Downregulation of inhibitory SRC Homology 2 Domain-containing Phosphatase-1 (SHP-1) leads to recovery of T cell responses in elderly
- Aurélie Le Page†1,
- Carl Fortin†1, 2,
- Hugo Garneau1,
- Nancy Allard1,
- Krassimira Tsvetkova1,
- Crystal Tze Ying Tan4,
- Anis Larbi4,
- Gilles Dupuis5 and
- Tamas Fülöp1, 3Email author
© Le Page et al.; licensee BioMed Central Ltd. 2014
Received: 12 July 2013
Accepted: 4 January 2014
Published: 9 January 2014
Immune responses are generally impaired in aged mammals. T cells have been extensively studied in this context due to the initial discovery of their reduced proliferative capacity with aging. The decreased responses involve altered signaling events associated with the early steps of T cell activation. The underlying causes of these changes are not fully understood but point to alterations in assembly of the machinery for T cell activation. Here, we have tested the hypothesis that the T cell pool in elderly subjects displayed reduced functional capacities due to altered negative feedback mechanisms that participate in the regulation of the early steps of T cell activation. Such conditions tip the immune balance in favor of altered T cell activation and a related decreased response in aging.
We present evidence that the tyrosine phosphatase SHP-1, a key regulator of T cell signal transduction machinery is, at least in part, responsible for the impaired T cell activation in aging. We used tyrosine-specific mAbs and Western blot analysis to show that a deregulation of the Csk/PAG loop in activated T cells from elderly individuals favored the inactive form of tyrosine-phosphorylated Lck (Y505). Confocal microscopy analysis revealed that the dynamic movements of these regulatory proteins in lipid raft microdomains was altered in T cells of aged individuals. Enzymic assays showed that SHP-1 activity was upregulated in T cells of aged donors, in contrast to young subjects. Pharmacological inhibition of SHP-1 resulted in recovery of TCR/CD28-dependent lymphocyte proliferation and IL-2 production of aged individuals to levels approaching those of young donors. Significant differences in the active (Y394) and inactive (Y505) phosphorylation sites of Lck in response to T cell activation were observed in elderly donors as compared to young subjects, independently of CD45 isoform expression.
Our data suggest that the role of SHP-1 in T cell activation extends to its increased effect in negative feedback in aging. Modulation of SHP-1 activity could be a target to restore altered T cell functions in aging. These observations could have far reaching consequences for improvement of immunosenescence and its clinical consequences such as infections, altered response to vaccination.
Aging is generally associated with an impairment of the immune response, a phenomenon globally referred to as immunosenescence [1–4]. For instance, there is reduced production of IL-2 and impaired proliferation of T lymphocytes of elderly individuals , as well as a series of altered signaling events associated with the early steps of T cell activation reviewed in [6, 7]. Immunosenescence is observed even in healthy aged individuals  and has been shown to affect the innate and adaptive arms of the immune response [9, 10]. Although the mechanism of immunosenescence remains not fully understood, it has been clearly established that T cells of aged individuals display reduced clonal expansion that translates into increased susceptibility to infectious diseases , impaired responses to vaccination, [12, 13], increased susceptibility to cancer [6, 7, 14, 15] and autoimmune diseases [2, 16–18] and, chronic inflammatory diseases such as Alzheimer’s disease and atherosclerosis [19, 20].
T cell activation involves initial recognition of antigenic epitopes presented by professional APC within the context of MHC class I or class II molecules. Close contact between T cells and APC results in assembly of a supramolecular structure called the immune synapse (IS) [21–23]. IS formation is modulated through dynamic movements of lipid rafts [24–26] that facilitate local clustering of the essential components of T cell signaling . Interactions between TCR and epitope-loaded MHC I or II and with CD8 or CD4 respectively, provide the first signal that leads to a cascade of events resulting from the assembly of the signal transduction machinery or signalosome . However, a second signal has to be provided by occupation of co-stimulation molecules of activation [29–33]. Combination of TCR and CD28 signaling results in full T cell activation characterized by gene expression, cytokine production, cell proliferation, clonal expansion and, generation of effector and memory functions .
From a biochemical standpoint, one of the first events that follow engagement of the TCR by epitope-loaded MHC is the CD45-dependent removal of the phosphate group on tyrosine residue 505 (Y505) of the p56Lck (Lck) Src kinase . Dephosphorylation relieves its cAMP-regulated  Csk-dependent inhibition and allows its auto/trans-activation . Activated Lck then targets the TCR-associated ζ homodimer that provide sites for recruitment and activation of the SH2 domain-containing ZAP-70. Activated ZAP-70 tyrosine-phosphorylates several constituents of the signalosome, including scaffold and adaptor proteins, protein and lipid kinases which are essential for Ca2+ mobilization  and activation of the p21Ras (Ras)/MAP kinase and NF-AT pathways [28, 38, 39]. On the other hand, engagement of CD28 triggers activation of the NF-κB pathway [40, 41]. These activated pathways converge to allow nuclear translocation of the transcription factors AP1, NF-AT and NF-κB and, initiation of expression of a number of genes, including IL-2 and its receptor which are essential for T cell proliferation . Whereas this series of events works in a forward direction to trigger T cell activation and protection against antigenic aggression, complex mechanisms of T cell regulation of activation also operate to prevent uncontrolled responses and autoimmunity and/or immune catastrophe [43, 44]. A number of early cytoplasmic components including protein kinases and phosphatases and, adaptor proteins also act as negative feedback regulators of the TCR signalosome [28, 44]. A key target for the negative regulation of T cell signaling is Lck. Lck activity is finely tuned by a complex of proteins comprising plasma membrane-embedded protein tyrosine phosphatase CD45 and cytoplasmic protein tyrosine kinase Csk bound to scaffold protein PAG (CBP), and to adaptor protein TSAD [45, 46]. In addition, cytoplasmic phosphatases SHP-1 and Lyp (PTPN22) are thought to play a role in the regulation of Lck activity [28, 47–49]. Many of the components of the signaling machinery of T cell activation are targets of SHP-1. For instance, SHP-1 removes key phosphate groups on tyrosine residues of Lck  and ZAP-70 and that results in loss of activity of these essential protein kinases [50, 51]. Furthermore, this regulatory loop of Lck can discriminate between self and non-self as weakly binding ligands predominantly trigger a negative feedback loop leading to rapid recruitment of the tyrosine phosphatase SHP-1, followed by receptor desensitization through inactivation of Lck while, strongly binding ligands efficiently activate a positive feedback circuit involving Lck modification by ERK, preventing SHP-1 recruitment and allowing the long-lasting signaling necessary for gene activation [52, 53].
Beside decreased IL-2 production and T cell proliferation, we and others have shown that the composition and function of lipid rafts were altered with aging  and that the activation of Lck, Fyn, ZAP70 and LAT was impaired [55, 56]. Overall, these observations were consistent with the interpretation that T cell activation and early events in T cell signaling were altered in aged individuals . However, our data did not exclude the possibility that negative regulation of T cell activation through actions of protein tyrosine phosphatases or other mechanisms were also altered. Recently, it has been shown that by modulating later signaling events by phosphatases especially by the DUSP6 repression using miR-181a or specific siRNA and DUSP6 inhibition improved CD4 T cell responses, as evidenced by increased expression of activation markers, improved proliferation and supported preferential T helper type 1 cell differentiation [57, 58]. Here, we report that a deregulation of the Csk/PAG/CD45 loop in T cells of elderly subjects favors the maintenance of Lck inhibition through phosphorylation of Y505. We also observed an upregulation of SHP-1 activity and alterations in the dynamic movements of signaling proteins in lipid rafts. Of significance, pharmacological inhibition of SHP-1 resulted in recovery of TCR/CD28-dependent lymphocyte proliferation and IL-2 production in PBMCs of aged individuals. Our data suggest that the regulatory role of SHP-1 in the forward direction of T cell activation through TCR and CD28 extends to its involvement as negative feedback regulator of Lck activity in aging.
Differential levels of pLck (Y505) in T cells of young and aged subjects
Involvement of PAG phosphorylation in age-related T cell signaling dysfunction
Lipid raft distribution of pPAG and Csk in T cells of young and aged donors
Csk has been reported to transiently migrate out of lipid rafts following TCR engagement  concomitantly with pPAG migration out of lipid rafts. Confocal microscopy analysis showed that approximately 70% of Csk co-localized in lipid rafts of resting T cells of young donors (Figures 3C). Its distribution in lipid rafts decreased significantly (p < 0.006) 30 s after ligation of CD3-CD28 and it remained out of lipid rafts over the course of the experiments, without significant differences (Figures 3C). In marked contrast in the case of aged donors, Csk distribution in resting T cells is less abundant inside LR (Figure 3D). Activation through CD3-CD28 did not result in Csk migration in/out of lipid rafts and its distribution remained nearly unchanged over the course of the experiments (Figures 3D).
CD45, CD45RA And CD45RO expression, activity in T cells with aging
Expression, activity and distribution of phosphorylated SHP-1 in T cells with aging
Inhibition of SHP-1 leads to recovery of TCR/CD28-dependent proliferative response and IL-2 production in T cells of aged individuals
We next sought to determine whether the recovery of T cell proliferation of aged donors correlated with increased intracellular production of IL-2. Results from flow cytometry experiments showed an increase in the percentage of IL-2-producing T cells of young and elderly subjects measured 3 h, 6 h and 24 h after CD3-CD28 stimulation (Figure 7B). The percentage of IL-2-positive stimulated T cells of young donors was significantly higher than in the case of aged donors at 3 h and 6 h but was similar after 24 h of stimulation (Figure 7B). A brief (30 s) treatment with PTP-1 did not induce T cells of elderly donors to produce IL-2 to the same extent as T cells of young donors at 6 h only (p < 005). However, the presence of the SHP-1 inhibitor resulted in significant increases in the percentage of IL-2-positive T cells with respect to the absence of inhibitor in stimulated T cells of aged donors at 3 h, 6 h and 24 h following activation (Figure 7B). A similar effect was observed in T cells of young donors except at 3 h. Extended exposures to PTP-1 (30 min) resulted in IL-2 production that was not significantly different between the two groups of donors when measured 3 h, 6 h and 24 h following T cell stimulation (Figure 7B).
Effect of SHP-1 inhibition on Lck phosphorylation in T lymphocytes of young and elderly individuals
Here, we have tested the hypothesis that the T cell pool in elderly subjects displayed reduced functional capacities due to altered negative feedback mechanisms that are involved in the regulation of the early steps of T cell activation. Data presented here are based on purified T cells and total T cell populations of young and elderly individuals. Immune responses become less efficient as humans get older and this phenomenon contributes to the overall state of immunosenescence. In this connection, accumulated data [36, 40, 41, 68] have established that Lck is a pivotal regulator of the TCR- and CD28-associated early events of T cell activation and subsequent signal transduction, gene expression and T cell proliferation . Data presented here provide evidence that SHP-1 exerts a greater negative feedback effect on Lck-mediated activation of T lymphocytes of aged humans than in T cells of young individuals. This accrued effect of Lck in aging may, at least in part, be responsible for the characteristic impaired proliferation of these cells in response to stimulation.
The levels of the inactive phosphorylated form (Y505) of Lck relative to total Lck were moderate in the resting state of T cells of young donors, as reported in T cells and the Jurkat T cell line . The levels of pLck (Y505) decreased over time of stimulation, suggesting that the levels of inactive kinase decreased presumably at the expense of the active form. In marked contrast, the levels of Lck Y505 only slightly decreased over the time course of stimulation in the case of elderly individuals, suggesting a lack of modulation or maintenance of the inhibitory status of Lck. This differential behavior of T cell response in young and elderly donors suggested an alteration of T cell regulation in the control loop of Lck activation involving the PAG-Csk complex [35, 46, 61] in elderly subjects. An alternative but not exclusive interpretation was an alteration in the efficiency of CD45 to remove the phosphate group at position Y505 of Lck. Engagement of TCR triggers dephosphorylation of PAG and the subsequent migration of Csk out of the lipid rafts, a situation which facilitates CD45-dependent removal of the phosphate group on Y505  and pLck-Y394 upregulation. We showed here that several aspects of the PAG/Csk regulation loop were altered in T cells of elderly subjects. First, Western blot analysis clearly showed that the levels of pPAG were elevated in resting T lymphocytes of elderly subjects and remained at higher levels than those observed in young donors over the course of the experiments (Figure 2). These observations suggested that in T cells of aged subjects, the elevated levels of pPAG kept Lck in its inactive phosphorylated form, in agreement with the results of Western blots (Figures 1A). Second, this interpretation was further supported by analysis of location of pPAG in lipid rafts which clearly showed a differential distribution in T cells of elderly and young individuals (Figure 3). Kinetically, pPAG migrated out of lipid rafts in response to stimulation in the case of T cells of young donors but the levels of pPAG in lipid rafts remained unchanged in resting and activated T cells of aged donors (Figure 3). The inability of pPAG to migrate out of lipid raft microdomains in the case of T cells of aged donors was likely related to the reported decreased function of lipid rafts of T cells of elderly individuals due to the increased cholesterol level . Our data fit current models [28, 68, 71] whereby TCR/CD28 activation induces cellular phosphatases to dephosphorylate Csk-associated pPAG (Y317) during concurrent trafficking out of lipid rafts, resulting in the release of Csk to the cytoplasm. Interruption of Csk-pPAG association results in abrupt decline of phosphorylated (Y505) Lck. Our data showed differential levels of pLck (Y505) in young and elderly subjects that explained the decreased response in T cells of aged individuals .
CD45 activity controls the upregulation of Lck and Fyn activation [71–73]. Here, an alteration in CD45 activity did not appear to be the major cause of the differential activation of Lck in the two groups of individuals. Unexpectedly, the levels of CD45 or its activity were higher in activated T cells of elderly subjects (Figure 4) but there was no differences in lipid raft location of both groups of donors. Also, unexpectedly CD45RA expression and activity did not depend on the age of the subjects, while the expression of CD45RO (marker of memory T cells) was higher in T cells of elderly. This may contribute to the increase of the total CD45 expression and activity at least at 30 s of stimulation (Figure 4A through C). As there is no clear dichotomy in the role of CD45 isoforms in reduced T cell activation with aging, we can hypothesize that the isoform itself has a minimal importance while the associated signalosome, starting with Lck activation is more important. We have previously shown that the formation and composition of membrane lipid rafts, an important step of cell activation, are altered in T cells from elderly individuals.
SHP-1 and CD45 play gatekeeper functions as fine regulators of T cell activation [62, 64, 73]. Whereas CD45 is generally considered as having a positive and essential role in T cell activation, SHP-1 acts as a negative feedback mechanism by targeting tyrosine-phosphorylated components associated with the early events of T cell signaling such as LAT , Lck , ZAP-70 and the ξ homodimer . SHP-1-dependent dephosphorylation of these substrates leads to inhibition of T cell activation. Here, the levels of the active form (Y536) of SHP-1 significantly decreased 30 s after TCR-CD28 stimulation of T cells of young donors (Figure 5), in keeping with positive Lck upregulation and T cell activation. However, the active form of SHP-1 returned to elevated levels after 5 min, in agreement with the fact that T cells become rapidly committed to activation following engagement of the TCR and CD28. In marked contrast, the levels of SHP-1-Y536 remained elevated in T cells of elderly individuals, suggesting that SHP-1 played a negative role in the early sequence of events leading to activation of T cells in aged donors. This observation suggested that SHP-1 remained active under TCR/CD28 stimulation leading to maintenance of Lck in an inhibitory state. The interpretation of a persistent dominant negative role of SHP-1 in T cells of aged donors was further supported by determination of its activity. SHP-1 activity was similar in resting T cells of both groups of donors but rapidly increased and remained high in stimulated T lymphocytes of elderly individuals. In contrast, SHP-1 activity first significantly decreased and became elevated later (5 min) after activation in lymphocytes of young individuals (Figure 5). SHP-1 is mostly located outside of lipid rafts in Jurkat T cells but recruitment increases in response to protein tyrosine phosphatase inhibition . Furthermore, targeted recruitment of SHP-1 in lipid rafts results in dephosphorylation of LAT and subsequent defects in downstream events of TCR signaling in Jurkat T cells . Here, results of confocal analysis revealed that SHP-1 was present to the same levels in resting T cells of young and elderly donors, migrated out of lipid rafts more importantly in T cells from young subjects than in T cells of elderly after activation, but returned to lipid rafts over time (Figure 6). Thus, partitioning dynamics of SHP-1 in lipid rafts may to some extent also contribute to the decreased proliferative response of T cells of aged individuals, concomitantly with the much pronounced age-related differences of SHP-1 activity.
The bulk of the data pointed toward SHP-1 as a key protein phosphatase that negatively modulated T cell proliferation in aged individuals. We tested whether inhibition of SHP-1 would improve the proliferative response of lymphocytes of elderly individuals to CD3-CD28 stimulation. As previously reported, T cells of elderly donors displayed an impaired proliferative response to TCR-CD28-dependent activation and to mitogenic stimulation (Figure 7A). In marked contrast, treating the cells with an SHP-1 inhibitor (PTP-1) for 30 s or 30 min upregulated T cell proliferation to levels that were not statistically different (one-way ANOVA) than those found in cells of young donors. The observation that the presence of PTP-1 did not further increase CD3-CD28-dependent T cell response of young subjects can be explained by the absence of an effect on pLck-Y394 levels thus, on the already maximal proliferation of these cells. IL-2 production was increased in lymphocytes of aged donors in experiments using PTP-1 (Figure 7B). These observations suggested that SHP-1 was a key negative regulator of the proliferative response of T cells associated with aging (Figures 7 and 8) and that its inhibition could efficiently restore two of the most altered functions observed with aging in T cells.
Age-related changes in T cell subset composition involve a reduced frequency/number of naïve T cells, increased number of memory T cells and increased proportion of CD28- T cells, especially in the CD8+ subpopulation . A decrease in CD28 expression would affect the efficiency of the co-stimulatory pathway and this situation has been suggested to be in part responsible for impaired T cell activation with aging . On the other hand, data from our laboratories have provided evidence for a direct correlation between the state of activation of Lck and LAT and their association/recruitment with lipid rafts of CD4+ and CD8+ T cells . In relation with these reports, recent data have provided evidence that, in addition to being a required component of TCR/CD3 signaling, Lck is an obligatory link for T cell activation through the CD28 co-stimulatory pathway. Kong et al. have put forward a model whereby activated Lck associates with pY207 of the C-terminal portion of CD28 through its SH2 domain. This association retains Lck in lipid rafts and allows its concomitant interaction with GLK-dependent phosphorylated PKCθ through its SH3 domain. The model predicts that Lck therefore provides an essential molecular bridge between CD28 and PKCθ. Our data suggest that interfering with the negative regulatory effect of SHP-1 would have a beneficial effect on signals 1 and 2 of T cell activation and would help to restore T cell proliferation in aging, as shown here (Figure 7). Furthermore, it has been recently reported that impairment of early signalling events in activated T cells allows prediction of the levels of expression of the co-stimulatory molecules CD28 and CD27. This situation further predicts the number of population divisions in culture from a limited subset of signalling molecules such as Lck .
Materials and methods
Antibodies and reagents
Polyclonal antibodies (pAbs) anti-PAG (sc-25748), -Lck (sc-28882), -Csk (sc-286), -SHP-1 (sc-287), -CD45 (sc-1178), -CD45RA (sc-19664), -CD45RO (sc-70712) and -β-actin (sc-1616) were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-CD3 (clone UCHT1) and anti-CD28 (clone CD28.2) monoclonal antibodies (mAbs) were from BD Biosciences (Mississauga, ON). pAbs anti-phospho-PAG (Y763) (ab18030) and anti-PAG (ab56521) were from Abcam (Cambridge, MA). An anti-phospho-Tyr505 (2751S) Lck Ab was from Cell Signaling Technology Inc. (Pickering, ON) whereas an anti-phospho-Tyr394 (SAB4300118) Lck Ab was from Sigma-Aldrich. The anti-phospho-Tyr536 SHP-1 mAb was from ECM Biosciences (Versailles, KY). A FITC-conjugated anti-IL-2 mAb (clone M1Q_17H12) was purchased from BD Biosciences. The SHP-1 PTP inhibitor I (PTP-I, product #540200) was purchased from Calbiochem (EMD Chemicals Inc., Gibbstown, NJ). Alexa Fluor 488-labelled cholera toxin B subunit and Alexa Fluor 568-labelled goat anti-rabbit IgG were purchased from Invitrogen (Carlsbad, CA). RPMI-1640 culture medium was obtained from Wisent Inc. (St Bruno, QC), whereas Ficoll Paque plus and Dextran T-500 were from GE Healthcare (Piscataway, NJ). Protein A/G-bound Sepharose was obtained from BioVision Inc. (Milpitas, CA). A Western lightning Plus ECL kit and PVDF membranes were purchased from PerkinElmer (Waltham, MA). Reagents for SDS-PAGE were from Bio-Rad (Richmond, CA) and Fisher Scientific (Montreal, QC).
Twenty five healthy elderly volunteers aged 65 to 78 years (mean, 73 years) participated in the study. The cohort of 25 young healthy subjects was 19 to 25 year old (mean, 22 years). The research protocol was approved by the local institutional ethics committee of the Research Center on Aging. All subjects gave written informed consent. The volunteers were in good health, normolipemic and satisfied the inclusion criteria of the SENIEUR protocol for immune investigations of human elderly subjects . Individual experiments were performed in parallel (on the same day), that is by collecting blood samples from the same number of healthy young donors and healthy aged donors.
Isolation of PBMCs
Blood obtained by venipuncture was collected in heparinized tubes and diluted two-fold with phosphate-buffered saline (PBS). PBMCs were isolated by Ficoll Paque plus density sedimentation, as described . The buffy coat was recovered, the cells were washed (PBS) and counted. Cell viability was greater than 95% (Trypan blue exclusion). Identical numbers of cells from young and aged donors were used in each comparative experiment.
T cells purification
PBMCs were freed of monocyte by adhesion to plastic tissue culture flasks coated with autologous serum (1 h, 37°C) and, B cells and phagocytic cells by nylon wool retention, as described . Purified T cells were greater than 98% CD3+ cells with less than 1% surface IgM (B cells)-, CD16 (NK cells)- and CD14 (monocytes)-positive contaminating cells as verified by FACScan (FACSCalibur). Cell viability was greater than 97% (Trypan blue exclusion). Identical numbers of T cells from young and aged donors were used in each comparative experiment.
PBMCs (2 × 105 cells/well) were exposed to anti-CD3 (5 μg/ml) and/or anti-CD28 (5 μg/ml) for 72 h in 96 well flat-bottomed microcultures (Microtest, Becton Dickinson) in a final volume of 200 μl of RPMI 1640 medium containing 10% foetal bovine serum (FBS), streptomycin (100 μg/ml) and penicillin G (100 U/ml) at 37°C in an atmosphere of 95% air, 5% CO2 and 90% relative humidity. Cell proliferation was quantitated by measuring [3H]thymidine incorporation, as described .
Isolation of lipid rafts
T cells were kept for 1 h in RPMI medium at 37°C. The cells (20 × 106 lymphocytes) were then exposed to a combination of anti-CD3 (5 μg/ml) and anti-CD28 (5 μg/ml) mAb for various periods of time at 37°C, as described  or were left untreated (control). Lipid raft isolation on sucrose density gradients (9 fractions) was done as already described . Lipid rafts were distributed in fractions 1 to 3 of the gradient whereas non-lipid rafts corresponded to fractions 7 to 9. Western blotting analyses were done using pooled lipid raft and non-lipid raft fractions.
Proteins from total cell lysates (20 μg) or pooled lipid raft fractions (30 μl) were sized by SDS-PAGE under reducing conditions, transferred to PVDF membranes and revealed by Western blotting, as described [63, 87]. Acrylamide concentration was 10% in SDS-PAGE analysis performed under reducing conditions, according to Laemmli . To reveal β-actin, PVDF membranes were stripped of antibody by washing (twice, 5 min) with TBST buffer (20 mM TRIS, 150 mM NaCl, 0.1% Tween 20, pH 7.4), incubation with PBS (pH 2.0, ajusted with HCl) for 40 min at 65°C, followed by washing (twice, 5 min) with TBST. The membrane was then probed with an anti-actin antibody. Densitometric analyses were performed using the image analyzer Chemigenius2 Bio Imaging System (Syngene, Frederick, MD) or the Java-based ImageJ freeware (http://rsbweb.nih.gov/ij/). All blots were first normalized towards actin and then to the non-phosphorylated form of the studied protein.
Confocal microscopy analysis
T cells in RPMI 1640 medium containing 10% FBS were exposed to a combination of anti-CD3 (5 μg/ml) and anti-CD28 (5 μg/ml) mAbs for various periods of time at 37°C. Stimulation was terminated by centrifugation, the cells were washed with culture medium and then incubated for 30 min (4°C) with Alexa Fluor 488-conjugated cholera toxin B subunit. They were then fixed with 4% paraformaldehyde in PBS for 15 min on ice. After a wash with cold PBS, the cells were incubated for 1 h at room temperature in the presence of the relevant primary antibodies in PBS containing 1% skimmed powdered milk, 1.4% bovine serum albumin (BSA) and 0.1% Triton X-100. T cells were then washed with PBS and incubated in the same solution containing the secondary Ab (Alexa Fluor 568-labelled goat anti-rabbit IgG) for 1 h at room temperature. After washing with ice-cold PBS, the cells were placed on microscope slides and mounted with Vectashield Mounting Medium (Vector Laboratories, Burlington, ON). Scanning confocal microscope analysis were done as published using a FV1000 instrument (Olympus, Tokyo, Japan) coupled to an inverted Olympus microscope with a 63X oil immersion objective . Specimens were laser-excited at 488 nm (40 mW argon laser) and 563 nm (helium-neon laser). Serial horizontal optical sections of 512 x 512 pixels were taken at 0.5 μm intervals through the entire thickness of the cell. Images were acquired typically from 10–15 cells of similar size from each experimental condition using identical settings of the instrument. In the case of Alexa 488/Alexa 563-merged fluorescence images, dot fluorograms were obtained by plotting pixel values of each fluorochrome toward the horizontal and vertical axis, respectively. Quadrant markers were used to separate staining in background pixels (C, lower left), red-only pixels (A, upper left), green-only pixels (D, lower right) and co-localizing pixels (B, upper right). Percentage of colocalization was assessed as follows: (number of pixels in quadrant B)/(number of pixels in quadrant B + number of pixels in quadrant D). Images were contrast-enhanced, pseudocolored according to their original fluorochromes, merged (co-localizing green and red pixels are in yellow pseucolor in the Figures), cropped and then assembled using the FluoView version 3.1 software (Olympus, Tokyo).
Protein tyrosine phosphatase assays
T cells (5 x 106 cells/ml) in RPMI 1640 medium were left untreated or treated with PTP-1 for different periods of time, as follows. In the cases of short time experiments (30 s and 5 min), the cells were distributed in Eppendorf tubes (2 ml of cell suspension/tube) and PTP-1 was added to a final concentration of 50 ng/ml. For each set of experiments, T cells were centrifuged, washed and suspended in RPMI 1640 medium (10 x 106 cells/ml) and exposed to a mixture of anti-CD3 (5 μg/ml) and anti-CD28 (5 μg/ml) mAbs for the times indicated in the relevant Figures. In the cases of long time experiments (30 min), PTP-1 treatments were done with cells resting in 6-well culture plates (10 x 106 cells/well) containing 2 ml of culture medium in each well. At the end of the treatment, lymphocytes were retrieved, centrifuged and washed. Anti-CD3-CD28 stimulation was done as described above. The cells were then washed with PBS, and resuspended in ice-cold lysis buffer (150 mM NaCl, 10 mM EGTA, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM sodium polyphosphate, 1% Nonidet P-40 (NP-40) and antiproteases cocktail in 50 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4) for 30 min at 4°C, with periodic gentle shaking. Lysates were cleared by centrifugation (13,000 rpm). Samples containing 120 μg of proteins were retrieved and volume completed to 350 µl Protein A/G-bound Sepharose (20 μl) beads were added for preclearing. The mixture was gently rocked for 30 min at 4°C mAbs directed against SHP-1 or CD45 or CD45RA or CD45RO (2 μg/experimental condition) were added to the supernatants, followed by incubation overnight at 4°C under gentle rocking (2 μg/experimental condition). Protein A/G-bound Sepharose (35 μl) beads were added, and incubations under gentle rocking were performed for 4 h at 4°C. The beads were washed with buffer (150 mM NaCl, 10 mM EGTA, 5 mM EDTA and 0.1% NP-40 in 5 mM Tris.HCl buffer, pH 7.5) and subjected to phosphatase assays. Briefly, the beads were washed once with assay buffer (0.5 mM EGTA in 25 mM HEPES, pH 7.0) and then incubated with 200 μl of the same buffer containing 10 mM 4-nitrophenyl phosphate at 37°C for 4 h with periodic mixing. Reactions were stopped by addition of 0.2 M NaOH (800 μl), beads were removed by brief centrifugation, supernatants were distributed in 96-well plates and absorbance was read at 405 nm using a Victor X5 2030 Multilabeled reader (PerkinElmer (Waltham, MA). Equal amounts of proteins (Bradford’s reagent, Bio-Rad, Richmond, CA) from young and elderly donor aliquots were used in CD45, CD45RA, CD45RO and SHP-1 phosphatase assays, as already described  as verified also by Ponceau staining.
Measurement of intracellular IL-2
PBMCs (1 x 106 cells/ml) in RPMI 1640 medium were left untreated or treated with PTP-1 for different periods of time, as described for protein phosphatase assays. They were stimulated using a combination of anti-CD3 (5 μg/ml) and anti-CD28 (5 μg/ml) mAbs for the times indicated in the legend of the Figure. In the case of each experiments, the cells were placed in Eppendorf tubes, washed by brief centrifugation with cold PBS, fixed by treatment (20 min in the dark, 4°C) with 250 μl of 4% (w/v) paraformaldehyde (BioLegend, Burlington, ON) and then washed with a mixture of PBS (1 ml) and diluted (PBS) permeabilization buffer (250 μl) containing FBS and saponin (PermWash buffer, BD Pharmingen). After washings, staining was done with permeabilization wash buffer (200 μl) containing a FITC-conjugated rat anti-human IL-2 Ab (0.2 μg/ml) for 30 min at 4°C, in the dark as already described . The stained cells were washed once with permeabilization wash buffer and resuspended in PBS (250 μl). The cells were analyzed within 24 h by flow cytometry using a FACSCalibur instrument (Beckton Dickinson). A minimum of 10,000 events were acquired in each analysis.
Flow Cytometry measurement of pLck-Y505 and pSHP1-Y536 in T cells after TCR/CD28 stimulation
After stimulation for 0, 30 s and 5 minutes, cells (1 x106) were suspended into 500 μL of PBS 1X at room temperature (RT), and fixed 10 minutes in the dark at room temperature (RT) with 1% paraformaldehyde pre-warmed (Biolegend, Burlington, ON). Then cells were saturated by an incubation of 10 minutes with 10% PBS-AB-human serum (Life technologies). After two washings, a 30 minutes surface staining was made with anti-CD3 Alexa700, CD45RA BV421, and CD45R0 FITC (BD Biosciences, Mississauga, ON). After two washings and vortex of the sediment, 1 mL of BD Phosflow Perm Buffer III was added and incubated for 30 minutes at 4°C. Three washing with 3 mL of PBS was performed before starting 1 hour of intracellular staining at RT in the dark. Antibodies used were: pLckY505 Alexa Fluor 647 (BD Biosciences, Mississauga, ON) and pSHP1 (ECM Biosciences, Versailles, KY) with anti-rabbit IgG PE (Ebioscience, San Diego, CA) were used. The second intracellular staining with anti-rabbit IgG PE was made 1 hour at RT in the dark. After washing, cells were ready to be analysed on a FACS Aria III cytometer (BD Biosciences).
Data were analyzed using Student’s t-test and one-way ANOVA (SigmaStat software, Systat Software Inc., Chicago, IL).
The authors thank Ms Sarra Baëhl for helpful discussions and acknowledge the technical assistance of Ms Annie Larouche and Ms Naheed Azimy. This work was supported by grants from the Canadian Institutes of Health Research (CIHR) (No. 106634 and No. 106701), the Université de Sherbrooke, and the Research Center on Aging.
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