By
From * Department of Human Immunology and Department of Molecular Biology, DNAX Research
Institute of Molecular and Cellular Biology, Palo Alto, California 94304
In this study it is shown that both membrane-bound and soluble forms of signaling lymphocytic activation molecule (SLAM) induce proliferation and Ig synthesis by activated human B cells. Activated B cells express the membrane-bound form of SLAM (mSLAM), the soluble (s) and the cytoplasmic (c) isoforms of SLAM, and the expression levels of mSLAM on B cells are rapidly upregulated after activation in vitro. Importantly, recombinant sSLAM and L cells transfected with mSLAM efficiently enhance B cell proliferation induced by anti-µ mAbs, anti-CD40 mAbs or Staphylococcus aureus Cowan I (SAC) in the presence or absence of IL-2, IL-4, IL-10, IL-12, or IL-15. sSLAM strongly enhances proliferation of both freshly isolated B cells and B cells derived from long-term in vitro cultures, indicating that SLAM acts not only during the initial phase of B cell activation but also during the expansion of preactivated B cells. In addition, sSLAM enhances production of IgM, IgG, and IgA by B cells activated by antiCD40 mAbs. SLAM has recently been shown to be a high affinity self-ligand, and the present data suggest that signaling through homophilic SLAM-SLAM binding during B-B and B-T cell interactions enhances the expansion and differentiation of activated B cells.
Bcell activation is initiated after recognition of specific
antigen by cell surface Ig, which in naive, unprimed B
cells is of IgM or IgD isotype. Cytokines and membranebound costimulatory molecules expressed by activated
CD4+ T cells are required for subsequent proliferation, Ig
isotype switching and differentiation of activated B cells.
Cytokines produced by Th2-type cells, such as IL-4, IL-5,
IL-13, are considered to be primarily involved in providing
help for B cells, whereas Th1-type cytokines, IL-2 and
IFN- The CD40 ligand (CD40L)1 on activated CD4+ T cells,
which interacts with CD40 on B cells (12), was shown to
be a potent membrane-bound costimulatory molecule for
proliferation and differentiation of both human and murine
B cells (13). The B cell antigen CD19, the ligand of
which remains to be characterized, has also been shown to
mediate maturation, proliferation and differentiation of B
cells (18). In addition, CD70, a member of the TNF family expressed by activated B and T cells, and the membrane
form of TNF- Signaling lymphocytic activation molecule (SLAM) is a
70-kD glycoprotein expressed by CD45RO+ T cells, immature thymocytes, and a proportion of B cells (29). SLAM
is a member of the immunoglobulin gene superfamily and
belongs to the CD2 family of cell surface molecules (29, 30).
SLAM has limited homology to CD48 (26%), CD58/LFA-3
(17%), and 2B4 (28%), a signaling molecule expressed on
murine NK and cytotoxic T cells (31). Activated human
T cells express, in addition to membrane-form of SLAM
(mSLAM), mRNA encoding a soluble, secreted form of
SLAM (sSLAM) lacking 30 amino acids (aa) encompassing the entire 22-aa transmembrane region (29). In addition, a
cytoplasmic (c) form lacking the leader sequence and a
variant membrane (vm)SLAM with a truncated cytoplasmic
tail were identified (29). SLAM was recently shown to be a
high-affinity self-ligand with an affinity constant of 0.1 nM
(Cocks, B.G., G. Aversa, S. Balasubramanian, C.-C.J.
Chang, B. Bennett, D. Peterson, J.M. Carballido, J. Punnonen, and J.E. de Vries, manuscript submitted for publication). SLAM-Ig fusion protein specifically bound to SLAM
transfectants and to surface-immobilized SLAM as shown
by surface plasmon resonance. Engagement of SLAM by
specific mAb or sSLAM-induced proliferation of activated
human T cells in a CD28-independent manner, suggesting that the T cell responses observed in CD28-deficient mice
may be partially or completely mediated via SLAM (29). In
addition, SLAM engagement induced IFN- In the present study, the expression and function of
SLAM on B cells was investigated. We demonstrate that
mSLAM is rapidly induced on B cells after activation with
anti-µ or anti-CD40 mAbs or Staphylococcus aureus Cowan
I (SAC). A recombinant form of sSLAM and murine L cells
transfected with mSLAM induced B cell proliferation in
the absence of other stimuli, and they strongly enhanced B
cell proliferation induced by anti-µ or anti-CD40 mAbs or
SAC in the presence or absence of cytokines IL-2, IL-4,
IL-10, IL-12, IL-13, or IL-15. These results indicate that
SLAM is a novel B cell growth and differentiation promoting molecule.
Reagents.
Purified human recombinant (r) IL-4 and IL-10
were provided by Schering Plough Research Institute (Kenilworth, NJ). Purified rIL-2, rIL-13, and rIL-15 were kindly provided by Drs. Gerard Zurawski (DNAX), Satish Menon (DNAX)
and Robert Kastelein (DNAX), respectively. rIL-12 was purchased from R&D Systems, Inc. (Minneapolis, MN). Unconjugated mAbs specific for CD3, CD4, CD8, CD14, CD16, and
CD56, PE- and FITC-conjugated mAb specific for CD20, and
control antibodies with irrelevant specificities were obtained from
Becton-Dickinson (San Jose, CA). The purified anti-CD40 mAb
89 (IgG1) (32) was kindly provided by J. Banchereau (ScheringPlough France, Dardilly, France). mAb A12 specific for SLAM
(IgG1) was generated and purified as described (29). Beads coated
with anti-µ mAbs were obtained from Irvine Scientific (Santa Ana,
CA), and Staphylococcus aureus Cowan I cells (Pansorbin Cells) were
purchased from Calbiochem Novabiochem (La Jolla, CA). FITCconjugated mAb anti-IgD was obtained from Nordic Immunology (Tilburg, The Netherlands).
Generation of sSLAM.
Recombinant sSLAM was expressed in
insect cells using the baculovirus expression system as described (33).
In brief, Spodoptera frugiperda (Sf 9) cells were transfected with cDNA
encoding the soluble secreted form of SLAM (pSECslam) (29),
which was subcloned into the vector pVL1393 (Invitrogen, San
Diego, CA) using PCR and primers 5 Preparation of sSLAM-coated Beads.
Polystyrene latex beads with
a mean diameter of 0.6 µm (Interfacial Dynamics Corporation,
Portland, OR) were first incubated with anti-Flag mAb M2 for
30 min at room temperature (0.3 µg/108 beads), and then an additional 30 min on ice. The beads were washed with PBS. Recombinant sSLAM with a Flag tail was added to the beads at 0.3 µg/108 beads, and the suspension was incubated at room temperature for 30 min. These sSLAM-coated beads were then washed
three times with PBS supplemented with 2% FCS, and finally resuspended in PBS plus 2% FCS.
Cell Preparations.
Blood samples and spleens were obtained
from healthy volunteers or from patients undergoing splenectomy
due to trauma, respectively. PBMC were isolated by centrifugation over Histopaque-1077 (Sigma Chem. Co., St. Louis, MO).
, mediate delayed-type hypersensitivity reactions and
activate macrophages (1). In addition to T cells, monocytes can regulate B cell proliferation and differentiation through release of cytokines, such as IL-6, IL-8, and IL-10
(4), and B cell proliferation and differentiation also appear to be regulated by autocrine mechanisms. IL-1, IL-6,
IL-10, TNF-
, and lymphotoxin have been shown to be
produced, among other cell types, by activated B cells and
they enhance proliferation and differentiation of these cells
(7).
, expressed by activated and HIV-infected
T cells, are involved in productive T-B cell interactions
(19). Although several surface molecules can mediate
T-B cell collaboration, the role of CD40L appears to be
crucial, as is clearly demonstrated in patients with hyperIgM syndrome, who are unable to produce IgG, IgA, or IgE because of mutations in their genes encoding CD40L
(22). However, the patients have normal or elevated
levels of IgM and normal numbers of B cells in the circulation indicating that a CD40L-independent pathway of B
cell proliferation and differentiation is operational in these
patients. Moreover, CD40L-deficient mice have normal Ab
responses against thymus-independent antigens (26, 27),
and some T cell clones from patients with mutated CD40L genes induce Ig isotype switching even in the presence of
neutralizing anti-TNF-
mAbs (28), supporting the conclusion that yet to be characterized costimulatory molecules
contribute to the expansion, Ig isotype switching and differentiation of activated B cells.
production
even in Th2-type T cell clones, suggesting a role for SLAM
in directing cytokine synthesis in T helper cells (29).
-GAC TCA GAC GAA
TTC ATG GAT CCC AAG GGG CTC-3
and 5
-AGC TAG
ATC AGA TCT GCT CTC TGG AAG TGT CAC-3
followed by a Bg1I/EcoRI digestion. A Flag sequence was fused to
the carboxy terminus of the protein, and the expressed recombinant sSLAM was purified from the supernatants using an M2 antiFlag mAb affinity matrix (Eastman Kodak Company, New Haven,
CT) and glycine elution according to the manufacturer's instructions.
Culture Conditions. Purified B cells were cultured in roundbottomed 96-well plates (Linbro, McLean, VA) in 0.2 ml Yssel's medium supplemented with 10% FCS and incubated at 37°C in a humidified atmosphere containing 5% CO2. Cultures were performed in triplicate when proliferation of B cells was studied and in quadruplicate when Ig synthesis was studied. Total negatively selected B cells purified by magnetic beads were cultured at 105 cells/well, whereas sorted CD20+ or CD20+, sIgD+ B cells were cultured at 3 × 104 cells/well.
Phenotypic Analysis of Cultured Cells. B cells were cultured as described above and they were harvested and washed after culture periods varying from 6 to 72 h. FITC- and PE-conjugated mAbs were added to the cell pellet at saturating concentrations and the cells were then incubated at 4°C for 30 min. B cells cultured in the presence of SAC were preincubated with 5% mouse serum at 4°C for 30 min due to nonspecific staining otherwise observed on these cells. FITC- and PE-conjugated mAbs with irrelevant specificities were used as negative controls. Propidium iodide (PI) (2 µg per 5 × 105 cells) was added to all samples to exclude dead cells. A total of 104 cells with light scatter characteristics of lymphocytes of each sample were analyzed using FACScan® flow cytometer and CellQuest software (Becton Dickinson).
Isolation of RNA and Reverse-Transcriptase PCR.
Cultured cells
were harvested and washed in PBS. RNA was isolated using
RNAzol B (CNNA; Biotech, Friendswood, TX) according to manufacturer's instructions. The samples were stored at 80°C until
used. Reverse transcription was performed using Superscript R/T
enzyme, and the Superscript system protocol I (GIBCO BRL, Gaithersburg, MD). The resulting cDNA was diluted 10-fold in
water and stored at
70°C until used. 1-5 ng of cDNA was amplified with 1 U of Taq polymerase (Perkin Elmer Cetus, Norwalk, CT) and using 40 cycles of PCR (initial denaturation at
94°C for 2 min, then 30 s at 94°C; annealing 30 s at 55°C; extension 1 min, 72°C). To detect various isoforms of SLAM, specific
primers were used as follows: 5
-ATC ACT GGA GAA CAG
TGT-3
and 5
-CCC AGC ATA CAC TGC CC-3
, to amplify
mSLAM, cSLAM and vmSLAM; 5
-ATC ACT GGA GAA
CAG TGT-3
and 5
-TTC GTT TTA CCT GAG GGG TCT
G-3
to amplify sSLAM; 5
-ACT TTG GAC TGC CTG TGT
GAG-3
and 5
-CAG CCC AGT ATC AAG GTG CA-3
, to
amplify cSLAM; and 5
-CAG CTG GAG TGA AAA GGC-3
and 5
-CCG CAC CGG TCT TGG CGG-3
, to amplify
vmSLAM.
Measurement of Ig Production. Purified B cells were cultured in Yssel's medium supplemented with 10% FCS as described above for 12 d. IgM, IgG, IgA, and IgE levels in culture supernatants were determined by ELISA as described previously (34). The sensitivities of these ELISAs were 0.5 ng/ml.
The expression of mSLAM on peripheral blood (PB) and splenic B
cells was studied by double immunofluorescence using mAbs
specific for CD20 and SLAM (29). The expression level of
SLAM was generally higher on PB B cells than on splenic
B cells (Fig. 1). The percentages of SLAM+ cells among PB
B cells ranged between 20 and 50%, whereas those among
splenic B cells were generally less than 20%. B cells were rather homogenous in terms of their SLAM expression, and
staining with SLAM-specific mAb generally caused a shift
in the staining pattern rather than resulted in the detection
of two separate populations of positive and negative cells
(Fig. 1). Because of the low proportion of B cells in PB
mononuclear cells (MC), in some experiments B cells were
first enriched by depleting T cells, NK cells and monocytes
by magnetic beads. The results using total splenic MC or
PBMC were comparable to those using B cell enriched cell
populations (Fig. 1 and data not shown), indicating that cell
purification method did not affect the results.
mSLAM Is Upregulated on B Cells after Activation, and Activated B Cells Express Soluble, Cytoplasmic and Membrane Forms of SLAM.
We next studied the effect of activation
on SLAM expression on B cells. As shown in Fig. 2 A,
mSLAM expression was moderately upregulated on B cells
cultured in the presence of medium alone. The expression
level of mSLAM was not significantly modulated by IL-2,
IL-4, or IL-10 (100 U/ml each), when compared to B cells cultured in the presence of medium alone for up to 72 h
(Fig. 2 A and data not shown). In contrast, SAC, anti-µ
mAbs and anti-CD40 mAbs induced high levels of mSLAM
expression on >85% of the B cells during a culture period
of 20 h and the high expression levels were sustained for up
to 3 d. The most rapid effect was observed in response to
anti-µ mAbs, which induced high levels of mSLAM expression on B cells already during a culture period of 6 h,
whereas optimal enhancement of mSLAM expression by
SAC and anti-CD40 mAbs was observed after 20 h. These
data indicate that mSLAM is strongly upregulated on B cells
after activation, and suggest that mSLAM is rapidly induced
after recognition of antigen by surface Ig.
Activated human T cells were previously shown to express two alternatively spliced isoforms of mSLAM (29). In addition, they expressed a cytoplasmic and a soluble, secreted form of SLAM (29). Sorted, highly purified B cells activated by anti-CD40 mAbs and IL-4 transcribed the same isoforms of SLAM that are detectable in PBMC activated with PMA and Ca2+ ionophore (Fig. 2 B), including the secreted soluble form of SLAM. These data indicate that, in addition to T cells, B cells are capable of producing sSLAM, and suggest that the expression of SLAM on B and T cells are regulated by similar mechanisms.
sSLAM Induces Proliferation of Highly Purified B Cells.Since SLAM is a high-affinity self-ligand, we investigated
the role of SLAM-SLAM interactions in the regulation of
B cell function. As shown in Fig. 3, recombinant sSLAM
directly induced B cell proliferation of freshly isolated B cells
in a dose-dependent manner. sSLAM-induced B cell proliferation was in the same range as that induced by IL-2
(Fig. 3), which has been previously described to directly induce proliferation and differentiation of freshly isolated human B cells (35). IL-4 had no effect on B cell proliferation under similar culture conditions (Fig. 3). Optimal effects by sSLAM were observed at high concentrations of ~20 µg/ml,
but it has to be taken into account that gel filtration analysis
has indicated that >99% of sSLAM is in dimeric form (data
not shown), and that only monomers are available for
binding to mSLAM.
The function of SLAM on B cells was also studied by
adding anti-SLAM mAb A12 to B cell cultures stimulated
with SAC or anti-CD40 in the presence or absence of IL-2,
IL-4, or IL-10. This mAb was previously shown to induce
proliferation of preactivated human T cells and T cell clones
(29). Similar effects on T cells were observed irrespective
whether mAb A12 or its F(ab)2 or Fab fragments were used,
suggesting that cross-linking does not play a major role in
the signaling of SLAM. mAb A12 or its F(ab
)2 or Fab
fragments at concentrations 0.01-100 µg/ml had no enhancing effects on spontaneous or anti-CD40 mAb and IL-4-
induced B cell proliferation in six separate experiments
(data not shown). In fact, anti-SLAM mAb slightly inhibited B cell proliferation induced by SAC or anti-CD40 mAbs plus IL-4 (data not shown). In addition, no stimulatory effects by mAb A12 were observed on B cells that had
been preactivated with SAC or anti-CD40 mAbs and IL-4
or IL-10 for 3-5 d, further indicating that mAb A12 cannot
replace sSLAM in induction of B cell proliferation.
sSLAM also strongly enhanced
proliferation of B cells cultured in the presence of other B
cell growth promoting signals. sSLAM had additive or
slightly synergistic effect on B cell proliferation induced by
anti-CD40 mAbs and IL-4 (Fig. 4 A), and it also enhanced proliferation of B cells cultured in the presence of anti-µ
mAbs or SAC (Fig. 4 B). These effects were observed irrespective of whether the cells were negatively selected using
magnetic beads or positively sorted using cell sorting, suggesting that contaminating cells did not play a role in sSLAM-
induced B cell proliferation (Fig. 4 B). Moreover, sSLAM
had strong B cell growth promoting effects when saturating
concentrations (100 U/ml) of IL-2, IL-4, IL-10, IL-12, or
IL-13 were added to cultures stimulated with anti-CD40
mAbs, suggesting that the signaling pathway of sSLAM is
independent of that of these cytokines (Fig. 4 C ). Importantly, the level of sSLAM-induced proliferation of CD40stimulated B cells was in the same range as that induced by
IL-2, IL-4, IL-12, or IL-13 under the same conditions (Fig.
4 C ). In addition, although IL-10 was more potent than
sSLAM in inducing B cell proliferation in the presence of
anti-CD40 mAbs, sSLAM still enhanced IL-10-induced response in a synergistic fashion. The B cell growth-promoting effect of sSLAM was specific, because a control
protein that binds the human obese gene product (36), produced and purified in a manner similar to sSLAM, failed to
induce B cell proliferation when tested at concentrations
up to 25 µg/ml (data not shown).
Previously, mSLAM was shown to have two naturally occurring isoforms with different cytoplasmic tails (29). Myelin P0 is another homophilically interacting member of the Ig supergene family, and, interestingly, it was demonstrated that the cytoplasmic region of the molecule is required for optimal homophilic interactions of the extracellular portion (37). To study the role of the carboxy-terminal portion of sSLAM, which corresponds to the cytoplasmic tail of mSLAM, a truncated form of sSLAM lacking this region was generated. As shown in Fig. 4 C, the two forms of sSLAM had virtually identical effects on B cell proliferation, indicating that the carboxy-terminal tail of sSLAM is not required for the B cell proliferation inducing effect of the molecule.
Kinetics of sSLAM-induced B Cell Proliferation.The time
point of optimal effect of sSLAM on B cell proliferation
was dependent on the costimulus used in the cultures.
When B cells were activated by IL-10 and anti-µ mAbs,
which typically induce rapid B cell activation (38), sSLAM
induced its optimal effects already after a culture period of
2 d (Fig. 5 A). However, when B cells were activated by
IL-10 and anti-CD40 mAbs, optimal effects were generally
observed on day 4 and enhancing effects by sSLAM were
still detectable after a culture period of 8 d (Fig. 5 A). The
kinetics were primarily dependent on whether anti-µ or
anti-CD40 mAbs were used to costimulate the cells, because the results were essentially the same irrespective of
whether IL-2, IL-4, or IL-10 were included in these cultures (data not shown). IL-2 significantly enhanced sSLAMinduced B cell proliferation (Fig. 5 A), whereas IL-4 had
no or minimal effects (data not shown), which is consistent
with the fact that IL-2, but not IL-4, induces proliferation
of freshly isolated B cells (Fig. 3). sSLAM alone or in combination with IL-2 induced its optimal effect after a culture
period of 6 d (Fig. 5 A).
Anti-CD40 mAbs and IL-4 have previously been shown to support long-term growth and survival of human B cells (32). To investigate the effects of sSLAM on B cells derived from long-term cultures, B cells were cultured for 5 wk in the presence of anti-CD40 mAbs and IL-4, whereafter the cells were recultured in the presence or absence of sSLAM. As shown in Fig. 5 B, sSLAM significantly enhanced proliferation of these B cells. sSLAM was also effective when IL-2, IL-4, IL-10, or anti-CD40 mAbs, or combinations of these were added to the cultures. Like sSLAM, IL-10 significantly enhanced proliferation of these B cells, but the effect of IL-10 could be further enhanced by sSLAM (Fig. 5 B). Anti-CD40 mAbs and IL-4 did not significantly affect B cell proliferation, which is likely to be due to their presence during the primary cultures. These results indicate that SLAM enhances B cell proliferation both during the initial phase of B cell activation as well as during expansion of preactivated B cells.
L Cells Transfected with mSLAM- and sSLAM-coated Polystyrene Latex Beads Induce B Cell Proliferation.To study the
effects of mSLAM on B cell proliferation murine L cells were
transfected with cDNA encoding mSLAM. Importantly, the
transfectants expressed mSLAM at levels comparable to those on activated T cells (29) and B cells (Fig. 2 A), whereas untransfected L cells were completely mSLAM negative (Fig.
6, A and B). Irradiated mSLAM+ L cells strongly enhanced
B cell proliferation induced by anti-CD40 mAbs in the
presence of IL-4, IL-10 (Fig. 6 C), IL-2, or IL-15 (data
not shown). Like sSLAM, mSLAM transfectants also enhanced proliferation of anti-CD40-activated B cells in the
absence of exogenous cytokines.
Because normal L cells slightly enhanced B cell responses in some experiments, we examined whether the B cell growth promoting effects of mSLAM transfectants were due to B cell growth factors released by L cells activated through SLAM-SLAM interactions. Supernatants from both untransfected L cells and SLAM transfectants did have B cell growth promoting activities, but the responses were always <15% of those induced by SLAM transfectants, and no significant difference between supernatants derived from normal and transfected L cells could be observed (data not shown). In addition, supernatants from irradiated L cells, irrespective of whether they were transfected or not, had no effect on B cell proliferation induced by anti-CD40 mAbs and IL-4 (data not shown). To further investigate the effects of surface-bound SLAM, we also studied proliferation of B cells cultured in the presence of polystyrene latex beads coated with sSLAM using mAb specific for the Flagsequence that was attached to the carboxy-terminus of sSLAM. Similar to mSLAM transfectants, sSLAM-coated beads enhanced B cell proliferation induced by anti-CD40 mAbs and IL-4 (Fig. 6 D), supporting the conclusion that SLAM activates B cells not only in its soluble form, but also in its surface-bound form. These data also further indicate that SLAM-SLAM interactions between the L cells did not play a role in the capacity of SLAM transfectants to induce B cell proliferation.
Effects of sSLAM on Ig Synthesis by B Cells.Finally, we
studied the effects of sSLAM on Ig-synthesis by purified B
cells cultured in the presence or absence of anti-CD40 mAbs.
sSLAM significantly enhanced spontaneous IgM, IgG and
IgA synthesis produced by highly purified total B cells, but
the effects of sSLAM were generally more potent when
anti-CD40 mAbs were added to these cultures (Fig. 7 A).
The concentrations required for optimal induction of Ig
synthesis were comparable to those required for induction
of optimal B cell proliferation (~20 µg/ml). No IgE synthesis was observed in these cultures (data not shown). These results indicate that SLAM-SLAM interactions enhance not only B cell proliferation, but also Ig synthesis by
B cells.
To investigate whether sSLAM induces Ig isotype switching, sorted sIgD+ naive B cells were cultured in the presence of sSLAM. As shown in Fig. 7 B, sSLAM induced in a dose-dependent manner IgM synthesis by sIgD+ B cells cultured in the presence of anti-CD40 mAbs. However, no IgG, IgA, or IgE was produced by these cells, indicating that sSLAM induces human B cell differentiation, but fails to induce Ig isotype switching.
In the present study it is demonstrated that sSLAM and mSLAM transfectants promote growth and differentiation of human B cells. These effects were partially or completely mediated by homophilic SLAM-SLAM interactions, because mSLAM was induced or upregulated on virtually all B cells after activation in vitro, and because SLAM was recently shown to be a high-affinity self-ligand (Cocks, B.G., G. Aversa, S. Balasubramanian, C.-C.J. Chang, B. Bennett, D. Peterson, J.M. Carballido, J. Punnonen, and J.E. de Vreis, manuscript submitted for publication). Thus, SLAM is an unusual molecule in that it acts both as a signaling membrane receptor and as a soluble and membrane-bound growth promoting molecule. Other members of the CD2 family of cell surface molecules have previously been shown to mediate both B and T cell activation. Engagement of CD2 by a pair of agonistic mAbs induces T cell proliferation in the absence of T cell receptor signaling (39,40), and CD58, the natural ligand for CD2 (41), on antigen-presenting cells reduces the concentration of antigen required for T cell activation (42). CD2+ human B progenitor cell lines also proliferate in response to agonistic mAbs specific for CD2 (43), which is expressed on a subset of CD20+ cells in human fetal thymus and bone marrow (43, 44). Furthermore, blocking of CD2 on the surface of T cells inhibits the capacity of these cells to mediate productive T-B cell interactions (45), and mAbs specific for CD58 induce IgE isotype switching and IgE synthesis in a CD40-independent manner by purified B cells cultured in the presence of IL-4 (46). Recombinant sSLAM and murine L cells transfected with cDNA encoding mSLAM induced proliferation and IgM, IgG, and IgA production by unfractionated B cells, but only IgM production by sorted sIgD+ B cells was observed, indicating that SLAM acts as a B cell growth and differentiation promoting molecule, but not as an Ig isotype switch factor.
sSLAM had B cell growth promoting effects even in the absence of other stimuli, but the most potent effects were observed when B cells were cultured in the presence of polyclonal B cell stimuli and cytokines. Importantly, the levels of [3H]thymidine incorporation induced by sSLAM were comparable to those induced by IL-2, IL-4, IL-12, or IL-13 in B cell cultures costimulated by anti-CD40 mAbs. In addition, although sSLAM was less potent than IL-10 in inducing B cell proliferation, it also strongly enhanced IL-10- induced proliferative responses. Both sSLAM and murine L cells transfected with cDNA encoding mSLAM had growth promoting effects irrespective whether IL-2, IL-4, IL-10, IL-12, IL-13, IL-15, anti-µ mAbs, anti-CD40 mAbs, or SAC were added to the cultures, suggesting that the signaling pathway of SLAM is independent of that of these B cell activators. The exact mechanisms of SLAM signaling remain to be studied, but it seems that tyrosine kinases that contain Src homology 2 (SH2) domains are involved, because the intracellular portion of mSLAM has four tyrosine residues (29), three of which conform to the consensus sequence phosphotyrosine-hydrophobic-X-hydrophobic, determined for binding to SH2 domains (47). Because sIgM and CD40 mediate activation signals at different stages of B cell responses, SLAM appears to costimulate B cells not only during the initial antigen-induced activation phase, but also during T cell-dependent, antigen-independent expansion of preactivated B cells. This notion is supported by our kinetics studies, which indicated that sSLAM enhances B cell proliferation at all time points during culture periods of 2-8 d. Moreover, sSLAM enhanced proliferation of B cells, which had been maintained in culture with antiCD40 mAbs and IL-4 for 5 wk.
It is well established that recognition of a specific antigen results in vigorous B cell proliferation giving rise to the dark zones of germinal centers in the lymph nodes. Despite these regions are relatively devoid of T cells, recognition of antigen by a single B cell may give rise to 104 antigen-specific B cells in 3 d, suggesting that the expansion of B cells in germinal centers predominantly occurs in a T cell- independent manner (17). The fact that SLAM is rapidly induced on B cells after activation, and that the high expression levels are sustained for several days in vitro suggest that SLAM-SLAM interactions may play a major role in mediating homotypic B cell contacts in germinal centers resulting in expansion of antigen-specific B cells.
SLAM was previously shown to preferentially stimulate
production of Th1-type cytokines. Engagement of SLAM
by the specific mAb A12 during antigen-specific T cell activation induced IFN- production even in Th2-like T cell
clones (29), suggesting that SLAM is primarily involved in
delayed-type hypersensitivity responses. However, despite
that Th2-type cytokines are generally considered to be the
main factors involved in B cell activation and Ig synthesis, Th1 cytokines IL-2 and IFN-
have also been shown to
have direct effects on B cell function. IL-2 directly enhances
proliferation and Ig synthesis of in vitro and in vivo preactivated B cells (35, 48), and IFN-
has been shown to induce Ig isotype switching in murine B cells (49). IL-2 also
significantly enhanced sSLAM-induced proliferation of freshly
isolated B cells, whereas IL-4 was ineffective. Organ specific and systemic autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus, are characterized by both increased production of Th1-type cytokines,
including IL-2, and hyperactivity of B cells with elevated
production of autoantibodies (50). In addition, patients
with rheumatoid arthritis (RA) have elevated levels of
CD45RO+ T cells, which were shown to coexpress SLAM
in healthy individuals (29), suggesting that expression of
SLAM may be elevated in T cells from patients with RA.
Indeed, we have recently demonstrated that synovial fluid
CD4+ T cells from patients with rheumatoid arthritis express significantly higher levels of SLAM than PB CD4+ T
cells (Isomäki, P., G. Aversa, B.G. Cocks, R. Luukkainen, P. Toivanen, J.E. de Vries, and J. P., manuscript submitted
for publication). These data, together with the observations
that SLAM interacts homophilically, suggest that SLAM
may contribute to the productive T-B cell interactions and
B cell hyperactivity observed in patients with autoimmune
diseases.
Although our results clearly indicate that both mSLAM and sSLAM are capable of inducing B cell proliferation, the relative contribution of these two forms of SLAM in the regulation of B cell function remains to be determined. The fact that the expression level of mSLAM on L cells transfected with cDNA encoding mSLAM was comparable to that on activated B or T cells strongly suggests that mSLAM acts as a B cell costimulatory molecule under physiological conditions in vivo. Whether SLAM is in solution or expressed on cell surface does not appear to affect the function of the molecule, because mSLAM transfectants and sSLAM had essentially similar effects on B cell proliferation. In addition, the region that corresponds to the cytoplasmic portion of mSLAM was not required for the function of sSLAM, because the effects of a truncated sSLAM lacking this region were virtually identical to those of the naturally occurring sSLAM. However, the high concentrations of sSLAM required for induction of B cell proliferation suggest that mSLAM may be the dominant active form in physiological situations. On the other hand, because SLAM is a self-ligand, sSLAM primarily exists in a dimeric form in solution. Based on gel filtration analysis, the proportion of monomeric sSLAM is <1% of the total concentration of sSLAM (data not shown), and, therefore, the concentrations of monomeric sSLAM available for homophilic binding to mSLAM are far less than the total concentration of sSLAM.
Like activated T cells (29), activated B cells expressed sSLAM and the potential cytoplasmic form, which lacks the leader sequence. The relative contributions of these, and the two different alternatively spliced isoforms of mSLAM, in the regulation of B cell function remain to be determined. Nevertheless, sSLAM had potent growth promoting effects on B cells also when cross-linked to polystyrene latex beads using mAb specific for the Flag sequence attached to the molecule. These data are in line with the notion that sSLAM primarily acts in its monomeric form through binding to mSLAM, because the Flag sequence was attached to the carboxy-terminal end of sSLAM, and, therefore, the region corresponding to the extracellular portion of mSLAM was available for homophilic binding. Accordingly, transfection of mSLAM into murine pre-B cell line Baf/3 triggered aggregation of these cells, indicating that mSLAM also mediates cell adhesion (Cocks, B.G., G. Averso, S. Balasubramanian, C.-C.J. Chang, B. Bennett, D. Peterson, J.M. Carballido, J. Punnonen, and J.E. de Vries, manuscript submitted for publication). Consequently, it may also be possible that sSLAM, through binding to mSLAM, functions as an inhibitor of cell adhesion mediated by mSLAM- mSLAM interactions. However, this does not seem to be the case, because sSLAM did not block B cell proliferation induced by SLAM transfectants (unpublished observation).
The SLAM-specific mAb 12 did not induce or enhance B cell proliferation or Ig synthesis, indicating that it does not act as a surrogate SLAM ligand on B cells. These data are partially different from the data obtained on T cells, because both the mAb A12 (29) and sSLAM (Cocks, B.G., G. Averso, S. Balasubramanian, C.-C.J. Chang, B. Bennett, D. Peterson, J.M. Carballido, J. Punnonen, and J.E. de Vries, manuscript submitted for publication) induced proliferation of preactivated human T cells. Therefore, the functions of SLAM on activated B and T cells may not be completely comparable. However, because the mAb A12 recognizes both isoforms of mSLAM with different cytoplasmic tails, it remains possible that differential expression of these isoforms on the surface of B and T cells results in distinct signaling events after SLAM engagement. Moreover, there may be differences in B and T cells in the molecules that associate with the cytoplasmic tails of mSLAM isoforms. In addition, we cannot rule out the possibility that SLAM may also signal through molecule(s) other than SLAM on B cells, which might explain the differential effects of the mAb A12 on B and T cells. Unfortunately, preincubation of SLAM+ cells in the presence of mAb A12 did not block the binding of sSLAM to mSLAM (data not shown), indicating that this mAb cannot be used in blocking experiments to address this question.
Taken together, the present study indicates that activated B cells, like T cells, express four alternatively spliced isoforms of SLAM, and that both the soluble and the membrane-bound forms of SLAM induce proliferation and Ig synthesis by highly purified B cells, suggesting an important role for this molecule in the regulation of T-B cell interactions. In addition, because B cells are in close contact to each other in germinal centers, homophilic SLAM interactions during homotypic B cell contacts are likely to contribute to the rapid antigen-driven expansion of B cells in vivo, and may play a role in hyperactivity of B cells in disease situations.
Address correspondence to Jan E. de Vries, DNAX Research Institute, Department of Human Immunology, 901 California Avenue, Palo Alto, CA 94304.
Received for publication 5 December 1996 and in revised form 22 January 1997.
DNAX Research Institute is supported by Schering-Plough, Inc.We thank Jim Cupp, Eleni Callas, and Josephine "Dixie" Polakoff for their expert FACS® assistance, and JoAnn Katheiser for her careful secretarial help.
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