CD40 signaling induces B cell responsiveness to multiple members of the
chain-common cytokine family
Philip Griebel,
Terry Beskorwayne,
Anne Van den Broeke1 and
Giorgio Ferrari2
Veterinary Infectious Disease Organization, 120 Veterinary Road, Saskatoon, Saskatchewan S7N 5E3, Canada
1 Institut Bordet, University of Brussels, 121 Boulevard de Waterloo, 1000 Brussels, Belgium
2 Basel Institute for Immunology, 487 Grenzacherstrasse, Basel 4005, Switzerland
Correspondence to:
P. J. Griebel
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Abstract
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CD40 signaling induces B cell proliferative and differentiation responses that can be modulated by many different cytokines. Cytokines in the IL-2 receptor
chain (
c)-common family are known to play an integral role in B cell development. Therefore, we investigated the possibility that CD40 signaling induced B cell responsiveness to multiple
c-common cytokines and that individual
c-common cytokines induced distinct B cell responses. B cells were isolated from lymphoid follicles of sheep Peyer's patches (PP) and co-cultured with murine CD40 ligand (mCD40L). CD40 signaling induced PP B cell responsiveness to recombinant human IL-2, IL-4, IL-7 and IL-15. mCD40L-induced B cell growth was enhanced by combining IL-4 with a second
c-common cytokine and sustained B cell growth required co-stimulation with IL-4 plus IL-2, IL-7 and IL-15.
c-common cytokine responsiveness remained dependent upon CD40 signaling, and removal of mCD40L resulted in B cell differentiation and cell death. Similar proliferative responses to mCD40L and
c-common cytokines were observed for both immature (ileal) and mature (jejunal) PP B cells. Finally, the capacity of CD40-activated B cells to respond to multiple
c-common cytokines was analyzed with individual PP B cell clones. All B cell clones displayed similar proliferative responses to IL-2 but quantitatively different responses to IL-4, IL-7 and IL-15. The biological significance of B cell responsiveness to multiple
c-common cytokines is discussed.
Keywords: B lymphocytes, CD40, CD40 ligand,
chain-common cytokines, IL-2, IL-4, IL-7, IL-15, Peyer's patch, sheep
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Introduction
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The CD40 molecule and its ligand, CD40L, play a central role in T cell-dependent B cell development (reviewed in 1). The use of mAb, specific for the CD40 molecule, revealed that co-signaling by CD40 and IL-4 could support the long-term growth of human B cells (2). Subsequently, the CD40L gene was cloned and recombinant CD40L protein has been used to characterize the role of CD40 signaling during B cell development. This culture system has provided a model to investigate T cell-dependent B cell development. These investigations determined that many different cytokines modulate B cell proliferation or differentiation following CD40 signaling. However, the activity of each cytokine may vary depending upon the population of responding B cells or the provision of co-signals by other cell surface receptors (1).
Several cytokines have been grouped as a family on the basis of a common receptor complex protein, the IL-2 receptor
chain (
c). This cytokine family is referred to as the
c-common family, and includes IL-2, IL-4, IL-7, IL-9, IL-13 and IL-15 (reviewed in 3). The
c protein belongs to the hematopoietin receptor superfamily and the
c gene is located on the X chromosome. An essential role for the
c protein in lymphopoiesis was clearly demonstrated by human X-linked severe combined immunodeficiency, which is associated with a defect in the
c gene (4), and
c gene-targeted mice, which display a severe disruption of normal lymphopoiesis (5). Within this family of cytokines, IL-2, IL-4, IL-7, IL-13 and IL-15 influence either B cell growth or differentiation. IL-7 is essential for the T cell-independent growth of pre-B cells (6), while IL-2 (7), IL-4 (2), IL-13 (8) and IL-15 (9) influence T cell-dependent B cell proliferation and differentiation. The receptor for each
c-common cytokine may activate more than one intracellular signaling pathway and some cytokines may signal independently of the
c protein (10,11).
Cytokines may function either synergistically or in an additive manner to support B cell proliferative responses induced by CD40 signaling (reviewed in 1). Thus, we investigated the possibility that CD40 signaling may induce co-expression of receptors for multiple cytokines. The present investigation focused specifically on the
c-common cytokines that play a critical role in B cell proliferation (5,12). The B cells used for this investigation were isolated from the ileal and jejunal Peyer's patch (PP) of young lambs. The ileal PP consists of a large number of lymphoid follicles that contain immature, rapidly dividing, sIgM+ B cells and <0.5% CD4+ T cells (reviewed in 13). In contrast, lymphoid follicles of the jejunal PP of lambs contain mature, isotype-switched B cells and numerous CD4+ T cells (14). Lymphoid follicles can be isolated from the sheep PP and cell suspensions containing only sIgM+ B cells can be prepared from the ileal PP (15). The majority of cultured ileal PP B cells die by apoptosis within 24 h (16) but CD40 signaling, induced by recombinant murine CD40L (mCD40L), prevents much of this cell death (14). Furthermore, CD40 signaling induced IL-2 receptor (IL-2R; CD25) expression and recombinant human IL-2 supported the short-term growth of ileal PP B cells (14).
The cross-species activity of each human
c-common cytokine was confirmed before we investigated the capacity of the cytokines, either alone or in combination, to induce B cell proliferation. To determine if the state of B cell differentiation altered the effects of CD40 signaling we compared the responses of immature (ileal PP) B cells and mature, isotype-switched (jejunal PP) B cells. Finally, cloned PP B cell populations were used to clearly demonstrate that CD40 signaling induced B cell responsiveness to multiple
c-common cytokines.
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Methods
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Animals and cells
All experiments were conducted using 6 - to 10-week-old, Swiss White Alpine (Versuchbetrieb Sennweid, Olsburg, Switzerland) or Suffolk (Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, Canada) lambs. Peripheral blood mononuclear cells (PBMC) were isolated following the protocol described by Dudler et al. (17), and lymphoid follicles were isolated from the ileal and jejunal PP of lambs following the protocol described by Griebel (15). Cell suspensions prepared from ileal PP follicles consisted of >97% sIgM+ B cells and <0.5% T cells, and cell suspensions prepared from jejunal PP follicles consisted of ~80% B cells and 20% CD4+ T cells (14). Jejunal PP B cells are a mixture of sIgM+, sIgG1+ and sIgA+ B cells, and 57% plasmablasts (14). Cell number was counted electronically with a Coulter Counter ZM (Coulter, Hialeah, FL). Transfected J558L cells, expressing mCD40L on the cell surface, were provided by Peter Lane (Basel Institute for Immunology, Basel, Switzerland). mCD40L interacts specifically with CD40 on sheep B cells and
-irradiated mCD40L cells do not proliferate but maintain high levels of surface mCD40L (14). Jejunal PP B cells that had been co-cultured for 3 months with mCD40L and recombinant human IL-2 + IL-4 + IL-7 + IL-15 were cloned by limiting dilution, using similar culture conditions. Each B cell clone expressed a single Ig heavy chain and light chain isotype, and a unique heavy chain V gene (Griebel and Hein, in preparation).
Cytokines
Recombinant human IL-2, IL-4, IL-7 and IL-15 were purchased from Peprotech (London, UK). Cross-species activity with sheep lymphocytes has been reported for human IL-2 (18) and IL-7 (19). Cross-species activity was confirmed by assaying human cytokines for the induction of a dose-dependent proliferative response by sheep PBMC. Recombinant human IL-2, IL-7 and IL-15 induced maximal proliferative responses at concentrations between 1 and 5 ng/ml. Biological activity of human IL-4 was detected as a dose-dependent suppression of an IL-2-induced proliferative response (data not shown). Maximum IL-4 activity was observed at a final concentration of 10 ng/ml. Therefore, the recombinant human cytokines were used at a final concentration of 10 ng/ml in all subsequent experiments as this was deemed to be a saturating concentration of cytokine.
Cell culture
PP B cells and B cell clones were cultured in AIM-V (Gibco/BRL, Burlington, ON) supplemented with 2x105 M 2-mercaptoethanol (Sigma) and 2% FBS (Gibco/BRL). The mCD40L cells were cultured as described previously (14). All cultures were incubated at 37°C in a humidified atmosphere with 7% CO2 in air. Proliferative response assays with sheep PBMC (2x105 cells/well) and cloned PP B cells (2x104 cells/well) were conducted in flat-bottom, 96-well tissue culture plates (Costar, Cambridge, MA) in a final volume of 200 µl. During the last 4 h of incubation the cultures were pulsed with 0.4 µCi [3H]thymidine (Amersham, Little Chalfont, UK). [3H]Thymidine incorporation was determined using standard methods for cell harvesting and liquid scintillation counting. Long-term cell cultures were maintained in six-well culture dishes (Nunc, Roskilde, Denmark) in a final volume of 5 ml AIM-V and cytokines were added at a final concentration of 10 ng/ml. The PP B cells were transferred to a new culture every 24 days, and re-stimulated with fresh medium,
-irradiated mCD40L cells and cytokines. At the beginning of each culture period the irradiated mCD40L cell was added at a 1:10 ratio (mCD40L:B cells). This frequency of CD40L cells in ileal PP B cell cultures induced optimal proliferative responses, IL-2 responsiveness and rescue from apoptosis (14).
Flow cytometry and immunohistochemistry
mAb specific for sheep IgM (PIg45A), IgG1 (BIg715A), IgA (BIg312D3) and MHC class I (H58A) mAb were purchased from VMRD (Pullman, WA). The CD5 [ST1a (20)] and CD4 [17D-13 (21)] mAb were produced from hybridomas maintained at the Basel Institute for Immunology (Basel, Switzerland). The SIC4.8R mAb recognizes a molecular complex expressed on immature PP B cells and was produced in our laboratory (22). FITC- and phycoerythrin (PE)-conjugated, isotype-specific goat anti-mouse Ig antibodies were purchased from Southern Biotechnology Asociates (Birmingham, AL). FITC- and PE-conjugated, isotype-specific secondary antibodies were used for single- and dual-color analyses of cell-surface antigens with a FACScan flow cytometer, using the Lysys II program (FCM; Becton Dickinson, Mountain View, CA). Cell viability was analyzed by FCM using propidium iodide (PI) exclusion (2.5 µg/ml; Calbiochem, La Jolla, CA) and forward angle light scatter (FALS) as described previously (14). Phenotype analysis of cultured cells was restricted to the viable cell population. The preparation of cytospots and the detection of cytoplasmic and surface Ig by indirect immunoperoxidase with biotinylated, isotype-specific goat anti-mouse Ig antibodies (Southern Biotechnology) and the Vectastain Elite ABC kit (Dimension, Mississauga, Canada) was as previously described (23). Non-specific staining of cells was evaluated with an isotype-matched, irrelevant mAb (CD4; IgG1 isotype).
Statistics
Data for proliferative responses are presented as the mean ± SD of values from five replicate cultures. Data for viable cell number are presented as the mean of values from at least three independent experiments. Statistical analysis of differences in viable cell number and proliferative responses was performed using GraphPad Prism 2.01 software (Graphpad Software, San Diego, CA). A one-way ANOVA followed by Tukey's comparison of the means was used for comparison of multiple groups. Where applicable, differences between two groups were analyzed by using a two-tailed t-test.
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Results
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c-common cytokines do not directly influence ileal PP B cell growth or survival
Human IL-2 is not directly mitogenic and does not alter sheep ileal PP B cell survival (24) but there was no information regarding these activities for IL-4, IL-7 or IL-15. A 48 h proliferative response assay revealed that none of the
c-common cytokines, either alone or in combination, were directly mitogenic for ileal PP B cells (Fig. 1a
). Furthermore, none of the
c-common cytokines altered ileal PP B cell survival since viable B cell number declined throughout a 6-day culture period (Fig. 1b
). Thus, as observed for the IL-2R (14), ileal PP B cells probably did not express functional receptors for
c-common cytokines and were not responsive to
c-common cytokines.

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Fig. 1. Ileal PP B cells do not respond to c-common cytokines. (a) All cytokines were assayed at a final concentration of 10 ng/ml with 2x105 B cells/well during a 48 h incubation period. Dose titration experiments with peripheral blood lymphocytes indicated that this was a saturating concentration. Data presented are the mean ± SD of values from five replicate cultures and are representative of data from three independent experiments. (b) All cytokines were assayed at a final concentration of 10 ng/ml and cultures were initiated with 10x106 B cells/well. Triplicate cultures were established, and cells were harvested after 2, 4 and 6 days. Total cell number was counted with a Coulter counter and the percent viable cells determined by flow cytometric analysis of FALS and PI exclusion. Data presented are the mean of values from three independent experiments and the coefficient of variation for the SD of mean values was <18%.
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mCD40L induces B cell responsiveness to
c-common cytokines
Ileal PP B cells express functional IL-2R [CD25 (14)] following co-culture with mCD40L. Therefore, we used the same system to determine if mCD40L induced responsiveness to other
c-common cytokines. Co-culture with mCD40L induced responsiveness to all
c cytokines assayed but two distinct B cell responses were evident (Fig. 2a
). IL-2 and IL-15 supported a transient increase in viable B cell number that persisted for 20 days. In contrast, IL-4 and IL-7 did not support increased B cell numbers but, when compared to mCD40L stimulation alone, the addition of IL-4 and IL-7 prolonged B cell survival for an additional 10 days. These observations suggested that CD40 signaling induced responsiveness to multiple
c-common cytokines. Further support for this conclusion was provided by the interaction between IL-4 and other
c-common cytokines (Fig. 2b
). Co-stimulation with mCD40L and IL-2 + IL-4 induced a 25-fold increase in B cell number and enhanced B cell growth was also observed when IL-4 was combined with either IL-7 or IL-15. This apparent interaction between
c-common cytokines was further explored by combining multiple cytokines with IL-4. No further enhancement of B cell growth was observed except when IL-4 was combined with IL-2, IL-7 and IL-15 (Fig. 2c
). Following a 1520 day lag period, this combination of cytokines supported a 25-fold increase in viable B cell number. This complex interaction among the four
c-common cytokines provided further evidence that each cytokine induced a unique response in the ileal PP B cell population.
IL-4 is an essential co-factor for CD40L-induced growth of ileal PP B cells
IL-4, in conjunction with CD40 signaling, can sustain the long-term growth of human B cells (2) but this was not evident with sheep PP B cells. Therefore, we investigated the possibility that IL-4, when combined with multiple
c-common cytokines, could sustain ileal PP B cell growth. Co-stimulation with mCD40L and IL-2 + IL-7 + IL-15 induced a transient increase in viable B cell number. However, the addition of IL-4 to these co-cultures resulted in >100-fold increase in viable B cells number during a 70 day culture period (Fig. 3
). Repeated CD40 signaling was essential to maintain this B cell proliferative response to
c-common cytokines. The transfer of B cells, without the addition of mCD40L, arrested B growth and after 36 days viable B cell number declined rapidly despite the addition of
c-common cytokines (Fig. 3
). These observations confirmed that CD40 signaling was essential to maintain B cell responsiveness to
c-common cytokines.
The phenotype of the B cells responding to mCD40L and
c common cytokines was characterized with immunohistochemistry and flow cytometry. The majority of cells in the long-term cultures were lymphoblasts with a high nuclear:cytoplasmic ratio (Fig. 4a
) and most cells expressed surface IgM (Fig. 4b
). However, 6 days after culturing B cells, in the absence of mCD40L cells, the majority of B cells had differentiated to plasmablasts with a prominent clear perinuclear zone (Fig. 4e
) and cytoplasmic staining for IgM (cIgM) [Fig. 4f
; 86.8 ± 6.4% cells were cIgM+ (mean ± SD of three cultures)]. B cells co-cultured with CD40L were also stained for IgG1 and IgA (Fig. 4c and d
respectively) to confirm the specificity of IgM staining. However, only rare B cells were stained by these isotype-specific mAb. Staining for IgG1 (Fig. 4g
) and IgA (Fig. 4h
) after CD40L was removed from B cell cultures revealed that <3.0% plasmablasts had switched Ig isotype. Thus, co-stimulation with mCD40L and
c-common cytokines did not induce significant levels of isotype-switching, and loss of CD40 signaling was associated with B cell differentiation to plasmablasts and cell death (Fig. 3
).

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Fig. 4. Phenotype of ileal PP B cells cultured in the presence and absence of mCD40L. (a) Ileal PP B cells co-cultured with mCD40L and c-common cytokines (IL-2 + IL-4 + IL-7 + IL-15) were lymphoblasts characterized by a large nucleus and a narrow margin of basophilic cytoplasm. (b) Most B cells present in co-cultures with mCD40L and c-common cytokines display surface membrane staining specific for IgM. (c) Few B cells present in co-cultures with mCD40L and c-common cytokines display surface membrane staining specific for IgG1. (d) No B cells present in co-cultures with mCD40L and c-common cytokines had detectable surface membrane staining specific for IgA. (e) Many of the ileal PP B cells, when cultured for 6 days without mCD40L but in the presence c cytokines, displayed abundant cytoplasm and a prominent clear, perinuclear zone (arrows), typical of plasmablasts. (f) Most B cells, when deprived of mCD40L, display cytoplasmic staining specific for IgM. (g) Few B cells, when deprived of mCD40L, displayed cytoplasmic staining specific for IgG1. (h) Rare B cells, when deprived of mCD40L, displayed cytoplasmic staining specific for IgA. Cytospots for (a)(d) were prepared with ileal PP B cells co-cultured for 37 days with mCD40L and IL-2 + IL-4 + IL-7 + IL-15. Cytospots for (e) and (f) were prepared using the same B cell culture incubated for a further 6 days in the absence of mCD40L but in the presence of IL-2 + IL-4 + IL-7 + IL-15. Cytospots for cell morphology were stained with MayGrunwald Giemsa (a and e). Cytopsots stained with an isotype-matched, irrelevant mAb (CD4; IgG1 isotype) were negative (data not shown). Magnification: x50.
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Mature and immature B cells proliferate in response to
c-common cytokines
B cell populations isolated from different lymphoid tissues or at different stages of development display distinct responses to co-stimulation by CD40L and exogenous cytokines (1). Thus, the responsiveness of ileal PP B cells to multiple
c-common cytokines may reflect the unique developmental state of this immature B cell population (13). To test this hypothesis we compared the growth response of ileal PP B cells with B cells isolated from lymphoid follicles of the jejunal PP. Jejunal PP B cells are a heterogeneous population with a large number of isotype-switched B cells (Table 1
). Preliminary experiments confirmed that co-stimulation with mCD40L and human IL-2 + IL-4 + IL-7 + IL-15 was required to sustain jejunal PP B cell growth for >60 days (data not shown). A direct comparison of jejunal and ileal PP B cell growth responses was then made using cells isolated from the same animal. Similar growth curves were observed for both B cell populations throughout a 4-month culture period (Fig. 5
).
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Table 1. Phenotype of ileal and jejunal PP B cells before and after long-term co-culture with mCD40L and IL-2 + IL-4 + IL-7 + IL-15
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Flow cytometric analysis, using a mAb specific for sheep MHC class I, confirmed that the viable cells in long-term cultures were of sheep origin and not CD40L-expressing myeloma cells (Table 1
). These analyses also revealed substantial phenotypic differences between the ileal and jejunal PP B cell populations maintained in long-term cultures. Cells in the ileal PP cultures were predominantly sIgM+ B cells but the jejunal PP cultures consisted of an equal mix of sIgM+ and sIgG+ B cells (Table 1
). sIgA+ B cells were not maintained in the jejunal PP culture. B cells in both ileal and jejunal PP cultures also displayed some similar phenotypic changes, such as increased CD5 expression and loss of the SIC4.8R antigen, which is expressed on immature PP B cells. Furthermore, in both cultures there was a complete absence of CD4+ T cells. This observation supported the conclusion that the
c-common cytokines were directly stimulating B cell proliferation.
B cells respond to multiple
c-common cytokines
The experiments with ileal and jejunal PP cells indicated that CD40 signaling induced B cell responsiveness to multiple
c-common cytokines. However, it could be argued that functionally distinct B cell subpopulations were proliferating in response to individual
c-common cytokines. Cloned B cells provided a tool to confirm a direct interaction between B cells and
c-common cytokines, and to clearly establish that a single B cell could respond to multiple
c-common cytokines. B cell clones were established from a jejunal PP culture that had been co-stimulated with mCD40L and IL-2 + IL-4 + IL-7 + IL-15 (Fig. 5
). The proliferative responses of sIgM+ (Clone 2 and 13) and sIgG1+ (Clone 1 and 4) B cells were assayed for individual
c-common cytokines, either alone or in combination with IL-4. There was not a significant difference among clones when responses to IL-2 were compared but significantly different proliferative responses to IL-4 (Clone 2), IL-7 (Clone 1) and IL-15 (Clone 13) were observed (Fig. 6
). These experiments confirmed that B cells respond to multiple
c-common cytokines and can differ in their capacity to respond to individual
c-common cytokines. This was most apparent for IL-7 which induced a significant proliferative response in only one (Clone 1) of the four clones. Finally, stimulation of B cell clones with IL-4 and a second
c-common cytokines did not significantly increase the level of B cell proliferation. This observation confirmed that human IL-4, by itself, induced maximal PP B cell proliferation. Thus, the sustained ileal PP B cell growth response, induced by multiple
c-common cytokines, cannot be explained by an additive mitogenic activity or by cytokine cross-reactivity with a single cytokine receptor.
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Discussion
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Recombinant CD40L protein has been used in a variety of culture systems to demonstrate that CD40 signaling alters B cell responsiveness to numerous cytokines. These cytokines have included IL-1 (25), IL-2 (7,25), IL-4 (2,7,25), IL-5 (7,25), IL-10 (7, 26), IL-13 (8) and IL-15 (9). Several of these studies demonstrated that combining IL-4 with other cytokines, such as IL-1, IL-5, IL-10 or IFN-
, had additive or synergistic effects on B cell proliferation. These observations suggested that CD40 signaling might induce co-expression of receptors for multiple cytokines. Previously, this possibility had not been rigorously examined for a single cytokine family such as the
c-common cytokines. IL-4 can induce the expression of IL-2R (CD25) on B cells (27) and this may explain, in part, the synergistic growth response of sheep PP B cells when co-stimulated with IL-2 + IL-4. However, the present investigation provides evidence that CD40 signaling, in the absence of exogenous cytokines, induced responsiveness to at least four
c-common cytokines (Fig. 6
). Each
c-common cytokine had a distinct effect on B cell growth or survival and the
c-common cytokines acted synergistically to support B cell growth (Figs 2 and 3
). These observations provide indirect evidence that each
c-common cytokine interacted with a specific receptor and that signaling from multiple
c-common cytokine receptors was required to sustain B cell growth. Further studies will be required to determine if these
c-common cytokine receptors activate the same or distinct second messenger signaling pathways.
PP B cell responsiveness to multiple
c-common cytokines raises intriguing questions regarding the possible coordinated regulation of
c-common receptor expression and the biological relevance of this co-expression. CD40 signaling activates multiple protein kinases (28) in human B cells and alters numerous PP B cell functions, including the regulation of cell death, the induction of cytokine responsiveness and altered signaling by surface Ig (14). Thus, co-expression of multiple cytokine receptors may be a reflection of this complex signaling pathway rather than coordinated gene regulation. Alternatively, as suggested by the experiments with B cells clones (Fig. 6
), there may be B cell subpopulations with varying capacities to respond to individual
c-common cytokines. Banchereau et al. (1) suggested that there may be multiple ligands for CD40 and that CD40 signaling may play a much broader role in normal hematopoiesis. CD40L induction of multiple
c-common cytokine receptors on PP B cells may be consistent with this hypothesis. The PPs of young sheep have been characterized as a site of intense B lymphopoiesis with a mitotic index 5 times greater than the thymus (29). The cytokines assayed in the present experiments may be derived from T cells or non-lymphoid sources such as mast cells or stromal cells. Thus,
c-common cytokines may function to amplify both the T cell-dependent and T cell-independent B cell proliferation that is thought to occur in PP follicles (13). IL-2- and IL-4-producing cells have been identified in the corona of germinal center follicles (30), and stromal cells can produce soluble factors that amplify the proliferation of CD40-activated B lymphocytes (31). Thus, it would be of considerable interest to determine if multiple
c-common cytokines are produced within lymphoid follicles of the PP and to identify the cellular source of these cytokines.
IL-4 was an essential cytokine for maintaining the long-term growth of PP B cells, and CD40 signaling was required to induce IL-4 responsiveness in freshly isolated PP B cells (Fig. 2
) and long-term cultured B cells (Fig. 3
). CD40 signaling induces human B cells to proliferate in response to IL-4 and the mitogenic activity of IL-4 can be amplified by the addition of other cytokines. IL-2 enhancement of IL-4-induced proliferation has been noted for human B cells isolated from germinal centers (32) and tonsils (7,25). There are no previous reports of IL-4 enhancing either IL-7- or IL-15-induced B cell proliferation but IL-15 and IL-2 have very similar effects on CD40-activated B cells (9). A major difference between human and sheep B cells was the observation that IL-4 was not mitogenic for freshly isolated PP B cells that were co-stimulated with mCD40L (Fig. 2
). IL-4 also lacked mitogenic activity when assayed with mCD40L-stimulated jejunal PP and blood B cells of sheep (data not shown). However, cloned PP B cells displayed marked proliferative response to recombinant human IL-4 that were equal to or exceeded the responses to other
c-common cytokines (Fig. 6
). Thus, the lack of IL-4 activity with freshly isolated B cells cannot simply be explained by limited cross-species activity of this cytokine. The B cell proliferative responses to IL-4 and IL-7, together with changes in B cell phenotype during long-term culture (Table 1
), suggest that repeated CD40 signaling may alter B cell function and/or there may be a biased selection of B cells during the cloning procedure. Thus, caution should be exercised when extrapolating observations from the cloned B cells to cells present within lymphoid follicles of the PP.
The present investigation indicated that CD40 signaling prevented terminal B cell differentiation. B cell differentiation to plasmablasts occurred only after mCD40L cells were no longer present in the cultures. The sIgM+ ileal PP B cells did not undergo isotype-switching following mCD40L stimulation (Fig. 4
). Thus, there was no evidence that CD40 signaling, in conjunction with
c-common cytokines, induced isotype-switching. A large number of cIgG1+ plasmablasts were only observed when mCD40L cells were removed from jejunal PP cultures that contained numerous sIgG1+ B cells (data not shown). These observations may be consistent with the suggestion that CD40 signaling plays an important role in the generation of memory B cells and that memory B cells and naive B cells display very different responses to CD40 signaling (33,34). This hypothesis suggests that CD40 signaling induces naive B cells to become lymphoblasts and induces memory B cell differentiation to plasma cells. If this is correct, then the B cells proliferating in long-term sheep PP cultures should have arisen from naive B cells. The expansion of sIgM+ B cells from the ileal PP would seem to support this hypothesis. However, the growth of both sIgM+ and sIgG+ B cells from jejunal PP follicles is more difficult to explain (Table 1
). It may be that the
c-common cytokines used in these experiments play an important role in supporting the growth and survival of both naive and memory B cells.
In conclusion, the present experiments indicate that a cognate B cellT cell interaction may potentiate B cell development through the induction of receptors for multiple
c-common cytokines. Individual
c-common cytokines may have distinct effects on B cell growth and individual B cells may differ in their capacity to respond to each
c-common cytokine. The biological relevance of these observations may be evaluated by determining which
c-common cytokines are expressed or produced within specific microenvironments, such as PP lymphoid follicles, that are sites of intense B lymphopoiesis.
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Acknowledgments
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The Basel Institute for Immunology was founded and is supported by F. Hoffman La Roche & Co., Ltd. This research was supported by the Alberta Agriculture Research Institute and the Saskatchewan Health Services Utilization and Research Commission. Published with permission of the Director of VIDO as journal series no. 243
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Abbreviations
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c | IL-2R chain |
FALS | forward angle light scatter |
IL-2R | IL-2 receptor |
mCD40L | recombinant murine CD40 ligand |
PBMC | peripheral blood mononuclear cell |
PE | phycoerythrin |
PI | propidium iodide |
PP | Peyer's patch |
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Notes
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Transmitting editor: C. J. Paige
Received 19 April 1998,
accepted 29 April 1998.
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