Shb deficient mice display an augmented TH2 response in peripheral CD4+ T cells
© Gustafsson et al; licensee BioMed Central Ltd. 2011
Received: 10 December 2010
Accepted: 11 January 2011
Published: 11 January 2011
Shb, a ubiquitously expressed Src homology 2 domain-containing adaptor protein has previously been implicated in the signaling of various tyrosine kinase receptors including the TCR. Shb associates with SLP76, LAT and Vav, all important components in the signaling cascade governing T cell function and development. A Shb knockout mouse was recently generated and the aim of the current study was to address the importance of Shb deficiency on T cell development and function.
Shb knockout mice did not display any major changes in thymocyte development despite an aberrant TCR signaling pattern, including increased basal activation and reduced stimulation-induced phosphorylation. The loss of Shb expression did however affect peripheral CD4+ TH cells resulting in an increased proliferative response to TCR stimulation and an elevated IL-4 production of naïve TH cells. This suggests a TH2 skewing of the Shb knockout immune system, seemingly caused by an altered TCR signaling pattern.
Our results indicate that Shb appears to play an important modulating role on TCR signaling, thus regulating the peripheral CD4+ TH2 cell response.
The primary aim of T cell development is to create a fully competent T lymphocyte population in the periphery, capable of quickly identifying pathogens yet non-responsive to self-tissue [1, 2]. To maintain this delicate balance, the immature thymocytes are subjected to rigorous control, where signaling through the T cell receptor (TCR) is of utmost importance [3–5]. Mature, peripheral T cells are equally dependent on the TCR, as activation and effector cell development are decided by TCR signaling strength and duration [6–8].
Upon receptor engagement, a host of adaptor and effector proteins assemble at numerous phosphotyrosines within the cytoplasmic segment of the TCR signaling complex. Among the key players in this signaling system are the adaptors SLP76 [9, 10] and linker for activation of T cells (LAT) [11, 12], their association with the activated TCR and their subsequent phosphorylation that activates signaling through phospholipase C-γ (PLC-γ) , as well as Ras and the Rho family of GTPases [14, 15], which in turn enable activation of more distal pathways such as the various variants of mitogen activated kinases (MAPKs) [16–18]. Loss of early signaling elements, such as LAT or SLP76 therefore has profound effects on T cell development and function, with an almost complete block at the very first thymocyte development step requiring TCR signaling, the β- selection, and with severely impaired peripheral responses as a consequence [11, 19–21].
Shb is a widely expressed adaptor protein, known to associate with a variety of different tyrosine kinases, including receptors important for hematopoiesis in general and lymphopoiesis in particular, including the activated TCR, VEGFR- 2 and PDGFR [22–24]. In the case of TCR signaling, the SH2-domain of Shb binds to the ζ-chain of the CD3 complex, where it partakes in the LAT- SLP76 signaling complex [24, 25]. Shb has been demonstrated to associate with both LAT and SLP76, and to facilitate the phosphorylation of numerous downstream signaling targets such as Vav-1 and PLC-γ, accordingly playing an important role in proper TCR signal transduction [24–26].
A Shb knockout mouse has recently been generated. No viable Shb knockout offspring could be generated on the C57Bl/6 background owing to an early embryonic defect. However Shb null mice were obtained on a mixed background (129Sv/C57Bl6/FVB) . The animals display abnormalities in their reproduction, vasculature and glucose homeostasis [27–30]. We employed the Shb knockout to assess thymus and T cell function. Although early T cell development appears unaffected, we observe hyperproliferation and a skewing towards a TH2 response in peripheral T cells.
The generation of Shb knockout mice has been described previously . The animals were bred on a mixed background (129Sv/C57Bl6/FVB). All experiments were approved by the local animal ethics committee at Uppsala University.
Freshly isolated thymi and spleens were gently crushed through a 70 μm cell strainer (BD Bioscience, Erembodegem, Belgium) and thereafter treated with Red cell lysis buffer (Sigma Aldrich, St. Lois, MO) in order to remove erythrocytes.
For flow cytometry 1 × 106 cells in a final volume of 100 μl 1% BSA in PBS were stained with the following antibodies CD4-FITC, CD8-FITC, CD4-PE-Cy5, CD8- PE, CD44-PE-Cy5, CD25-PE, and CD62L-PE (all antibodies were purchased from eBioscience, Hartfield, UK). Flow cytometry was performed on a FACSCalibur (BD Bioscience) using CellQuest software (BD Bioscience, Franklin Lakes, NJ).
Single cell suspensions with 1 × 107 thymocytes or 1 × 106 splenocytes in RPMI 1640 (Gibco, Paisley, UK) supplemented with 10% FBS and 50 μM β-mercaptoethanol (Sigma Aldrich) were stimulated with CD3-antibody (BD Bioscience) at a concentration of 10 μg/ml in 37°C. Thymocytes were stimulated for either 2 or 5 minutes whereas splenocytes were stimulated for 2 minutes.
Immunoprecipitation and immunoblotting
Cell lysates were prepared by addition of lysis buffer (20 mMTris -HCl pH 7.8, 150 mM NaCl, 2 mM EDTA, 1%Triton X- 100, 2 mM PMSF, 10 μg/L aprotinin and 10 μg/L leupeptin). Immunoprecipitation was performed by incubating the samples with either α-phosphotyrosine antibody (Millipore, Watford, UK) or with Shb antibody  at 0.5 μg/ml or 5 μg/ml, respectively, for 1 hour at 4°C. This was followed by incubation with Protein A sepharose (GE Healthcare, Uppsala, Sweden) under the same conditions. Samples were washed 3 times in lysis buffer and resuspended in SDS-sample buffer.
Protein denaturation was achieved by boiling the samples for 5 minutes followed by separation on SDS- PAGE. Proteins were transferred to Hybond-P membranes (GE Healthcare) and subsequently blocked in 5% BSA over night at 4°C.Immunodetection The membranes were probed with the following antibodies: α-phosphotyrosine (Millipore), phospho-ERK (Cell Signaling Technology), PLCγ (Milipore), c-Cbl (BD Bioscience), Vav1 (Millipore), phospho-p38 (Cell Signaling Technology), ZAP70 (BD Bioscience), p38 (Cell Signaling Technology), Shb and ERK (Cell Signaling Technology). Immunodetection was performed using HRP-conjugated secondary antibodies (GE Healthcare) and ECL detection solution (GE healthcare) according to manufacturer's instructions. The autoradiographic film (GE healthcare) was afterwards exposed to the membrane for 2 seconds up to 2 minutes, depending on the strength of the signal.
T cell purification, proliferation and cytokine production assay
Single cell suspensions of splenocytes were fractioned into either CD4+ or CD8+ cells or into naïve CD4+ cells using MACS magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), following the manufacturer's instructions. 5 × 105 CD4+ or CD8+ splenocytes were plated on 24-well plates precoated with 2.5 μg/ml α-CD3 and 3 μg/ml α-CD28 antibodies (BD Bioscience) in a volume of 1.5 ml F-DMEM (SVA, Uppsala, Sweden) supplemented with 10% FBS and 50 μM β-mercaptoethanol. Supernatants were harvested every 24 hours for 6 days and cytokine production was estimated by sandwich immunoassays using Gyrolab Bioaffy (Gyros Biotech, Uppsala, Sweden) following the manufacturer's instructions . On the fourth day of stimulation, 10% of the cells were incubated with 1 μCi of 3H-thymidine (GE Healthcare) 4 hours, in order to estimate proliferative activity. The amount of radioactivity was thereafter determined with a Wallac 1409 scintillation counter (Perkin Elmer, Waltham, MA, USA). Naïve CD4+ cells were cultured under the same conditions as described above, but the cells were plated on 96-well plates at a concentration of 5 × 104 cells per well in 200 μl for 2 day cultures and 2.5 × 104 cells per well for 3 to 5 day cultures.
Real-time reverse transcription- PCR
PCR primer sequences and Tm used for the RT-PCR analysis
CAC TAT TTG GCA ACG AGC GG
TCC ATA CCC AAG AAG GAA GGC
TTG AGT GCC AAT TCG ATG ATG
AGA TGA TGC TTT GAC AGA AGG CTA
CGG AGA TGG ATG TGC CAA AA
GCA CCT TGG AAG CCC TAC AG
CCT GGG GCC TAG CTC TGA
CAG CCA GGA ACA GCC ATG AG
CGA GAT GGT ACC GGG CAC TA
GAC AGT TCG CGC AGG ATG
TTC CCA TTC CTG TCC TTC ACC
TGC CTT CTG CCT TTC CAC AC
Cell cycle analysis
The cell cycle status of naïve CD4+ T cells was analyzed at the indicated time points by adding 5-bromo-2-deoxyuridine (BrdU) (Sigma Aldrich) in a final conctration of 50 μM followed by a 2-hour incubation. The cells were subsequently fixed and permeabilized using the BD Cytofix/Cytoperm kit (BD Bioscience) following the instructions provided by the manufacturer. Cell cycle status was determined by staining with anti-BrdU-APC (Invitrogen Ltd, Paisley, UK) and 7-amino-actinomyocin D (7AAD) (eBioscience) followed by analysis on a FACSCalibur (BD Bioscience) using CellQuest software (BD Bioscience).
Data is presented as mean ± Standard error of the mean (SEM). For comparison of difference between two groups with normal distributed data, unpaired Students t-tests were used unless otherwise stated. For paired comparisons, one wild type and one age and sex matched knockout sample was analyzed simultaneously, under identical conditions, and the wild type and the knockout values were set as one observation each for the comparison. All p-values less than 0.05 were considered statistically significant.
Effect of Shb null allele on blood cell numbers
Aberrant signal transduction in Shb knockout thymocytes
Unaltered thymocyte development in the Shb knockout mouse
Shb knockout TH cells display altered signaling and proliferation
To further study the function of peripheral T cells, splenocytes were fractionated into CD4+ TH cells and CD8+ TK cells and stimulated with CD3 and CD28 antibodies for 96 hours after which proliferation was assessed by estimating 3H thymidine incorporation. When compared to the wild type cells, the Shb null CD4+ cells exhibited a modest proliferation increase in response to stimulation (WT 70 ± 9; KO 98 ± 2; p < 0.05) (Figure 4B). CD8+ Shb knockout cells also displayed a slightly elevated proliferative response, although this effect did not reach statistical significance (Figure 4B).
An increased proliferative rate is likely to be caused by changes in TCR signal transduction. T cell signaling was consequently examined by stimulating CD4+ TH cells with CD3 antibody for 2 minutes. In immunodetection with phosphotyrosine antibody the Shb knockout samples displayed an overall increased protein phosphorylation in the absence of stimulation (Figure 4C), a pattern similar to what was observed in thymocytes. More specifically there was a difference between wild type and knockout in the phosphorylation of a 150 kDa band under basal conditions as well as in stimulated cells (Figure 4C). In addition, bands of molecular weights 120 kDa, 100 kDa and 36/38 kDa exhibited a higher degree of phosphorylation in unstimulated Shb knockout samples, but phosphorylation levels did not appear to increase with stimulation.
In order to address the effect on other signaling pathways downstream of Shb, ERK and p38 MAPK activity was measured in the samples, as both are important for T cell survival and function. p38 MAPK stimulation was poor in the knockout, partly as a consequence of increased basal signal, whereas there was a clear increase in the amount of phospho-p38 MAPK in the wild type after stimulation (Figure 4C). ERK signaling on the other hand seemed normal (Figure 4C).
TH2 skewing in Shb knockout T cells after stimulation
To further confirm that Shb knockout CD4+ TH cells have a tendency to develop a type 2 cytokine response, naïve CD4+CD62L+ T cells were isolated and stimulated. BrdU incorporation and 7AAD staining revealed that naïve knockout T cells appear to progress faster through the cell cycle when stimulated, as demonstrated by the modest but consistent increased percentage (p < 0.05) of Shb knockout cells in the later stages of the cell cycle at 72 and 96 hours of stimulation (72 h WT 43 ± 5%, KO 48 ± 4%; 96 h WT 32 ± 2%, KO 36 ± 1%) (Figure 6B and 6C). Additionally, when cell numbers were determined at the given time points the relative increase in cell numbers were higher in Shb knockout samples compared to the wild type (72 h WT 1.7 ± 0.5 fold increase, KO 2.4 ± 0.5 fold increase; 96 h WT 2.4 ± 0.6 fold increase, KO 4.0 ± 0.7 fold increase; 120 h WT 3.1 ± 0.8 fold increase, KO 6.0 ± 2.0 fold increase, all compared with the starting number of cells; 72 h p < 0.01, 96 h p < 0.05, 120 h p < 0.01).
The differentiation of a newly activated T cell into a TH2 or a TH1 cell is governed by the transcription factors GATA3 and T-bet, respectively [43, 44]. To further ascertain whether the Shb knockout displays a TH2 skewing the expression of these transcription factors was studied. The lack of Shb resulted in an elevated expression of GATA3 after 120 hours of stimulation (120 h WT 4.4 ± 2.2, KO 6.4 ± 1.6; p < 0.05) (Figure 7D) whereas T-bet levels appeared unaffected (Figure 7E). The altered CT-value corresponds to a 17.7 ± 8.5 fold increase in GATA3 mRNA in the Shb KO.
We have previously demonstrated the involvement of the adaptor protein Shb in TCR signal transduction in Jurkat cells. The current study expands these observations by examining T cell development and T cell function in a Shb knockout mouse. Our data establish that Shb is dispensable for thymocyte development but that it exerts effects on peripheral CD4+ T cell signaling. Consequently, unfractionated CD4+ and purified naïve T cells proliferate at a higher rate. An increased number of memory T cells is most probably not the cause of the Shb knockout phenotype since no difference in the proportions of memory and naïve CD4+ T cells was noted and the hyperproliferative effect was also observed on purified naïve T cells. The aberrant TCR signaling is a more likely candidate for the observed elevation in proliferation.
Shb knockout CD4+ T lymphocytes displayed accelerated levels of tyrosine phosphorylation under basal conditions. Several signaling components, putatively Vav-1, LAT and p38 MAPK, were markedly more phosphorylated in the absence of TCR activation. Stimulation-independent signaling has been implicated as an important factor in determining TCR signaling responsiveness [45, 46]. For instance, microRNA-181, a negative regulator of several protein phophatases involved in TCR signaling, causes increased basal signaling and a lowered activation threshold when over-expressed . The elevated basal phosphorylation of important TCR pathway targets in Shb knockout CD4+ T cells could make them prone to respond quicker and more vigorously to a given stimulus than their wild type counter parts.
Even though modifications in basal signaling might in part explain the high proliferative rate displayed by Shb knockout mice, the phosphorylation pattern of LAT is also important to take into consideration. Shb and LAT association, in response to TCR activation, has been demonstrated in Jurkat cells , and mice expressing the LATY136F mutant show a phenotype reminiscent of the one present in the Shb knockout, with TH2 skewing and lymphoproliferation [21, 40]. As already mentioned, a product probably corresponding to LAT (p36/38) exhibited a high level of basal phosphorylation in Shb null T cells, without becoming additionally phosphorylated by stimulation. LAT was originally identified as a key adaptor protein in the TCR signaling cascade, responsible for signals essential to T cell activation. Recent studies have revealed that LAT is not only a mediator of positive signals but also an important negative regulator of TCR signaling . The defective stimulation-induced LAT phosphorylation displayed by Shb knockout T lymphocytes could result in a suboptimal assembly of the LAT signalosome. Consequently, negative feedback loops acting on the TCR machinery might be affected augmenting the proliferative response. On the other hand, LATY136F mice have increased numbers of CD4+ memory cells [21, 40], and as above mentioned, no such increase could presently be detected in the Shb knockout.
The changes in peripheral CD4+ signaling resemble those observed in the Shb knockout thymus. In Shb knockout thymocytes Vav-1 displayed increased phosphorylation under basal conditions without any signs of further amplified phosphorylation after TCR stimulation. Cbl is a ubiquitin ligase that exerts a role in down- regulating ZAP70 activity upon TCR stimulation . Since no difference in ZAP70 activity was noted upon TCR stimulation between wild type and Shb knockout, it seems unlikely that the altered Cbl phosphorylation pattern observed plays any major role in affecting the Shb knockout T cell phenotype. Instead the changes in Cbl phosphorylation are probably a mere reflection of the overall effects of Shb deficiency on the TCR signaling complex.
T cell proliferation is dependent on TCR signaling that in turn indirectly promotes cell division by activation of IL-2 transcription . IL-2 production was, however, normal in knockout cells despite the appearance of an increased responsiveness to TCR stimulation excluding this as an explanation for the Shb knockout hyper-responsiveness.
In addition, Shb appears to affect more than proliferation, since the cytokine production was also different from that observed in wild type T cells. Upon activation, CD4+ T cells have the choice of maturing into different classes of effector cells, each characterized by their own cytokine profile. TH1 and TH2 cells are the two major subsets, responsible for cellular and humoral immune responses, respectively [39, 49]. Signaling from the TCR and cytokine receptors are of great importance in the development of these effector cell responses. Alterations in the activities of targets downstream of the TCR might therefore also affect cytokine production. Vav-1 has for instance been demonstrated as an important factor in IL-4 production and the generation of a TH2 response. Vav-1 knockout mice preferentially develop a TH1 response and Vav-1 in synergy with protein kinase C-Θ (PKC-Θ) has been implicated in the promotion of IL-4 transcription [50, 51]. As already noted Shb knockout thymocytes as well as CD4+ T cells exhibited a slight alteration in their Vav-1 activation. The increased basal activity of Vav-1 in knockout cells may alter the intracellular signaling conditions in favor of a TH2 response.
Moreover, the development kinetics of TH1 and TH2 cells are quite different. The hallmark cytokine of TH1 cells, IFN-γ is produced within hours of activation. Transcripts from typical type 2 cytokines such as IL-4 are on the other hand detected at the earliest on day 2 of stimulation in any significant amounts and a full-fledged TH2 response can take weeks to develop [52, 53]. The main reason for the slow development of TH2 cells is thought to be the extensive chromatin remodeling that is required to fully open the il4 gene locus for transcription [54, 55]. A critical part of the remodeling process is cellular proliferation and it has even been suggested that a certain number of cell divisions are required before IL-4 transcription occurs . Naïve and unfractionated CD4+ T cells from Shb null mice produce IL-4 more promptly after stimulation. Since absence of Shb appeared to lead to an increased cell division rate and faster cell cycle progression it may well result in a more accessible il4 locus thus contributing to a slight TH2 skewing in the immune system of Shb knockout mice.
In the present work, we observe that CD4+ naïve T lymphocytes lacking Shb exhibit increased proliferation due to alterations in important TCR signaling pathways also resulting in a bias towards developing a TH2 cytokine response. Further studies of the effects of Shb on the immune system may therefore prove useful in the elucidation of TH2 driven pathologies such as allergies.
We are grateful to Ing-Britt Hallgren and Eva Törnelius for expert technical assistance. The study was supported by grants from The Swedish Research Council, The Swedish Cancer Foundation, The Swedish Diabetes Association, The Medical Faculty at Uppsala University, Sweden, The Wallenberg Foundation, the Family Ernfors Fund, the Anna Maria Lundin Stipendfund and the Sederholm Fund.
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