B cell receptor
Protein phosphatase 2A
Ulex europaeus agglutinin-1
α4-mediated signaling is involved in a variety of functions in mammalian cells. To determine whether this is true for immunocompetent cells, we generated mutant (Lck-α4–) micein which the α4 gene was deleted in a T cell-specific manner using the Cre/loxP system. These mice showed impaired early T cell development. Thymi at most ages were small and their architecturewas disorganized. This defect was not due to increased thymocyte apoptosis but to decreased cell proliferation. T cell development was found to be severely arrested at the CD4/CD8 double-negative 3stage and the thymus contained very few double-positive or single-positive (SP) mature thymocytes. The mutant thymocytes showed impaired proliferative responses to anti-CD3 monoclonal antibody (mAb) stimulation or to the cytokines IL-2, IL-1 or TNF. In the spleen, the numbers of mature SP T cells were decreased and their proliferative responses to anti-CD3 plus IL-2 or to anti-CD28 mAb were impaired. A severe impairment of CD3-induced expression of CD25 was also observed. These data suggest that α4 plays a critical role in the proliferation of thymocytes, which is necessary for early T cell development.
α4 was initially identified as a signal transduction molecule that associates with the Igα component of the B cell receptor (BCR) complex 1, 2. Thatα4 transiently associates with a tyrosine-phosphorylated molecule after BCR cross-linking suggested its involvement in BCR-mediated signal transduction. Alpha4 associates with the catalytic subunit of protein phosphatase 2A (PP2A) and enhances its catalytic activity 3. This association was disrupted by treating cells with the immunosuppressant rapamycin, which inhibits the proliferation of lymphocytes by decreasing PP2A activity. Alpha4 homologues are highly conserved in evolution in yeast as well as among mammalian species 3–6.Mutation of the yeast homologue Tap42 is lethal due to impaired signal transduction through the rapamycin-sensitive Tor pathway, and the same is true for α4 in the mouse.
For complete understanding the mTor/α4-associated pathway, it is necessary to determine the role of α4 in immune-competent cells. Global deletion of α4 gene in all tissues revealedthat α4 is essential for mouse development. Initially, we deleted the α4 gene specifically in B cells using the Cre/loxP system 7. CD19-α4– mice had reduced numbers of splenic B cells and bone marrow pre-B cells. The proliferative response of α4-deficient B cells to different stimuli including BCR was impaired and rapamycin sensitivity was decreased. These results demonstrated that α4 is a pivotal signal transduction molecule involved in BCR-mediated signaling and that it is involved in the rapamycin-sensitive signal transduction pathway.
While the Ag recognition mechanism of T cells is clearly different from that of B cells (because T cells recognize peptides from degraded proteins which are bound to MHC molecules), T and B cells do show certain similarities in Ag receptor signal transduction. The CD3 complex is necessary for TCR expression as are CD79α and CD79β for the BCR 8–11]. Dynamics of T and B cell responses are regulated by the similar cellular mechanisms yielding intermediates, effectors, and memory cells [12–15. Similar molecules are involved in signal transduction through the Ag receptor in both types of cells; for example, adaptor molecules such as SLP76 and BLINK, and src- and Syk-type tyrosine kinases 16–19.
However, the immunosuppressant rapamycin apparently exerts different effects on the different cell types 20. In particular, rapamycin was initially reported to block T cell proliferation rather by inhibiting IL-2-induced signaling 21, 22. However, according to data from α4-deficient B cells, in which primarily the BCR-mediated signal transduction was impaired, we hypothesized that α4 plays an important role in TCR-mediated signal transduction. To determine the role of α4 in T cells, we established a mutant mouse in which the α4 gene was specifically deleted only in T cells (Lck-α4– mice). Lck-α4– mice had reduced numbers of thymocytes and splenic T cells. The number of immature double-negative (DN) thymocytes was reduced and the proliferative response to CD3 stimulation was also impaired. These results show that the α4-mediated signal transduction plays an important role in early T cell development as well as for mature T cell function.
2.1 Lck-Cre/flox-α4 mice
Mice with α4 conditionally targeted in T cells only (Lck-α4– mice) were created by mating α4-floxed females with male transgenics expressing Cre under the control of the mouse Lck promoter 23. The mutant mice showed normal development but marked impairment of thymocyte production (Table 1). The number of thymocytes was less than two million per mouse, averaging only 4.3% of the number in wild-type (WT) littermates. Expression of α4 was not detected by Western blot analysis of thymocytes from Lck-α4– mice (Fig. 1a), and this effect was extended to the peripheral spleen T cells in comparison with B cells of both WT and mutant mice (Fig. 1b). The extent of α4 deletion was shown (as estimated less than 5% remained) by reverse transcription (RT)-PCR analysis of DN3 thymocytes in comparison with WT littermates (Fig. 1c). The results demonstrate that T cell development in the thymus is severely impaired in Lck-α4– mice. However, a substantial number of T cells could be found in the spleen of Lck-α4– mice (30% of the number in WT littermates).
2.2 T cell development in the thymus
The thymi of Lck-α4– mice contained a higher percentage (but reduced numbers) of immature DN cells (92.3%) compared to control littermates (2.5%) (Fig. 2a). The numbers and percentages of double-positive (DP) and single-positive (SP) thymocytes were markedly reduced in Lck-α4– mice (1.2% versus 86.7% of WT littermates for DP and 6.5% versus 10.8% for SP cells). These results suggest that T cell differentiation from DN to DP was impaired in Lck-α4– mice. To determine more precisely at which stage T cell development was impaired in the thymus of Lck-α4– mice, DN thymocytes were analyzed for expression of CD25 and CD44, which define distinct immature thymocyte subsets 24, 25. The earliest subset is CD44+CD25– (DN1). Subsequent subsets are defined as CD44+CD25+ (DN2), CD44–CD25+ (DN3), and CD44–CD25– (DN4). In Lck-α4– mice, the proportion of the DN3 population was increased (78.3% versus 55.6% of WT mice) and the DN4 population was decreased (14.1% versus 29.6% of WT mice), indicating that DN differentiation is blocked at the DN3 stage (Fig. 2a). Regardless of this impairment of thymus cell differentiation, there were some mature CD3+ T cells in the spleen (5.9% versus 22.5% of WT mice; Fig. 2b), with the comparable ratio of CD4 and CD8 SP cells.
We further investigated T cell development in Lck-α4– mice by immunohistochemical analysis of the thymus. Hematoxylin/eosin (H/E) staining of control littermate thymi revealed a clear demarcation between the cortex and the medulla (Fig. 3a). However, the thymi of Lck-α4– mice maintained no clear distinction between the cortex and the medulla. Because the fucose-binding lectin Ulex europaeus agglutinin-1 (UEA-1) reacts with thymic medullary epithelial cells, thymic sections were double-stained with TCRVβ and UEA-1 to visualize the cortico-medullary junction (Fig. 3b). Thymi of control littermates showed a clear distinction between TCRVβ-positive and UEA-1-positive regions. Thymi from Lck-α4– mice possessed a few UEA-1-positive or TCRVβ-positive cells.
CD4/CD8 double staining of thymic sections showed that the cortex contained mostly DP cells in the control littermates, while the thymus of Lck-α4– animals contained almost no DP cells (Fig. 3c). This is in agreement with the flow cytometric analysis, which indicated the presence of a small number of DP thymocyte in Lck-α4– mice (Fig. 2a). Next, the DN thymocyte population was analyzed by CD25/CD117 double staining. CD117 is normally expressed on DN1 and DN2 cells in much the same way as CD44. CD117-positive and CD25-positive cells were detected brightly at the thymic periphery in control littermate thymi (Fig. 3d), but this population was not detected in Lck-α4– mice.
2.3 Proliferative capacity of α4-deficient thymocytes
There are two possibilities to explain the reduced number of thymocytes in Lck-α4– mice: decreased proliferation and increased apoptosis. We first examined the proliferation of thymocytes by the BrdU method (Fig. 4a). In the thymus from control littermates, most proliferating cells were DP, CD25-negative, BrdU-positive cortical cells. In contrast, in Lck-α4– mice, only a small number of BrdU-positive cells were observed. Thymocyte apoptosis was visualized by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) method (Fig. 4b). Similar numbers of TUNEL-positive cells were found in Lck-α4– mice and littermate controls, indicating that apoptosis was not enhanced in the thymus of the Lck-α4– animals. These results suggest that impairment of proliferation rather than increased apoptosis was responsible for the decreased number of thymocytes in Lck-α4– mice.
To further investigate the proliferative response of α4-deficient thymocytes, we cultured total thymocytes in the presence of different stimuli (Fig. 5a). Thymocytes from Lck-α4– mice showed severe impairment of proliferation induced by anti-CD3 mAb or concanavalin A, presumably due to the fewer SP thymocytes in the mutant mice. The DN cells of the mutant mice, containing γ δ T cells (14.1% versus 8.1% of WT mice; Fig. 5b), showed a decrease of the proliferation after stimulation with IL-2, IL-2 + IL-1β, and IL-2 + TNF (Fig. 5c). These results confirmed that impairment of proliferation was a major contributor to disrupted T cell development.
2.4 Splenic T cells in Lck-α4– mice show impaired CD3 signaling
There are substantial numbers of T cells (30% of control littermates) in the spleen of Lck-α4– mice (Table 1 and Fig. 2b). To examine the molecular mechanism of impaired thymocyte proliferation, splenic T cells were stimulated with anti-CD3 mAb in vitro (because only the spleen contained a sufficient amount of cells to analyze) (Fig. 6). Alpha4-deficient B cells showed severe impairment of cell proliferation induced by anti-BCR cross-linking 7. However, the α4-deficient T cells responded well to anti-CD3 cross-linking, albeit slightly reduced in comparison with the response of control littermates. Addition of IL-2 or anti-CD28 mAb as costimulators, together with anti-CD3 cross-linking, did not result in an increase of T cell proliferation to the level seen in control littermates. Because higher doses of IL-2 failed to enhance anti-CD3-induced proliferation, we investigated whether the IL-2 receptor was up-regulated after anti-CD3 stimulation of splenic T cells. As shown in Fig. 7, anti-CD3 did not induce IL-2R expression by splenic T cells from Lck-α4– mice. On the other hand, anti-CD3 stimulation did induce CD28 expression, although also to a lesser degree than in control littermates. These results suggest that CD3 signal transduction pathways leading to IL-2 receptor expression are impaired in Lck-α4– mice and therefore T cells cannot be optimally activated.
We developed mutant mice in which the α4 gene was deleted in a T cell-specific manner using the Cre/loxP system. These mice had impaired T cell development in the thymus and impaired CD3 signal transduction, demonstrating that α4 plays an important role in T cell immunity.
In Lck-α4– mice, the proliferative response to different stimuli was compromised, as was also observed previously in CD19-α4– mice. However, the pattern of defects was not identical in the two types of mutant mice. In Lck-α4– mice, CD3-induced proliferation of splenic T cells was not greatly impaired, but in contrast, BCR-induced proliferation of splenic B cells from CD19-α4– mice was dramatically reduced. In CD19-α4– mice, α4-deficient B cells showed a less drastic impairment of the response to anti-CD40, LPS or IL-4 costimulation. In Lck-α4– mice, however, anti-CD28 or IL-2 second-signal-induced proliferation was completely abrogated. This defect was due to impaired CD3-induced CD25 expressionrather than compromised second-signal transduction. The anti-CD28-induced proliferation signal was possibly also compromised via the failure of CD25 up-regulation. Therefore, α4 is required in CD3-induced TCR signaling as is also the case for BCR-induced signaling, indicating a critical role of α4 in Ag receptor signaling. The results obtained with thymocytes indicate that cytokine-induced signaling is also compromised in Lck-α4– mice. However, it does not follow that α4 is involved only in general proliferation mechanisms, because CD3-induced events, such as CD25 up-regulation, were impaired, but CD28 up-regulation or thymidine uptake was not.
In the peripheral lymphoid organs, the decrease in number of T cells was not as drastic as in the thymus and there were substantial numbers of T cells in the spleen. Because γ/δ-positive T cells can develop extrathymically 26, we tested whether these splenic T cells were γ/δ-positive. However, most of the T cells in the spleen were α β-positive (data not shown); this is in accordance with the finding that these T cells were CD4 or CD8 SP cells.
Defined cortical and medullary zones were absent and most of the cellular components of the thymus, i.e. DP and SP cells, were also drastically decreased in Lck-α4– mice.Thymic epithelial cells in the medulla defined by UEA-1 positivity were essentially unidentifiable. Therefore, factors produced by these thymocytes and the interdependent nonlymphoid thymic stroma are missing in the Lck-α4– thymus. Intercellular interactions between precursor cells and thymic stroma are critical for the proliferation and differentiation of thymocytes in the normal thymus 27. It is therefore possible that gross disruption of thymic architecture and composition results in secondary effects on the proliferation of residual thymocytes. We speculate that accumulation of T cells that have survived the thymic developmental stage could be maintained in the splenic environment and account for recovery of the peripheral T cell number.
The Lck-α4– mice maintained a markedly reduced number of T cells during thymocyte development, which is quite different from the case of CD19-α4– mice. CD19-α4– mice had a decreased number of cells both in the spleen and bone marrow, but the decrease was less severe in the latter. The CD19 promoter becomes active in the pre-B cell stage in the bone marrow and its activity increases later in development, which is most pronounced in the spleen 28. On the other hand, the Lck proximal promoter becomes active between the DN2 and DN3 stage and is most active in the thymus 23. This may be one reason why the α4 deficiency phenotype was manifested at an earlier stage in the T cell-specific as opposed to the B cell-specific gene targeting system.
Another possibility is that α4 is involved in the pre-TCR signal transduction. The signal transduction pathways of pre-TCR and mature TCR share many similarities 29, 30. Genetic inactivation of many different genes has been shown to cause defects in T cell development, especially at the DN3 stage, characterized by a CD44-negative and CD25-positive phenotype. It is at this stage that the pre-TCR complex is first expressed, and is believed to exert its function in a subset of DN3 cells 31. The DN3-to-DN4 transition requires signals from the pre-TCR complex. Targeted disruption of genes such as TCRβ, RAG1 and RAG2, CD3 subunits, Lck or CD45 resulted in blockade of T cell development at this stage 32. In the case of our mice, α4 gene deletion is regulated by the Lck proximal promoter, which is activated at an early stage of T cell development, between the DN2 and DN4 stages 23. The DN3-to-DN4 transition is impaired in Lck-α4– mice, which might suggest that α4 is required for pre-TCR signal transduction.
4 Materials and methods
4.1 Antibodies and reagents
Anti-mouse α4 Ab was prepared as previously described 2. Anti-PP2Ac Ab was purchased from Upstate Biotechnology Inc (Lake Placid, NY). Phycoerythrin (PE)-B220, PE-CD8, PE-CD25, PE-anti-TCRδ, biotin-CD3, biotin-CD44, fluorescein isothiocyanate (FITC)-CD4 and FITC-CD28 Ab were purchased from BD PharMingen (San Diego, CA) for flow cytometric analysis. FITC-TCRV β, FITC-CD8, FITC-CD25, biotin-UEA-1, biotin-CD4 and biotin-CD117 were purchased from BD PharMingen for immunohistochemistry. Streptavidin-Cy3 was purchased from Molecular Probes (Eugene, OR). CD3mAb and CD28 mAb were purchased from BD PharMingen for proliferation assays. IL-2 was a kind gift from Dr. Hamuro (Ajinomoto, Japan). IL-1β and TNF were purchased from Sigma Aldrich Japan (Tokyo, Japan).
4.2 RT-PCR analysis
To determine the extent of α4 deletion at various thymocyte stages, we carried out RT-PCR analysis using specific primers for α4. Poly(A) RNA from sorted DN3 thymocytes was isolated using the MicroPoly (A) Pure from Ambion (Austin, TX). The cDNA was synthesized with the GeneAmp RNA PCR kit (Applied Biosystems Japan Ltd., Tokyo, Japan). The primers for α4 are 5′-ATGGCAGCGTCTGAAGAC-3′ and 5′-TGATAGCAATGACACTGA-3′; and for β–actin 5′-GTTGCTATCCAGGCTGTGCT-3′ and 5′-CGGATGTCCACGTCACACTT-3′. Each PCR cycle consisted of denaturation for 1 min at 94°C, annealing for 1 min at 60°C and extension for 3 min at 72°C. After 35 cycles of amplification, PCR products were analyzed by electrophoresis on 1.5% agarose gels stained with ethidium bromide.
4.3 Western blot analysis
Cells were lysed in lysis buffer containing 1% Nonidet-P40, 150 mM NaCl, 10 mM Tris-HCl (pH 7.8), 1 mM EDTA, 0.05% NaN3, 100 mM NaVO4, 1 mM phenylmethylsulfonyl fluoride, and10 μg/ml aprotinin. The lysates were centrifuged for 5 min at 12,000×g at 4°C to remove nuclei. Proteins were separated by SDS-PAGE and were transferred onto nitrocellulose filters by electroblotting. PP2Ac and α4 were detected by anti-PP2Ac and anti-α4 Ab at a dilution of 1:1,000. The blots were developed using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Little Chalfont, UK) according to the manufacturer's protocol.
4.4 Flow cytometric analysis and sorting
Cell surface markers were analyzed with mAb by flow cytometry. Single-cell suspensions of lymphoid tissues were incubated with a saturating amount of each mAb conjugated either to fluorochromeor to biotin. Two-color analyses were performed with the fluorochromes FITC and PE or with Red670 and PE. Lymphoid cell populations were analyzed after gating by forward and side scatter on a FACScan using CellQuest software (BD Biosciences, San Jose, CA). For analysis of DN cell population, thymocytes were first purified with the mixed beads conjugated with anti-CD4, anti-CD8 and anti-B220 mAb (Dynal, Oslo, Norway). DN3 cell acquisition was performed on a FACSCalibur flow cytometer (BD Biosciences).
Mice carrying three identically-oriented loxP sites in the α4 gene (α4-flox) have previously been reported 7. Lck-Cre transgenic mice, in which the Cre gene is underthe control of the p56lck proximal promoter, were as described 23.
4.6 In-situ proliferation and apoptosis assays on thymocytes
Mice were injected intraperitoneally with BrdU (1 mg in 200 μl of PBS), and thymi were taken 6 h after injection. Thymus tissue sections (6 mm thick) were prepared and immunohistochemical analysis was performed as previously described 7. BrdU staining was done with anti-5–2′-deoxyuridine mAb (Amersham Pharmacia Biotech) and counterstained with Alexa Fluor 546(Molecular Probes) and FITC-CD25. Double-stained sections were analyzed using a confocal laser scanning microscope (FV300, Olympus, Japan). TUNEL assays in the thymus were carried out using an in-situ cell death detection kit (Roche, Indianapolis, IN). Thymus tissues were fixed in 4% paraformaldehyde for 20 min at room temperature and then permeabilized with 0.1% Triton X-100, 0.1% sodium citrate for 2 min at 4°C. The tissues were then incubated with the solution containing TdT enzyme and FITC-labeled dUTP. After incubation for 60 min at 37°C, the samples were analyzed using confocal laser scanning microscope as above.
4.7 In-vitro cell proliferation assay
Single-cell suspensions of splenocytes were treated with 0.1 M ammonium chloride solution to lyse red blood cells and then incubated at 37°C for 1 h to allow macrophages to adhere. Nonadherentcells were recovered and incubated with Dynabeads anti-mouse B220 (Dynal) to remove B cells by magnetic sorting. More than 90% of the purified cells were positive for the CD3 marker. For proliferation assays, purified T cells were cultured at 1×105/well in 96-well microtiter plates with or without stimulation in 200 μl of RPMI 1640 culture medium containing 10% heat-inactivated fetal calf serum, 2 mM LL-glutamine, and 5×10–5 M 2-mercaptoethanol. After 2 days, cells were pulsed with 0.5 μCi=18.5 kBq of [3H]thymidine/well (Amersham Pharmacia Biotech) for the last 16 h. Cells were harvested onto glass-fiber filters, and the incorporated radioactivity was measured.