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*
Laboratory of Cellular Immunology, Alton Ochsner Medical Foundation, New Orleans, LA 70121; and
Department of Immunology and Cell Biology, Forschungszentrum Borstel, Borstel, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(7), LT
, and LTR (8, 9, 10), TNF, and TNFR
(11, 12) lack the ability to form GC and their secondary
immune responses were abrogated, underscoring the important roles of
these signaling molecules in GC reaction. Recent experiments with LT
and LTR gene-targeted mice have further demonstrated that the
initial cluster formation of LT-producing B cells with FDC is
essential for GC formation (13, 14). Although the in vivo experimental model generated by the gene-targeting method has revealed the essential requirement of these individual factors, it is not clear how these factors interact in GC reaction. In addition, the signals for survival, proliferation, and differentiation of GC-B cells are poorly understood because of the lack of proper in vitro models to analyze the cellular and molecular interaction between B cells and FDC. Hence, an experimental model has been developed to mimic the in vivo GC reaction (15, 16). In this model, an FDC line, HK, was used to culture with ex vivo GC-B cells in the presence of CD40L, IL-2, IL-4, and IL-10. GC-B cells indeed proliferated and differentiated into memory B cells and PC (16, 17). By using this unique culture system, the functions of individual factors and cytokines produced by activated T cells or FDC/HK cells that regulate GC-B cell differentiation can be investigated in detail.
To identify FDC-signaling molecules required for GC-B cell differentiation, FDC-specific mAbs were generated and screened for their ability to block FDC-mediated GC-B cell growth and differentiation (18). With one of the inhibitory mAbs, 8D6, the cDNA encoding FDC-signaling molecule 8D6 (FDC-SM-8D6) was cloned from a cDNA library of HK cells, and this molecule was identified as a novel protein of 282 aa. However, the specific function of FDC-SM-8D6 is not clear.
In this paper, we report the distinct functions of FDC-SM-8D6 and T cell signals in the regulation of B cell differentiation in the GC. The differentiation pathway of GC-B cells is not determined by CD40 stimulation, but rather by T cell cytokines, i.e., IL-4 and IL-10. In the presence of IL-4, centroblasts differentiate into memory B cells, whereas IL-10 induces centrocytes to differentiate into PC secreting IgG. In the process of GC-B cell differentiation, FDC-SM-8D6 is required in PC generation but not in memory B cell expansion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Abs used in this work were DRC-1 (clone R4/23; Dako, Carpinteria, CA); isotype-matched control mAb 3C8 (IgG1; Ref. 15); biotin-conjugated goat anti-mouse Ig, HRP-conjugated goat anti-human IgG, and goat anti-human IgG (BioSource International, Camarillo, CA); streptavidin-biotinylated HRP complex (Amersham, Piscataway, NJ); HRP-conjugated goat anti-human IgG (Cappel Pharmaceuticals, Malvern, PA); anti-CD44 (NKI-P1, IgG1; Dr. C. G. Figdor, University Hospital Nijmegen, Nijmegen, The Netherlands); rat anti-mouse IgG1 microbeads (Miltenyi Biotech, Sunnyvale, CA); and FITC-conjugated anti-CD20, PE-conjugated anti-CD38, PE-conjugated or purified anti-CD27, and FITC-conjugated goat anti-mouse Ig (BD PharMingen, San Diego, CA).
Cytokines and reagents
The culture medium for GC-B cells was IMDM (Irvine Scientific, Santa Ana, CA) supplemented with 10% FCS (Life Technologies, Grand Island, NY), 2 mM glutamine, 100 U/ml penicillin G, and 100 µg/ml streptomycin (Irvine Scientific). The culture medium for CD27+ and CD27- B cells was RPMI 1640 medium (Irvine Scientific). Cytokines were IL-2 (Hoffman-La Roche, Nutley, NJ), IL-4 (a generous gift from Schering-Plough, Union, NJ), and IL-10 (R&D Systems, Minneapolis, MN). Soluble human CD40L was generously provided by Dr. R. Armitage (Immunex Corporation, Seattle, WA). Percoll and Ficoll were purchased from Pharmacia LKB Biotechnology (Uppsala, Sweden).
Preparation of B cell subsets
GC-B cells were purified from tonsillar B cells by magnetic cell separation (MACS; Miltenyi Biotec) as described previously (15). The purity was >98%, as assessed by the expression of CD20+CD38high. CD27+ and CD27- B cells were isolated from high-density B cells by the same method with anti-CD27 mAb.
Culture of B cell subsets with HK cells
GC-B cells were cultured in 24-well plates with or without mAb 8D6 or 3C8 (50 µg/ml) in the presence of irradiated HK cells (2 x 104 cell/well; 5000 rad), CD40L (100 ng/ml), IL-2 (30 U/ml), IL-4 (50 U/ml), or IL-10 (50 ng/ml). Every 3-5 days, cells were collected, washed, and recultured (1 x 105 cell/well) with irradiated HK cells and cytokine combinations. Purified CD27+ and CD27- cells were cultured in the same condition as GC-B cells for 6 days. At the end of each culture, viable cells were counted by trypan blue exclusive assay and stained with FITC-conjugated anti-CD20 and PE-conjugated anti-CD38 mAbs for FACS analysis. The culture supernatant was harvested and IgG concentration measured by ELISA as described previously (19).
Coculture of GC-B cells with FDC-SM-8D6-expressing COS cells
COS cells in six-well plates were transfected with 2 µg of FDC-SM-8D6 cDNA by using LipofectAMINE (Life Technologies). After 24 h, transfected COS cells were cultured with tonsillar GC-B cells with or without mAb 8D6 in the presence of CD40L, IL-2, IL-10, and 25% HK cell conditioned medium for 16 h. GC-B cells then were separated from COS cells and recultured in the presence of the above cytokines for 7 days. Then cells were harvested for viable cell number count by trypan blue exclusive assay and culture supernatant for IgG concentration measurement by ELISA.
Immunohistological staining
Cryostat sections of human tonsils were fixed in cold acetone and chloroform. The slides were blocked with 1% (w/v) BSA-PBS and then incubated with mAbs 8D6 and DRC-1 and then with FITC-conjugated goat anti-mouse IgG1 and PE-conjugated anti-mouse IgM. After washing with Tris buffer, the slides were fixed with 4% paraformaldehyde, embedded in DABCO (Sigma, St. Louis, MO), and examined under the confocal microscope.
Flow cytometry
Cells were stained with FITC-conjugated CD20, PE-conjugated CD38, or CD27 mAb. Flow cytometric analysis was conducted on a FACScan (Becton Dickinson, San Jose, CA) with CellQuest software (17). The absolute number of PC or memory B cells were determined by multiplying viable cell count with CD20-CD38high or CD20+CD38low cell frequency.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Both FDC/HK cells and T cells are essential for GC-B cell growth and differentiation because GC-B cells do not survive >3 days in the absence of either component (15). However, the role of FDC/HK cells and T cell cytokines (e.g., IL-2, IL-4, and IL-10) has not been clearly identified.
To investigate the effect of individual cytokines, purified GC-B cells
with the centroblast phenotype
(CD20+CD38high) were
cultured in the presence of HK cells, CD40L, and different cytokine
combinations (IL-2 plus IL-4 vs IL-2 plus IL-10) for 15 days as
described in Materials and Methods. The cells were harvested
every 3-5 days and analyzed by FACS (Fig. 1
A). On day 3, CD20 and CD38
expression of GC-B cells were down-regulated to centrocyte phenotype
regardless of whether the cultures contain either IL-2 plus IL-4 or
IL-2 plus IL-10. On day 7, 40% of the recovered cells were
CD20-CD38high, PC
phenotype (16), in the culture containing IL-10. In
contrast, the phenotype of cells in the culture containing IL-4
remained CD20+CD38low. The
CD20+CD38low cells were
indeed memory B cells as indicated by CD44 expression as defined
previously (17, 20). On day 15, 72% of the recovered
cells were PC in the culture containing IL-10, and 88% of the
recovered cells were memory B cells in the culture containing
IL-4.
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B). Such remarkable growth of GC-B cells requires HK cells
as there was no cell growth in the absence of HK cells despite of the
presence of T cell components, i.e., CD40L, IL-2, IL-4, or IL-10. In
addition, PC generation and IgG secretion were not observed in the
cultures without HK cells even if they contained IL-10 (Fig. 1, C and D, right). By day 10, the
absolute number of PC in the culture containing IL-10 increased from
1 x 104/ml to 1.35 x
106/ml whereas no PC were detected in the culture
containing IL-4 (Fig. 1C). The generation of PC was
substantiated by 86-fold increase of IgG secretion in culture with HK
cells, compared with culture without HK cells. There was no detectable
IgG secretion in the culture containing IL-4 (Fig. 1D).
The critical role of IL-10 was further demonstrated by the following
experiments. The GC-B cells were cultured in the presence of IL-4 for 5
days. There was no significant increase of
CD20-CD38high PC during
this culture period (Fig. 2). If IL-4 was
replaced by IL-10 on day 5 and the cells were recultured for an
additional 7 days, a population of
CD20-CD38high PC (31%)
emerged. In simultaneous culture where IL-4 was present throughout,
only a few PC were detected. These results underscore the critical
function of activated T cells in determining the differentiation
pathway by providing either IL-4 or IL-10 to Ag-activated B cells in
the GC. In contrast, the important function of FDC/HK cells appears to
costimulate B cell proliferation in the GC.
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With one of the FDC-staining mAbs, 8D6, prepared by immunizing
mice with freshly isolated FDC, we recently have identified a novel
molecule, FDC-SM-8D6 (18). To explore the tissue
distribution of this molecule, immunohistochemical staining of
tonsillar tissue section was performed and imaged by confocal
microscopy. As shown in Fig. 3A, FDC-SM-8D6 was abundantly
expressed by FDC in the GC, exhibiting a strong staining of the
reticular network (Fig. 3A, in green). The diffuse staining
of mAb 8D6 was characteristic of FDC surrounding mononuclear cells in
the GC. Such expression was not observed in the mantle zone where
CD20+ B cells are found. There was a clear
demarcation between the GCs, including T cell zones. The specific
staining of mAb 8D6 was confirmed by staining the tissue section with a
known FDC-specific mAb, DRC-1 (Ref. 21 ; Fig. 3B, in red). There was significant overlapped staining of
mAbs 8D6 and DRC-1 (Fig. 3C, in yellow), indicating
FDC-SM-8D6 is colocalized with DRC-1. Such FDC staining in the GC was
not observed with the isotype matched control mAbs (data not shown). In
addition, freshly isolated single FDC and HK cells strongly expressed
FDC-SM-8D6 as detected in the cytospin preparations and FACS analysis
(18).
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B,
right). Of total cells recovered, 49% were PC as determined
by the number of
CD20-CD38high cells (Fig. 4A). By day 7, IgG concentration in the supernatant of the
culture containing IL-10 was 18.3 µg/ml (Fig. 4D).
Compared with the control cultures, the addition of mAb 8D6 reduced the
cell recovery from 920 to 435% (Fig. 4B) and the percentage
of PC from 49 to 24% (Fig. 4A). The absolute number of PC
was decreased by 78% when mAb 8D6 was present (Fig. 4C).
The decrease of PC number also was reflected by 61% reduction of IgG
secretion if mAb 8D6 was added (Fig. 4D). Such inhibitory
effect was not observed in the culture containing isotype-matched
control mAb 3C8 that was prepared by immunizing mice with HK cells
(15). Although mAb 3C8 binds FDC, it does not inhibit the
FDC/HK cell-mediated GC-B cell growth (18). However, mAb
8D6 did not affect memory B cell proliferation in the culture
containing IL-4 (Fig. 4, B–D, left). The
inhibitory effect of mAb 8D6 was observed in at least three
reproducible experiments. These data suggest that the addition of mAb
8D6 selectively inhibits PC generation and reduces IgG secretion in the
cultures containing IL-10.
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, B–D, gray bars).
To confirm the direct inhibitory effect of mAb 8D6, we investigated
whether recombinant FDC-SM-8D6 was capable of stimulating PC
generation. FDC-SM-8D6 was expressed in COS cells by transfection with
its cDNA. Twenty-four hours after transfection, 20% of the COS cells
expressed FDC-SM-8D6 (Fig. 5A). In the coculture of COS
cells with GC-B cells as described in Materials and Methods,
FDC-SM-8D6-transfected COS cells enhanced GC-B cell proliferation and
differentiation. Compared with mock cDNA-transfected COS cells,
FDC-SM-8D6-expressing COS cells increased the viable cell number of
GC-B cells by 204% and IgG secretion by 185% (Fig. 5, B
and C). The augmenting activity of transfected COS cells is
mediated by FDC-SM-8D6 because such enhancement was completely
abrogated by mAb 8D6. Thus, the stimulatory function of FDC-SM-8D6 in
augmenting PC generation from GC-B cells was confirmed by the
recombinant protein expressed in COS cells.
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Because FDC-SM-8D6 enhanced PC generation but not memory B cell
proliferation, its specific function in PC generation was further
investigated. Centrocytes were generated by culturing centroblasts in
the presence of HK cells, CD40L, IL-2, and IL-4 for 5 days in the
first-step culture (Fig. 2). In the second-step culture, IL-4 was
replaced by IL-10, and cells were cultured for an additional 7
days. In this culture condition, 31% of recovered cells were
CD20-CD38high PC. In the
same experiment, 7.4 µg/ml IgG was detected in the culture
supernatant (Fig. 6). Meanwhile, the
culture containing IL-4 did not produce PC or IgG. The addition of mAb
8D6 inhibited viable cell number by 38% in the second-step culture
containing IL-10, whereas it did not affect cell growth in the culture
containing IL-4 throughout. The selective inhibition of PC generation
by mAb 8D6 was confirmed by 43% reduction of IgG secretion in the
culture with IL-10, but not in the culture with IL-4. These results
suggest that the target population of FDC-SM-8D6 is PC precursors in
the GC.
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In human B cell culture, IgG-secreting cells are derived from
memory B cells but not from naive B cells (22, 23).
Recently, it has been reported that a member of the TNFR family, CD27,
is expressed on memory B cells (24, 25).
CD27+ B cells are capable of differentiating into
PC, whereas CD27- B cells do not differentiate
in vitro (26, 27, 28). Hence, it was speculated that mAb 8D6
would affect the differentiation of CD27+ B cells
but not the proliferation of CD27- B cells. To
investigate this hypothesis, tonsillar high-density B cells were
separated into CD27+ and
CD27- subsets. These two subsets were cultured
with HK cells, CD40L, IL-2, and IL-10. By day 6, 63% of recovered
cells were CD20-CD38high
PC (Fig. 7A), and 21 µg/ml
IgG (Fig. 7D, left) was secreted in the culture
of CD27+ B cells. Much fewer PC (13%) were
generated (Fig. 7A), and only 3.9 µg/ml IgG was secreted
in the culture of CD27- B cells (Fig. 7C, right). This small number of PC might result
from a few CD27+ B cells contaminated in the
latter culture. The addition of mAb 8D6 reduced viable cell number by
36% in the culture of CD27+ B cells, while no
decrease of cell number in the culture of CD27-
B cells was observed (Fig. 7B). The reduction in cell number
was accompanied by decreased IgG secretion (39%), indicating the
inhibition of PC generation from CD27+ B cells.
Such effect on PC generation is specific to mAb 8D6 because the
addition of control mAb 3C8 did not affect the cell numbers or IgG
secretion in the cultures with either CD27+ or
CD27- B cells. Cellular proliferation was
comparable in the cultures of both subsets as shown by recovered cell
numbers.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Both CD40L and FDC/HK cells rescue GC-B cells from spontaneous apoptosis and support proliferation of centroblasts and centrocytes (29, 30). A distinct function of T cells is to provide cytokines in addition to CD40L that induce differentiation of centroblasts to centrocytes. Centrocytes continued to differentiate into cells of memory B cell phenotype (CD20+CD77-CD44+CD27+) in long-term cultures with FDC/HK cells (17, 20). The cellular proliferation is sustained by IL-2, IL-4, and HK cells. However, in the presence of IL-10, a majority of cells differentiated into PC, suggesting the critical role of IL-10 in PC generation. The generation of PC was accompanied by a remarkable increase in IgG secretion. Such increase was not observed in the cultures with IL-4. This selective generation of either memory B cells or PC was not attributed to death of either cell type during the culture, as viable cell numbers recovered at the end of culture were comparable.
The important function of IL-10 also was demonstrated in two-step culture experiments. GC-B cells were cultured for 5 days with IL-4 to generate memory B cells, and then the cells were recultured for another 7 days in the presence of either IL-4 or IL-10. The replacement of IL-4 by IL-10 in the second culture led to PC generation, confirming the specific function of IL-10 in directing centrocytes to PC. These results reveal the distinct functions of IL-4 and IL-10 produced by activated T cells in the GC. IL-4 stimulates proliferation of memory B cells, whereas IL-10 steers centrocytes to differentiate into Ig-secreting PC. The addition of IL-2 was necessary because recovered cell numbers were much less in the cultures with IL-4 or IL-10 alone (15). IL-2 synergizes with either IL-4 or IL-10. This study shows that a potential physiological function for IL-4 is the continuous cellular proliferation of B cells in the GC, allowing affinity maturation of the immune response to occur. The cellular proliferation is interrupted by IL-10, which induces the terminal differentiation.
Because FDC/HK cells are essential for sustaining GC-B cell proliferation and differentiation in vitro, we recently have identified one of the growth factors produced by FDC (18). FDC-SM-8D6 is a novel protein of 282 aa. Its cysteine-rich type A domain has 44-62% homology to the amino acid sequence of low-density lipoprotein receptors that are known to bind multiple ligands (31, 32). The expression of FDC-SM-8D6 was confined in the GC and not detectable outside of the GC as shown in immunohistochemical staining of tonsillar tissue sections. To date, six similar human FDC-specific mAbs, namely 3C8, 7D6, DRC-1, HJ2, GP93, and Ki-M4 have been obtained by various investigators (15, 21, 33, 34, 35, 36). However, these mAbs have failed to affect FDC/HK cell stimulatory activity in GC-B cell proliferation assays (data not shown).
The important function of FDC-SM-8D6 in differentiation of GC-B cells is demonstrated in our experiments. When the specific neutralizing mAb 8D6 was added to the long-term cultures as described above, it consistently inhibited the HK cell-mediated GC-B cell differentiation into Ig-secreting PC, but not memory B cell proliferation. mAb 8D6 inhibited both cellular proliferation and Ig secretion in cultures containing IL-10 but not in cultures containing IL-4. Furthermore, the addition of mAb 8D6 at the beginning of cultures is critical, as there was no inhibition if it was added 3 days after the initiation of culture. These results suggest that FDC-SM-8D6 is required in the early stage of PC generation when centrocytes make the commitment to differentiate into PC. This conclusion is supported by the fact that the inhibition of PC generation by mAb 8D6 was 36-78% but not complete. The residual PC generated in the presence of mAb 8D6 may result from PC precursors that have passed the critical time point in vivo when they require FDC-SM-8D6. Indeed, a significant fraction (15%) of the tonsillar GC-B cells is committed to become PC by expressing the transcription factor Blimp-1 (37). However, it is not ruled out that FDC/HK cells provide surface molecules or soluble factors other than FDC-SM-8D6 that regulate GC-B cell proliferation. The existence of other FDC-signaling molecules is suggested by the fact that HK cells are required for the proliferation of centrocytes in the presence of IL-2 plus IL-4. Currently, the identification of such additional FDC-signaling molecules is in progress in our laboratory.
FDC-SM-8D6 did not induce PC in the cultures without IL-10 (data not shown). The PC precursors are generated by IL-10 and then stimulated by FDC-SM-8D6 expressed by FDC in the GC. In the absence of FDC-SM-8D6, the PC precursors may not be able to expand. This conclusion is supported by the observation that PC generation increased when GC-B cells were cultured with COS cells transfected with FDC-SM-8D6 gene but not with mock. Such augmenting effect of the FDC-SM-8D6-expressing COS cells was specifically neutralized by mAb 8D6.
The function of FDC-SM-8D6 is further corroborated by the effect of mAb 8D6 on CD27+ B cells. CD27+ B cells are known to differentiate into PC in vitro, whereas CD27- B cells do not (26, 28, 38). Hence, a CD27+ B cell subset provides an ideal PC precursor population to study the specific function of FDC-SM-8D6. The addition of mAb 8D6 inhibited proliferation and IgG secretion in the culture of CD27+ B cells but not proliferation of CD27- B cells.
Taken together, our in vitro experimental results delineate the distinct functions of T cells and FDC. Centroblasts in the GC require FDC for their vigorous proliferation. When GC-B cells encounter Ag-activated T cells that produce CD40L and cytokines, CD40L induces differentiation of centroblasts to centrocytes as indicated by down-regulation of CD38 and CD77 and up-regulation of CD44 (17). Centrocytes proliferate in the presence of IL-2 plus IL-4 and differentiate into Ig-secreting PC when IL-4 is replaced by IL-10. A novel function of FDC-SM-8D6 is to augment proliferation of PC precursors generated by IL-10. The unique anatomical localization of FDC-SM-8D6 in the GC indicates that it is an important growth factor for PC.
Our observation may have a significant implication in understanding the pathogenesis of multiple myeloma, which usually is not curable. Although the origin of myeloma cells is not clear in the physiological conditions, PC are generated in the GC and migrate to bone marrow to expand in the presence of stroma cells (39). In the process of rapid cellular proliferation, translocation, and mutation of centroblasts in the GC, malignant transformation may occur as a consequence of the genetic mobility and mutability permitted to generate a diverse Ab repertoire. It is possible that FDC-SM-8D6 contributes to the survival and proliferation of the malignant PC. Thus, mAb 8D6 may be a promising therapeutic approach to achieve selective suppression of myeloma cell growth.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yong Sung Choi, Laboratory of Cellular Immunology, Alton Ochsner Medical Foundation, 1516 Jefferson Highway, New Orleans, LA 70121. E-mail address: ychoi{at}ochsner.org
3 Abbreviations used in this paper: GC, germinal center; PC, plasma cells; FDC, follicular dendritic cells; CD40L, CD40 ligand; FDC-SM-8D6, FDC-signaling molecule 8D6; LT, lymphotoxin.
Received for publication February 20, 2001. Accepted for publication April 26, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 K N Potter, C I Mockridge, A Rahman, S Buchan, T Hamblin, B Davidson, D A Isenberg, and F K Stevenson Disturbances in peripheral blood B cell subpopulations in autoimmune patients Lupus, December 1, 2002; 11(12): 872 - 877. [Abstract] [PDF]
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T. Beyer, M. Meyer-Hermann, and G. Soff A possible role of chemotaxis in germinal center formation Int. Immunol., December 1, 2002; 14(12): 1369 - 1381. [Abstract] [Full Text] [PDF]
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J. Jung, A.-K. Yi, X. Zhang, J. Choe, L. Li, and Y. S. Choi Distinct Response of Human B Cell Subpopulations in Recognition of an Innate Immune Signal, CpG DNA J. Immunol., September 1, 2002; 169(5): 2368 - 2373. [Abstract] [Full Text] [PDF]
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A. J. Marshall, Q. Du, K. E. Draves, Y. Shikishima, K. T. HayGlass, and E. A. Clark FDC-SP, a Novel Secreted Protein Expressed by Follicular Dendritic Cells J. Immunol., September 1, 2002; 169(5): 2381 - 2389. [Abstract] [Full Text] [PDF]
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B. Johansson-Lindbom and C. A. K. Borrebaeck Germinal Center B Cells Constitute a Predominant Physiological Source of IL-4: Implication for Th2 Development In Vivo J. Immunol., April 1, 2002; 168(7): 3165 - 3172. [Abstract] [Full Text] [PDF]
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)
or without (•) HK cells. Total viable cell number was counted by
trypan blue exclusive assay. The values represent mean ± SD in
triplicate cultures. The absolute number of PC was determined by
multiplying viable cell count with
CD20-CD38high cell frequency.
D, The supernatants in the cultures were harvested and
IgG concentrations measured by ELISA.
), mAb 8D6 (
), mAb 8D6 added 3 days after the initiation of
culture (
), or control mAb 3C8 (
).
