells stain with OKT11, an mAb against the E receptor, and their positivity cannot therefore be attributed to mitogen-induced nonspecific stickiness. The E positivity of the B cells was shown to be endogenous since passive acquisition of E receptor shed by T cells was excluded in a number of ways; no phenotypic changes were observed when supernatants from cultures containing many E⁺ sIg⁺ non-T cells were added to B cells, or when the B and T cells were separated by a Millipore membrane; and E receptor was re-expressed after stripping with trypsin or pronase. The intimate presence of T cells is essential for the expression of E receptors by B cells, and this helper capacity was shown to reside within the OKT4-defined helper T cell subpopulation. The significance of the expression of E receptor by B cells is discussed in relation to the recent in vitro and in vivo demonstration of E⁺ sIg⁺ cells in certain leukemias and in relation to the specificity of E rosetting as a marker of T cells.     

Involvement of GANP in B cell activation in T cell-dependent antigen response

Adaptive immunity is dependent on proliferation of antigen-driven B cells for clonal expansion in germinal centers (GCs) against T cell-dependent antigens (TD-Ag), accompanied with somatic hypermutation of variable-region gene and class switching of B cell antigen receptors. To study molecular mechanisms for B cell differentiation in GCs, we have identified and studied a 210kDa GANP protein expressed in GC-B cells. GANP has domains for MCM3-binding and RNA-primase activities and is selectively up-regulated in centrocytes surrounded with follicular dendritic cells (FDCs) upon immunization with TD-Ag in vivo and in B cells stimulated with anti-CD40 monoclonal antibody in vitro, which suggested that GANP plays a certain important role in the maturation of immunoglobulin or selection of B cells in GC during the immune response to TD-Ag. Since this up-regulation has not been detected in T cells in GCs and in Concanavalin A-stimulated T cells in vitro, selective function of GANP molecule on B cell proliferation and differentiation might exist.     

Equine gammaherpesvirus 2 (EHV2) is latent in B lymphocytes

Summary Peripheral blood leukocytes were collected from 5 Thoroughbred horses and examined for the presence of EHV2 in sub-populations of mono-nuclear cells. Peripheral blood mononuclear cells were separated on Percoll gradients and then enriched for plastic adherent cells (predominantly mono-cytes), surface immunoglobulin positive (sIg⁺) B lymphocytes and T lymphocytes, using panning techniques. The purity of each cell population was assessed by fluorescence activated cell scanning. In an infectious centre assay, each cell population was inoculated onto equine foetal kidney monolayer cell cultures which are fully permissive for the replication of EHV2. Only enrichment for sIg⁺ B lymphocytes resulted in a marked increase in the number of infectious centres, indicating that EHV2 is present in B lymphocytes. Freeze-thawing of sIg⁺ B lymphocytes, prior to inoculation onto EFK monolayer cell cultures, resulted in the complete abrogation of infectious centre formation, confirming that EHV2 is latent in B lymphocytes i.e., infectious free virus was not present in the cells. The number of EHV2 infected B lymphocytes varied considerably between horses from 4 to 780 per 10⁶ cells. Evidence was also obtained that direct cell to cell contact between the epithelial cells and sIg⁺ B lymphocytes was necessary for the production of infectious centres. The data indicate that EHV2, like other members of theGammaherpesvirinae, is latent within B lymphocytes.     

Somatic mutations and affinity maturation are impaired by excessive numbers of T follicular helper cells and restored by Treg cells or memory T cells

We previously reported that Cd3e‐deficient mice adoptively transferred with CD4⁺ T cells generate high numbers of T follicular helper (Tfh) cells, which go on to induce a strong B‐cell and germinal center (GC) reaction. Here, we show that in this system, GC B cells display an altered distribution between the dark and light zones, and express low levels of activation‐induced cytidine deaminase. Furthermore, GC B cells from Cd3e^–/– mice accumulate fewer somatic mutations as compared with GC B cells from wild‐type mice, and exhibit impaired affinity maturation and reduced differentiation into long‐lived plasma cells. Reconstitution of Cd3e^–/– mice with regulatory T (Treg) cells restored Tfh‐cell numbers, GC B‐cell numbers and B‐cell distribution within dark and light zones, and the rate of antibody somatic mutations. Tfh‐cell numbers and GC B‐cell numbers and dynamics were also restored by pre‐reconstitution of Cd3e^–/– mice with Cxcr5^–/– Treg cells or non‐regulatory, memory CD4⁺ T cells. Taken together, these findings underline the importance of a quantitatively regulated Tfh‐cell response for an efficient and long‐lasting serological response.     

Cellular origin of human B-cell neoplasms and Hodgkin's disease based on analysis of somatic hypermutations in the immunoglobulin variable region genes

In response to antigen stimulation, B cells undergo a germinal center(GC) reaction such as somatic hypermutations of the immunoglobulin variable region genes, which results in the production and selection of antigen-specific antibodies with increased affinity. Therefore, somatic hypermutations are considered to be a hallmark of GC B cells and their descendants. Pre-GC B cells(precursor B cells, immature B cells, naive B cells and CD5+ B cells) carry no somatic hypermutations, whereas GC B cells and post-GC B cells(memory B cells and plasma cells) express somatic hypermutations. This phenomenon is useful in identifying the cellular origin of various B-cell neoplasms. Precursor B-lymphoblastic leukemia/lymphoma, mantle cell lymphoma, and most B-CLL originate from pre-GC B cells, and follicular lymphoma, Burkitt's lymphoma, marginal zone B-cell lymphoma, diffuse large B-cell lymphoma and myeloma from GC B cells or post-GC B cells. Nodular lymphocyte-predominant Hodgkin's disease and most classical types of Hodgkin's disease are derived from GC B cells. Most human-B cell neoplasms including Hodgkin's disease are derived from GC B cells or their descendants. Molecular processes that modify the DNA of GC B cells, such as somatic hypermutation, class switching and receptor editing occur in the environment of the GCs, and increase the risk of malignant transformation.     

Germinal-center organization and cellular dynamics

Germinal centers (GCs) are important sites of antibody affinity maturation. In the classical model, the GC dark zone contains large centroblasts that are rapidly proliferating and undergoing somatic hypermutation of their antibody variable-region genes. Centroblasts give rise to smaller nonproliferating centrocytes in the light zone that compete for binding antigen on follicular dendritic cells. Recently, the approach of real-time imaging of GCs by two-photon microscopy of intact lymph nodes has provided new insights into GC dynamics that both support and challenge fundamental aspects of this model. Here we review recent and older findings on cell migration, proliferation, and interaction dynamics in the GC and discuss a model in which dark- and light-zone cells are morphologically similar, proliferation occurs in both zones, and GC B cells compete for T cell help as well as antigen.     

Sites of specific B cell activation in primary and secondary responses to T cell-dependent and T cell-independent antigens.

Techniques which identify hapten-specific B cells in tissues have been used to determine the sites of B cell activation in rat spleens in response to T cell-dependent (TD) antigens and T cell-independent type-1 (TI-1) antigens. Surface-associated hapten binding by specific memory B cells and B blasts was distinguished from the strong cytoplasmic hapten binding by specific plasma cells and plasmablasts. Blast cells in S phase were identified in tissue sections by staining cells which had been pulse labeled in vivo with 5-bromo-2'-deoxyuridine. Hapten-specific B blast cells are found in three sites: (a) around interdigitating cells in the T cell-rich zones; (b) in the follicular dendritic cell network and (c) in association with macrophages in the red pulp. Hapten-binding memory B cells, which are not in cell cycle, accumulate in the marginal zones and to a lesser extent the follicular mantles in response to TD and TI-1 antigens. The hapten-specific blast response in T zones is confined to the first few days after antigen is given and is low for primary responses to TD antigens, but massive on secondary challenge, when marginal zone memory B cells migrate to the T zones. Both the primary and secondary T zone responses to TI-1 antigens are impressive and in these responses hapten-specific B blasts are also found in the splenic red pulp. The follicular response to TD antigens starts with a small number of B blasts (fewer than five) entering each follicle. These increase in number exponentially so that by the 4th day after immunization they fill the follicle. The oligoclonality of the response is shown in simultaneous responses to two haptens where 6%-31% of the follicles on day 3 after immunization contain blasts specific for only one of the two haptens. During the 4th day classical zonal pattern of germinal centers develops. The surface immunoglobulin-positive B blasts are lost from the follicle center, while one pole of the follicular dendritic cell network fills with surface immunoglobulin-negative centroblasts. Centroblasts do not increase in numbers but divide to give rise to centrocytes, which re-express sIg and migrate into the follicular dendritic cell network. Cell kinetic studies indicate that the centrocyte population is renewed from centroblasts every 7 h. Centrocytes either leave the germinal center within this time or die in situ.(ABSTRACT TRUNCATED AT 400 WORDS)     

B cell development in mice with a defective λ5 gene

The surrogate light chain encoded by the two pre-B cell-specific genes V_preB and λ₅ plays a critical role in B cell development of the mouse. It has been shown that targeted disruption of the λ ₅gene results in a depletion of B220⁺ CD43^? IgM^? pre-B cells in bone marrow, and in a delayed appearance both of CD5⁺ as well as CD5^? surface immunoglobulin (slg)⁺ B cells in the periphery. In this report we show that D_HJ_H-rearranged B220^? and B220⁺, CD43⁺, c-kit⁺, sIgM^? pro- and pre-B-I cells with long-term capacity to proliferate in vitro on stromal cells in the presence of interleukin-7 are present in normal numbers in the bone marrow of λ ₅T/λ.₅T mice at various ages. They express normal levels of V_preB mRNA but, in contrast to normal pre-B-I cells, do not express surrogate light chain on their surface. Pre-B-I cells from fetal liver and bone marrow of λ ₅T/λ ₅T mice differentiate with normal kinetics and in normal numbers to sIg⁺, mitogen-reactive B cells. These results suggest that the delayed generation of sIg⁺ B cells in the peripheral, mature compartments of CD5⁺ and CD5^? cells could be accounted for by the daily production of approximately 5 × 10⁵ sIg⁺ B cells from the pre-B-I cell pool in the absence of a normal pool of pre-B-II cells.     

Subtle differences in antibody responses and hypermutation of λ light chains in mice with a disrupted χ constant region

Analysis of λ light chain use in normal mice is made difficult by the dominant x light chain repertoire. We produced mice rendered deficient in x light chain expression by gene targeting and focused on questions concerned with the generation of λ light chain diversity. Whilst these mice compensate the x deficiency with increased λ titers, and their Ig level is therefore not significantly reduced, they show major differences in immunization titers, germinal center (GC) development and somatic hypermutation. After immunization, using antigens that elicit a restricted IgL response in normal mice, we obtained in the x^?/? mice elevated primary antibody titers but a subsequent lack in titer increase after repeated antigen challenge. Analysis of the Peyer's patches (PP) revealed a dramatically reduced cell content with rather small but highly active GC. Flow cytometric analysis showed different cell populations in the PP with enriched peanut agglutinin (PNA)^hi/CD45R(B220)⁺ B cells, implying that the apparent compensation for the lack of x light chain expression involves the GC microenvironment in cell selection, the initiation of hypermutation and high affinity expansion. The three Vλ genes, V1, V2 and Vx, are mutated in the GC B cells, but show no junctional diversity. In contrast, a reduced rate of Vλ hypermutation is found in the hybridoma antibodies, which appears to reflect a selection bias rather than structural constraints. However, mechanisms of somatic mutation and specificity selection can operate with equal efficiency on the few Vλ genes.     

p72syk protein tyrosine kinase: an early transducer of sIgG-triggered apoptotic signalling in human follicular lymphoma cells

Cross-linking of B cell antigen receptor (sIg) elicits different biological responses, including cell activation, proliferation, differentiation, anergy and cell death depending on the maturational stage of the cell. We established the tumor cell lines HF-1.3.4 and HF- 4-9 from two patients with follicular lymphoma. Both cell lines carry the characteristic t(14;18) chromosomal translocation and display constitutively overexpressed Bcl-2. HF-1.3.4 represents a mature B cell with sIgG and several somatic hypermutations in its Ig genes, while HF- 4-9 is a less mature B cell, expressing sIgM and only a few mutations in its Ig genes. Cross-linking of sIg with antibodies leads to apoptosis in HF-1.3.4 cells but not in HF-4-9 cells. Triggering of sIg induced, within seconds, identical tyrosine phosphorylation of p53/56lyn protein tyrosine kinase (PTK) and p55blk PTK in both of the cell lines; however, a prominent tyrosine phosphorylation and activation of p72syk PTK only in HF-1.3.4 cells. We conclude that p72syk PTK is of importance in relaying apoptotic signalling upon sIg cross-linking in the HF-1.3.4 cell line. Given the mature phenotype of the HF-1.3.4 cell line it serves as a model for the late negative selection during B cell ontogeny. Moreover, our results question the current concept that a constitutive overexpression of BcI-2 confers resistance to sIg ligation-induced apoptosis in lymphoma cells.      

Langerhans cells promote early germinal center formation in response to Leishmania‐derived cutaneous antigens

Efficient formation of early GCs depends on the close interaction between GC B cells and antigen‐primed CD4⁺ follicular helper T cells (T_FH). A tight and stable formation of T_FH/B cell conjugates is required for cytokine‐driven immunoglobulin class switching and somatic hypermutation of GC B cells. Recently, it has been shown that the formation of T_FH/B cell conjugates is crucial for B‐cell differentiation and class switch following infection with Leishmania major parasites. However, the subtype of DCs responsible for T_FH‐cell priming against dermal antigens is thus far unknown. Utilizing a transgenic C57BL/6 mouse model designed to trigger the ablation of Langerin⁺ DC subsets in vivo, we show that the functionality of T_FH/B cell conjugates is disturbed after depletion of Langerhans cells (LCs): LC‐depleted mice show a reduction in somatic hypermutation in B cells isolated from T_FH/B cell conjugates and markedly reduced GC reactions within skin‐draining lymph nodes. In conclusion, this study reveals an indispensable role for LCs in promoting GC B‐cell differentiation following cutaneous infection with Leishmania major parasites. We propose that LCs are key regulators of GC formation and therefore have broader implications for the development of allergies and autoimmunity as well as for future vaccination strategies.