Tim4 inhibition of Tcell activation and T helper ... - Wiley Online Library

IMMUNOLOGY

ORIGINAL ARTICLE

Tim-4 inhibition of T-cell activation and T helper type 17 differentiation requires both the immunoglobulin V and mucin domains and occurs via the mitogen-activated protein kinase pathway

Wei Cao,1 Michelle Ryan,2 Deirdre Buckley,2 Rosemary O’Connor2 and Michael R. Clarkson3 1

Immunology, Moffitt Cancer Center, Department of Biochemistry, and 3 Department of Renal Medicine, Cork University Hospital, University College Cork, Ireland 2

doi:10.1111/j.1365-2567.2011.03424.x Received 24 June 2010; revised 30 January 2011; accepted 16 February 2011. Correspondence: Dr M. R. Clarkson, Department of Renal Medicine, Cork University Hospital, University College Cork, Ireland. Email: [email protected] Senior author: Wei Cao, email: [email protected]

Summary Emerging experimental data suggest an important role for the T-cell immunoglobulin mucin 1 (Tim-1):Tim-4 pathway in autoimmune and alloimmune responses in vivo. Using a Tim-4 ectodomain human IgG Fc fusion protein we studied the role of Tim-4 in T-cell activation, signalling and differentiation responses in vitro. We demonstrate that Tim-4Fc can inhibit naive and pre-activated T-cell activation, proliferation and cytokine secretion via a Tim-1-independent pathway. Tim-4 contains immunoglobulin variable (IgV) and mucin domains; to identify which domain accounts for the inhibitory effect novel Tim-4 fusion proteins containing either the IgV or mucin domain were generated. We demonstrate that both IgV and mucin domains are required for the inhibitory effects and that they are mediated at least in part by inhibition of extracellular signal-regulated kinase pathway activity. Given the emerging interest in the role of the Tim family in T helper type 17 (Th17) cells, which play an important role in autoimmune disease and transplantation tolerance, our data show that Tim-4Fc can prevent polarization of CD4+ T cells to the Th17 phenotype. Collectively, our results highlight an inhibitory role for Tim-4Fc in vitro, which we propose is mediated by a receptor other than Tim-1. In addition, this study provides new insights into the role of Tim4Fc in regulating Th17 immune responses and may open a new avenue for autoimmune therapy. Keywords: alloimmune response; immune inhibition; T helper type 17; T-cell immunoglobulin mucin-4

Introduction T-cell immunoglobulin and mucin domain proteins (Tim) regulate T-cell activation and tolerance. The mouse gene family includes eight members (Tim-1–8) and the human gene family includes three members (Tim-1, -3 and -4). All Tim family protein members are type I cell surface glycoproteins and share common structural motifs including an immunoglobulin variable (IgV) domain with six cysteine residues, a mucin-like domain, a transmembrane domain and a cytoplasmic domain. All Tims except for Tim-4 contain a tyrosine-kinase phosphorylation motif in the cytoplasmic domain. Tim-4 is highly

expressed on Mac1+ cells in lymph nodes, dendritic cells and liver1 and Tim-4 message has been detected in activated human T cells (both CD4 and CD8 subsets).2 To date, two Tim-4 receptors have been identified: Tim-13 and phosphatidylserine (PtdSer).1 Exposure of PtdSer on the plasma membrane is a key signal for recognition of apoptotic cells by macrophages.4,5 Binding assays show specific binding of PtdSer to Tim-4.6 In addition, Tim monoclonal antibodies (mAbs) that blocked binding to PtdSer substantially reduced phagocytosis of apoptotic cells by mouse peritoneal macrophages.6 More recently, it has been proposed that although Tim-4 is a PtdSer tethering protein, it does not

2011 The Authors. Immunology 2011 Blackwell Publishing Ltd, Immunology, 133, 179–189

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W. Cao et al. mediate any direct signalling of its own.7 The mechanism (if any) by which Tim-4 transduces signals downstream of PtdSer recognition remains to be elucidated. It is possible that the extracellular region of Tim-4 might interact with other transmembrane protein(s) to transmit its signals into the cytoplasm. Tim-1 is another functional receptor for Tim-4 and was found to be transcribed during primary antigen stimulation in CD4+ T cells,8 a period of time that is crucial in influencing T helper type 1 (Th1) –Th2 orientation. Tim-1 is therefore suggested to act as a co-stimulatory molecule for T cells, but with potentially stronger effects on Th2 than Th1 cells. The published data suggest that Tim-4 exerts a bimodal regulation depending on the activation status of the T cell; inhibitory to naive T cells but stimulatory to pre-activated T cells (expressing Tim-1).9 Understanding the role of the Tim-1 pathway is further complicated by published data showing two monoclonal anti-Tim-1 antibodies that differentially regulate antigenspecific T cells depending on the affinity with which Tim1 is cross-linked.10 The importance of Tim proteins in Th17 cell biology is a relatively unexplored area. Recent evidence suggests that interleukin-17 (IL-17) -producing Th17 cells play a crucial role in the pathogenesis of autoimmune inflammation.11,12 Tim-1 and Tim-3 in particular have been linked to Th17 cells.13–15 In the absence of Th1 immunity, CD8 Th17 alloreactivity constitutes a barrier to transplantation tolerance.15 Therefore, targeting Tim-1 provides an approach to overcome resistance to tolerance in clinical transplantation. With the goal of further elucidating the role of Tim-4 in T-cell activation and Th17 differentiation, we generated Tim-4Fc and two Tim-4 mutants, Tim-4-IgV-Fc and Tim-4-mucin-Fc. In contrast to previously published data we provide direct evidence for an inhibitory role for Tim4-Fc. We demonstrate an inhibitory response in naive CD4+ T cells, pre-activated T cells and T-cell blasts. Furthermore, for the first time we demonstrate negation of Th17 polarization by Tim-4Fc. The effects of Tim-4Fc in our model system appear to be mediated via the mitogen-activated protein (MAP) kinase extracellular signalregulated kinase (ERK) pathway. Furthermore, using a blocking anti-Tim-1 antibody our data show that Tim-4 can mediate effects independently of Tim-1.

Materials and methods Mice BALB/c mice (6–8 weeks) were purchased from Harlan (Wyton, UK). All mice were housed and cared for in the animal facility at the Biological Services Unit, University College Cork, according to the guidelines established by the Animal Care Committee. 180

Antibodies and reagents The Tim-1-specific antagonistic mAb RMT 1-10 was kindly provided by Dr M.H. Sayegh. Anti-mouse CD3 mAb (145-2C11), functional anti-mouse CD28 mAb (37.51) and FITC-labelled anti-mouse CD4 mAb were obtained from eBiosciences (San Diego, CA). Phycoerythrin-labelled anti-mouse Tim-1 mAb or anti-human IgG (Fc-specific) -FITC were purchased from BioLegend (San Diego, CA) and Sigma (St Louis, MO), respectively. For Western blotting, anti-ERK 1/2 (monoclonal) and antiphospho-ERK 1/2 (polyclonal Thr202/Tyr204) were purchased from Cell Signaling Technology (Danvers, MA). Antibodies to total Akt (clone) and phospho-Akt (Ser473) and a-tubulin (Sigma) were also purchased from Cell Signaling Technology. All common chemicals and cell culture reagents were purchased from Sigma Chemicals (St Louis, MO) and were the best analytical grade except for transforming growth factor (TGF-b) which was purchased from Chemicon (Millipore, Billerica, MA).

Cell culture Peripheral blood mononuclear cells were isolated from lymph nodes (inguinal, axillary and cervical) and spleen following red blood cell lysis with Lysis Buffer (Lonza, Walkersville, MD). The CD4+ T cells were isolated using a negative selection isolation kit (Miltenyi Biotec, Auburn, CA). The purity of the T-cell population was determined by FACS. CD4+ T cells were maintained in RPMI-1640 medium (Sigma Chemicals) with 10% fetal calf serum, penicillin (100 lg/ml), streptomycin (100 U/ml), 2 mM HEPES and b-mercaptoethanol. T-cell blasts were maintained in the same culture medium with concanavalin A and IL-2 (both Sigma). For Th17 polarization cells, CD4+ T cells were isolated as previously described and incubated with complete medium containing anti-interferon-c (10 lg/ml), anti-IL-4 (10 lg/ml), IL-6 (20 ng/ml) and TGF-b (5 ng/ml) for 3 days and IL-23 (10 ng/ml) for 24 hr (added on day 4).

Polymerase chain reaction Tim-4Ig expression vector was kindly provided by Dr Shigekazu Nagata, while Tim-4-IgV-Ig (amino acids 24–135) and Tim-4-mucin-Ig (amino acids 136–281) expression vectors were constructed separately. The sequences of the oligonucleotide primers used to amplify the desired sequences by PCR are available upon request. In brief, human IgG1-Fc DNA fragment was cloned from human peripheral blood mononuclear cells and then cloned into pcDNA 3 vector. Tim-4 IgV or Tim-4 mucin domain was inserted into the pcDNA 3 containing human IgG1 Fc fragment at the N terminus. Positive clones were confirmed by DNA sequencing.


Inhibitory effect of Tim-4 to make two novel fusion proteins expressing only the IgV domain or mucin domains, respectively. The Fcfusion proteins were then purified by protein G agarose bead separation. Under reducing SDS–PAGE both fusion proteins ran at approximately 55 000 molecular weight (Fig. 3a–c).

Production of Fc-fusion proteins

CFSE proliferation assays Purified naive CD4+ T cells were labelled with 5 lM carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes, Carlsbad, CA) and then stimulated with platebound anti-CD3 (1 lg/ml) and/or CD28 mAbs (5 lg/ml) for 72 hr. In some experiments, the cells were also treated with Tim-4Fc, anti-RMT 1-10, Tim-4-IgV-Fc or Tim-4200

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Fusion proteins of Tim-4 with the human immunoglobulin Fc region were produced essentially as described elsewhere.16 In brief, human 293T cells were transfected with the resulting expression vectors by the calcium-phosphate co-precipitation method, and cultured for another 2 days in Dulbecco’s modified Eagle’s medium without serum. The fusion proteins secreted into the culture supernatant were purified by chromatography on Protein G beadmediated affinity purification. The human IgG1-tagged protein molecular weight and purity were confirmed by 8% SDS–PAGE. To determine which part of the Tim-4Fc protein was responsible for inhibition of T-cell activation we decided

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Figure 1. Tim-4Fc binds to naive CD4+ T cells independent of Tim-1. (a, b) Purified CD4+ T cells were stained with FITC-labelled anti-CD4 monoclonal antibody (mAb) and the corresponding control antibody. (c, d) The same cells were stained with phycoerythrin-labelled anti-mouse Tim-1 mAb and the isotype control antibody. (e, f) The cell population was also stained with Tim-4Fc or mock-Fc control and FITC-labelled anti-human IgG1 Fc fragment antibody.


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Figure 2. Inhibition of CD4+ T-cell activation by Tim-4Fc. (a) Unlabelled naive CD4+ T cells. (b) Naive CD4+ T cells were labelled with 5 lm CFSE and analysed after 72 hr. (c) Proliferation of anti-CD3 plus anti-CD28 stimulated cells is manifested by a series of peaks with diminished fluorescence represented cellular division cycles. (d) Cell proliferation is not inhibited by soluble (S) Tim-4Fc. (e) Cell proliferation is severely disrupted by plate-bound Tim-4-Fc. (f) The Tim-1 antagonistic antibody RMT 1-10 does not abrogate the anti-proliferative effect. (g) Soluble RMT 1-10 has no effect on T-cell receptor stimulation by anti-CD3 and anti-CD28. (h) Plate-bound anti-RMT 1-10 has no effect on T-cell receptor stimulation by anti-CD3 and anti-CD28. (i) Plate-bound anti-RMT 1-10 has no effect on the cell cycle response in the presence of soluble Tim-4Fc. (j) ELISA showing interleukin-2 secretion by naive CD4+ T cells stimulated with anti-CD3 and anti-CD28 with and without Tim4Fc and RMT 1-10. *P = 00003; **P = 00001 compared with positive control.

mucin-Fc (75 lg/ml), separately. The cell division history of CFSE-labelled CD4+ T cells was analysed using a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA) and CELLQUEST software (BD Biosciences).

anti-mouse Tim-1 mAb or iso-type control, or Tim-4Fc/ mock-Fc and anti-human IgG1 (Fc specific)-FITC at 4. The excess probe was washed with phosphate-buffered saline, and the cell-associated fluorescence was analysed by CELLQUEST software.

Flow cytometry The expression profiles of Tim-1 or unknown Tim-4 receptor on CD4+ T cells were assayed by flow cytometry. In brief, cells were incubated with phycoerythrin-labelled 182

ELISA Purified naive CD4+ T cells were stimulated with platebound anti-CD3 (1 lg/ml) and/or CD28 mAbs (5 lg/ml).


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Figure 3. The generation of two novel fusion proteins, Tim-4-IgV-Ig and Tim-4-mucin-Fc. (a) Purified Tim-4-Fc was separated by SDS–PAGE under non-reducing (lane 1, far left panel) and reducing (lane 2, far left panel) and stained with Coomassie Brilliant Blue. Molecular weight markers are shown on the left of the panel. (b, c) Purified Tim-4-IgV-Fc and Tim-4-mucin-Fc are shown separated under reducing SDS–PAGE conditions. The molecular weight of both mutant proteins is 55 000. (d) A schematic for the construction of Tim-4-Fc, Tim-4-IgV-Fc and Tim4-mucin-Fc (taken from Miyanishi et al.1). (e) ELISA showing interleukin-2 secretion by stimulated T cells is inhibited in the presence of fulllength Tim-4FC and but not by either of the two Tim4 both mutants.*P = 0001 compared with positive control.

In some experiments, the cells were also treated with Tim-4Fc, RMT 1-10, Tim-4-IgV-Fc or Tim-4-mucin-Fc (75 lg/ml), separately. The supernatants were collected for IL-2 analysis 24 hr after stimulation. The concentration of IL-2 was detected using mouse IL-2 ELISA kits (R&D Systems, Minneapolis, MN). The supernatants for IL-17 analysis were collected 96 hr after stimulation and

IL-17 was detected using an IL-17A ELISA kit (eBiosciences).

Intracellular cytokine staining Cells (1 · 106) were re-suspended in complete medium and re-stimulated with PMA (10 ng/ml; Sigma) and


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W. Cao et al. ionomycin (500 ng/ml; Sigma). Brefeldin A (10 lg/ml; Sigma) was added. Cells were incubated for 4 hr at 37. After staining for the surface marker CD4, cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences), according to the manufacturer’s instructions, and incubated with phycoerythrin-conjugated IL-17 for 30 min at 4. A gate was set on CD4+ cells and % of IL-17 cells was determined by flow cytometry analysis.

T-cell activation assays The required number of wells of a 48-well plate were coated overnight with anti-CD3 (2 lg/ml) and anti-CD28 (5 lg/ml) ± Tim-4Fc (2 lg/ml) ± RMT 1-10 (75 lg/ml). The plate was washed twice with sterile Dubecco’s-PBS without Ca2+ or Mg2+ (Lonza). One million CD4+ T cells and T-cell blasts were added per well, the plate was spun for 1 min at 200 g and then incubated for 4 min at 37. Whole cell lysates were prepared by transferring the cells from each well to an eppendorf and boiling in 20 ll of SDS–PAGE loading buffer for 10 min, samples were stored at )20. For Th17 polarization cells, CD4+ T cells were isolated as previously described and incubated with complete medium containing IL-6 (20 ng/ml) and TGF-b (5 ng/ml) for 15, 30, 60 and 180 min, respectively followed by stimulation with anti-CD3 and anti-CD28.

Western blot analysis of Akt and MAPK (p44/p42) phosphorylation Whole cells lysates were prepared from T cells activated as described. Proteins were resolved by SDS–PAGE and transferred to Immobilon (Millipore, Bedford, MA) membrane. The blots were probed with antibodies specific to phosphorylated Akt (Ser473) or phosphorylated MAPK p44/p42 ERK 1/2 followed by secondary antibodies (LICOR). Immunoreactive bands were visualized with infrared fluorescence detection using a LICOR Odyssey detection system. Data for Akt and ERK 1/2 phosphorylation are representative of three experiments with similar results.

Results Tim-4Fc binds to an unknown inhibitory receptor on naive CD4+ T cells We used flow cytometry to investigate the specificity of Tim-4 binding to naive CD4+ T cells. Purified CD4+ T cells were isolated and labelled with FITC anti-CD4 to establish the purity of the isolated population (> 97%). A single CD4-positive peak was detected by flow cytometry denoting a T-cell population (Fig. 1b). This naive T-cell population did not express appreciable amounts of Tim-1 (Fig. 1d). We then incubated naive CD4+ T cells with 184

Tim-4Fc and FITC-labelled anti-human Fc secondary antibody (Fig. 1f). Binding of Tim-4Fc to the naive T cells was increased compared with mock Fc protein. (mean fluorescence intensity 2003 versus 306; representative of three separate experiments) (Fig. 1e,f). Collectively, these data suggest that in naive CD4+ T cells, Tim-4Fc binds to the cells although they do not express Tim-1. This suggests an alternative receptor and is in keeping with previously published data that show that Tim-1 is only expressed on pre-activated cells but not on naive T cells.9

Tim-4Fc inhibits CD4+ T-cell activation via a Tim-1independent pathway We evaluated the proliferation of naive CD4+ T cells in the presence of plate-bound Tim-4Fc and soluble Tim4Fc. Using CFSE proliferation assays we demonstrate prompt proliferative response to anti-CD3/CD28 stimulation (Fig. 2c). This proliferative response was abrogated by plate-bound, but not soluble Tim-4Fc (Fig. 2d,e). To investigate whether the anti-proliferative effect was a result of Tim-4Fc:Tim-1 interactions, we used the antiTim-1 blocking antibody, RMT 1-10, which has been shown to inhibit antigen-specific T-cell proliferation.10 Neither plate-bound nor soluble RMT 1-10 had any influence on T-cell proliferation nor did it prevent platebound Tim-4Fc-mediated inhibition of T-cell proliferative responses to anti-CD3/CD28 (Fig. 2f–h). We correlated the anti-proliferative effect of Tim-4Fc (as demonstrated by CFSE proliferation assay) with IL-2 secretion. By ELISA we found IL-2 secretion by naive CD4+ T cell in response to T-cell receptor (TCR) signalling to be significantly diminished in the presence of plate-bound Tim-4Fc after 24 hr incubation (P = 00003) (Fig. 2j). This result was more pronounced in the presence of plate-bound Tim-4Fc compared with soluble Tim-4Fc (Fig. 2j). The RMT 1-10 had no effect on Tim4Fc inhibition (P = 00001) (Fig. 2j).

The IgV and mucin domains of Tim-4 are required for inhibition of T-cell activation by Tim-4Fc To determine which domain of the Tim-4Fc protein was responsible for inhibition of T-cell activation (Fig. 1e,f), we constructed two fusion proteins expressing only the IgV domain or mucin domains, respectively. As expected, Tim-4Fc inhibited IL-2 secretion by stimulated CD4+ T cells (P = 0001), however, neither of the Tim-4-IgV or Tim-4-mucin proteins had any effect on IL-2 secretion (Fig. 3e). This suggests first that the inhibitory effect of Tim-4Fc requires both the extracellular IgV and mucin domains and second that the effects are not the result of non-specific binding of the Fc fragment and hence disruption of the TCR-complex.


Inhibitory effect of Tim-4 Tim-4Fc inhibits the expansion of Th17-differentiated CD4+ T-cells Having demonstrated an inhibitory role for Tim-4Fc in T-cell activation in naive CD4+ T cells, we next investigated the role of Tim-4Fc in the generation of Th17 cells. Th17 cells play an important function in immunoregulation, host defence and pathogenesis of autoimmune disease.17 However, the role of Tim proteins in Th17 immunobiology has yet to be delineated. We first isolated naive CD4+ T cells and polarized them to the Th17 phenotype over 5 days in vitro. Supernatants were collected from the cultures at 96 hr to verify that a Th17 phenotype had been established. Analysis of the supernatants by ELISA demonstrated IL-17 secretion to be inhibited in the presence of Tim-4Fc (Fig. 4b). In addition, the antiTim-1-treated cultures produced similar levels of IL-17 as seen in the control cultures (Fig. 4b). Intracellular cytokine staining was used to determine if Th17 polarization was skewed by plate-bound Tim-4Fc. We observed a 50% reduction in IL-17-secreting cells when cultured with 75 lg/ml Tim-4Fc for 5 days (Fig. 4a, P < 005, n = 3 experiments). However, comparable (with control populations) numbers of IL-17-secreting cells were present when cultured with RMT 1-10 antibody (1368% versus 1570%, P > 005). Intriguingly, there was no effect on the generation of Th1 cells when naive T cells were cultured under Th1 polarizing conditions in the presence of Tim-4Fc, suggesting a specific effect on the cellular pathways controlling Th17 generation (Fig. 4e). As Tim-4 inhibited the activation of naive CD4+ T cells, to observe the effect of Tim-4 on pre-differentiated Th17 cells, we pre-polarized Th17 cells for 5 days first and then treated with Tim-4Fc and re-polarized for another 5 days. Not surprisingly, the number of IL-17-secreting cells increased approximately twofold with an additional 5 days in vitro (Fig. 4c). As expected, the number of Th17-positive cells in control cultures and cultures treated with RMT 1-10 were comparable (Fig. 4c). However, interestingly, Tim4Fc still inhibited the further differentiation of pre-polarized Th17 cells (Fig. 4c). In accordance with intercellular staining data, analysis of IL-17 by ELISA in supernatants taken from re-polarized cultures showed significantly decreased cytokine levels in these cultures when they were cultured with plate-bound Tim-4Fc (P = 0035) (Fig. 4d). The data indicate that Tim 4-Fc prevents the differentiation of Th17 cells and blocks IL-17 production in Th17polarized cultures.

Tim-4Fc inhibits activation of the MAP kinase pathway It is well established that the MAP kinase pathway is crucial to T-cell proliferation and differentiation.18–20

Furthermore, the MAP kinase pathway is implicated in a number of autoimmune diseases such as rheumatoid arthritis.21 To date, Tim-4 has been shown to induce phosphorylation of Akt and ERK in CD3+ T cells supposedly via cross-linking with Tim-1.22 Similarly, inducible Akt phosphorylation has been shown in primary T-cell blasts in response to Tim-1.23 We focused on the MAP kinase pathway as a possible target for Tim-4 inhibition. We used the T-cell mitogen concanavalin A to generate T-cell blasts expressing Tim-1. The concanavalin A was then removed and cell cultures were grown in IL-2 for a further 24 hr. In addition, if cell numbers were insufficient after 5 days, cultures were allowed to proliferate for an additional time up to 2 weeks. Subsequently, all cultures were stimulated with plate-bound anti-CD3 and anti-CD28 with and without Tim-4Fc. We found consistent transient inhibition of ERK phosphorylation in the presence of Tim-4Fc at a 5-min time-point (Fig. 5a–c). Tim-4Fc on its own appeared to have no effect on ERK activation. We next added RMT 1-10 to our assay, this antibody did not interfere with T-cell activation in the presence of the TCR stimulatory signals (Fig. 5b,c). We previously demonstrated that Tim-4 acted independently of the Tim-1 pathway; therefore we subjected our T-cell blasts to a combination of Tim-4Fc and RMT 1-10. In keeping with the above data, Tim-4Fc inhibited ERK phosphorylation in these cultures, whereas RMT 1-10 failed to block the inhibitory effect of Tim-4. Taken together, the data suggest that Tim-4Fc can inhibit the MAP kinase pathway in T cells even when the cells have been pre-activated (expressing Tim-1) and that the inhibitory effect of Tim-4Fc must be occurring via some other as yet unknown receptor or through physical blockade of the TCR by Tim-4Fc.

Tim-4Fc has no effect on STAT3 activation We next asked if Tim4-Fc affects other downstream signalling responses, we focused specifically on signal transducer and activator of transcription 3 (STAT-3) because this transcription factor has emerged as a key regulator of the pro-inflammatory response during induction of several experimental autoimmune diseases.24,25 In addition, STAT3 is a requirement for IL-17 production by Th17 cells, IL-6 activates Jak and then induces STAT3phosphorylation.24 However, when CD4+ T cells were briefly polarized to the Th17 phenotype (for up to 3 hr) with TGF-b and IL-6, Tim-4Fc appeared to have no effect on STAT-3 phosphorylation (Fig. 6a,b). As IL-6 can activate both Erk and STAT3, it is possible that Tim4-Fc inhibits generation and proliferation of IL-17 through the Erk-TF alternative pathway independent of STAT3.


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Figure 4. Tim-4Fc inhibits CD4+ T-cell polarization to T helper type 17 (Th17) phenotype. (a) Intracellular cytokine staining for interleukin-17 (IL-17) in the presence of mock-Fc (control), Tim-4-Fc and RMT 1-10, 5 days after Th17 polarization. (b) ELISA analysis of IL-17 secretion from culture supernatant harvested after 96 hr. (c) Intracellular cytokine staining for IL-17 in cultures re-polarized for an additional 5 days after the initial 5-day polarization. (d) ELISA analysis of IL-17 secretion from re-polarized culture supernatant harvested after 96 hr re-polarization. *P = 0001; **P = 0035 compared with positive control. (e) Intracellular cytokine staining for interferon-c in cultures re-polarized for an additional 5 days after the initial 5-day polarization.

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Figure 5. Inhibition of extracellular signal regulated kinase (ERK) phosphorylation by Tim-4Fc in T-cell blasts. (a) Whole cell lysates were prepared from 5-day-old T-cell blasts that were stimulated with anti-CD3 (2 lg/ml) and anti-CD28 (5 lg/ml) plus Tim-4-Fc (3 lg/ml) for 0 and 5 min, respectively. Approximately 1 million cells were separated per well and blots were immunostained with antibodies to anti-ERK 1/2 and anti-phospho-ERK 1/2. Anti-tubulin acted as a loading control. (b) Five-day-old T blasts stimulated with anti-CD3 and anti-CD28 plus the Tim1 antagonistic antibody RMT 1-10 (75 lg/ml). (c) Eleven-day-old T-cell blast stimulated with anti-CD3 and anti-CD28 plus Tim-4Fc and RMT 1-10. Representative blots of four separate experiments.

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In this study we focused on Tim-4 and re-evaluated its effect on T-cell activation and further examined the role of Tim-4 in intracellular signalling. Our Tim-4 fusion protein was inhibitory in three different cell culture models including naive CD4+ T cells, T-cell blasts and Th17 cells. Furthermore, Tim-4 mediated the inhibitory response via a Tim-1-independent pathway in all three cell culture models tested. Proliferation of antibody-activated naive CD4+ T cells and their secretion of IL-2 were dramatically reduced in the presence of Tim-4 with intact ectodomain. The use of intracellular cytokine staining avoids any confounding effect that would occur as the result of a reduction in total cell number, as might occur with assays of soluble cytokine levels. Here we clearly enumerate the total number of cytokine-secreting cells and show a clear reduction in cytokine IL-17-secreting activated CD4 cells. Tim-4 with intact ectodomain fusion protein was a prerequisite for in vitro T-cell inhibition as neither the IgV nor mucin domain alone has an inhibitory effect. Similarly, the number of IL-17-secreting Th17-polarized naive CD4+ T cells was substantially reduced in the presence of Tim-4. In addition, the levels of IL-17 secreted by Th17-polarized cells were substantially reduced by Tim-4. To elucidate the biochemical mechanism underlying Tim-4 inhibition in vitro, we generated T-cell blasts from naive CD4+ T cells and used them for a signalling study. Our data demonstrated that Tim-4 inhibition occurs specifically via the MAP kinase pathway. Throughout our study Tim-4-Fc was inhibitory to T cells in vitro. We were unable to elicit a stimulatory

response with our Tim-4 fusion protein, which may be attributed to both the activation status of the T cell itself and/or the binding affinity of our Tim-4 protein. Indeed, as highlighted in other studies, the activation status of the target T cell is a crucial parameter in its response to Tim4 protein. For example, Mizui et al.,9 have shown that Tim-4 inhibits naive T cells but not pre-activated cells through a ligand other than Tim-1 (naive CD4+ T cells do not express Tim-1). As a comparison, our data demonstrated that Tim-4 strongly inhibited CD4+ T cells even

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Figure 6. Tim-4Fc has no effect on signal transducer and activator of transcription 3 (STAT3) activation. CD4+ T cells polarized to the T helper type 17 (Th17) phenotype (with transforming growth factor-b1 and interleukin-6) for the times indicated and stimulated with anti-CD3 plus anti-CD28+/) Tim-4Fc. Whole cell lysates separated by SDS–PAGE, transferred to nitrocellulose and immunoblotted with anti-phospho-STAT3.


187

W. Cao et al. these cells have been pre-activated by CD3/CD28 or concanavalin A. In keeping with our study, Mizui et al. could not demonstrate clear evidence for the stimulatory activity of Tim-4 on either naive or pre-activated T cells. We are not quite sure of the reason for this difference. An alternative explanation is that the inhibitory nature of our Tim-4 fusion protein may be partly the result of its molecular structure, for example, its glycosylation status in a different expression system, and hence its binding affinity for the unknown receptor. In addition, Meyers et al. found that a lower concentration of Tim-4 (1 lg/ ml) exerts an inhibitory effect while a higher concentration of Tim-4 (25–10 lg/ml) exerts a stimulatory effect on CD3+ T cells. Interestingly, even using higher concentrations, Tim-4 still inhibits the proliferation and IL-2 secretion of CD4+ T cells in our system (data not shown). The difference may be because CD3+ and CD4+ T cells may have different expression levels of the unknown inhibitory receptor. In addition, it should be noted that Tim-1 also inhibited the T-cell response in vitro and in vivo.2 Hence, it is reasonable to propose that Tim-1 and Tim-4 may share the same inhibitory receptor expressed on CD4+ T cells. It is also worth noting that the in vitro effects mediated by Tim-4 do not necessarily mirror Tim-4 behaviour in vivo.9 Although the bimodal nature of Tim-4 (enhancement or inhibition of proliferation of responding CD3+ T cells) depends upon the dose of Tim-4 used for in vitro proliferation assays, when given in vivo, Tim-4-Fc enhanced proliferation and cytokine production from responding CD3+ T cells. Similarly, Meyers et al.,3 have shown that Tim-4 administered in vivo causes high background proliferation and cytokine production in splenocytes. Collectively, these data support a bimodal function for Tim-4 in T-cell regulation that is dependent on the activation profile (related with the expression level of the unidentified inhibitory receptors) of the responding cells. The binding affinity of Tim-4 may also contribute to the response of the T cell as well. Heterogonous glycosylation on Tim-4 in a different expression system may account for the variant binding affinity. Tim-4 is a highly glycosylated protein with an average of 43 O-linked carbohydrates on its mucin domain in mice (compared with 38 in humans).8 In addition, human Tim-4 has two N-linked glycosylation sites which are not present in murine Tim-4. Rodriguez-Manzanet et al.,22 generated three anti-Tim-4 antibodies, which they designated 5G3, 3A1 and 3H11. All three anti-Tim-4 mAbs specifically bound Tim-4 as demonstrated by ELISA. In addition, the antibody 5G3 was used to determine the size of the O-deglycosylated Tim-4 protein from lysates of CHO-Tim-4 transfectants by immunoprecipitation. Under these conditions, Tim-4 was shown to have a molecular weight (MW) of approximately 70 000. However, immunoprecipitation of Tim-4 without deglycosylation resulted in 188

unique bands of approximately 100 000–85 000, 70 000 and 55 000 MW. In our study system, Tim-4 human IgG Fc fusion protein generated in 293T cell gives an approximately 65 000 MW single band, and the molecular weight under non-reducing conditions was 150 000 MW as a single band. Therefore, it is possible that in generating Tim-4-Fc molecules the number of carbohydrates may vary slightly, which in turn may influence Tim-4’s affinity for different receptors. We also sought to explore the intracellular signalling downstream (if any) of Tim-4 when it did engage with the unknown receptor(s). We focused on the MAP kinase pathway as a possible target for Tim-4 inhibition. Our study shows that Tim-4-Fc inhibits ERK phosphorylation in pre-activated CD4+ T cells irrespective of the stimulus (anti-CD3/CD28 or concanavalin A) and furthermore that inhibition is occurring via a non-Tim-1 pathway. Other studies have shown Tim-4 induction of ERK in T cells, however, CD3+ T cells were used and they were stimulated by Tim-4-Fc-coated beads.26 In contrast, Mizui et al.,9 demonstrated Tim-4 down-regulation of the ERK kinase pathway in naive T cells but not in activated T cells. In this instance, splenocytes were cultured with ovalbumin peptide as a stimulant or alternatively, naive CD4+ T cells were incubated with anti-CD3 and antiCD28 for 4 days. Once again, this highlights the importance of the activation status of the T cell, the stimulus employed to activate the T cell and the nature of the Tim-4-Fc used in the experiments. Recently, Tim-4-deficient mice have been generated to address Tim-4 function in vivo.26,27 Rodriguez-Manzanet et al.26 showed that Tim-4)/) peritoneal macrophages and B-1 cells do not clear apoptotic bodies in vivo. In addition, Tim-4-deficient-mice have hyperactive T and B cells, elevated levels of serum immunoglobulin and develop antibodies to double-stranded DNA (dsDNA). Tim-4)/) T cells had a higher proliferative response and also produced elevated levels of the pro-inflammatory cytokines IL-17 and interferon-c when reactivated in vitro. However, the absolute cell numbers in spleen, lymph nodes and thymus were not significantly affected. In a parallel study, Wong et al.,27 also showed that the ability of Tim4)/) resident peritoneal macrophages to bind and engulf apoptotic cells is significantly compromised in vitro and in vivo. Although resident peritoneal macrophages caused elevated tumour necrosis factor-a production in vitro, no such increase could be detected in vivo probably because of its relatively low level. More importantly, Tim-4 deficiency results in increased cellularity in the peritoneum. Interestingly, the cytoplasmic tail of Tim-4 was found to be important in mediating the engulfment step in 293T cells. However, in this study, the levels of anti-dsDNA in aged or apoptotic cell-injected mice did not differ significantly between wild-type and Tim-4 knockout animals.27 These observations using Tim-4-deficient mice would


Inhibitory effect of Tim-4 suggest that, although Tim-4 is capable of inducing bimodal signals to responding T cells, Tim-4-mediated inhibition is predominant in vivo. In summary, our Tim-4Fc is inhibitory to CD4+ T-cell activation via a non-Tim-1 receptor. Therefore, one of the principle aims of our future work is to determine what other Tim-4 receptors are present on CD4+ T cells. Actually, a single band round 70 000 MW has been pulled down with Tim-4 Fc protein but not Tim-4-Igv or Tim4-mucin Fc fusion protein. Next, we hope to identify the protein by mass spectrometry. In addition, the importance of STAT3 and STAT5 to T-cell alloactivation and Th17 differentiation make these transcription factors possible targets of Tim proteins and an attractive focus for future Tim-4Fc studies.28–30 The paucity of information regarding Tim proteins and Th17 cells is an area that we hope to address in the near future. In particular, the role of Tim-4 in Th17 signal transduction will be investigated. The identification of the inhibitory receptor and the development of its antibody will help us make a thorough investigation.

Acknowledgements We thank Dr M.H. Sayegh, Harvard Medical School, Boston, USA for generously providing the RMT 1-10 antibody and Prof. Shigekazu Nagata, Osaka University Medical School, Japan who kindly donated the plasmid expressing Tim-4Fc. This work was funded by a Health Research Board of Ireland grant (RP/2006/75) to M.C. and R.O’C.

Disclosures No conflict of interest reported.

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