Metalloproteases regulate T-cell proliferation and effector function via LAG-3
2007; Springer Nature; Volume: 26; Issue: 2 Linguagem: Inglês
10.1038/sj.emboj.7601520
ISSN1460-2075
AutoresNianyu Li, Yao Wang, Karen Forbes, Kate M. Vignali, Bret S.E. Heale, Paul Säftig, Dieter Hartmann, Roy A. Black, John J. Rossi, Carl Blobel, Peter J. Dempsey, Creg J. Workman, Dario A.A. Vignali,
Tópico(s)Cancer Immunotherapy and Biomarkers
ResumoArticle24 January 2007free access Metalloproteases regulate T-cell proliferation and effector function via LAG-3 Nianyu Li Nianyu Li Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USAPresent Address: Department of Investigative Toxicology, Amgen Inc., 1201 Amgen Court West, Seattle, WA 98119, USA Search for more papers by this author Yao Wang Yao Wang Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Karen Forbes Karen Forbes Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Kate M Vignali Kate M Vignali Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Bret S Heale Bret S Heale Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, CA, USAPresent Address: MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, Scotland Search for more papers by this author Paul Saftig Paul Saftig The Biochemical Institute, Christian-Albrechts University, Kiel, Germany Search for more papers by this author Dieter Hartmann Dieter Hartmann Department for Human Genetics, KU Leuven and Flanders Interuniversity Institute for Biotechnology (VIB4), Leuven, Belgium Search for more papers by this author Roy A Black Roy A Black Department of Inflammation, Amgen Inc., Seattle, WA, USA Search for more papers by this author John J Rossi John J Rossi Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, CA, USA Search for more papers by this author Carl P Blobel Carl P Blobel Arthritis and Tissue Degeneration Program, Hospital for Special Surgery at Weill Medical College of Cornell University, New York, NY, USA Search for more papers by this author Peter J Dempsey Peter J Dempsey Pacific Northwest Research Institute, Seattle, WA, USA Department of Medicine, University of Washington, Seattle, WA, USAPresent Address: Departments of Pediatrics and Molecular and Integrative Physiology, University of Michigan, 1150 W. Medical Ctr Drive, Ann Arbor, MI 48109-0656, USA Search for more papers by this author Creg J Workman Creg J Workman Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Dario A A Vignali Corresponding Author Dario A A Vignali Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Nianyu Li Nianyu Li Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USAPresent Address: Department of Investigative Toxicology, Amgen Inc., 1201 Amgen Court West, Seattle, WA 98119, USA Search for more papers by this author Yao Wang Yao Wang Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Karen Forbes Karen Forbes Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Kate M Vignali Kate M Vignali Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Bret S Heale Bret S Heale Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, CA, USAPresent Address: MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, Scotland Search for more papers by this author Paul Saftig Paul Saftig The Biochemical Institute, Christian-Albrechts University, Kiel, Germany Search for more papers by this author Dieter Hartmann Dieter Hartmann Department for Human Genetics, KU Leuven and Flanders Interuniversity Institute for Biotechnology (VIB4), Leuven, Belgium Search for more papers by this author Roy A Black Roy A Black Department of Inflammation, Amgen Inc., Seattle, WA, USA Search for more papers by this author John J Rossi John J Rossi Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, CA, USA Search for more papers by this author Carl P Blobel Carl P Blobel Arthritis and Tissue Degeneration Program, Hospital for Special Surgery at Weill Medical College of Cornell University, New York, NY, USA Search for more papers by this author Peter J Dempsey Peter J Dempsey Pacific Northwest Research Institute, Seattle, WA, USA Department of Medicine, University of Washington, Seattle, WA, USAPresent Address: Departments of Pediatrics and Molecular and Integrative Physiology, University of Michigan, 1150 W. Medical Ctr Drive, Ann Arbor, MI 48109-0656, USA Search for more papers by this author Creg J Workman Creg J Workman Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Dario A A Vignali Corresponding Author Dario A A Vignali Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Author Information Nianyu Li1, Yao Wang1, Karen Forbes1, Kate M Vignali1, Bret S Heale2, Paul Saftig3, Dieter Hartmann4, Roy A Black5, John J Rossi2, Carl P Blobel6, Peter J Dempsey7,8, Creg J Workman1 and Dario A A Vignali 1 1Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USA 2Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, CA, USA 3The Biochemical Institute, Christian-Albrechts University, Kiel, Germany 4Department for Human Genetics, KU Leuven and Flanders Interuniversity Institute for Biotechnology (VIB4), Leuven, Belgium 5Department of Inflammation, Amgen Inc., Seattle, WA, USA 6Arthritis and Tissue Degeneration Program, Hospital for Special Surgery at Weill Medical College of Cornell University, New York, NY, USA 7Pacific Northwest Research Institute, Seattle, WA, USA 8Department of Medicine, University of Washington, Seattle, WA, USA *Corresponding author. Department of Immunology, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA. Tel.: +1 901 495 2332; Fax: +1 901 495 3107; E-mail: [email protected] The EMBO Journal (2007)26:494-504https://doi.org/10.1038/sj.emboj.7601520 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Tight control of T-cell proliferation and effector function is essential to ensure an effective but appropriate immune response. Here, we reveal that this is controlled by the metalloprotease-mediated cleavage of LAG-3, a negative regulatory protein expressed by all activated T cells. We show that LAG-3 cleavage is mediated by two transmembrane metalloproteases, ADAM10 and ADAM17, with the activity of both modulated by two distinct T-cell receptor (TCR) signaling-dependent mechanisms. ADAM10 mediates constitutive LAG-3 cleavage but increases ∼12-fold following T-cell activation, whereas LAG-3 shedding by ADAM17 is induced by TCR signaling in a PKCθ-dependent manner. LAG-3 must be cleaved from the cell surface to allow for normal T-cell activation as noncleavable LAG-3 mutants prevented proliferation and cytokine production. Lastly, ADAM10 knockdown reduced wild-type but not LAG-3−/− T-cell proliferation. These data demonstrate that LAG-3 must be cleaved to allow efficient T-cell proliferation and cytokine production and establish a novel paradigm in which T-cell expansion and function are regulated by metalloprotease cleavage with LAG-3 as its sole molecular target. Introduction Metalloproteases have long been considered viable therapeutic targets for a variety of important human diseases such as cancer, cardiovascular disease, arthritis and multiple sclerosis (Baker et al, 2002; Overall and Kleifeld, 2006). However, many of the clinical trials using broad-range metalloprotease inhibitors have produced disappointing results, in part owing to unexpected side effects. This is complicated by the broad range of molecules targeted by these metalloproteases. Matrix metalloproteases (MMP), membrane-tethered MMPs and the zinc-dependent a disintegrin and metalloproteinases (ADAM), have all been shown to shed proteins from the cell surface (Black and White, 1998; Becherer and Blobel, 2003; Seals and Courtneidge, 2003; Parks et al, 2004; Blobel, 2005). Among these, two members of the ADAM family of metalloproteases, ADAM10 (Kuzbanian) and ADAM17 (TACE), are known to be important cell surface sheddases for a diverse array of transmembrane proteins of immunological importance, such as Notch, EGFR ligands, TNF-α, TNF-α receptor, CD44, CD62L (L-selectin) and CD23 (Black and White, 1998; Becherer and Blobel, 2003; Blobel, 2005; Maretzky et al, 2005; Reiss et al, 2005; Weskamp et al, 2006). For some time, metalloprotease inhibitors have been known to inhibit T-cell proliferation but the target molecule and mechanism that is inhibited remain unknown. T-cell proliferation and function following antigenic stimulation is a tightly regulated process. Inappropriate or uncontrolled expansion of activated T cells is regulated by activation-induced cell death, downregulation of stimulatory molecules and/or upregulation of inhibitory molecules. Lymphocyte activation gene-3 (LAG-3; CD223) has recently been shown to be a novel inhibitory molecule that is required for maximal regulatory T-cell function, and controls effector T-cell expansion and homeostasis (Huang et al, 2004; Workman et al, 2004; Workman and Vignali, 2005). Importantly, these studies clearly show that LAG-3 has cell-intrinsic regulatory activity, but the physiological importance of this is unclear. LAG-3 is related to CD4 in chromosomal location, exon organization and structure (Triebel et al, 1990; Bruniquel et al, 1997). They also share the same ligand, MHC class II, although LAG-3 binds with a much higher affinity (Triebel et al, 1990; Bruniquel et al, 1998; Workman et al, 2002a, 2002b). We have shown that binding to MHC class II molecules and a conserved KIEELE motif in the LAG-3 cytoplasmic domain are essential for its function. LAG-3 clearly possesses both cell-intrinsic and cell-extrinsic regulatory activity (Huang et al, 2004; Workman and Vignali, 2005). Ectopic expression of LAG-3 on effector T cells controls their proliferation and cytokine production. It is noteworthy that all activated T cells and NK cells express LAG-3. It is unclear what effect the presence of this negative regulatory pressure might have on their ability to proliferate and function, and if there are any mechanisms present that modulate LAG-3 activity. We recently observed that LAG-3 is cleaved within the short connecting peptide (CP) between the membrane-proximal D4 domain and the transmembrane domain, resulting in the release of a soluble form of LAG-3 (sLAG-3) (Li et al, 2004). Indeed, significant amounts of sLAG-3 are found in murine sera (∼200 ng/ml), which increases following T-cell stimulation in vivo. LAG-3 is known to have inhibitory activity, yet is expressed by all activated T cells. In this study, we tested our hypothesis that there was a direct link between the ability of metalloproteases to regulate T-cell proliferation and effector function and the possibility that LAG-3 may be the target of metalloprotease activity. Results Metalloprotease inhibition reduced wild-type but not LAG-3−/− T-cell proliferation We first tested whether the metalloprotease inhibitor GM6001, which is stable in long-term cultures (Ethell et al, 2002), can reduce T-cell proliferation. As anticipated, GM6001 significantly reduced the peptide-induced proliferation of wild-type CD4+ OTII T-cell receptor (TCR)-transgenic T cells (Figure 1A). We have previously shown that LAG-3 is cleaved from the cell surface and speculated that this might be mediated by metalloproteases as they have been shown to cleave many cell surface proteins of immunological importance (Black and White, 1998; Becherer and Blobel, 2003; Seals and Courtneidge, 2003; Parks et al, 2004; Blobel, 2005). This was confirmed by our observation that GM6001 blocked the generation sLAG-3 in a dose-dependent manner (Figure 1B). We then asked if GM6001 affected the proliferation of LAG−/− T cells. To our surprise it did not, suggesting that the GM6001-mediated suppression of T-cell proliferation is LAG-3-dependent (Figure 1C and D). Taken together, these data demonstrate that the metalloprotease inhibitor GM6001 can reduce T-cell proliferation and that a candidate molecular target in mediating this effect is LAG-3. These data also raise the possibility that prevention of LAG-3 cleavage may inhibit T-cell proliferation. Figure 1.Blocking LAG-3 cleavage by metalloprotease inhibitor GM6001 inhibits T-cell activation. (A, C, D) LAG-3 wild-type OTII TCR-transgenic T cells (A) or LAG-3 knockout OTII TCR-transgenic T cells (C) were MACS purified and activated with OVA326−339 in the presence of irradiated splenocytes for 3 days. Cultures were pulsed with [3H]thymidine for the last 8 h. Data are representative of three independent experiments. The reduced [3H]thymidine incorporation seen with LAG−/− T cells is due to increased activation-induced cell death that often occurs following in vitro stimulation (Workman and Vignali, 2003). (D) Percentage of inhibition on T-cell proliferation by GM6001 treatment was calculated from three individual experiments. (B) Whole splenocytes from OTII TCR transgenic mice were activated with OVA326−339 in the presence of various concentrations of GM6001 (GM) for 3 days. All cells and supernatants were collected and tested as indicated. Download figure Download PowerPoint ADAM10 mediates constitutive LAG-3 cleavage To ensure that LAG-3 cleavage was restricted to metalloproteases, we first tested the effect of various protease inhibitors on sLAG-3 production by a LAG-3+ CHO transductant. These included an alternate metalloprotease inhibitor (TAPI-1) and inhibitors of serine proteases (Pefabloc and leupeptin), aspartyl proteases (pepstatin), cysteine proteases (E64), gamma-secretases (DAPT) and proteasome proteases (MG-132 and LLNL). Of these, the only inhibitor that blocked sLAG-3 production was the metalloprotease inhibitor TAPI-1, consistent with our previous results (Figure 2A). Figure 2.ADAM10 and ADAM17 cleave LAG-3. (A) LAG-3+-transduced CHO cells were cultured in medium for 1 h±various protease inhibitors (0.5 mM Pefabloc, 100 μM TAPI, 10 μM leupeptin, 10 μM pepstatin A, 10 μM E-64, 25 μM MG-132, 1 μM DAPT or 25 μM LLNL) as indicated in the figure. Supernatants were collected and cells were lysed. Both were immunoprecipitated with the anti-LAG-3 mAb and eluted proteins were separated by SDS–PAGE and blotted with anti-LAG-3.D1 antibody. (B, C, E, F) All cells (3A9T cell hybridomas (B), ADAM10−/+ or ADAM10−/− MEFs (C), ADAM17+/+ or ADAM17ΔZnΔ/Zn fibroblasts (E), CHO and CHO.M1 cells (F)) were transduced with LAG-3 retrovirus. Cells were cultured in medium with DMSO (control), 100 μM TAPI, 1 μM PMA or TAPI and PMA for 0.5 h (E) or 1 h (C). Supernatants and lysates were tested as above. (B–F) The concentration of sLAG-3 in cell culture medium was also assessed by ELISA. LAG-3 concentration was calculated using a standard curve generated with affinity-purified sLAG-3. Data are presented as the mean±s.e. of three separate experiments with P-values shown. (D) ADAM10−/− MEFs were cotransfected with LAG-3 in pMIC (LAG-3) and an IRES-GFP vector pIRES (Vec), a dominant negative bADAM10 cDNA in pIREScg (bADAM10E−A) or wild-type bADAM10 in pIREScg. CFP and GFP double-positive cells were sorted and cultured for another 2 days. Cells (1 × 106) were cultured in 12-well plates. Supernatant and lysate were tested as above. ADAM10 expression was confirmed by blotting whole-cell lysate with anti-ADAM10. The concentration of sLAG-3 in cell culture medium was measured by ELISA. Data are the mean±s.e. of three independent experiments. Download figure Download PowerPoint We focused our attention on the ADAM family of metalloproteases, ADAM10 (Kuzbanian) and ADAM17 (TACE), as they are known to cleave many transmembrane proteins of immunological importance (Black and White, 1998; Becherer and Blobel, 2003; Blobel, 2005). Although the proteolytic activity of ADAM10 is generally constitutive, cleavage by ADAM17 can be induced by PMA (Sahin et al, 2004). Therefore, we first tested whether sLAG-3 production by the LAG-3+ 3A9T cell hybridoma was altered by PMA treatment. Production of sLAG-3 was significantly increased by 1 h PMA treatment (Figure 2B), implicating a role for ADAMs in LAG-3 shedding. This increase was not due to enhanced LAG-3 synthesis, as the total protein in whole-cell lysates was unchanged. It should be noted that sLAG-3 can be generated by multiple transduced or transfected cell types including T cells, CHO and 3T3 cells (Li et al, 2004), suggesting that the sheddase is ubiquitously expressed. Furthermore, shedding does not require LAG-3 ligation, cellular activation or the presence of serum-derived proteases or cofactors (Li et al, 2004) (Supplementary Figure S1A and B). Initial analysis of serum sLAG-3 concentration in mice lacking ADAM9, 12, 15 and/or 17 suggested that these were not responsible for constitutive LAG-3 cleavage (Supplementary Figure S1C and D). As ADAM10 deficiency results in embryonic lethality (Hartmann et al, 2002), we assessed its role in LAG-3 cleavage using ADAM10−/− and ADAM10+/− MEFs transduced with LAG-3 encoding retrovirus. Strikingly, there was a 90% reduction in sLAG-3 production by ADAM10−/− MEFs compared with heterozygous control cells (Figure 2C). However, when the LAG-3+ ADAM10−/− MEFs were treated with PMA, sLAG-3 was still generated, suggesting that a different protease was responsible for PMA-induced LAG-3 cleavage. It is noteworthy that this increase was comparable to that seen following PMA induction of the ADAM10+/− control MEFs (increase in sLAG-3 production in the presence of PMA over control untreated cells: ADAM10+/−=0.81 μg/ml, ADAM10−/−=0.80 μg/ml) (Figure 2C). To confirm that ADAM10 was responsible for constitutive LAG-3 cleavage, LAG-3.pMIC was cotransfected into ADAM10−/− MEFs with either bovine ADAM10 (bADAM10) in the GFP containing plasmid pIRES, an enzymatically inactive mutant (bADAM10E−A) or the empty vector control (Vec). Analysis of sLAG-3 production by CFP+/GFP+ MEFs demonstrated that LAG-3 cleavage was restored in the presence of active but not inactive bADAM10 (Figure 2D). In addition, surface LAG-3 expression was drastically reduced in bADAM10-expressing MEFs (Supplementary Figure S1E). Taken together, our data clearly show that ADAM10 is responsible for constitutive LAG-3 cleavage. ADAM17 mediates PMA-induced LAG-3 cleavage We established that ADAM17 was the PMA-inducible LAG-3 sheddase with two experiments. First, ADAM17ΔZn/ΔZn and wild-type Ras/Myc-transformed fibroblasts (Reddy et al, 2000) were transduced with retrovirus encoding LAG-3. sLAG-3 production by transduced ADAM17ΔZn/ΔZn fibroblasts was not increased after PMA treatment compared with the wild-type fibroblasts (Figure 2E). Second, we expressed LAG-3 on the ADAM17-deficient CHO-M1 cell line (Li and Fan, 2004; Villanueva de la et al, 2004) and assessed sLAG-3 production following PMA treatment. An ∼2.5-fold increase in sLAG-3 production by ELISA was seen with the wild-type CHO LAG-3 transfectant, whereas PMA treatment had no effect on the sLAG-3 production by the LAG-3+ CHO-M1 cell line (Figure 2F). In both experiments, constitutive LAG-3 cleavage was unaffected. Taken together, these data demonstrate that ADAM17 is responsible for the PMA-induced cleavage of LAG-3. Furthermore, our data show that there are two distinct metalloproteases (ADAM10 and ADAM17) that independently cleave LAG-3. TCR signaling increases LAG-3 cleavage via two distinct pathways Our observation that PMA treatment induced LAG-3 cleavage by ADAM17 suggested that this process might be controlled by a protein kinase C (PKC)-dependent signaling pathway. As PKC had been shown to play important roles in T-cell activation, we questioned whether ADAM17-mediated LAG-3 cleavage was regulated by the TCR signaling pathway. MACS-purified CD4+ OTII T cells were activated with OVA326−339 for 2 days. LAG-3 expression was confirmed by flow cytometry and sLAG-3 production was verified by Western blot. As shown above, constitutive sLAG-3 shedding was again increased by PMA treatment and inhibited by TAPI-1 (Figure 3A). Interestingly, TCR crosslinking by anti-CD3ε antibody (2C11) also stimulated T cells to produce more sLAG-3, which could be inhibited by TAPI-1 and the tyrosine kinase inhibitor genistein. This increase was not due to induction of LAG-3 synthesis, as the total protein in whole-cell lysates was unchanged. Figure 3.TCR signaling induces ADAM17-mediated LAG-3 cleavage. (A) OTII TCR-transgenic T cells were MACS purified and activated with OVA326−339 in the presence of irradiated LAG-3−/− splenocytes for 3 days. Activated T cells were then treated as indicated (as above plus 2 μg/ml anti-CD3 (2C11), anti-CD3 and TAPI, or anti-CD3 and 50 μg/ml Genistein) for 1 h. (B) Splenocytes from ADAM17ΔZn/ΔZn or ADAM17+/+ littermates were stimulated with 10 μg/ml of SEB for 4 days. Activated splenocytes were then treated and tested as indicated and described above. (C) OTII TCR-transgenic T cells were MACS purified and activated with OVA326−339 in the presence of irradiated LAG-3−/− splenocytes for 3 days. Activated T cells were then treated as indicated (‘Rot’ as Rottlerin) for 1 h. (D) Splenocytes from PKCθ+/+ or PKCθ−/− mice were stimulated with 0.25 ng/ml of PMA for 1 day. Activated splenocytes were then treated and tested as indicated and described above. (E, F) CD4+ T cells from OTII TCR transgenic mice were activated with OVA326−339 for 0, 24 or 48 h. ADAM17 and ADAM10 expression was detected by real-time PCR (E) or Western blot (F). Download figure Download PowerPoint We then asked if TCR-induced LAG-3 cleavage was absent in ADAM17-deficient T cells. As expected, low-level constitutive sLAG-3 shedding was seen with unstimulated wild-type and mutant T cells, whereas sLAG-3 production was increased following PMA and anti-CD3 stimulation of wild-type T cells (Figure 3B). However, CD3 crosslinking induced minimal sLAG-3 production by ADAM17ΔZn/ΔZn T cells. The same observation was also made with T cells from Rag-1−/− mice reconstituted with bone marrow from ADAM17ΔZn/ΔZn mice, eliminating the possibility that this phenotype was due to the development of T cells in an ADAM17-deficient environment (Supplementary Figure S2A). Although we cannot rule out the possibility that the absence of ADAM17 has affected T-cell responsiveness in general and/or the function of ADAM10, these data do suggest that ADAM17 is responsible, at least in part, for the TCR-induced cleavage of LAG-3. To determine if PKC was required for CD3-induced sLAG-3 production, we stimulated LAG-3+ T cells with anti-CD3 in the presence of rottlerin, a broad-spectrum PKC inhibitor. LAG-3 cleavage was effectively blocked by rottlerin, even at 5 μM (Figure 3C). PKCθ and PKCδ are particularly sensitive to rottlerin, having an ID50 of 5–30 μM (Gschwendt et al, 1993; Villalba et al, 1999). PKCθ is known to be activated by p56lck and recruited to the immunological synapse following TCR ligation (Arendt et al, 2002; Isakov and Altman, 2002). Furthermore, PKCθ−/− T cells are poorly responsive to TCR ligation but respond normally to PMA (Pfeifhofer et al, 2003). Thus, we asked if PKCθ was required for CD3-induced LAG-3 cleavage. While PMA-induced LAG-3 cleavage was intact in PKCθ−/− T cells, CD3 ligation failed to increase LAG-3 shedding (Figure 3D). The simplest explanation for these data is that PKCθ participates directly by phosphorylating ADAM17 and inducing its activation. However, we cannot exclude the possibility that TCR signaling induces ADAM17 activity via a different pathway that indirectly utilizes PKCθ. Although these data suggest that anti-CD3-induced LAG-3 cleavage is mediated by ADAM17 in a PKCθ-dependent manner, it is also possible that TCR signaling (via PKCθ) increases ADAM17 and/or ADAM10 expression. To assess this, we determined the amount of ADAM10/17 mRNA in resting and activated T cells by real-time PCR. There was essentially no change in ADAM17 mRNA, whereas there was a significant increase in ADAM10 mRNA (∼12-fold) 24 h post T-cell activation (Figure 3E). A substantial increase in pro-ADAM10 and ADAM10 protein was also seen 24 and 48 h post T-cell activation (Figure 3F and Supplementary Figure S2B). It is noteworthy that LAG-3 expression is detectable by flow cytometry in activated T cells and transduced cells lines, suggesting that ADAM expression and activity are limiting. This suggests that changes in ADAM10 expression may significantly affect LAG-3 cleavage. Indeed, this is evident from our MEF overexpression experiments in which high ADAM10 expression by transfection led to the complete cleavage of LAG-3 (Figure 2D). Taken together, these data suggest that there are two pathways by which TCR signaling can induce LAG-3 cleavage: PKCθ-dependent activation of ADAM17 and increased production of constitutively active ADAM10. What is the physiological function of LAG-3 cleavage? The physiological function of LAG-3 cleavage is unknown. First, sLAG-3 may have a suppressive ‘cytokine-like’ function that blocks CD4 and/or TCR interaction with MHC class II molecules, or compete with membrane-bound LAG-3 to limit its function as a consequence of its high affinity for MHC class II molecules (Huard et al, 1997). Second, it may serve to terminate LAG-3 signaling and thus provide a mechanism for the rapid cessation of LAG-3 regulatory function. Third, ectodomain shedding may be a prerequisite for initiating further intramembrane cleavage (commonly referred to as RIPping) in a manner similar to that required for Notch signaling (McDermott et al, 1999; Brou et al, 2000; Yan et al, 2002; Sahin et al, 2004). We first asked if sLAG-3 could alter T-cell proliferation. Initial in vitro studies clearly showed that LAG-3+ and LAG-3− T cells were unaffected by the addition of purified sLAG-3 (Supplementary Figures S3A and B). Given that mouse serum contains significant amounts of sLAG-3 (Li et al, 2004), we asked whether this endogenous protein could influence T-cell expansion in vivo. CFSE-labeled CD4+ splenic T cells from LAG-3+/+ and LAG-3−/− Thy1.1+ OTII TCR transgenic mice were adoptively transferred into Thy1.2+ LAG-3−/− or wild-type C57BL/6 mice, stimulated with OVA326–339 and cell division was analyzed 6 days later. While a clear difference in the extent of cell division was seen between the LAG-3−/− and LAG-3+/+ OTII T cells, whereas the presence of sLAG-3 in the serum of recipient mice had no effect on the antigen-induced division of either T-cell population (Supplementary Figure S3C and D). It is possible that the local sLAG-3 concentration in the microenvironment of T-cell activation and/or in the presence of LAG-3+ T cells might be much higher than the serum sLAG-3 concentration. To address this possibility, we generated mice expressing ∼1000-fold higher levels of sLAG-3 than normal serum concentrations by retroviral-mediated stem cell gene transfer (Supplementary Figure S3E) (Workman and Vignali, 2003). These mice then served as recipients for purified, CFSE-labeled, LAG+/+ or LAG−/− Thy1.2+ CD4+ OTII T cells and were treated as above. Despite the presence of substantial quantities of sLAG-3, T-cell proliferation was surprisingly unaffected, and the ability of membrane-associated LAG-3 to control this expansion was also unperturbed (Supplementary Figure S3F and G). Taken together, these results suggest that sLAG-3 has no effect on antigen-driven T-cell activation and proliferation in vitro or in vivo, and does not serve to limit or control LAG-3 function. Prevention of LAG-3 cleavage blocks T-cell proliferation and cytokine production We reasoned that if LAG-3 cleavage was required to attenuate its negative regulatory function, a noncleavable version of LAG-3 would be predicted to have enhanced regulatory activity. In contrast, reduced regulatory activity would be observed if cleavage was required to release a ‘functional’ sLAG-3 molecule or if LAG-3 RIPping initiated signaling. We had previously shown that LAG-3 cleavage occurs within membrane-proximal CP (Li et al, 2004). To generate noncleavable LAG-3 mutants for functional analysis, we first analyzed the influence of CP length and amino-acid composition on LAG-3 shedding. A series of LAG-3 CP mutants were expressed in a LAG-3/CD4 double loss variant 3A9 T-cell hybridoma by retroviral transduction (Supplementary Figure S4). Constitutive shedding by unstimulated cells was assessed by detection of sLAG-3 using Western blot and ELISA. Analysis of these mutants demonstrated that cleavage requires a long CP (>8 amino acids) and that the protease(s) that mediates this shedding are relatively promiscuous, as indicated by some tolerance for amino-acid substitutions within the CP (Supplementary Figure S4). Two noncleavable LAG-3 mutants were chosen for functional analysis: LAG-3mCD4CP in which the 20–amino-acid LAG-3 CP has been replaced with the eight-amino-acid CD4 CP, and LAG-3ESCP which has a 12-amino-acid deletion of the LAG-3 CP (Supplementary Figure S4A) (Li et al, 2004). Splenic LAG-3−/− CD4+ OTII T cells were transduced with vector alone (pMIC), wild-type LAG-3 or LAG-3mCD4CP or LAG-3ESCP-encoding retrovirus. Physiological levels of LAG-3 expression, that were comparable to that seen o
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