Stimulation Reveals the Contribution of to Negative Regulation of T Activation

This information is current as Gabriel Criado and Joaquín Madrenas of September 27, 2021. J Immunol 2004; 172:222-230; ; doi: 10.4049/jimmunol.172.1.222 http://www.jimmunol.org/content/172/1/222 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Superantigen Stimulation Reveals the Contribution of Lck to Negative Regulation of Activation1

Gabriel Criado and Joaquõ«n Madrenas2

The conventional paradigm of T cell activation through the TCR states that Lck plays a critical activating role in this signaling process. However, the T cell response to bacterial does not require Lck. In this study we report that not only is Lck dispensable for T cell activation by superantigens, but it actively inhibits this signaling pathway. Disruption of Lck function, either by repression of Lck or by selective pharmacologic inhibitors of Lck, led to increased IL-2 production in response to superantigen stimulation. This negative regulatory effect of Lck on superantigen-induced T cell responses required the activity of Lck and correlated with early TCR signaling, but was independent of formation and TCR internalization. Our data demonstrate that the multistage role of Lck in T includes the activation of a negative

regulatory pathway of T cell activation. The Journal of Immunology, 2004, 172: 222Ð230. Downloaded from

ngagement of the Ag receptor on T triggers And third, CD4-Lck fusion molecules without kinase activity en- a complex signaling network that culminates in the acti- hance T cell responses (14, 15). Together, these findings are con- E vation and differentiation of these cells. One of the earliest sistent with the existence of an Lck-mediated negative feedback on detectable events in this process is the of T cell activation. residues in motifs within the TCR-associated CD3 chains by the More direct evidence for Lck as a negative regulator of T cell http://www.jimmunol.org/ Src-related Lck (1). Tyrosine phosphorylation of activation is provided by three separate observations. The first is these motifs leads to the recruitment and activation of the ZAP-70 that Lck activity increases after TCR engagement with some an- kinase and phosphorylation of the linker for activated T cells tagonist ligands (16). The second observation is that Th2 T cell (LAT),3 which then provides multiple docking sites for the assem- lines rendered Lck deficient by transfection of Lck antisense DNA bly of signalosomes that activate downstream signaling pathways show enhanced production (17). The third observation is (2). The importance of Lck in this activation process is highlighted that peripheral T cells from patients with deficient Lck activity by the abnormalities in cells and animals lacking Lck. For exam- show hyper-responsiveness to allostimulation (6). These observa- ple, the JCaM1.6 line, a Lck-deficient variant of the Jurkat T cell tions prompted us to hypothesize the existence of a Lck-dependent

line, does not respond to TCR ligation by Abs, and this defect is negative feedback pathway in T cell activation. by guest on September 27, 2021 reversed upon transfection with full-length Lck cDNA (3). More To test this hypothesis, it was first necessary to identify an ex- dramatically, Lck knockout mice have arrested thymocyte devel- perimental system in which Lck is not essential for TCR-depen- opment at the double-negative stage that translates into a signifi- dent T cell activation. Such a system would allow us to determine cant loss of peripheral T cells (4). Similarly, patients with deficient the degree to which Lck contributes to down-regulation of T cell Lck activity show CD4ϩ T lymphopenia (5, 6). activation. Among the conditions of T cell activation that can oc- In contrast to the above-mentioned evidence, several observa- cur in the absence of functional Lck (4, 6, 18Ð20), we chose T cell tions suggest that Lck may play a negative regulatory role in TCR- stimulation with bacterial superantigens (SAg) and APCs. After dependent signal transduction. First, Lck is involved in ligand- this type of stimulation, biochemical (e.g., extracellular signal-reg- induced TCR internalization and thus may regulate the availability ulated kinase (ERK) activation) and functional (e.g., IL-2 produc- of TCR and the magnitude of T cell signaling (7Ð11). Second, tion) responses are observed in Lck-deficient T cells; thus, it serves separate ligation of TCR and CD4 causes Lck activation and in- as a model of Lck-independent T cell activation. Given the value hibits T cell responses by priming CD4ϩ T cells for death (12, 13). of Jurkat T cells to characterize the events involved in early TCR- mediated signaling and the availability of genetic mutants of these cells, we measured the response of wild-type Jurkat T cells, Lck- FOCIS Center for Clinical Immunology and Immunotherapeutics, Robarts Research deficient (JCaM1.6), and several Lck-reconstituted Ju- Institute, and University of Western Ontario, London, Ontario, Canada rkat variants to bacterial SAg. Using this system, we provide for Received for publication July 1, 2003. Accepted for publication October 20, 2003. the first time genetic, pharmacological, and functional evidence of The costs of publication of this article were defrayed in part by the payment of page the contribution of Lck to the down-regulation of T cell activation charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. and cytokine production. 1 This work was supported by the Canadian Institutes of Health Research, the Kidney Foundation of Canada, and the Ontario Research and Development Challenge Fund. Materials and Methods G.C. is an Ontario Research and Development Challenge Fund postdoctoral fellow, Cells and J.M. holds a Canada Research Chair in Transplantation and Immunobiology. 2 Address correspondence and reprint requests to Dr. Joaquõ«n Madrenas, Robarts Re- Human PBMC were isolated from healthy volunteers by Ficoll gradient search Institute, Room 2.05, P.O. Box 5015, 100 Perth Drive, London, Ontario, Can- centrifugation (Ficoll-Paque Plus; Amersham Pharmacia Biotech, Piscat- ada N6A 5K8. E-mail address: [email protected] away, NJ). Peripheral blood T cells were isolated from PBMC by negative 3 Abbreviations used in this paper: LAT, linker for activated T cells; ERK, extracellular selection with a human T cell enrichment cocktail (StemCell Technologies, signal-regulated kinase; HE, diethidium; MD-DC, monocyte-derived dendritic cell; SAg, Vancouver, Canada). Mitogen-activated T cells and monocyte-derived superantigen; SEE, staphylococcal enterotoxin E; PP2, 4-amino-5-(4-chlorophenyl)-7- dendritic cells (MD-DC) were generated from PBMC that were allowed to (t-butyl)pyrazolo[3,4-d]pyrimidine; PP3, 4-amino-7-phenylpyrazol[3,4-d]pyrimidine. adhere to six-well plates for2hat37¡C. Nonadherent cells enriched in

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 223

lymphocytes were collected and treated with PMA (1 ng/ml) and ionomy- washed, stained with FITC-labeled anti-CD3 or PE-labeled anti-CD69, and cin (100 ng/ml; Sigma-Aldrich Canada, Oakville, Canada). After 72 h, analyzed by flow cytometry. cells were washed four times with fresh medium and allowed to rest for at least 24 h. For MD-DC generation, adherent cells were treated with GM- Confocal microscopy CSF (1000 U/ml) and IL-4 (1000 U/ml; R&D Systems, Minneapolis, MN) as previously described (21, 22) for 4Ð6 days. Jurkat cells (1.0 ϫ 106/group) were mixed with .45-gfp cells that had been E6.1 cells, Lck-deficient JCaM1.6 cells, and ZAP-70-deficient P116 preincubated overnight with SEE (100 ng/ml), pelleted, and incubated at cells were purchased from American Type Culture Collection (Manassas, 37¡C for 30 min. Stimulation was stopped by adding 1 ml of cold PBS. VA). JCaM1.6 cells expressing kinase-dead (K273R) Lck (JCamLck KD) Cells were transferred to poly-L-lysine-coated plates, stained with anti- cells (23), JC1/Lck cells (24), LAT-deficient JCaM2.5 cells, and LAT- CD3-PE-Cy5 and anti-LFA-1-PE Abs, and fixed with PBS containing 4% reconstituted JCaM2.5 B3 cells (25) were provided by Dr. R. Wange (Na- paraformaldehyde. Image capture was performed with a confocal micro- tional Institutes of Health, Baltimore, MD). The lymphoblastoid line scope (Carl Zeiss, New York, NY) and analyzed with the LSM 510 soft- LG2 was provided by Dr. E. Long (National Institutes of Health, Bethesda, ware (Carl Zeiss; Microsoft, Redmond, WA). MD). The lymphoblastoid B cell line .45 retrovirally transfected with gfp was a gift from Dr. B. Carreno (Wyeth Research, Cambridge, MA). All cell lines were maintained in RPMI 1640/10% FCS and standard supplements. Results and Discussion Bacterial SAg cause polyclonal T cell activation due to their ca- Abs and reagents pacity to bind different TCR V␤-chains (30). Although Lck is ac- The following Abs were used for biochemical experiments: anti-active tivated after TCR engagement with SAg (31), it is not required for ERK mAb E10 (Cell Signaling Technology, Beverly, MA), anti-ERK rab- SEE-induced T cell activation, as illustrated by the ability of the bit polyclonal antiserum (StressGen, Victoria, Canada), antiphosphoty- Lck-deficient T cell line JCaM1.6 to respond to SEE (19). To rosine mAb 4G10 (provided by Dr. B. Drukker, Oregon Health Sciences University, Portland, OR), anti-LAT and anti-Lck rabbit polyclonal Abs analyze the contribution of Lck to the response to SEE, we exam- Downloaded from (Upstate Biotechnology, Lake Placid, NY), anti-ZAP-70 rabbit polyclonal ined the effect of blocking the activity of this kinase in PBMC Ab (265), provided by Dr. J. M. Rojo (Centro de Investigaciones Biolo«gi- using the pharmacological inhibitor PP2, a compound that selec- cas, Madrid, Spain), goat anti-rabbit Ig HRP conjugated (Bio-Rad, Her- tively inhibits the tyrosine Lck and Fyn (32). As a control, cules, CA), and sheep anti-mouse Ig HRP-conjugated (Amersham Phar- macia Biotech). The following Abs were used for flow cytometry and cells were stimulated with anti-CD3 Ab, a TCR ligand that triggers confocal microscopy: FITC-, PE-, and PE-Cy5-labeled anti-CD3; PE-la- a response that is entirely dependent on Lck activation (3, 4). Pre-

beled anti-CD69; PE-labeled anti-CD25; and PE-labeled anti-LFA-1 treatment with PP2, but not the control compound PP3, inhibited http://www.jimmunol.org/ (eBioscience, San Diego, CA). 4-amino-5-(4-chlorophenyl)-7-(t-bu- anti-CD3-induced IL-2 production in PBMC in a dose-dependent tyl)pyrazolo[3,4-d]pyrimidine (PP2) and 4-amino-7-phenylpyrazol[3,4- manner, with an 50% inhibitory concentration of 1.46 ␮M and a d]pyrimidine (PP3) tyrosine kinase inhibitors were purchased from Cal- ␮ biochem (San Diego, CA). Staphylococcal enterotoxin E (SEE) was complete block at 12.5 M. In marked contrast, IL-2 production purchased from Toxin Technology (Sarasota, FL). Doxycycline and di- by PBMC in response to SEE showed only marginal inhibition at ethidium (HE) were purchased from Sigma-Aldrich Canada. low concentrations of PP2 and was blocked only at concentrations ␮ Cell stimulation, immunoprecipitation, and Western blotting of PP2 higher than 10 M (Fig. 1), which are toxic to these cells. The apparent PP2 resistance of the response of PBMC to SEE Jurkat T cells were stimulated with LG2 cells (5:1 ratio) preincubated with could be interpreted as indicative that Src kinases are differentially

SEE for 40 min at 37¡C. Cells were subsequently lysed in 1% Triton X-100 by guest on September 27, 2021 lysis buffer, and the lysates were analyzed directly by Western blot or after involved in the responses of specific T cell subsets to bacterial immunoprecipitation with Abs on A/G agarose beads (27, 28). SAg, inhibiting the activation of some, while enhancing the re- sponse of others. As it has been reported that Lck-defective pa- IL-2 production tients have an over-representation of activated T cells in an oth- PBMC (5 ϫ 104) were stimulated with SEE or anti-CD3 OKT3 mAb, T erwise lymphopenic background (5), we hypothesized that the ϫ 4 ϫ 3 cell blasts (5 10 ) were stimulated with MD-DC (2.5 10 ) and SEE SEE-responsive primary T cell subset that did not require Lck (10 ng/ml), and Jurkat T cells (5 ϫ 104) were stimulated with LG2 cells (2.5 ϫ 104) and SEE in 96-well plates. supernatants were would be mostly a primed/activated subset. To test this hypothesis, collected after 16Ð24 h and assayed for IL-2 content by ELISA using human T cell blasts were generated, and their response to SEE OptEIA human IL-2 Set (BD PharMingen, San Diego, CA). Data were presented by autologous MD-DC in the absence or the presence of analyzed with MPM III 1.34 (Bio-Rad, Hercules, CA) and GraphPad PP2 was analyzed. We found that human T cell blasts responded PRISM software (GraphPad, San Diego, CA). well to SEE. Moreover, in these cells, the presence of PP2 during Stimulation and analysis of T cell-APC conjugation stimulation resulted in a dose-dependent enhancement of the SEE- The presence of T cell-APC conjugates after SEE-mediated stimulation induced IL-2 production (Fig. 1B), revealing the presence of a was determined by flow cytometry as previously described (29) with minor negative role for Lck in T cell activation. Under the same exper- modifications. Briefly, Jurkat T cells were resuspended at 2 ϫ 106 cells/ml, imental conditions, the response of non-blast T cells showed the treated for 30 min at 37¡C with HE (3 ␮g/ml) in serum-free RPMI 1640, same marginal inhibition of IL-2 production by PP2 as that ob- ϫ washed three times with serum-free RPMI 1640, and resuspended at 2 served with unfractionated PBMC (Fig. 1, A and B). 106 cells/ml. .45-gfp cells, used as APC, were collected, resuspended at 2 ϫ 106/ml in serum-free RPMI 1640, and incubated for 40 min at 37¡C The observation of NFAT activation and IL-2 production in re- with SEE (100 ng/ml). For conjugation, .45-gfp (106 cells/sample) and sponse to SEE by Lck-deficient T cells has been interpreted as a Jurkat T cells (106 cells/sample) were mixed, pelleted, and incubated for 30 reflection of Lck dispensability (19). However, our observation min at 37¡C. Stimulation was stopped by adding 1 ml ice-cold PBS, and that PP2 enhances the response of some primary T cell subsets to cells were fixed with PBS/1% paraformaldehyde for 20 min. Forty thou- sand events were acquired using a FACScan flow cytometer (BD Bio- SEE argues that not only is Lck dispensable in this response, but sciences, Mississauga, Canada) and analyzed with CellQuest computer it may play a negative regulatory role. This possibility is suggested software (BD Biosciences). The percentage of conjugated T cells was de- by recent observations of the apparent lack of inhibition of T cell termined as the number of dual-labeled events divided by the sum of the responses using novel Src kinase inhibitors (33). To avoid prob- dual-labeled events and the unconjugated Jurkat cells as previously de- lems in experimental interpretation that could arise from the poly- scribed (29). clonal nature of primary T cells and the variability in the levels of Analysis of TCR/CD3 internalization and CD69 up-regulation expression of Src kinases in these cells, we took advantage of the by flow cytometry availability of Jurkat T cells and their Lck-deficient and Lck-re- T cells (2 ϫ 106) were incubated at 37¡C for with LG2 cells (2 ϫ 106)in constituted counterpart lines to address the question of whether the presence or the absence of 100 ng/ml SEE. Cells were collected, Lck is involved in signaling to negative regulatory pathways. 224 NEGATIVE REGULATION OF T CELL ACTIVATION BY Lck

FIGURE 1. Src kinase inhibition differentially affects SAg-induced activation of freshly isolated T cells or mitogen primed human T cell blasts. A, PBMC were stimulated with SEE (1 ng/ml) or anti- CD3 OKT3 Ab (1 ng/ml) in the presence of differ- ent concentrations of the Src kinase inhibitor PP2 or the control compound PP3. B, Peripheral blood T cells and T cell blasts were stimulated with au- tologous MD-DC (ratio, 20 T cells/1 DC) and SEE (10 ng/ml) in the presence of the indicated amounts of PP2. After 16Ð24 h of culture, supernatants were collected and assayed for IL-2 content by ELISA. Results are representative of two separate experi- Downloaded from ments for each cell condition. http://www.jimmunol.org/

To test the effect of Lck expression on Jurkat T cell activation affect IL-2 production when added to nontransfected JCaM1.6 by staphylococcal SAg, we analyzed the SEE-induced response of cells. In addition, the increased IL-2 production observed in Lck- Lck-deficient T cells stably reconstituted with full-length Lck repressed cells was not due to less activation-induced cell death, as cDNA under a Tet-repressible promoter (hereafter called JC1/Lck the level of activation-induced cell death, as determined by pro- cells) (24). When JC1/Lck cells were stimulated with SEE in the pidium iodide incorporation, was comparable in Lck-replete

presence of 10 ng/ml doxycycline, conditions that correlate with and Lck-deficient T cells (data not shown). These data demon- by guest on September 27, 2021 almost complete inhibition of Lck expression, we observed a sig- strate that repression of Lck expression enhances T cell respon- nificant enhancement of IL-2 production for each concentration of siveness to SEE, and thus illustrate for the first time that in SEE (Fig. 2A). The enhancing effect on IL-2 production in re- addition to its conventional activating role, Lck has a negative sponse to SEE was inversely proportional to the amount of Lck regulatory function on T cell activation in response to TCR present in the cell, and maximal enhancement of IL-2 production engagement by SAg. was detected at concentrations of doxycycline that repressed Lck Next, we tested whether the inhibitory role of Lck on SEE- expression below detection levels (Fig. 2B). Doxycycline did not induced activation of Jurkat T cells required its kinase activity. If

FIGURE 2. Lck plays a negative regulatory role in SEE-induced T cell activation in Jurkat T cells. A, Lck-deficient T cells (JCaM1.6; E and F) and doxycycline-repressible Lck-reconstituted T cells (JC1/Lck; f and Ⅺ) were cultured overnight without (E and Ⅺ) or with doxycycline (10 ng/ml; F and f) and subsequently stimulated with APC and different concentrations of SEE. B, Jurkat JC1/Lck cells were cultured overnight without or with the indicated concentrations of doxycycline and stimulated with APCs and SEE (100 ng/ml) in the presence of the indicated concentrations of doxycycline. After 24 h, culture supernatants were collected and assayed for IL-2 content. B, Doxycycline-treated JC1/Lck cells were lysed, and Lck levels were assayed by immunoblot (bottom panel). The image was acquired in a Fluorchem 8800 station (Alpha Innotech, San Leandro, CA) and quantitated with Phoretix 1D software (Non Linear, Durham, NC). The Journal of Immunology 225

FIGURE 3. Pharmacological inhibi- tion of Lck enhances SEE-induced T cell activation in Jurkat T cells. A, Jurkat E6.1 cells were pretreated for 30 min with different concentrations of PP2 or PP3 and stimulated with APCs and SEE (10 ng/ml). B, Jurkat E6.1 cells were left untreated, pretreated for 30 min with PP2 (20 ␮M), or treated at different times dur- ing the stimulation with APCs and SEE (10 ng/ml). C, Jurkat E6.1 and Lck-defi- cient JcaM1.6 cells were cultured for 30 min without or with the Src kinase inhib- itor PP2 (20 ␮M) and stimulated with APCs and different amounts of SEE. Su- pernatants were collected after 24 h of Downloaded from culture and were assayed for IL-2 by ELISA. The inhibitor was maintained in the culture for the entire stimulation pe- riod in all experimental groups. http://www.jimmunol.org/

so, this might explain the increased Lck kinase activity in response enhancing potency when added after1hofstimulation (Fig. 3B). to TCR antagonists (16) and the enhanced responses of T cells Analysis of IL-2 production upon titration of SEE stimulation expressing a kinase-dead CD4-Lck fusion protein (14, 15). We showed that the enhancing effect of PP2 on T cell activation was examined the effect of pharmacological inhibition of the Src ki- functional along the full range of SEE concentrations tested (Fig. nases with PP2 on the response of Jurkat T cells to SEE and APC. 3C, left panel). In sharp contrast, PP2 pretreatment completely by guest on September 27, 2021 In this cell line, in which Lck is the predominant Src kinase (24, inhibited the response of Lck-deficient T cells to the same type of 34), we found that pretreatment with PP2 enhanced the production stimulation, corroborating that the enhancing effect of PP2 re- of IL-2 in response to SEE and APC in a dose-dependent manner. quired the Lck kinase activity (Fig. 3C, right panel). It is important Such an enhancing effect was specific to PP2 and was not detected to note that these results are not unique to SEE, as the same results with PP3 (Fig. 3A). Furthermore, the effect of PP2 on IL-2 pro- were obtained when T cells were stimulated with the streptococcal duction was detected when cells were pretreated or treated shortly SAg streptococcal mitogenic exotoxin-Z (data not shown). How- after the initiation of TCR stimulation, but PP2 lost most of its ever, this effect was not seen with Jurkat T cells stimulated with

FIGURE 4. The inhibitory effect of Lck on T cell activation requires its kinase activity. A, Jurkat E6.1 cells, Lck-deficient JcaM1.6 cells, and JcaM1.6 cells stably transfected with wild-type Lck (JC1/Lck) or with a kinase-dead Lck (JLckKD) were cultured for 30 min without or with PP2 (20 ␮M) and stimulated with APCs and SEE (10 ng/ml). B, JC1/Lck cells were left untreated or were treated overnight with doxycycline (1000 ng/ml) to repress Lck expression or for 30 min with PP2 (20 ␮M) or with a combination of doxycycline and PP2, and stimulated with APCs and SEE (10 ng/ml). After 24 h, culture supernatants were collected and assayed for IL-2. A, The level of IL-2 production for each cell line was normalized to that reached in the absence of the inhibitor to account for interclonal variations. When used, PP2 and doxycycline were maintained in the culture during the stimulation. 226 NEGATIVE REGULATION OF T CELL ACTIVATION BY Lck bead-immobilized anti-CD3 Abs and soluble anti-CD28 Abs, con- hancing effect of PP2 on IL-2 production in response to SEE was ditions under which PP2 inhibited IL-2 production in both Lck- detected in Lck-deficient T cells reconstituted with functional Lck replete and Lck-deficient T cells (data not shown). Together, these (JC1/Lck), but not in Lck-deficient T cells reconstituted with a results show that pharmacological inhibition of Lck activity en- kinase-dead Lck (JLckKD; Fig. 4A). Thus, the kinase activity of hances IL-2 production by Jurkat T cells in response to bacterial Lck is critical for the negative regulatory pathway that down-reg- SAg and imply that the inhibitory signals delivered by Lck upon ulates T cell activation in response to SEE. This was also demon- SEE stimulation are mediated by its kinase activity. strated in the JC1/Lck cell line by comparing the effects of PP2 To further demonstrate that the negative regulatory function of under conditions of Lck expression and Lck deficiency. In these Lck on SAg-induced T cell activation requires the kinase activity cells we observed that when Lck is expressed, i.e., without doxy- of Lck, we undertook a genetic approach. We compared the IL-2 cycline in the culture medium, PP2 had an enhancing effect on response to SEE and APC of multiple Lck-deficient T cell clones IL-2 production in response to SEE and APC (Fig. 4B). However, stably reconstituted with functional Lck to that of T cell clones when the same T cells did not express Lck, i.e., with doxycycline reconstituted with a kinase-dead Lck. We observed that the en- in the medium, PP2 inhibited IL-2 production in response to SEE Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 5. T cell-APC conjugation and immunological synapse formation in the absence of functional Lck. A, HE-loaded Jurkat E6.1 and JCaM1.6 cells (106 cells/sample) were stimulated for 30 min with .45-gfp APC (106 cells/sample) in the absence or the presence of SEE (100 ng/ml) and analyzed by flow cytometry. APCs were detected in the FL1 channel, T cells in the FL2 channel and APC-T cell conjugates were detected as dual-color events. A representative experiment is shown, indicating the percentage of conjugates for each condition. B, Data from four experiments were collected and analyzed p Ͻ 0.01). Similar results were obtained when CFSE-loaded T cells and ,ءء) for statistical significance by ANOVA (p Ͻ 0.0001) and Student’s t test HE-loaded LG2 cells were assayed. C) Jurkat E6.1 and JCaM1.6 cells (106 cells/sample) were stimulated for 30 min with .45-gfp cells (106 cells/sample; signal in green) in the absence or the presence of SEE (100 ng/ml), stained with anti-CD3⑀-PE-Cy5 (blue signal) and anti-LFA-1-PE (red signal), and analyzed by confocal microscopy. D, Jurkat E6.1 (106 cells/sample), untreated or treated with PP2 (20 ␮M), were stimulated for 30 min with LG2 cells in the presence of 100 ng/ml SEE, stained with anti-CD3⑀-PE-Cy5 (blue signal) and anti-LFA-1-PE (red signal), and analyzed by confocal microscopy. Note that for D, the LG2 APC were not labeled. White arrowheads point toward molecular clusters at the T cell:APC interface. The Journal of Immunology 227 and APC (Fig. 4B). This result conclusively demonstrates that it is by the colocalization of TCR/CD3 and LFA-1 at a flat interface the kinase activity of Lck that plays a negative regulatory role on between T cell and APC (Fig. 5, C and D). Therefore, the negative SAg-induced T cell activation. It also implies that the inhibition of role of Lck does not involve a gross alteration of the T cell:APC IL-2 production by PP2 in T cells that do not express Lck is due interaction. to inhibition of Fyn, which then is essential for activation of these Ligand-induced internalization and degradation of TCR can cells (19). down-regulate T cell responses by decreasing the number of avail- Next, we examined whether the negative regulatory role of Lck able TCR, thus reducing the magnitude of TCR-dependent signal- in the response to SEE was associated with changes in the ability ing (8, 11, 37Ð39). This type of TCR internalization is dependent to form T cell:APC conjugates and immunological synapses. After on Src kinases (7, 9, 10, 39, 40). One could argue that the en- 30 min, we observed that the basal number of T:APC conjugates hancement of T cell activation in the absence of Lck activity could was lower in Lck-deficient T cells than in parental T cells (Fig. result from more TCR complexes available at the cell surface due 5A). However, SEE stimulation consistently induced a 3-fold in- to a decreased rate of TCR/CD3 internalization. However, this is crease in the number of conjugates in both parental T cells and not the case, because we found that TCR/CD3 internalization in Lck-deficient JCaM1.6 T cells ( p Ͻ 0.01; four experiments; Fig. the early stages of T cell activation (at 30 min, when inhibition of 5B). This finding contrasts with the lack of conjugate formation in Lck activity had an enhancing effect) was comparable between Lck-deficient T cells reported by Morgan et al. (29). However, it Lck-deficient T cells and parental T cells (Fig. 6A). This result was should be noted that these investigators examined early time points confirmed in PP2-treated T cells (Fig. 6B) and in Lck-repressed T (10Ð15 min), whereas we studied later time points (30 min). Pro- cells (data not shown) and was apparent at two different concen- longed stimulation may allow the formation of stable conjugates trations of SEE, confirming previous reports of SEE-induced TCR Downloaded from and subsequent mature immunological synapses between T cells internalization in the absence of Lck (11, 41). These data show that and APCs (35, 36). This claim is corroborated by the observation the Lck-dependent negative feedback on SEE-induced T cell ac- that the lack of Lck or the inhibition of Lck activity by PP2 did not tivation is not due to differences in the availability of the TCR. prevent the formation of putative immunological synapses defined Interestingly, at later times of stimulation (15 h), we consistently observed increased CD3 expression in T cells lacking functional

Lck to levels that were only seen in Lck-expressing T cells at http://www.jimmunol.org/ higher concentrations of SEE, suggesting a potential effect of Lck on TCR re-expression. The previous set of experiments demonstrated that membrane- associated events in response to bacterial SAg (e.g., T cell-APC conjugation, immunological synapse formation, TCR internaliza- tion) are comparable between Lck-expressing and Lck-deficient T cells and pointed to an intracellular signaling event(s) as the tar- get(s) of Lck-mediated inhibition of T cell activation. Therefore, we examined the key early TCR signaling steps upon T cell stim- by guest on September 27, 2021 ulation by SEE under inhibition of Lck by PP2 (Fig. 7). As ex- pected, SEE stimulation of Lck-expressing Jurkat T cells induced early tyrosine phosphorylation of TCR-␨ and association with phospho-ZAP-70. In contrast, SEE stimulation in the presence of Lck inhibition induced tyrosine phosphorylation of TCR-␨ with delayed kinetics, and under these conditions, ZAP-70 associated

FIGURE 6. SEE-induced TCR internalization in the absence of func- tional Lck. A, Jurkat E6.1 and JCaM1.6 cells (106 cells/sample) were stim- ulated for 30 min with .45gfp cells (106 cells/sample) in the absence or the presence of SEE (100 ng/ml). B, Jurkat E6.1 (106 cells/sample), untreated or treated with PP2 (20 ␮M), were stimulated for different times with LG2 FIGURE 7. Tyrosine phosphorylation and association of TCR-␨ and cells (106 cells/sample) in the absence or the presence of different SEE ZAP-70: effect of Lck inhibition. Parental Jurkat cells (E6.1) were stimu- concentrations. After stimulation, cells were stained with anti-CD3-PE- lated with APCs and SEE (10 ng/ml) in the presence or the absence of PP2 labeled (A) or anti-CD3-FITC-labeled (B) Abs and analyzed by flow cy- (20 ␮M) for the indicated times. ZAP-70 immunoprecipitates from these tometry. Data from three independent experiments were collected for each cells were prepared and sequentially blotted for phosphotyrosine and ZAP- experimental condition, and the mean Ϯ SD is shown. Similar results were 70. An APC sample containing the same number of APCs used to stimulate obtained when staining with an anti-TCR Ab. LG2 and .45gfp cells are the T cells was analyzed (APCs), and a sample containing the eluate of the both APC expressing similar levels of HLA-DR1 and costimulatory beads incubated with the Ab and lysis buffer in the absence of cellular molecules. extracts (Beads) was included. 228 NEGATIVE REGULATION OF T CELL ACTIVATION BY Lck Downloaded from

FIGURE 8. LAT phosphorylation-independent activation of ERK1/2 in Lck-deficient Jurkat T cells responding to SEE. A, Parental Jurkat cells (E6.1) and their Lck-deficient counterparts (JCaM1.6) were stimulated with APCs and different concentrations of SEE for 2 min (left panel) or with APCs and 10 ng/ml SEE for the indicated times (right panel). Lysates were immunoprecipitated with anti-LAT Ab and sequentially blotted for phosphotyrosine and LAT. B, Jurkat E6.1 and JCaM1.6 cells were stimulated with APCs and SEE for 2 min at the indicated concentrations (left panel) or with APCs and 10 ng/ml SEE for the indicated times (right panel). Whole cell lysates were prepared and sequentially immunoblotted for active ERKs and total ERK1/2. http://www.jimmunol.org/ with phospho-␨, but was not phosphorylated, consistent with the expression of CD69, although to a lesser extent than LAT-recon- lack of Lck activation. stituted JCaM2.5 cells (Fig. 9). As expected, SEE stimulation in- Next, we analyzed the tyrosine phosphorylation of LAT, a cor- duced CD69 up-regulation in the parental E6.1 Jurkat cell line and nerstone event for the assembly of signalosomes connecting the the ZAP-70 deficient Jurkat cell line (P116), as we have previously TCR-proximal events to effector signaling pathways (2, 25), and reported (20). These results were further corroborated when CD25 the kinetics of ERK activation, which may reflect distinct signaling up-regulation was analyzed in the same cell lines (data not shown). patterns from the TCR (42). Remarkably, T cell stimulation with These data demonstrate that the Jurkat T cell response to SEE does SEE induced LAT phosphorylation only in E6.1 cells (detectable not require ZAP-70 or LAT. by guest on September 27, 2021 after 1 min of stimulation and maximal after 15 min of stimula- The specific biochemical pathway that mediates the negative tion), not in the Lck-deficient counterparts (Fig. 8A). Comprehen- regulatory function of Lck in vivo is currently under investigation. sive dose-response and time-course analyses did not reveal any It may involve several mediators that can act as in vitro substrates tyrosine phosphorylation of LAT in response to SEE in Lck-defi- cient T cells (Fig. 8A). This observation is remarkable because it occurred under conditions that induced maximal levels of IL-2 production by Lck-deficient T cells (Fig. 2). Activation of T cells by SEE in the absence of LAT phosphorylation confirms our pre- vious findings of the ability of certain TCR stimuli to induce signal transduction leading to ERK activation and CD69 up-regulation in a LAT-independent manner (20, 28). Analysis of the kinetics of ERK activation in response to SEE: APC revealed that Lck deficiency did not prevent ERK activation (Fig. 8B). Although Lck-deficient T cells required higher concen- trations of SEE to activate ERK1/2 to levels similar to those seen in Lck-expressing T cells (Fig. 8B, left panel), the temporal pro- files of ERK activation in response to SEE:APC were comparable in Lck-expressing T cells and Lck-deficient T cells (Fig. 8B, right panel). The observation of ERK activation in the absence of Lck activity and tyrosine phosphorylation of LAT in Jurkat cells is reminiscent of the phenotype of mice homozygous for a LAT mu- tation in which tyrosine 316 is replaced by phenylalanine (43, 44). These mice develop a lymphoproliferative disorder, and their T lymphocytes show intact ERK activation despite impaired phos- pholipase C␥-1 activation. FIGURE 9. ZAP-70-independent and LAT-independent T cell activa- The ability of Jurkat cells to respond to SEE in the absence of tion in response to SEE. Parental (E6.1), ZAP-70-deficient (P116), LAT- LAT was assessed by analyzing the up-regulation of the activation deficient (JCaM2.5), and LAT-reconstituted (JCaM2.5B3) Jurkat cells marker CD69 in the LAT-deficient variant JCaM2.5 cell line and were stimulated with APCs in the presence or the absence of SEE (100 the LAT-reconstituted counterpart JCaM2.5B3. Upon stimulation ng/ml) for 15 h. Cells were collected and analyzed for CD69 expression by with SEE, LAT-deficient Jurkat cells were able to up-regulate the flow cytometry. The Journal of Immunology 229 of Lck. An obvious candidate for this role is Csk, a kinase that 2. Wange, R. L. 2000. LAT, the linker for activation of T cells: a bridge between T down-regulates TCR signaling upon relocation to lipid rafts in a cell-specific and general signaling pathways. Sci. STKE 2000:RE1. 3. Straus, D. B., and A. Weiss. 1992. Genetic evidence for the involvement of the Lck-dependent fashion (45Ð47). However, we have failed to no- lck tyrosine kinase in signal transduction through the T cell receptor. Cell tice a significant difference in the amount of Csk in lipid rafts of 70:585. parental or Lck-deficient T cells upon stimulation with SEE (G. 4. Molina, T. J., K. Kishihara, D. P. Siderovski, W. van Ewijk, A. Narendran, E. Timms, A. Wakeham, C. J. Paige, K. U. Hartmann, A. Veillette, et al. 1992. Criado and J. Madrenas, unpublished observations). Another po- Profound block in thymocyte development in mice lacking p56lck. Nature tential mechanism to account for the negative role of Lck de- 357:161. 5. Hubert, P., F. Bergeron, V. Ferreira, M. Seligmann, E. Oksenhendler, P. Debre, scribed in this study may be the recently reported Src homology and B. Autran. 2000. Defective p56Lck activity in T cells from an adult patient protein tyrosine phosphatase-1-mediated negative feedback on T with idiopathic CD4ϩ lymphocytopenia. Int. Immunol. 12:449. cell activation (48). Such an effect is based on the regulation of 6. Goldman, F. D., Z. K. Ballas, B. C. Schutte, J. Kemp, C. Hollenback, N. Noraz, and N. Taylor. 1998. Defective expression of p56lck in an infant with severe tyrosine-phosphorylated Src homology protein tyrosine phospha- combined immunodeficiency. J. Clin. Invest. 102:421. tase-1 binding to Lck by activated ERK. However, to be active, 7. D’Oro, U., M. S. Vacchio, A. M. Weissman, and J. D. Ashwell. 1997. Activation this mechanism requires early Lck-mediated tyrosine phosphory- of the Lck tyrosine kinase targets cell surface T cell antigen receptors for lyso- somal degradation. Immunity 7:619. lation of SHP-1 combined with delayed and transient activation of 8. Lee, J. E., M. B. Cossoy, L. A. Chau, B. Singh, and J. Madrenas. 1997. Inacti- ERK. These features of ERK activation are not those reported in vation of lck and loss of TCR-mediated signaling upon persistent engagement this study to be associated with the negative role of Lck, i.e., early with complexes of peptide:MHC molecules. J. Immunol. 159:61. 9. Luton, F., M. Buferne, J. Davoust, A. M. Schmitt-Verhulst, and C. Boyer. 1994. and sustained ERK activation (Fig. 7). Evidence for protein tyrosine kinase involvement in ligand-induced TCR/CD3 Together, our data provide for the first time functional evidence internalization and surface redistribution. J. Immunol. 153:63. for the contribution of Lck to the negative regulation of T cell 10. Luton, F., V. Legendre, J. P. Gorvel, A. M. Schmitt-Verhulst, and C. Boyer. Downloaded from 1997. Tyrosine and serine activities associated with ligand-induced activation by SAg and offer biochemical evidence to explain the internalized TCR/CD3 complexes. J. 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Today 14:177. with Ag and APC correlates with a loss of TCR signaling and late 14. Xu, H., and D. R. Littman. 1993. A kinase-independent function of Lck in po- inactivation of Lck (8), suggesting that the negative regulatory role tentiating antigen-specific T cell activation. Cell 74:633. 15. Collins, T. L., and S. J. Burakoff. 1993. Tyrosine kinase activity of CD4-asso- of Lck may be active under these conditions of stimulation. The ciated p56lck may not be required for CD4-dependent T-cell activation. Proc. capacity of some SAg to dimerize may translate into oligomeriza- Natl. Acad. Sci. USA 90:11885. tion of the TCR to a degree similar to that seen with very potent 16. Racioppi, L., G. Matarese, U. D’Oro, M. De Pascale, A. M. Masci, S. Fontana, and S. Zappacosta. 1996. The role of CD4-Lck in T-cell receptor antagonism: Ag-induced stimulation, but in a unique way that facilitates the evidence for negative signaling. Proc. Natl. Acad. Sci. USA 93:10360. involvement of Fyn and plays down the need for Lck for the ini- 17. al-Ramadi, B. K., T. Nakamura, D. Leitenberg, and A. L. Bothwell. 1996. De- lck ficient expression of p56 in Th2 cells leads to partial TCR signaling and a by guest on September 27, 2021 tiation of TCR signaling (49). dysregulation in lymphokine mRNA levels. J. Immunol. 157:4751. The negative regulation of SEE-induced T cell activation by Lck 18. Yamada, H., Y. Y. Kong, K. Kishihara, T. W. Mak, and K. Nomoto. 1997. p56lck is probably coordinated with the other roles of Lck, i.e., initiation is not essential for the T-cell response to allo-MHC . Immunology 92:33. 19. Yamasaki, S., M. Tachibana, N. Shinohara, and M. Iwashima. 1997. Lck-inde- of signaling from nascent immunological synapses, stabilization of pendent triggering of T-cell antigen receptor signal transduction by staphylococ- T:APC conjugates through integrin affinity maturation, and con- cal enterotoxins. J. Biol. 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