1 AstraZeneca R & D Lund, Department of Bio & Molecular Sciences, Scheelevägen 2, 221 87 Lund, Sweden 2 Section for Immunology, BMC I:13, Lund University, 221 84 Lund, Sweden 3 Center of Transplantations, Foundation for Biomedical Research of the Academy of Athens, Soranou tou Efesiou 4, 10310 Athens, Greece
Correspondence to: A. Rosendahl; E-mail: alexander.rosendahl{at}astrazeneca.com
Transmitting editor: R. A. Flavell
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Abstract |
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Keywords: cytokine, cellular development and differentiation, TCR, thymus
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Introduction |
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The transforming growth factor (TGF-)-ß family is composed of three major subgroups, i.e. TGF-ß, Activins/Inhibins and bone morphogeneic proteins (13,14). These proteins regulate cellular growth and differentiation, control morphogenesis and angiogenesis, affect adhesion and chemotaxis, regulate immune responses, and play a determinative role during tissue remodeling and fibrogenesis (1519). These cytokines induce the formation of heteromeric complexes between type II and type I receptors. The constitutively active type II kinase recruits and activates the type I receptor which subsequently propagates the signal downstream by phosphorylating specific receptor-regulated Smad proteins (R-Smads) (2025). Phosphorylated R-Smads form heterocomplexes with the common partner Smad4 (Co-Smad) and translocate to the nucleus where they participate in the regulation of transcription of target genes (14,21).
The significance of different TGF-ß family members during T cell development has only recently started to be elucidated. Elegant thymic organ culture and in vitro studies have shown that thymocytes themselves and thymic epithelial cells express all TGF-ß isoforms, which seem to induce expression of CD8 on DN cells, inhibit their proliferation and control transition of CD4CD8low to DP cells in vitro (2630). Further more, the ßß chain heterodimeric Activins inhibit thymocyte proliferation through inhibition of IL-6 synthesis, while the ß chain heterodimeric Inhibins antagonize the Activins and thus promote thymocyte proliferation (31,32).
In the present study, presence of Activin/TGF-ß signaling was monitored by analyzing phosphorylation patterns of Smad2 in thymocyte subpopulations. Expression of pSmad2 was noted in medulla and subcapsular cortex. Interestingly, the pSmad2 expression was most pronounced in the most immature DN thymocytes and in SP thymocytes.
The demonstration of (i) in situ cytokine production (Activins and TGF-ß), (ii) ongoing TGF-ß/Activin signaling (as visualized by the presence of pSmad2+ cells in the thymus) and (iii) the transition from a more Activin-responsive phenotype during early thymopoiesis to a more TGF-ß-responsive phenotype as thymocytes progress to more mature stages, strongly suggest that TGF-ß and/or Activins could play non-redundant roles during T cell development in the thymus.
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Methods |
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Immunohistochemistry
Immunohistochemistry was conducted as described elsewhere (33). Briefly, dissected thymuses were frozen in pre-chilled 2-methylbutane isopentane (Sigma-Aldrich, Steinheim, Germany), and then covered by Tissue-Tek OCT (Sakura Fintek, Torrance, CA) on dry ice and stored at 70°C until used. Sections (7 µm thick) were fixed in 2% paraformaldehyde (Sigma, Steinheim, Germany) for 10 min. After quenching of the endogenous peroxidase activity by 15 min incubation with 1% H2O2 (Sigma) dissolved in methanol (Histolab, Gothenburg, Sweden), and blocking with avidin/biotin (Vector, Burlingame, CA) and normal goat serum (5%) for 60 min, the sections were incubated with primary antibody dissolved in 0.5 M NaCl EBSS/saponin buffer for 45 min (Life Technologies, Paisley, UK/ICN Biomedicals, Aurora, OH). Biotinylated goat anti-rabbit IgG (Vector) secondary antibodies were added for 30 min in EBSS/saponin buffer supplemented with 2% normal goat serum. Vectastain Elite kit (Vector) was used to enzymatically label the secondary antibody. Slides were developed for 5 min in diaminobenzidine (Vector) and counter-stained with Mayers hematoxylin (Histolab, Gothenburg, Sweden). Four to five mice were analyzed for expression of the investigated markers. Images were analyzed using the Leica IM1000 and the number of positively stained cells was determined ± SEM with the Leica Qwin computer program (Leica Microsystems, Heerbrugg, Switzerland).
Antibodies
Polyclonal antisera were raised in rabbits against synthetic peptides as previously described (34,35). Briefly, for antibodies against Activin type IB receptor (ActRIB) also termed Activin receptor-like kinase (ALK)-4, TGF-ß type I receptor (TßRI) also known as ALK5 and Activin type IIB receptor (ActRIIB), peptides that corresponded to the divergent intracellular juxta-membrane domains, were used. For antibodies against the TGF-ß type II receptor (TßRII) and Activin type IIA receptor (ActRIIA), peptides that corresponded to the divergent C-termini were used. For antibodies against Smad2, the peptide corresponding to the variable proline-rich linker region were used and for pSmad2, KKK-SpSMpS (where pS stands for phosphorylated serine residue). All peptides were coupled to keyhole limpet hemocyanin with glutaraldehyde and mixed with Freunds adjuvant before immunization of rabbits. All the antisera were tested for specificity by immunoprecipitation and western blotting on COS cells transfected with different receptors and Smads. The antisera were purified using the ImmunoPure IgG (Protein A) purification kit (Pierce, Rockford, IL) according to the manufacturers instructions. Alternatively, antibodies were affinity purified as described elsewhere (33). Polyclonal antibodies against pSmad2 (Upstate Biotechnology, Lake Placid, NY) and normal rabbit IgG (Dako, Alvsjo, Sweden) were used as positive or negative controls in the experiments respectively. Antibodies against Activin ßA, ßB and ßC were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
ALK4, ALK5 and pSmad2 antibodies were coupled with FluoroLink-Cy2/5 according to the manufacturers instructions (Amersham Pharmacia Biotech, Uppsala, Sweden). Briefly, 1 mg/ml antibody was incubated for 30 min with Cy2 or Cy5 dyes at room temperature. Fluorescent-labeled antibody was separated by gel filtration from unconjugated dyes and analyzed, demonstrating approximately two dye molecules per antibody.
MACS
Single-cell suspensions of thymocytes were prepared and subpopulations were enriched by magnetic separation (Miltenyi Biotec, Auburn, CA) as previously described (36). Briefly, erythrocytes were lysed by hypotonic shock and the remaining thymocytes were passed through a nylon filter and suspended in 0.5% BSA in PBS. To enrich for each subpopulation in the thymus, cells were incubated for 20 min at 4°C with anti-CD4 magnetic beads (10 µl/10 x 106 cells). After washing, CD4-expressing cells were positively enriched by passing the cells twice over a MidiMACS column (Miltenyi Biotec). Captured cells were either CD4+ SP or DP cells, while the flow through was CD8+ SP, DN or antigen-presenting cells. The CD4 beads, on the captured cells, were then released using the MultiSort Kit (Miltenyi Biotec) and the cells were incubated with anti-CD8 magnetic beads for 20 min at 4°C. After two wash steps, the cells were passed twice over a MidiMACS column (Miltenyi Biotec) leaving the captured cells DP and the flow through CD4+ SP.
To enrich for DN cells, a cocktail of CD4, CD8, CD11b, CD11c, CD19 and I-A antibody-conjugated magnetic beads (Miltenyi Biotec) was added to unseparated thymocytes. After two wash steps, the cells were passed twice over a MidiMACS column (Miltenyi Biotec) leaving the flow through true DN thymocytes. Enrichment purity was routinely >90%, estimated by flow cytometry.
Flow cytometry
Single-cell suspensions of thymocytes were prepared and expression of antigens was analyzed using the Cytofix/Cytoperm kit according to the manufacturers description with minor changes (PharMingen, San Diego, CA). Briefly, cells were stained for surface expression of CD4, CD8, CD3, CD25 and CD44 after blocking of the Fc receptor with CD16CD32 mAb in PBS supplemented with 1% BSA (Sigma-Aldrich) buffer for 30 min at 4°C. After two wash steps, cells were fixed with Fix/Perm solution for 15 min at 4°C, washed twice and incubated with 0.1% Triton for 90 s on ice followed by two additional washes. Purified or fluorescent-labeled rabbit anti-TGF-ß receptor family or anti-Smad antibodies diluted in 1 x Perm/Wash buffer were added to the cells and incubated for 30 min at 4°C. Cells were washed twice in 1 x Perm/Wash buffer, and cells stained with purified receptor/Smad antibodies were stained with FITC-labeled anti-rabbit antibody (Dako) for 30 min at 4°C and analyzed with a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) where the geometric mean value was determined ± SD using the CellQuest Pro program (Becton Dickinson, Mountain View, CA). CellQuest Pro converts the acquired logarithmically amplified value to the corresponding linear fluorescence intensity by the formula: linear value = 10(channel number/scaling factor). The geometric mean value was chosen as statistical formula due to the skewed population and is expressed as the anti-log of the linear fluorescent value. mAb directed against CD3, CD4, CD8
, CD25, CD44 and CD16CD32 were all purchased from PharMingen. The pSmad2 signal was competed efficiently by a 30-fold excess of pSmad2 peptide and similar results were noted with the receptor antibodiespeptides, thus further verifying the specificity of these reagents. Briefly, 10 µl of the antibody was incubated 60 min with the peptide at room temperature followed by addition of 1 ml buffer and then centrifuged for 1 min at 14,000 r.p.m. to remove complexes. The supernatant was then used to stain the cells as described above.
In vitro stimulation
The magnetically purified cells were washed twice in RPMI 1640 medium without FCS supplemented with 0.1 mM Na3VO4, counted and 0.2 x 106 cells/well were seeded into 96-well plates. Recombinant TGF-ß1 (2 ng/ml) or ActivinA (2 ng/ml) (R & D Systems, Minneapolis, MN) was added to the cells followed by incubation at 37°C water bath for 5, 30 or 60 min. Maximum Smad2 phosphorylation was noted at 60 min and all results shown are from this time point. Cells were harvested and stained for pSmad2 as described above, analyzed by flow cytometry, and compared to medium-stimulated cells. Percentage increase of pSmad2 expression was calculated according to the formula: 100 x [(pSmad2 MFIafter stimulation isotype MFIafter stimulation)/(pSmad2 MFIbefore stimulation isotype MFIbefore stimulation)].
RNA isolation and RT-PCR
Total RNA was isolated from whole thymus tissue and from sorted thymocytes with RNAqueous (Ambion, Austin, TX) according to the manufacturers instruction. cDNA was synthesized from 2 µg total RNA using the First-Strand cDNA synthesis kit (Amersham Pharmacia, Piscataway, NJ), according to the protocol provided by the manufacturer. Semi-quantitative RT-PCR amplifications were performed using different concentrations of cDNA template (1:3, 1:9, 1:27, 1:81, 1:243, 1:729 and 1:2187). Sufficient amplification of the templates was obtained with the 1:9 dilution, which fell into the linear range, thus all data shown are derived using this dilution of the template. Amplification was performed in 25 µl PCR reaction mixtures containing 0.5 µl cDNA template, 1 µl 10 mM dNTP, 2.5 µl 10 x PCR reaction buffer, 15 mM MgCl2, 0.5 µl 250 U Taq polymerase (Boehringer Mannheim, Mannheim, Germany), 10 pmol of each primer (DNA Technology, Aarhus, Denmark) and 18.5 µl H2O. The reactions were performed using the DNA Engine/Tetrad (MJ Research, Watertown, MS), and the amplification conditions were 10 min at 95°C followed by 30 cycles each consisting of 60 s at 95°C, 90 s at 55°C, 60 s at 72°C and, finally, followed by 10 min at 72°C. The PCR products were separated on 1% agarose gels and visualized by ethidium bromide staining, and the relative intensity of the obtained products was measured. The gels were scanned using the Fluor-S MultiImager (Bio-Rad, Hercules, CA). The PCR primers used were: ALK-1 forward: 5'-GAGCCGTGTTCATGGTAGT-3'; ALK-1 reverse: 5'-GGAGGAGCCAGAAGTTGAT-3'; ALK-2 forward: 5'-GGCGGGGTCTTACACGTAA-3'; ALK-2 reverse: 5'-CTGGACCAGAGGAACAAAGG-3'; ALK-4 forward: 5'CTGAGGACTGCTACGGGAA-3'; ALK-4 reverse 5'-TAAGCGTGCAGGAAGATGT-3'; ALK-5 forward: 5'-ATGGGCAATAGCTGGTTTT- 3'; ALK-5 reverse; 5'- GCCATAACCGCACTGTC-3'; TßRII forward: 5'-AATTTCTGGGCGCCCTC GGTCT-3'; TßRII reverse: 5'-CCCGGGGCATCGCTCATCT-3'; ActRIIA forward: 5'-ACACAGCCCACTTCAAATCC-3'; ActRIIA reverse: 5'-CTGACAGTGAGCCCTTTTC-3'; ActRIIB forward: 5'-GCTTAAAGGAGTCCGCACA-3'; ActRIIB reverse: 5'-TCAATTGCTACGGGCA-3'; Smad2 forward: 5'-GCCGTCTTCAGGTTTCACA-3'; Smad2 reverse: 5'-TAGTATGCGATTGAACACC-3'; Smad3 forward: 5'-CGCCAGTTCTACCTC CAGTG-3'; Smad3 reverse: 5'-AAAGACCTCCCCTCCGATGT-3'; Smad4 forward: 5'-AGCCGTCCTTACCCACTGA-3'; Smad4 reverse: 5'- CTCAATCGCTTCTGTCCTG-3'; Activin ßA forward: 5'-GAGGACGACATTGGCAGGA-3'; Activin ßA reverse: 5'-GCACTAGACTGGCACCACT-3'; Activin ßB forward: 5'-GCAGGCAACAGTTCTTCAT-3'; Activin ßB reverse: 5'-CTCCCTCTGGTCCTGACTG-3'; Activin ßC forward: 5'-CACAATGCCACCCAGACC-3'; Activin ßC reverse: 5'-CAGCCAATCTCACGGAAGT-3'; Activin ßE forward: 5'-CAGCCG TCCCAGAATAACT-3'; Activin ßE reverse: 5'-CAACATAAGGGGGTCTCAG-3'.
Western blotting
Proteins were extracted from 10 x 106 MACS-sorted SP, DN and DP thymocytes respectively, and from frozen whole thymus. The proteins were extracted in 1% Triton X-100, 25 mM TrisHCl (pH 7.6), 0.1 M NaCl, 1 mM EDTA, 5 mM NaF, 0.1 mM Na3VO4, 1 mM PMSF and complete protease inhibitor cocktail diluted 1:25 (Boehringer Mannheim). Proteins from 1 x 106 cells from each subpopulation or alternatively 2 µg/lane with the whole thymus extracts were run under reduced conditions on a 412% NuPage Bis/Tris gel (NOVEX/Invitrogen, Groningen, Netherlands) and transferred to PVDF membranes (NOVEX/Invitrogen) by electroblotting using transfer buffer supplemented with 20% methanol. Blots were blocked overnight at 4°C in PBS/0.1% Tween 20/1% BSA and then incubated with 1 µg/ml of the primary antibody for 1 h at room temperature using the DecaProbe system (Hoefer; Amersham Pharmacia). Thereafter, the membranes were washed 6 x 10 min with blocking buffer, incubated for 1 h with the secondary horseradish peroxidase-linked donkey-anti-rabbit antibody at 1:5000 (Santa Cruz Biotechnology), washed 6 x 10 min with blocking buffer, incubated with ECL substrate (Amersham Pharmacia) and, finally, exposed to radiographic films.
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Results |
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Both Activin and TGF-ß type I receptors are expressed in the thymus
The observation that thymocytes expressed pSmad2 protein indicated that either TGF-ß and/or Activin ligands had signaled through a functional receptor complex. Therefore, the expression patterns of type I and type II receptors responsible for propagating TGF-ß (ALK1, ALK5 and TßRII) or Activin (ALK4, ActRIIA and ActRIIB) signals were analyzed by immunohistochemistry.
The TGF-ß-reactive, pSmad1/5-signaling ALK1 (37) was only detected in a few thymocytes that were scattered both in the medulla and in the cortex (Fig. 3A). In contrast, pronounced staining was observed in the endothelium of both small and large vessels (Fig. 3A), further strengthening our previous results suggesting this receptor to be important for blood vessels (37). The majority of the cells in the medulla expressed high levels of ALK5 (70 ± 11%), while a more heterogeneous expression pattern was obtained in the cortical areas, where 40 ± 8% expressed high ALK5 levels (Fig. 3B). Several of the subcapsular thymocytes were brightly ALK4+ (45 ± 5%) (Fig. 3C). Moderate ALK4 expression was also recorded in medulla, while the cortical cells showed weak expression of ALK4 (Fig. 3C and D). In addition, fibroblast-like cells in the medulla had moderate expression of ALK4 (data not shown).
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These data demonstrate that the type I as well as the II receptors for TGF-ß and Activin are expressed in the thymus, both with distinct expression patterns.
pSmad2 expression is confined to SP and DN thymocytes
In order to distinguish which T cell compartments and which maturation stages contain pSmad2+ cells, we developed conditions that allowed detection of the Smad and receptor proteins by flow cytometry. To address if pSmad2 expression changes during development, pure DN cells (CD4, CD8, CD19, CD11b, CD11c and I-A) were magnetically enriched. Cells stained with isotype control antibody were analyzed in parallel for each thymocyte subset (Fig. 4A). The specificity of these antibodies in FACS was demonstrated by the addition of 30-fold molar excess cognate peptide, which efficiently competed the binding of all antibodies used in this study (Fig. 4A).
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In vitro challenge with ActivinA and TGF-ß1 induces specific Smad2 phosphorylation patterns dependent on maturation stage
To confirm that the DN and the SP cells were truly competent to respond to stimulation via TGF-ß family receptors and to identify which ligand-signaling pathway they utilize, magnetically sorted cells were cultured in vitro with either biologically active TGF-ß1 or ActivinA and induction of pSmad2 was analyzed. All cell subsets responded to in vitro stimulation with TGF-ß1 and ActivinA by phosphorylating Smad2, compared to medium alone (Fig. 4D). The response to ActivinA was markedly higher in the CD44+CD25 and CD44+CD25+ subpopulations of the DN cells compared to the other subsets of thymocytes and significantly higher compared to medium alone (P = 0.009). A partial, but not significant, induction was noted in the CD44CD25+ in response to ActivinA. All the other thymocytes showed marginal responses. TGF-ß1 induced phosphorylation of Smad2 in the CD44+CD25, CD44+CD25+ and CD44CD25+ subsets, but to a lower extent than ActivinA (P = 0.02). In sharp contrast, in both the CD4+ and CD8+ SP subsets a marked and significant (P = 0.008/ P = 0.007 to medium and P = 0.02/P = 0.01 to ActivinA respectively) induction of pSmad2 was noted after TGF-ß1 stimulation.
DN thymocytes express ALK4 and pSmad2, while SP expresses ALK5 and pSmad2
To monitor whether this maturation-dependent ligand- selective phosphorylation was due to distinct expression of the respective receptors during thymic selection, magnetically sorted thymocytes were stained either with antibodies towards type I or II Activin/TGF-ß receptors and analyzed by FACS. A similar cellular distribution as for pSmad2 was obtained with antibodies towards the Activin-responsive type I and II receptors, ALK4 and ActRIIA respectively (Fig. 5A), as well as the TGF-ß-responsive ALK5 (Fig. 5B). Interestingly, the expression of TßRII increased gradually as the thymocytes matured, as visualized by a 2-fold higher mean expression in SP compared to the CD44+CD25 DN (Fig. 5B). ALK1 was expressed at a low level, indicating that the positive cells obtained with immunohistochemistry most likely corresponded to endothelial and other stromal cells (data not shown and Fig. 3A).
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These data demonstrate that thymocytes can receive signals from members of the TGF-ß family in vivo during both early and late thymic selection. Our in vitro studies demonstrate that the responsiveness, measured as phosphorylation of Smad2 by recombinant ligand, changes during the maturation from predominately ActivinA-reactive DN cells to more TGF-ß1-reactive SP thymocytes.
Activin ßA, ßB and ßC are produced in the thymus, and are present in cleaved biological active forms
To further support the results derived at protein level, mRNA from whole thymus was analyzed by semiquantitative RT-PCR. The analyses demonstrated presence of low levels of ALK1, ALK2 and ActRIIB in thymic specimens (Fig. 6A). In contrast, a significant expression of mRNA transcripts for Smad2, Smad3, Smad4, ALK4, ALK5, ActRIIA and TßRII was evident, confirming the protein results obtained. Activin ßA, ßB and ßC ligand mRNAs were produced in the thymus, and the mature biologically active forms of these proteins were present. Purified thymocytes themselves produce only Activin ßB mRNA and protein (Fig. 6B and C). However, Activin ßE mRNA was neither detected in total thymus nor in the thymocyte extracts (Fig. 6B).
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Discussion |
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Smad2 is phosphorylated by various stimuli other than TGF-ß family ligands (40,41). In an elegant study, Mamura et al. showed that ligation of the TCR complex resulted in phosphorylation of Smad2 in peripheral T lymphocytes (41). However, when phosphorylated via TCR signaling alone, Smad2 failed to interact with Smad4 and thus did not migrate into the nucleus. Only when Smad2 was phosphorylated by the type I receptor was it translocated to the nucleus (41). Thus, the nuclear expression of pSmad2, observed in the medulla and in a fraction of the subcapsular thymocytes in our experiments, indicates that these cells have responded to the ligand rather than TCR signaling alone. In fact, since early DN populations do not express ß or
TCR (42), the pSmad2-expressing cells within the CD44+CD25+ and CD44+CD25 subpopulations most likely received signals derived from the active TGF-ß/Activin receptor complex and not through the TCR/CD3 complex.
Our study identifies the putative target cell populations for TGF-ß family cytokines in the thymus. Our data indicate that signaling triggered either by TGF-ß or Activin can take place in DN and SP thymocytes, since both these cell compartments express significant levels of the corresponding signaling type I receptor, ALK4 and ALK5 respectively. In contrast, the DP cells showed a more heterogeneous expression varying from completely negative to weakly positive. Interestingly, all three isoforms of TGF-ß (TGF-ß13) are expressed in vivo in subcapsular and cortical thymic epithelial cells (27,30). Moreover, in contrast to other tissues, the TGF-ß might be produced as active dimers rather than inactive latent forms in the thymus (43). Our results extend these findings, and demonstrate that Activin ßA, ßB and ßC mRNA and protein can also be found within the thymus.
During maturation, the DN cells progress into CD4CD8low and subsequently down-regulate the CD44 surface antigen in the subcapsular region, a process that is independent of TGF-ß (27). These immature thymocytes are arrested at the G1 phase in the cell cycle by TGF-ß and do not progress to the DP stage until they have completed one cell cycle (27). The pSmad2+ cells detected at this location in the thymus could thus represent the most immature DN thymocytes (CD44+CD25 and CD44+CD25+) before they down-regulate the CD44 antigen. Along this line, elegant experiments by Hedger et al. demonstrated that Activin interferes with production of IL-6 by thymocytes, which in turn leads to an arrest in the G1 phase described by Yamato et al. (32,45). Indeed, in our in vitro stimulation experiments, these cells responded significantly to ActivinA and partially to TGF-ß as monitored by induction of pSmad2 after stimulation. This raises the possibility that maturation of the immature DN cells to thymocytes could involve both Activin- and TGF-ß-mediated signaling by pSmad2. Thus, the local cytokine/growth factor network will influence the outcome of the Activin receptor signaling through Smad2, since in the presence of IL-6 Activin actually enhances proliferation of thymocytes, while in the absence of IL-6 Activin results in a cell cycle arrest at the G1 phase (32).
An array of evidence indicates that negative selection occurs mainly in the medulla where semi-immature SP thymocytes, recognizing self-peptides on hematopoetic/macrophages and dendritic cells, are eliminated via apoptosis (27,4649). TGF-ß has been shown both to induce and to protect from apoptosis in several different cell types (5053). Moreover, addition of TGF-ß to organ cultures induces a dramatic decrease in all compartments in the thymus with the exception of SP CD8+ cells, which seem to develop normally (44). Given the high expression of pSmad2 noted in our study in SP thymocytes in the medulla, which was further induced by exogenously added TGF-ß in vitro, members of the TGF-ß family may play important and regulatory roles during the final maturation and negative selection processes of T cell development. In contrast, positive selection in the cortex might be less dependent on the TGF-ß family, since only a few scattered cells are actively signaling through pSmad2 among the cortical DP thymocytes.
Recently, the role of TGF-ß in T cell development and homeostasis was investigated by two knockout strategies. T cells in mice expressing a dominant-negative form of TßRII, under the transcriptional control of the CD2 promoter, had severely disturbed lymphoid homeostasis in the periphery characterized by a CD8+ T cell hyperproliferative disorder in lymphoid organs (54). However, the thymic development was normal suggesting that TGF-ß signaling through the ALK5/TßRIISmad2 pathway in the thymus was redundant or did not play a determinative role. However, thymic development and shaping of the T cell repertoire could be more dependent on signals derived from Activins transduced via the ALK4/ActRIISmad2, a pathway that should work properly even in TßRII dominant-negative mice. This may be especially important during the very early steps in DN cell maturation in which, as shown in Fig. 4(D), the thymocytes responded vigorously to exogenous Activin.
A different approach to illustrate the importance of TGF-ß in T cell homeostasis is the TGF-ß1 knockout mouse. These mice, dependent on genetic background, show a massive peripheral lymphoproliferative, autoimmune-like disease, which is lethal within 4 weeks after birth (5659). It has been proposed that the failure to maintain peripheral tolerance in these mice could relate to a defective negative selection failing to eliminate the self-reactive thymocytes. In support of this assumption are several manifestations of autoimmunity in these animals such as autoantibodies to nuclear antigens similar to Sjögrens syndrome (60), Ig deposits in renal glomeruli (57,60), progressive tissue inflammation (61), and significantly increased MHC class II expression in which auto-peptides are presented and peripheral T cells activated (62). Our results demonstrating a strong in vivo pSmad2 expression and TGF-ß responsiveness in vitro further imply TGF-ß to play important signaling roles in the SP cells undergoing negative selection. The TGF-ßSmad2 signaling cascade in these SP cells could participate in either apoptosis of self-reactive cells (63) or result in growth arrest allowing the selection processes to take place properly (64).
Collectively, we have shown that subcapsular as well as medullary thymocytes express both type II and I receptors for Activins and TGF-ß, and most importantly pSmad2, demonstrating that TGF-ß family signaling actively participates during T cell development. Thus, our data support the view that pSmad2 signaling, induced by locally or systemically produced TGF-ß/Activin, takes place in the immature DN. This may possibly have a growth inhibitory effect to allow entry into the DP selection step in a controlled manner and possibly also to assist in the selection processes both acting growth inhibitory in the case of TGF-ß, but not in the case of Activin, which also promotes apoptosis (32).
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Acknowledgements |
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Abbreviations |
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ALKActivin receptor-like kinase
DNdouble-negative
DPdouble-positive
SPsingle-positive
TßRTGF-ß receptor
TGFtransforming growth factor
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References |
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