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Tanja Scheikl,1,
Bernhard Reis,2,
Norbert Hüser,1
Klaus Pfeffer,2 and
Bernhard Holzmann1*
Department of Surgery, Technische Universität München, 81675 Munich, Germany,1 Institute of Medical Microbiology, Universität Düsseldorf, 40225 Düsseldorf, Germany2
Received 4 July 2005/ Accepted 27 July 2005
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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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Both B and T lymphocytes use antigen receptor engagement to initiate distinct signal transduction pathways that direct ultimate cellular responses. It is well established that adaptor molecules regulate the coupling of receptor-proximal events, such as protein tyrosine kinase activation, with results such as inducible gene expression and cytoskeletal rearrangements. While adaptor molecules possess no intrinsic enzymatic activity, their ability to mediate protein-protein interactions is vital for the integration and propagation of signal transduction cascades in lymphocytes (12, 16, 29). Adaptor molecules may contain multiple phosphorylatable residues or proline-rich regions that allow them to be recognized by interaction domains such as SH2 and SH3 on other signaling proteins. Therefore, adaptor molecules are able to coordinate interactions among the signal transduction effector proteins. This characteristic suggests that adaptor molecules act as intracellular scaffolds around which signaling complexes are organized. The use of genetically manipulated mice has shown that adaptor proteins play essential roles in T and B lymphocyte development and in activation of adaptive immune responses (31).
Recently, we have identified the putative adaptor protein SLY1 (SH3 domain protein expressed in lymphocytes), which contains a bipartite nuclear localization signal as well as SH3 and SAM domains. It is preferentially expressed in T and Blymphocytes (4). Using a phosphopeptide-specific antiserum it was demonstrated that SLY1 is phosphorylated on Ser27 following T- or B-cell receptor ligation (2). Moreover, inhibitor studies implied that SLY1 represents a target for phosphorylation by phosphatidylinositol 3-kinase, protein kinase C, or a protein kinase C-dependent protein kinase in antigen receptor-stimulated lymphocytes. These studies therefore defined SLY1 as a previously unrecognized target for antigen receptor signal transduction and suggested that it may play a role in adaptive immunity.
In the present study,
Sly1 mutant mice were generated expressing a SLY1 protein that
lacks the Ser27 phosphorylation site and parts of the nuclear
localization signal (SLY1
aa 20-100). In contrast to
the wild-type SLY1 protein, the mutant SLY1 protein is not localized in
the nucleus. Sly1d/d mice reveal impaired lymphoid
organ development and antigen receptor-mediated lymphocyte activation.
Sly1 mutant mice exhibit severe defects in humoral
immune responses. Finally, Sly1d/d mice show
extended allograft survival. Thus, expression of intact SLY1 protein
appears crucial for adaptive immune
responses.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
were obtained from Cell Signaling,
whereas antiphosphotyrosine antibody 4G10 was from Upstate Signaling.
Rabbit polyclonal antisera against SLY1 were produced against peptides
LRRKPSNASDKEPTQ (anti-SLY1 residues 2 to 16) and
TEQEEREPPSL-SRQT (anti-SLY1 residues 131to 145)
according to standard protocols. Recombinant IL-2 and recombinant IL-4
were from R&D Systems, and lipopolysaccharide was from Sigma.
Concentrations of IL-2, IL-4, IL-10, tumor necrosis factor, and gamma
interferon in culture supernatants were measured using enzyme-linked
immunosorbent assay kits from R&D Systems. Trinitrophenol
(TNP)-CGG, TNP-Ficoll, and TNP-bovine serum albumin were
from Biosearch Technologies, and alum was obtained from Serva. phorbol
myristate acetate, ionomycin, Na3Ov4 and protease
inhibitory cocktail were purchased from
Sigma. Gene targeting and mice. E14.1 embryonic stem (ES) cells from 129/Ola micewere grown in Dulbecco's modified Eagle's medium (GIBCO BRL) supplemented with L-glutamine (2 mM; Seromed), leukemia inhibitory factor, penicillin/streptomycin (100 µg/ml; Seromed), 2-mercaptoethanol (0.05 mM; GIBCO BRL), and 15% heat-inactivated fetal bovine serum (Boehringer).
A murine genomic ES-129 bacterial artificial chromosome library (Genome Systems Inc.) was screened by hybridization with a 205-bp murine Sly1 cDNA fragment as a probe. The resulting bacterial artificial chromosome clone was mapped by Southern blot hybridization using murine Sly1 cDNA fragments. bacterial artificial chromosome fragments were cloned into pBluescript (Stratagene) and fully sequenced. The targeting vector was constructed in pBluescript such that a 600-bp fragment of the genomic Sly1 locus encoding exon 2 and 3 was replaced by lacZ and a neomycin resistance cassette. A herpes simplex virus thymidine kinase cassette was inserted 3 kb upstream of the targeted sequence. E14.1 ES cells were electroporated with the NotI-linearized targeting vector, and the transfected cells subjected to G418 and ganciclovir selection. Clones showing homologous recombination were detected by PCR and subsequently confirmed by Southern blot hybridization with the 3' flanking probe indicated in Fig. 1a after digestion of ES cell DNA with EcoRI. Single integration was verified by probing the Southern blot with the neomycin resistance cassette. Correctly targeted ES cell clones were injected into C57BL/6 blastocysts, which were transferred into pseudopregnant foster mothers. Resulting chimeric mice were backcrossed to C57BL/6 mice, and germ line transmission of the targeted allele was confirmed by Southern blot analysis.
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For reverse transcription-PCR analysis of SLY1 mRNA splicing, total RNA from thymus and spleen was extracted using the TRIZOL reagent (Invitrogen Life Technologies), and was reverse-transcribed to cDNA using oligo(dT) primer and SuperScript II (Invitrogen Life Technologies). The following primer set was used for exon skipping analysis: Ex1_for: 5'-GAGTAGCAGCCCCAGGC-3', and Ex4_rev: 5'-CTGTTGATGTCTGGCGGC-3'.
Generation of bone marrow chimeric mice. For generation of bone marrow chimeric mice, recipients were lethally irradiated with 6 Gy and intravenously injected with 107 donor bone marrow cells 24 h later. Analysis of mice was performed 4 weeks after adoptive bone marrow transfer.
B-cell proliferation. Splenocytes from wild-type and Sly1d/d mice were depleted of non-B cells using immunomagnetic beads conjugated with antibodies against CD90, CD11b, and CD11c (Miltenyi Biotech). Enriched B cells were stained with CD23 and CD21 antibodies and marginal zone (CD21hi CD23lo) and follicular B cells (CD21int CD23hi) were purified on a cell sorter (MoFlo; DakoCytomation). Purified B-cell subsets were stimulated with antibodies against immunoglobulin M and CD40 (10 µg/ml each) for 3 days. [3H]thymidine (Amersham Pharmacia Biotech) was added after 56 h of culture and incubated for 16 h.
T-cell proliferation and cytokine production.
Splenocytes were incubated at
2x 105 cells per well in 96-well
round-bottomed plates with the indicated soluble antibodies or reagents
in 200 µl medium. For proliferation assays, 1 µCi
[3H]thymidine (Amersham Pharmacia Biotech) was added after
66 h of culture and incubated for 16 h.
Incorporation of [3H]thymidine was measured with a Matrix 96
direct 
counter (Packard Instrument Co.). For analysis of
cytokine production, supernatants were harvested after 18 h
of stimulation and assayed by enzyme-linked immunosorbent assay (all
R&D Systems).
Allogeneic mixed lymphocyte reaction. For induction of mixed lymphocyte reactions, allogeneic splenocytes (H-2d) were used as stimulator cells and cocultured with responder cells at concentrations of 2 x 106 cells/ml. The stimulator population was irradiated with 30Gy prior to coculture. The responder cells were total splenocytes (1.2 x 106 cells/ml), CD4-depleted splenocytes (1.5 x 106 cells/ml), or CD8-depleted splenocytes (1.7 x 106 cells/ml). Cell depletion was performed using anti-CD4 or anti-CD8 microbeads according to the manufacturer's instructions (Miltenyi Biotec). Depletion efficiency as determined by flow cytometry analysis was >99%. Proliferation was measured after 84 h of MLR activation by addition of 1 µCi/well 3[H]thymidine (Amersham Pharmacia Biotec) during the last 10 h of culture.
Cytotoxic lymphocyte assay. Mice were immunized s.c. into the hind footpad with ovalbumin (150 µg/footpad; ovalbumin grade IV; Sigma) using CpG-DNA 1668 (5 nmol/footpad) as an adjuvant. Draining lymph nodes were removed 4 days later and cells were cultured for an additional 4 days in medium supplemented with 10 U/ml recombinant IL-2, which allows expansion of in vivo primed cytotoxic T lymphocytes precursors. 51Cr release assays were performed withEL4 target cells pulsed with 0.1 µM of the ovalbumin-derived peptide SIINFEKL. Serial dilutions of effector cytotoxic T lymphocytes were added to target cells and 51Cr release was measure after 4 h of coculture. Specific lysis was calculated according to the following formula: % specific lysis = [cpm (sample) - cpm (spontaneous release)/cpm (maximal release - cpm (spontaneous release)] x 100. For control, unpulsed EL4 cells were used as targets. Specific lysis of unpulsed EL4 cells was below 2% in all experiments.
Humoral immune responses. Mice were immunized intraperitoneally with 5µg TNP-CGG (NP19-CGG) adsorbed to alum or with 100 µg TNP-Ficoll in phosphate-buffered saline. Sera were collected before and 4, 7, 14 and 21 days after immunization. Total NP-specific immunoglobulin isotypes (IgM, IgG1, IgG2a, and IgG3) were detected employing an enzyme-linked immunosorbent assay with NP14-bovine serum albumin-conjugated plates (10 µg/ml in carbonate-buffer pH 9.5). Sera were diluted 1:1,000 for TNP-CGG/alum and 1:500 for TNP-Ficoll immunizations. Murine NP-specific antibodies were detected with alkaline phosphatase-conjugated isotype-specific detection antibodies, which were obtained from Pharmingen (anti-IgG1, anti-IgG2a, and anti-IgG3) or Jackson ImmunoResearch (anti-IgM).
Allograft transplantations. Vascularized cardiac grafts from donor mice were collected in anesthesia by dividing and excising the aorta and pulmonary artery as described (17). Donor hearts were placed in Brettschneider's solution (HTKKöhler Chemie, Germany) until grafting. Abdominal aorta and vena cava were prepared in recipient mice. Subsequently, the cardiac grafts were heterotypically transplanted by end-to-side anastomosis of donor aorta and pulmonary artery to the host's aorta and vena cava, respectively. Resumption of cardiac function of donor hearts was verified. Graft function was monitored daily by transabdominal palpation and scored. Rejection was defined as complete cessation of palpable heart beats and confirmed by direct inspection after laparotomy.
Signal transduction and subcellular protein localization. To prepare total cell lysate, CD90+ T cells were incubated with biotinylated anti-CD3 antibody and cross-linked with streptavidin. Stimulation was stopped with ice-cold PBS and cells were subsequently treated with lysis buffer (150 mM NaCl, 50 mM Tris, pH 8, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1% NP-40, 1 mM NaF, 2 mM Na3Ov4, and protease inhibitors) for 15 min on ice, and centrifuged at 13,000rpm for 10 min. The supernatants were supplemented with 5× Laemmli sample buffer, boiled for 5 min, and used for experiments. Nuclear and cytosolic fractions of cells were prepared according to standard procedures and the cytosolic fractions were precipitated with 10× volume acetone. Protein samples were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (4 to 12% gradient gel; Invitrogen) under reducing conditions, transferred to nitrocellulose membrane (Schleicher and Schuell), probed with the indicated primary antibodies, followed by reacting with horseradish peroxidase-conjugated secondary antibody, and developed by the ECL detection system (Amersham).
Ca influx analysis.
For
Ca2+ flux analysis, splenocytes were loaded with
2.5µM Fluo-4 (Molecular Probes) for 25 min
at 37°C and washed with RPMI 1640 (without phenol red)
containing 25 mM HEPES. Cells were stained on ice with
anti-CD8-allophycocyanin (53.6.7; eBiosciences),
anti-CD4-phycoerythrin-Cy5.5 (RM4 to 5; Caltag) and biotinylated
anti-CD3
. Cells were warmed to 37°C and analyzed for
20 seconds to establish baseline Ca2+ levels. Then,
CD3 was cross-linked by the addition of 10 µg/ml
streptavidin (Sigma). The flow cytometric analysis was performed with a
FACSCalibur (BD Biosciences) using FlowJo Software
(TreeStar).
Gel shift analysis.
For
electrophoretic mobility shift assays, nuclear extracts of
CD90+ purified T cells were prepared according to
standard procedures and the binding reaction was performed in the
presence of a 32P-labeled oligonucleotide containing the
specific recognition sites. Oligonucleotidess were from Santa Cruz:
AP-1 (sc-2501), NF-AT (sc-3577), NF-B (sc-2505), and Oct-1
(sc-2506).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although SLY1, SLY2, and SASH1 are all highly expressed in hematopoietic cells, only the expression of SLY1 appears restricted to the lymphoid lineage (4, 6, 24, 35, 39). SLY1 has been shown to be phosphorylated in a PI3-kinase- or protein kinase C-dependent fashion upon B- and T-cell receptor engagement (2), but the role of SLY1 for the generation of immune responses is not known so far. To investigate the physiological functions of SLY1 in vivo, we replaced the majority of coding sequences of exons 2 and 3 of the Sly1 gene by homologous recombination in embryonic stem cells and generated homozygous mutant mice (Fig. 1b). Western blot analyses with two independent antisera raised against the N-terminal (amino acids 2 to 16) and central regions (amino acids 131 to 145) of the SLY1 protein revealed that homozygous mutant mice express a truncated protein of 45 kDa as opposed to the 55-kDa wild-type SLY1 protein (Fig. 1c).
Based on these findings we examined the possibility that the residual SLY1 protein was generated by a splicing event involving exons 1 and 4, thereby skipping the inserted ß-galactosidase and neomycin resistance elements. Reverse transcription-PCR analyses using primers located in exons 1 and 4 detected a 574-bp fragment in wild-type mice and a 331-bp fragment in homozygous Sly1 mutant mice, which is consistent with an mRNA lacking exons 2 and 3 (Fig. 1d). Nucleotide sequence analysis of the 331-bp fragment directly demonstrated that it was derived from an in-frame splicing of exon 1to exon 4 of the mutant Sly1 allele (data not shown). Similar results were obtained when primers located in exons 1 and 5 were used for reverse transcription-PCR (data not shown). We conclude that the mutant Sly1 allele (hereafter designated Sly1d) encodes a truncated protein with a deletion encompassing amino acids Leu20 to Met100 (Fig. 1a, arrows). It should be noted that this deletion encompasses crucial structural elements of the SLY1 protein such as Ser27, which is phosphorylated upon antigen receptor engagement (2), and the C-terminal half of the putative bipartite nuclear localization sequence, rendering it functionally inactive (see below).
Altered lymphoid organogenesis and lymphocyte development in Sly1d/d mice. Homozygous Sly1 mutant mice were born at normal Mendelian ratios, were fertile, and did not exhibit any gross anatomical or behavioral abnormalities (data not shown). Analysis of different lymphoid organs revealed that total cell numbers in thymus, spleen, peripheral lymph nodes, and Peyer's patches were significantly reduced by about 33 to 60% in Sly1d/d mice compared to wild-type littermates, whereas cellularity of bone marrow and peritoneal cavity appeared to be normal (Fig. 2a). In contrast to peripheral lymph nodes, the Sly1 mutation did not affect cell numbers in mesenteric lymph nodes. These data therefore extend previous work showing that organogenesis of peripheral and mesenteric lymph nodes requires distinct developmental triggers (1, 15, 25). In addition to reduced cellularity, the number of macroscopically visible Peyer's patches was significantly lower in Sly1d/d mice than in wild-type littermate controls (Fig. 2b). When examined by immunohistochemistry, lymphoid organs of Sly1d/d mice exhibited a normal distribution of B and T lymphocytes as well as nonlymphoid cells such as macrophages and dendritic cells (data not shown).
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To further characterize the effects of the Sly1d/d mutation on the development of T and B cells we performed flow cytometry analyses of various lymphoid organs. The results depicted in Table 1 and Fig. 3a show that Sly1d/d mice had significantly lower absolute numbers of CD4 and CD8 double-positive and CD4 single positive thymocytes than wild-type controls. Therefore, experiments were performed to examine the influence of the Sly1d/d mutation on thymocyte differentiation in more detail.
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We observed comparable expression of the T-cell receptor ß-chain and CD5 on double-positive and single-positive thymocyte subsets from Sly1d/d and wild-type mice (Fig. 3c) suggesting that the Sly1d/d mutation does not affect thymic positive selection. To further investigate the role of SLY1 for thymic selection, Sly1d/d mice were crossed with transgenic mice expressing a T-cell receptor specific for the male antigen HY in the context of H-2Db. Thymocytes that express the HY-specific T-cell receptor are negatively selected in male H-2b mice resulting in a strong reduction of double-positive and CD8 single-positive thymocytes, whereas thymocytes from female transgenic mice are positively selected on an H-2b background and develop into mature CD8 single-positive cells (14). The data in Fig. 3d show that the Sly1d/d mutation did not influence the development of double-positive or CD8 single-positive thymocytes expressing the HY-specific T-cell receptor irrespective of whether analyses were performed in female or male mice. These results therefore show that thymic positive and negative selection is not altered in Sly1d/d mice.
Further analysis of peripheral T cells in spleen revealed that both the proportion and the absolute numbers of CD4+ cells were reduced in Sly1d/d compared to wild-type mice (Table 1 and Fig. 3e). In contrast, the proportion of CD8 T cells was increased in Sly1d/d mice and the absolute numbers were comparable in Sly1d/d and wild-type mice. Similar results were obtained upon analysis of T-cell subsets in peripheral lymph nodes (data not shown). Both CD4 and CD8 wild-type and Sly1d/d T cells did not differ in their surface expression of CD3, CD28, CD25, CTLA4, CD62L, and CD69 (data not shown). Moreover, SLY1 mRNA was found to be expressed in both CD4 and CD8 T-cell subsets (data not shown). The subset of CD4 T cells expressing CD25, which is known to include regulatory T cells (18, 28, 30), was not significantly altered by the Sly1d/d mutation (Table 1).
Within the B-cell lineage, the Sly1d/d mutation caused a marked reduction in the fraction of splenic marginal zone B cells, which were defined by their IgMhi IgDlo or IgM+ CD21hi CD23lo phenotype (Table 1 and Fig. 3f). Notably, the absolute numbers of splenic marginal zone B cells were reduced in Sly1d/d mice by about 63% when compared to wild-type controls. In contrast, Sly1d/d mice had normal proportions and numbers of splenic follicular IgMlo IgDhi B cells as well as peritoneal B1 cells (Table 1, Fig. 3f and 2a). Moreover, the proportions of precursor B-cell subsets in bone marrow were not influenced by Sly1 mutation (Table 1). Surface levels of IgM and IgD were not different between wild-type and Sly1d/d B cells (Fig. 3f, lower panel). Thus, the Sly1d/d mutation resulted in a marked hypoplasia of various lymphoid organs and impaired the development and/or maintenance of DN1, double-positive, and CD4 single-positive thymocytes, the peripheral CD4 T-cell subset, and splenic marginal zone B cells.
Effects of Sly1d/d mutation on B-cell activation and humoral immune responses. The Sly1d/d mutation affected generation of marginal zone, but not follicular, B cells. To examine the influence of the Sly1d/d mutation on B-cell function, B-cell subsets were purified and cell proliferation was measured after cross-linking of the antigen receptor by antibodies to IgM in the presence or absence of CD40 costimulation. The results in Fig. 4a show that antibody-mediated cross-linking of IgM and CD40 induced comparable proliferation of follicular B cells from Sly1d/d and wild-type mice, whereas anti-IgM alone was not sufficient to stimulate proliferation. Wild-type and Sly1d/d follicular B cells also exhibited comparable proliferation upon exposure to lipopolysaccharide, which is known to engage Toll-like receptor 4 (Fig. 4a). In contrast, proliferation of purified Sly1d/d marginal-zone B cells in response to IgM and CD40 ligation was strongly reduced when compared to wild-type cells (Fig. 4b).
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Mice expressing mutant SLY1 exhibit a marked reduction of marginal zone B cells, which are known to play an important role in humoral immune responses to T-cell-independent antigens (8, 19). We therefore examined the antibody response ofSly1d/d mice against the T-cell-independent antigen TNP- Ficoll. The results in Fig. 4e demonstrate that production of TNP-specific IgM antibodies was severely reduced in Sly1d/d mutant mice compared to wild-type littermates at all time points studied. At the peak of the T-independent antibody response (day 7), antigen-specific IgM levels were reduced in Sly1d/d mice by greater than 80%. In addition, TNP-Ficoll stimulated production of antigen-specific IgG3 antibodies was markedly attenuated in Sly1d/d compared to wild-type mice (Fig. 4e).
Sly1d/d mutant mice exhibited reduced basal levels of serum IgG1, whose production is dependent on T-cell help to B cells. We therefore investigated whether the Sly1d/d mutation might also influence T-dependent humoral immune responses. Mice were immunized with TNP-CGG (chicken gammaglobulin) adsorbed to alum as an adjuvant and TNP-specific antibodies were measured. Following TNP-CGG immunization very low levels of antigen-specific IgM antibodies were detected in both groups of mice (data not shown). The results in Fig. 4f demonstrate that the levels of IgG1 as well as IgG2a antibodies against TNP were substantially reduced in Sly1d/d mice compared to wild-type controls. Thus, T-cell-dependent humoral immune responses are also markedly attenuated in Sly1d/d mice. Because the Sly1d/d mutation did not affect surface IgM-triggered proliferation of follicular B cells, the data also suggest the possibility that, in addition to reduced cell numbers, CD4 T-cell function might be impaired in Sly1d/d mice.
Impaired antigen receptor-mediated activation of T cells from Sly1d/d mice. To directly investigate the influence of the Sly1d/d mutation on T-cell responses, we stimulated splenic Tcells in vitro by incubation with antibodies to CD3. The production of IL-2, IL-10, tumor necrosis factor, and the Th1- and Th2-type cytokines gamma interferon and IL-4 by Sly1d/d mutant splenocytes was severely impaired compared to wild-type cells (Fig. 5a). Cytokine production was reduced to a similar extent when Sly1d/d T cells were activated in the presence or absence of anti-CD28 antibodies suggesting that costimulation through CD28 cannot compensate for the defect caused by the Sly1d/d mutation. In contrast to T-cell receptor ligation, stimulation with phorbol myristate acetate and calcium ionophore resulted in a comparable production of IL-2 by wild-type and Sly1d/d T cells (Fig. 5a).
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The role of SLY1 for T-cell responses was further investigated in allogeneic mixed lymphocyte reactions using irradiated BALB/c splenocytes as stimulator cells. As shown in Fig. 5c the proliferation of Sly1d/d T cells was significantly reduced compared to wild-type T cells. To further distinguish the effects of the Sly1d/d mutation on CD4 and CD8 T-cell subsets, purified CD4 and CD8 T cells were used as responder cells. We found that the Sly1d/d mutation severely attenuated the proliferative response of CD4 T cells, whereas CD8 T-cell proliferation was only moderately affected (Fig. 5c). To investigate the function of SLY1 for CD8 T cells in more detail, mice were immunized with ovalbumin and CpG-DNA as adjuvant and cytotoxic T-cell activity was determined in vitro. Under these conditions of immunization, the generation of antigen-specific cytotoxic T cells is independent of CD4 T cells (5, 32). Sly1d/d mice and wild-type controls were able to mount comparable cytotoxic T-cell activity (Fig. 5d). These results therefore indicate that SLY1 is essential for the proliferation and induction of certain effector functions of T cells with more pronounced effects on the CD4 cell subset.
Prolonged allograft survival in Sly1d/d mice. The Sly1d/d mutation caused impaired T-cell cytokine production and attenuated the proliferative response of T cells to allogeneic stimulator cells in vitro. To investigate whether the mutation would also affect T-cell responses in vivo, a model of heterotopic heart transplantation was examined. The results in Fig. 6a demonstrate that Sly1d/d mutant mice (H-2b) rejected fully allogeneic heart transplants (H-2d) as efficiently as wild-type hosts (H-2b). Control syngeneic grafts were not rejected during the observation period.
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Functional T-cell receptor signaling in Sly1d/d lymphocytes. Considering that SLY1 is phosphorylated upon T-cell receptor triggering (2), we determined whether receptor-proximal signaling events would be altered in Sly1d/d mutant cells. Engagement of the T-cell receptor by antigen or antibody-cross-linking causes immediate increases of intracellular Ca2+ levels. However, the data in Fig. 7a show that stimulation of CD4 and CD8 T cells with CD3 antibodies results in comparable Ca2+ flux of wild-type and Sly1d/d cells. To examine the effects of the Sly1d/d mutation on T-cell receptor-triggered tyrosine-phosphorylation purified splenic T cells were stimulated with CD3 antibodies for the indicated time points and Western blot analyses were performed.
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B (Fig.
7c). In order to
investigate whether the translocation of transcription factors would be
altered, Sly1d/d mutant cells were stimulated with
anti-CD3 and anti-CD28 for 16 h and nuclear fractions were
prepared. Consistent with normal mitogen-activated protein kinase
activation and IB degradation, DNA binding by
NF-B, AP-1, and NF-AT was comparable in wild-type and
Sly1d/d T cells (Fig.
7d).
Nuclear localization of SLY1 is abolished by the Sly1d/d mutation.
The
Sly1d/d mutation results in the loss of the
C-terminal portion of a putative nuclear localization signal
(Fig.1a and
e). We therefore hypothesized that the
Sly1d/d mutation would abrogate nuclear
localization thereby impairing SLY1 function. To address this question,
cytosolic and nuclear extracts of T lymphocytes were prepared and the
presence of SLY1 protein was examined by Western blotting with specific
antisera. As shown in Fig.
7e, wild-type SLY1 protein
was detected in both the cytoplasm and the nucleus, whereas mutant
SLY1d protein was present exclusively in cytoplasmic
fractions. The purity of the subcellular fractions was confirmed by
Western blotting with antibodies against IB
(cytosolic fractions) and lamin A/C (nuclear fractions). Thus, the
nuclear localization of SLY1 and/or the phosphorylation of Ser27 are
essential for its physiological
function.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although cytokine
production upon T-cell receptor triggering in
Sly1d/d cells was strongly reduced,
membrane-proximal events such as Ca2+ influx,
tyrosine phosphorylation, and activation of mitogen-activated protein
kinases were not altered. Moreover, translocation of transcription
factors such as NF-B, NFAT, and AP-1 was comparable.
It is intriguing to speculate whether the physiological function of
SLY1 is to mediate transport of as yet unidentified interaction
partners from the cytoplasm to the nucleus or vice versa. Further
studies are required to address this question, in particular the nature
of the possible interacting molecule and the role of the SH3 and SAM
domains in this context. Furthermore, it has to be determined whether
the lack of Ser27 phosphorylation or the defective function of the
nuclear localization signal or both contribute to the observed
functional deficiencies in Sly1d/d mice.
Collectively, the results obtained in the Sly1d/d
mice indicate that the expression of intact SLY1 protein and its
localization to the nucleus are required for the generation of adaptive
immune responses.
CD4 T cells are known to be of paramount importance for allograft rejection (27). Activated CD4 T cells direct the immune response against allografts by secreting cytokines that activate and attract effector cells such as CD8 T cells, B cells, and NKT cells (37). Thus, the markedly prolonged survival of semi-identical allografts in Sly1d/d mice may be explained, at least in part, by the fact that the described mutation of SLY1 leads to attenuated proliferation and cytokine production of Tcells upon antigen receptor ligation and an impaired capacity of CD4 T cells to respond to allogeneic stimuli. Activation of allograft-reactive CD4 T cells may occur through multiple pathways that involve an intense interplay of innate and adaptive immune reactions. Thus, NK receptor-bearing cells become activated upon transplantation of fully allogeneic grafts and provide help to T cells, which subsequently mediate acute transplant rejection (17). Importantly, help provided by activated NK and/or NKT cells may be sufficient to overcome defects of T-cell costimulation such as those caused by CD28 deficiency (17).
These results clearly established that the pathways for the induction of T-cell responses to allografts are, at least in part, functionally redundant. Therefore, the influence of individual regulators of T-cell activation such as SLY1 on transplant rejection may only become apparent after reducing this complexity. To avoid help derived from NK-receptor-bearing cells, we transplanted semi-identical (H-2bxd) cardiac allografts into wild-type (H-2b) and Sly1d/d mice (H-2b). We show that the acceptance of semi-identical grafts was substantially extended in Sly1d/d hosts with 50% of the grafts surviving even after 60days. Fully allogeneic grafts, however, were vigorously rejected in Sly1d/d mice. The phenotype of Sly1d/d mice in transplantation is therefore similar to that of CD28-null mice, which also accept semi-identical cardiac transplants but efficiently reject fully allogeneic grafts (17).
In Sly1d/d mice, the antibody response to T-dependent as well as T-independent antigens was severely impaired thereby identifying SLY1 as a novel molecular regulator of humoral immune responses. T-dependent antibody responses may be affected by the Sly1d/d mutation at multiple levels. Efficient B-cell activation by T-dependent antigens requires signals delivered through binding of the antigen to the B-cell receptor as well as generation of antigen-specific T helper cells that provide costimulatory signals to B cells via CD40-CD40 ligand interactions and the secretion of cytokines. Following antigen receptor ligation, the proliferative response of CD4 T cells derived from Sly1d/d mice was markedly diminished, whereas proliferation of follicular B2 B cells was not affected. In addition, Sly1d/d T cells exhibited reduced secretion of cytokines that are required as growth and differentiation factors for proliferation and isotype switching of antigen-reactive B cells. Consistent with a diminished production of IL-4 and gamma interferon, both IgG1 and IgG2a responses to T-dependent antigen were diminished in Sly1d/d mice. It is therefore conceivable that T-dependent antibody responses may be impaired in Sly1d/d mice due to the lack of sufficient T-cell help.
T-independent antibody responses are considered important for host defenses as they bridge early innate to subsequently appearing adaptive immune reactions (8). In addition to B1 cells, marginal-zone B cells play a major role for the first-line responses against blood-borne T-independent antigens. Evidence for this notion is provided by analysis of Pyk-2-deficient mice, which exhibit a selective defect in marginal zone B-cell development that is associated with severely impaired T-independent antibody responses (11). Subsequent studies with B-cell receptor transgenic mice directly demonstrated that marginal zone B cells efficiently generate antigen-specific plasma blasts after immunization with T-independent antigens (20). It therefore appears likely that the impaired T-independent antibody response in Sly1d/d mice may result, at least in part, from severely reduced numbers and a substantial reduction of antigen receptor-driven proliferation of Sly1d/d marginal zone Bcells.
After emigration of newly formed
surface IgM+ B cells from the bone marrow to
peripheral lymphoid organs the antigen receptor signal strength
determines cell fate decision to follicular or marginal zone B cells
(23). Genetic ablation of
CD19, Lyn, or the p110
subunit of
phosphatidylinositol 3-kinase, all of which influence B-cell receptor
signaling, impairs generation and/or positive selection of marginal
zone B cells, whereas PTEN deficiency, which results in enhanced B-cell
receptor signaling, is characterized by increased numbers of marginal
zone B cells. It has been shown that other genes unrelated to B-cell
receptor signaling such as Pyk-2, Lsc, or NF-B p50 are also
indispensable for marginal-zone B-cell development, suggesting that, in
addition to quantitative aspects of B-cell receptor signaling,
qualitatively distinct pathways of B-cell activation may exist
(10,
23). It is unclear, if
the preferential reduction of marginal zone versus follicular B-cell
numbers in Sly1d/d mice is due to modified B-cell
receptor signaling, because downstream signals are not altered (data
not shown).
Few genes on the X chromosome encompassing Btk, SH2D1A, and CD40 ligand are linked to genetic immune disorders. Mutations in Btk result in X-linked agammaglobulinemia (34, 36), X-linked lymphoproliferative disease is a result of mutations in the SH2D1A gene, whereas CD40 ligand deficiency causes X-linked hyper-IgM syndrome (7, 26, 38). In addition, there are several X-linked immune diseases with unidentified corresponding genetic defects. The results of the present study demonstrate that mutations in the Sly1 gene can lead to multiple defects in B- and T-cell responses consistent with a common variable immunodeficiency syndrome. Gene mutations affecting the function of SLY1 may therefore represent a novel type of X-linked immunodeficiency.
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ACKNOWLEDGMENTS |
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We thank Bernadett Bartsch, Evelyn Schaller, Anja Conrad, Jennifer Meinecke, and Karin Buchholz for excellent technical assistance, Irmgard Förster for critical reading of the manuscript, Markus Moser for support with the generation of SLY1-specific antisera, and Tatjana Dorn and Cord Brakebusch for help with the Ca2+ flux assays.
We declare that we have no financial or other conflicts of interest.
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FOOTNOTES |
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These authors contributed equally to this work.
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