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*
Istituto Ricerche Farmacologiche "Mario Negri,"
Fondazione Matarelli, Hospital Fatebenefratelli e Oftalmico, and
Department of Pathological Anatomy, Hospital L. Sacco, Milan, Italy;
§
Department of Hematology, University College London Medical School, London, United Kingdom; and
¶
Department of Experimental Oncology A, Istituto Nazionale dei Tumori, Milan, and
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Department of Experimental Medicine and Pathology, Immunopathology Section, Università di Roma La Sapienza, Rome, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF-
, anti-CD19, and anti-CD20, failed
to induce A-myb expression, suggesting that proliferation
alone is not sufficient for A-myb expression in the absence
of induction of a true centroblast phenotype. Finally, we show that
differentiation of germinal center B cells in vitro toward either
memory or plasma cells is accompanied by rapid down-regulation of
A-myb expression. We conclude that A-myb is a
marker of centroblasts generated in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Germinal centers are central to the differentiation and function of B lymphocytes (24, 25, 26). Naive mature B lymphocytes migrate from the bone marrow to peripheral lymphoid organs. Upon Ag encounter in the T cell areas of spleen and lymph nodes, B cells migrate to the follicles where the activated cells will form GC. GC B cells undergo extremely rapid proliferation (estimated doubling time, 6–7 h) and somatic mutation of their Ig gene within the dark zone of GC, which contains mostly large proliferating B cells or centroblasts. Further differentiation of centroblasts to centrocytes is accompanied by migration to the light zone of GC and arrest of the proliferation. The light zone contains the follicular dendritic cells (FDC) and GC helper T cells, which are thought to be important in regulating the B cell differentiation processes taking place in this region of GC: apoptosis of B cells with low affinity for the Ag, leading to affinity maturation, and differentiation to memory cells or to preplasma cells, which will then migrate to the bone marrow for full differentiation to plasma cells. The latter processes also involve isotype switching for the production of different Ig classes. Thus, the very fine compartmentalization of the GC has allowed a precise understanding of the sequence of events that takes place in GC, through the study of cell markers and morphology, expression of specific genes, and the mutation and recombination of Ig genes in normal and genetically modified animals (24, 25, 26). In turn, knowledge of the localization of new gene products within GC can indicate the potential function of such products. Previous experiments using purified tonsillar B cell subpopulations had suggested that the A-Myb transcription factor is expressed by GC B cells, but its possible subcompartmentalization within this structure had not been investigated (20). Given the important role demonstrated for this transcription factor in B cell differentiation, our goal was to determine the pattern of A-Myb expression in GC in vivo and in models of B cell proliferation and differentiation in vitro. We show that A-Myb is specifically expressed in proliferating centroblasts generated in vivo and is not induced in B cells activated in vitro. Furthermore, it is down-regulated during differentiation of centroblasts to more mature cells. Thus, among known B cell-restricted transcription factors, A-Myb shows the most restricted pattern of expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tonsils were obtained from routine tonsillectomies. Axillary and mesenteric lymph nodes were obtained from specimens of patients with unrelated pathologies. All tissues used for in situ hybridization were formalin fixed and embedded in paraffin. Total tonsillar B lymphocytes were purified as described previously (20). Briefly, tonsils were minced to a single-cell suspension and centrifuged over a Ficoll-Hypaque gradient (Seromed, Berlin, Germany). T cells were removed by rosetting with aminoethylisothiouronium-treated SRBC and centrifuging through a Ficoll-Hypaque gradient. Total B cells at the interface were further separated into different fractions on a discontinuous gradient made of 80, 57, 50, 40, and 30% Percoll (Pharmacia, Uppsala, Sweden). The 80 to 57% Percoll interface consisted mostly of resting small B lymphocytes. The 40 to 50% interface was highly enriched (at least 80%) with GC B cells strongly positive for both CD38+ and CD20+. B cell purity of both the GC and resting B cell fractions was 96 to 98%, as judged by staining with an anti-CD40 or anti-CD19 Ab.
Daudi and Ramos cell lines were from the American Type Culture Collection (Rockville, MD). All cells were grown in RPMI 1640 (Seromed), supplemented with 10% FCS (HyClone Laboratories, Logan, UT), glutamine (Life Technologies, Paisley, Scotland), and 50 µg/ml gentamicin (Life Technologies). Cells were resuspended at 3 x 105/ml 24 h before sorting.
Plasmid constructions and probes
For A-myb-specific in situ hybridization, a fragment of A-myb spanning nucleotide (nt) 1190–1550 (2) was cloned in the pGem3 vector (Promega Corp., Madison, WI). Briefly the XbaI-HindIII 360-bp fragment from plasmid A-myb-PCR was subcloned in the same sites of pGem3. For B-myb in situ hybridization, a 390-bp SacI-PstI fragment (nt 742–1133) was subcloned in the same sites of the pGem4 plasmid. The probes for in situ hybridization were labeled with[35S]UTP (Amersham, Little Chalfont, Buckinghamshire, U.K.) using the Riboprobe kit (Promega). The A-myb, B-myb, and cyclin A probes used for Northern blot hybridization have been described elsewhere (19, 27) and were labeled with[32P]dCTP (Amersham) with the Megaprime DNA labeling system (Amersham).
In situ hybridization
In situ hybridization was performed essentially as described by Wilkinson (28) with minor modifications. Briefly, myb sense and antisense probes were generated by transcription with the T7 and SP6 polymerases (Riboprobe kit, Promega) in the presence of[35S]CTP (Amersham). The template was then degraded in RNase-free DNase (Pharmacia), and the labeled probe was purified through a Sephadex G-50 (Pharmacia) column followed by NH4 acetate precipitation. The probes were dissolved at a working concentration of 106 cpm/µl in hybridization mix. Before hybridization, the sections were treated with 3 µg/ml proteinase K, fixed in 4% paraformaldehyde in PBS, and finally washed in a solution of 0.25% acetic anhydride in 0.2 M triethanolamine. Then, 30 µl of the appropriate probe was added to each slide, and the hybridization was conducted overnight at 55°C. The slides were then washed under stringent conditions (65°C, 2x SSC, 50% formamide) and treated with 100 µg/ml RNase A (Sigma) to remove unhybridized and nonspecifically bound probe. Autoradiography was performed with Kodak NT/B2 emulsion. Exposure times were between 2 and 3 wk. After developing, the sections were mounted in DPX (Merck, Darmstadt, Germany). Adjacent sections were processed for histologic staining or immunohistochemistry.
Proliferation and differentiation assays in vitro
Recombinant trimeric human CD40 ligand (CD40L) was obtained from
Immunex (Seattle, WA) and used at 1 µg/ml. rIL-2 (Eurocetus,
Amsterdam, Holland) was used at 200 U/ml, rIL-4 (Immunex, Seattle, WA)
at 50 ng/ml, rIL-6 (Serono, Roma, Italy) at 5 ng/ml, rIL-10
(Shering-Plough, Milano, Italy) at 20 ng/ml, and rIL-13 (Sanofi,
Montpellier, France) at 40 ng/ml; IFN- was from Roussel-UCLAF
(Paris, France) at 200 U/ml and TNF- (BASF Pharma Knoll,
Ludwigshafen, Germany) at 10 ng/ml. F(ab')2 Goat
anti-human µ-chain (Cappel, Organon Teknika Corp., West Chester,
PA) was used at 25 µg/ml.
The purified anti-CD19 Ab, HD37, was a kind gift of Dr. G. Moldenhauer (Deutsches Krebsforschungszentrum, Heidelberg, Germany) and was used at 1 µg/ml for stimulation. The Ab against CD20, 1F5, was a kind gift of Dr. E. A. Clark (University of Washington, Seattle, WA) and used at 2 µg/ml. Resting or GC B cells were cultured at 1 to 2 x 106/ml with the indicated stimuli. Double immunofluorescence staining was performed with CD20-FITC and CD38-phycoerythrin Abs (Becton Dickinson, San José, CA).
Cell sorting
7 x 105 cells/ml were stained with 20 µg/ml Hoechst 33342 and sorted with a FACSVantage instrument (Becton Dickinson) equipped with a 5 W argon-ion laser emitting 20 mW of UV light (352 nm). Cells were lysed in SDS sample buffer containing protease and phosphatase inhibitors. In some experiments, cells were fixed in 80% EtOH and stained with propidium iodide before sorting. Both methods gave the same results. A fraction of sorted cells were reanalyzed and found to be at least 95% pure with respect to cell cycle phase.
Northern and Western blots
RNA was extracted by standard guanidium isothiocyanate and cesium chloride gradient purification. Total RNA (18–20 µg) was run in 1% formaldehyde-agarose gels and blotted onto GeneScreen Plus membrane (New England Nuclear, Boston, MA), and hybridization was performed according to the manufacturer’s instructions. The A-Myb-specific antiserum has been described previously (29) and was used a a 1/1000 dilution. The anti-cdk2, anti-p27, and anti-actin antisera were from Santa Cruz Biotechnology (Santa Cruz, CA). To control for A-Myb antiserum specificity, the A-Myb-negative myeloma cell line IM-9 was transfected with the complete A-mybcDNA cloned in the episomial pCEP4 vector (Invitrogen Leeks, The Netherlands) or with the empty vector, using electroporation (270 mV, 960 µF in a Bio-Rad Gene Pulser; Bio-Rad, Hercules, CA). Transfected cells were selected by culture in 500 µg/ml hygromycin B (Boehringer Mannheim, Mannheim, Germany). Pools of transfected hygromycin resistant cells were obtained after 3 to 4 wk of culture. Then, 1 to 2 x 106 transfected or untransfected B cells were lysed in SDS loading buffer and run in a 10% SDS-polyacrylamide gel. The gels were electroblotted onto nitrocellulose filters (Schleicher and Schuell, Dassel, Germany) for 5 h at 60 V, according to standard procedures. The blots were incubated with the Abs diluted in PBS containing 5% nonfat milk powder and washed in the same solution containing 0.5% Nonidet P-40. Detection was performed using the enhanced chemiluminescence system (ECL; Amersham).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous data both in the mouse and in human B cells had indicated
that A-Myb is expressed by GC B cells, but the localization of
A-myb gene expression in the different GC compartments had
not been investigated (15, 20). We decided, therefore, to use in situ
hybridization to answer this question. A 360-bp fragment of human
A-myb cDNA spanning a region not conserved with other
myb genes was subcloned in the pGem3 vector for generation
of antisense and control sense RNA probes using the SP6 and T7
polymerases, respectively. As a control, a similarly unique 390-bp
fragment of human B-myb cDNA was subcloned in the pGem4
vector. B-myb expression correlates with proliferation in
many cell types, including tonsillar B cells, and was therefore
expected to stain the proliferating B cells in the dark zone of GC (3, 4). 35S-labeled sense and antisense RNA probes were
generated with SP6 and T7 RNA polymerases and used to hybridize serial
sections of formalin-fixed human lymphoid tissues, including tonsils,
lymph nodes, and spleen. Adjacent sections were also stained with
hematoxylin or with the Ki67 Ab to identify the dark zone of GC (30, 31). Representative results obtained with a lymph node and a tonsil are
shown in Figures 1
and
2, respectively. In the lymph node
section of Figure 1, the compartmentalization of the GC is most
evident, with clear staining of dark zone B cells with the Ki67 Ag
(Fig. 1A). As expected, B-myb positivity
correlated with expression of the Ki67 Ag and therefore with
proliferating cells in the dark zone of GC (Fig. 1B).
The follicular mantle, T cell area, and light zone of GC were mostly
negative for B-myb expression. The pattern of A-mybexpression was very similar to that of B-myb and
correlated closely with Ki67 expression. Small differences in
A-myb, B-myb, and Ki67 positivity probably reflect the fact
that different probes were used on adjacent sections. Sections from the
same tissues were also hybridized with the sense probes as negative
controls and did not show any specific signal (data not shown). As
shown in Figure 2, the pattern of expression was similar in tonsil
sections, with a clear correspondence between A-myb (Fig. 2C), B-myb (2D), and
Ki67 (2B) expression within the GC. Of note, however,
was the fact that the layer of proliferating epithelial cells (E)
clearly marked by Ki67 also strongly hybridized with the B-mybprobe (marked with a double arrow in Fig. 2D)
but not with the A-myb (Fig. 2C). This
demonstrates the specificity of A-myb expression for the
proliferating centroblasts of the dark zone, whereas B-mybis expressed in all proliferating cell populations present,
including centroblasts. Also, in spleen sections A-myb was specifically
expressed in the dark zone of GC (data not shown).
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The association of A-myb expression with the dark zone
of GC and therefore with proliferating centroblasts suggested that
A-Myb may play a role in proliferation and may be regulated in the cell
cycle in these cells (24, 25). To verify this hypothesis, we stained
purified tonsillar B cells with DNA dyes and sorted the B cell
populations according to the cell cycle phase. Equivalent numbers of
sorted cells were then analyzed for A-myb expression in Western blots,
as verified by Ponceau red staining of the blot. It was demonstrated
that the anti-A-Myb antiserum was specific for A-Myb by Western
analysis of the IM-9 cell line transfected with either the complete
A-myb cDNA in an expression vector or with the empty vector.
The antiserum specifically recognized a 90-kDa band in
A-myb-transfected and not in vector-transfected cells (Fig. 3A). In sorted
tonsillar B cell populations, the 90-kDa A-Myb protein could be
detected only in the S and G2/M phases of the cell cycle
but not in G0/G1. To verify that sorting
according to cell cycle phase had been successful, the same blot was
stained with an anti-p27 Ab. As expected, p27 is strongly expressed
in the G0/G1 cell population and is efficiently
down-regulated in S/G2/M phase cells (Fig. 3A) (32). Furthermore, the blot was stripped and
reprobed with anti-Actin Ab, showing that each of the lanes
contained equivalent amounts of protein (Fig. 3A).
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A). Furthermore, the
purity of the different cell cycle phases was regularly determined on
the FACS and found to be >82% for all phases. One representative
example of such cell cycle analysis on the Ramos cell line, before and
after sorting, is shown in Figure 3B. We conclude that A-Myb
expression is restricted to the S and G2/M phases of the
cell cycle in both normal and BL cell lines. A-myb expression is not induced following in vitro-stimulated proliferation of resting tonsillar B cells
Resting tonsillar B cells can be induced to proliferate in vitro
by a number of agents, including crude polyclonal B cell activators
such as SAC (formalin-killed Staphylococcus aureus Cowan I
strain bacteria), drugs such as PMA and Ca2+
ionophores, Abs against cell surface molecules, and presumably more
physiologic signals such as different combinations of anti-µ,
trimeric CD40L, and a variety of cytokines (34, 35). We had previously
observed that the induction of B cell proliferation in vitro by
nonphysiologic signals (SAC, PMA + calcium ionophore, etc.) was
not accompanied by the induction of A-Myb (20). This was somewhat
puzzling in view of the finding described above that A-Myb is expressed
by the highly proliferating centroblasts and is cell cycle regulated in
these cells. We therefore set out to investigate whether more
physiologic means of inducing proliferation of resting B lymphocytes in
vitro could induce A-myb expression. As shown in Figure 4 and Table I, we used a wide combination of signals
including anti-µ or anti-CD20 Abs with trimeric CD40L and/or
different cytokines, i.e., IL-2, IL-4, IL-6, IL-10, IL-13, TNF-, and
IFN-, or an anti-CD19 Ab. The cytokines were chosen among those
known to induce either growth or differentiation of B cells or both.
TNF- was chosen because it is known to be required for GC formation
in vivo. IFN- as well as anti-CD19 were chosen because they have
been reported to induce a partial GC phenotype to resting B cells in
vitro (36). Proliferation was measured by thymidine incorporation and
in some cases cell cycle analysis. All combinations used stimulated
strong proliferation of resting B cells, the combination of
anti-CD20 and IFN- being the only relatively weak stimulus
(Table I). RNA was extracted from resting B cells at the beginning of
culture and 3 days after stimulation of proliferation and analyzed for
A-myb expression in Northern blots. As shown in Figure 4, whereas GC B cells express high levels of A-myb RNA, resting
B cells induced to proliferate did not express detectable levels of
A-myb. To verify that other cell cycle-regulated genes
characteristic of the S/G2/M phases of the cell cycle could
be induced in the same conditions, the blots were rehybridized with a
cyclin A cDNA probe and, in some cases, also with
B-myb. As shown in Figure 4, both B-myb and
cyclin A were efficiently induced by all of the stimuli
employed to levels corresponding roughly to the levels of thymidine
uptake or S/G2/M phase induction. Indeed the percentage of
cells in the S/G2/M phases of the the cell cycle obtained
with the most effective stimuli, such as anti-µ + CD40L +
IL-2 + IL-10, corresponded approximately to that of purified GC B
cells (Table I). However, the levels of A-myb expression
were clearly very different in the two populations (Fig. 4, lane
1 compared wiht lanes 3–15). Most stimulation
regimens were also tested for A-Myb protein expression in Western
blots. Again, A-Myb could be detected in purified tonsillar GC B cells
but not following stimulation of resting B cells (data not shown).
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did lead
to doubling of CD38 levels as reported (36), other markers of
centroblasts were not obtained during our culture conditions (in
particular, all activated B cell populations showed high CD39
expression, unlike GC B cells, which are CD39-; data not
shown). A-myb is rapidly down-regulated during in vitro-induced differentiation of GC B cells
CD38+CD20+ GC B cells can be induced
to proliferate and differentiate in vitro toward either memory or
plasma cells, a process that can be followed by a down-regulation in
CD38 or CD20, respectively (37, 38, 39). Given the restricted expression of
A-myb in centroblasts in vivo, we investigated whether
differentiation of these cells in vitro along both of the
differentiation pathways was accompanied by down-regulation of
A-myb expression. Purified GC B cells were cultured in the
presence of CD40L, IL-2, and IL-10for 3 days, washed, and replated in
medium containing IL-2 and IL-10 in either the presence or absence of
CD40L for an additional 4 days. As reported by others (37), these
culture conditions led to partial differentiation of the GC B cells
toward memory (CD20+CD38-) (MC) or plasma-like
cells (CD38+CD20-) (PC), respectively (Fig. 5A). Plasma-like cells
increased from 1 to 27% after 7 days of culture and memory cells from
20 to 36% (Fig. 5A). In addition, an early and
transient peak of proliferation was observed at 24 h, as
determined by thymidine uptake and cell count (data not shown). We then
investigated the time course of A-myb expression during the
same culture period. As shown in Figure 5B, A-mybexpression was rapidly down-regulated, having decreased at least
threefold after 24 h and disappearing completely by 66 h.
Differentiation toward either memory or plasma cells did not lead to
later induction of A-myb. Expression of cyclin A,
on the other hand, reflected the proliferative activity of the cells
during the in vitro culture (Fig. 5B). We conclude
that A-myb expression in centroblasts is rapidly
down-regulated both in vitro and in vivo during differentiation to
centrocytes and then to memory or plasma cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IFN-, anti-CD20, and anti-CD19 Abs in different
combinations. Thus, the simple induction of proliferation in vitro is
not sufficient for A-Myb expression but may require the acquisition of
a true centroblast phenotype. In vitro differentiation of GC B cells
with CD40L, IL-2, and IL-10, however, led to the rapid disappearance of
A-myb mRNA levels, in accordance with in situ hybridization
data showing that centrocytes are negative for A-mybexpression. We conclude that A-Myb is a transcription factor that
is specifically induced in centroblasts during the in vivo GC
reaction. We had previously investigated A-myb expression in different subpopulations of human tonsillar B cells using sorting by FACS and PCR analysis (20). These data had already suggested that A-Myb is expressed in GC B cells. Furthermore, studies of A-myb expression in mice by in situ hybridization had confirmed a GC localization for this gene (15). In neither case, however, had it been possible to analyze the localization of A-myb within the different GC subcompartments. In this report, we have demonstrated that A-mybis specifically expressed in the dark zone of GC and therefore in centroblasts, which show an extremely high rate of proliferation (with a cell cycle of about 7 h) and undergo somatic mutations of their Ig (24, 25, 40). Further differentiation of centroblasts to centrocytes, which move to the light zone of GC, leads to the elimination of cells with low affinity for the Ag through apoptosis, resulting in affinity maturation (24, 25, 41). Also, the processes of isotype switching and further differentiation to memory and plasma cells is thought to take place in the light zone of GC, which in addition to centrocytes contains FDC and some T cells (24, 41). Thus expression of A-Myb in centroblasts suggests that this transcription factor may be associated with the process of somatic mutation or with the particularly high rate of proliferation of these cells.
A role for A-Myb in centroblast proliferation is also supported by its
regulation during the cell cycle. Also, in bovine smooth muscle cells,
A-myb has been shown to be induced during the
G1/S phase transition (42). Furthermore, these data are
reminiscent of the specific A-myb expression only in
proliferating populations of cells in the mouse (immature spermatocytes
and neuronal and breast ductal epithelium) (15, 18). However, simple
proliferation of mature human B cells, induced either by polyclonal B
cell activators (20) or by more physiologic signal as reported here, is
not sufficient to induce A-myb expression, even though the
percentage of S/G2/M phase cells obtained after 3 days of
culture with many combinations of stimuli was similar to that of
purified GC B cells that expressed high levels of A-myb. It
is known, however, and has been verified in our culture conditions,
that in vitro stimulation of resting B cells does not to lead to the
formation of true centroblast-like cells (36). Anti-µ together with
IFNs induce CD38 and CD95 but not carboxypeptidase-M, a marker of in
vivo centroblasts; nor do they down-regulate CD44, which is normally
absent from centroblasts (36). We found that whereas anti-µ +
IFN- was indeed the most effective stimulus to induce CD38 on
resting B cells, none of the culture conditions used led to a
down-regulation of CD39 (data not shown). We suggest, therefore, that
A-Myb is a marker of proliferating centroblasts produced in vivo and
that culture conditions that do not induce a true centroblast phenotype
cannot up-regulate A-Myb expression. Similarly, in vitro
differentiation of GC B cells led to a rapid disappearance of
A-myb expression, in accordance with the in situ data
showing that centrocytes do not express this gene. Thus, A-Myb is the B
cell-restricted transcription factor with the narrowest pattern of
expression during B cell differentiation. As shown in Figure 6, Bcl-6 (43) is most similar to A-Myb,
but is expressed in both centroblasts and centrocytes. Blimp-1 (44) is
present only during the latest stages of B cell differentiation,
whereas BSAP (pax-5) (45) and the coactivator OCA-B (OBF-1/Bob-1) (46)
show a more widespread expression pattern in B cells.
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It is interesting to note that all members of the Myb family in mammals have been shown to play a direct role in the control of either proliferation or differentiation or both. C-myb knockout mice die in utero due to the complete loss of fetal hemopoiesis (10). C-myb is cell cycle regulated in mature hemopoietic cells and is required for their proliferation (9, 47). Furthermore, c-myb is a crucial regulator of hemopoietic cell differentiation (6, 7). B-myb is induced in the S/G2/M phases of the cell cycle and is required for the proliferation of many cell types (11, 13). More recent evidence also suggests a role for B-myb in differentiation. Finally, A-myb expression is restricted to a very few cells and during a restricted stage of their differentiation: it is expressed in immature proliferating spermatocytes, mammary gland ductal epithelial cells, and some neuronal cells (15, 18). It has been demonstrated recently, using A-myb knockout and transgenic animals, that A-myb plays an important role in these restricted cell types (18, 23). The former show defects in spermatocytes and breast duct development (18). Transgenic animals expressing ectopic A-mybin most tissues show a restricted effect on B cells. These animals show hyperplasia of the follicles of peripheral lymphoid organs with an accumulation of CD38+ B cells in the follicles (23). These data demonstrate that A-Myb regulates the proliferation and/or differentiation of GC B cells. Thus, the present report further defines the role of A-Myb as being specific for centroblasts.
A further understanding of A-Myb function in centroblasts will require the definition of its molecular targets in the context of B lymphocytes. Recently, we and others have shown that the c-mycpromoter is up-regulated by A-Myb (48). Furthermore, we have recently shown that A-Myb may be active as a transcriptional factor in B cells only and not in other hemopoietic cells, probably through interaction with a B cell-specific cofactor (48). Thus, the definiton of the target genes, cofactors, and specific signals that induce A-Myb in vivo should prove crucial elements for the understanding of A-Myb function during the GC reaction in response to Ags and in B cell pathologies such as Burkitt’s lymphoma.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Josée Golay, Istituto Ricerche Farmacologiche "Mario Negri," via Eritrea 62, 20157 Milano, Italy. E-mail address:
3 Abbreviations used in this paper: GC, germinal center; CD40L, CD40 ligand.
Received for publication July 28, 1997. Accepted for publication November 18, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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