Cancer Research Landon Prizes for Basic and Translational Cancer Research Translational Medicine Conference in Israel
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Cancer Research 67, 3555-3559, April 15, 2007. doi: 10.1158/0008-5472.CAN-06-2763
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Swoboda, R. K.
Right arrow Articles by Herlyn, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Swoboda, R. K.
Right arrow Articles by Herlyn, D.

Priority Reports

Shared MHC Class II–Dependent Melanoma Ribosomal Protein L8 Identified by Phage Display

Rolf K. Swoboda1, Rajasekharan Somasundaram1, Laura Caputo1, Elizabeth M. Ochoa1, Phyllis A. Gimotty2, Francesco M. Marincola7, Patricia Van Belle4, Stephen Barth4, David Elder3,4, DuPont Guerry3,5, Brian Czerniecki3,6, Lynn Schuchter3,5, Robert H. Vonderheide3 and Dorothee Herlyn1

1 The Wistar Institute; 2 Center for Epidemiology and Biostatistics and 3 Abramson Cancer Center, University of Pennsylvania; Departments of 4 Pathology, 5 Medicine, and 6 Surgery, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania; and 7 Department of Transfusion Medicine Clinical Center, NIH, Bethesda, Maryland

Requests for reprints: Dorothee Herlyn, Immunology Program, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. Phone: 215-898-3962; Fax: 215-898-0980; E-mail: dherlyn{at}wistar.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Antigens recognized by T helper (Th) cells in the context of MHC class II molecules have vaccine potential against cancer and infectious agents. We have described previously a melanoma patient's HLA-DR7–restricted Th cell clone recognizing an antigen, which is shared among melanoma and glioma cells derived from various patients. Here, this antigen was cloned using a novel antigen phage display approach. The antigen was identified as the ribosomal protein L8 (RPL8). A peptide of RPL8 significantly stimulated proliferation and/or cytokine expression of the Th cell clone and lymphocytes in four of nine HLA-DR7+ melanoma patients but not in healthy volunteers. The RPL8 antigen may represent a relevant vaccine target for patients with melanoma, glioma, and breast carcinoma whose tumors express this protein. [Cancer Res 2007;67(8):3555–9]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD4+ T helper (Th) lymphocytes play a central role in the development of protective immunity against tumors. Adoptively transferred CD4+ Th cells, in the absence of CD8+ CTL, inhibit tumor growth in mice. In melanoma patients, spontaneous tumor regression was associated with CD4+ lymphocyte infiltrates (reviewed in ref. 1, 2).

A few human CD4+ Th cell lines and clones directed against various tumors have been described (3–7). Th antigens are usually recognized by MHC class II–restricted CD4+ Th cells after processing by antigen-presenting cells (APC) through the exogenous pathway.

Although peptides derived from tumor antigens originally defined by MHC class I–restricted CTL have been shown to stimulate Th cells (reviewed in ref. 8), peptide vaccines have not held promise in clinical trials (reviewed in ref. 9). Antigen vaccines expressing multiple potentially immunogenic epitopes may be superior to peptide vaccines expressing only a single epitope (10). Antigens recognized by noncytolytic MHC class II–restricted CD4+ T cells have been cloned using the Ii-cDNA fusion approach (11–16).

In the present study, we have cloned, using a novel antigen phage display approach, a Th antigen shared between melanomas and gliomas, each derived from various patients. A peptide derived from this antigen is recognized by the lymphocytes of several melanoma patients but not healthy donors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Melanoma patients. Patient 35 has no evidence of disease after excision of primary lesion ~23 years ago. All other melanoma patients included in this study had metastatic lesions excised between 2002 and 2005. Peripheral blood mononuclear cells (PBMC) were obtained from the patients' peripheral blood on the day of surgery (3522, 3523, 3626, and 3704) or up to 24 months after surgery (all other patients) with informed consent and under approved protocols.

Cell lines. Melanoma cell line WM35, EBV-B35 cell line, and Th35-1A Th cell clone were established and maintained in culture as described (7). COS-7L cells (Life Technologies-Invitrogen, Carlsbad, CA) were maintained in DMEM (Life Technologies-Invitrogen) supplemented with 10% fetal bovine serum.

Antibodies. Anti-HLA class II antibody B33.1 directed to HLA-DR was obtained from B. Perussia (Thomas Jefferson University, Philadelphia, PA) and normal mouse IgG from Cappel-ICN (Costa Mesa, CA). FITC-conjugated antihuman CD4 antibody (clone RPA-T4) and APC-conjugated antihuman interleukin-2 (IL-2) antibody (clone MQ1-17h12) were obtained from eBioscience (San Diego, CA).

Th antigen cloning approach using antigen phage display—overview. The currently used molecular cloning approach of HLA class II–dependent human melanoma and colon carcinoma antigens is based on fusing cDNA tumor libraries to MHC invariant chain (Ii) fragments with the aim of targeting the fusion proteins to the endosomal and lysosomal compartments (6). Fused libraries were transfected into 293 cells genetically engineered to express DR{alpha}, DRß, DMA, DMB, and Ii and screened for reactivity with CD4+ T cells (Fig. 1, left ).


Figure 1
View larger version (16K):
[in this window]
[in a new window]

  		Figure 1. Schematic presentation of the Ii-cDNA fusion approach (15) and our bacteriophage-cDNA fusion approach.

 
We have expressed a melanoma cDNA library in bacteriophages followed by phage-library protein presentation to Th lymphocytes by APCs and identification of the relevant Th antigen by its capacity to induce proliferation and cytokine release in Th cells (Fig. 1, right). This strategy, in contrast to the cDNA library-Ii fusion approach, does not require prior knowledge of the MHC class II restriction element and it involves natural processing of the antigen by APC.

cDNA library construction and screening. mRNA was isolated from cultured WM35 cells using the FastTrack 2.0 kit (Invitrogen, Carlsbad, CA). PolyA+ RNA was converted to cDNA using the OrientExpress system (EMD Biosciences Novagen, La Jolla, CA) and ligated into T7Select10-3b vector (EMD Biosciences Novagen) according to the manufacturer's instructions. The ligated DNA was packed in vitro using T7 packing extract. The library was 1.2 x 106 phages and was amplified on plates once and divided into 100 phages per pool. For screening, each pool was amplified once in liquid culture, and released phages were purified twice by polyethylene glycol/NaCl precipitation. The phage titers were determined, and ~3 x 103 phages were used per well of 96-well microtiter plate (392 wells screened) in lymphocyte proliferation and IFN-{gamma} release assays (see below). Phages from one pool stimulated proliferation and IFN-{gamma} release in Th35-1A cells. Stimulatory pools were subdivided until a single positive phage was identified.

Lymphocyte proliferation assay. This assay was done as described (17). For screening of Th35-1A cell reactivity with phage libraries, Th cells (1 x 104 to 2 x 104 per well of 96-well round-bottomed microtiter plates; Corning, Corning, NY) were cultured with irradiated autologous EBV-B cells (104 per well) prepulsed with 1 x 103 to 3 x 103 phages. To determine Th or PBMC reactivity with peptide, adherent monocytes (5 x 104 per well, obtained from PBMC) prepulsed with various concentrations (3.1–50 µmol/L) of peptide were incubated with Th35-1A cells or PBMC (5 x 104 per well). Th cells or PBMC were stimulated with peptide-pulsed monocytes once or twice. Proliferative responses of lymphocytes were determined by standard [3H]thymidine ([3H]Tdr) incorporation assay. All determinations were done in triplicate. The lymphocyte proliferation inhibition assay with anti-HLA class II antibody B33.1 was done as described (17).

IFN-{gamma} release assay. Supernatants obtained 48 h after Th cell stimulation with phage-pulsed EBV-B cells were tested for the presence of IFN-{gamma} using an ELISA kit (Endogen, Rockford, IL).

Intracellular staining of lymphocytes for IL-2. Peptide-pulsed monocytes (see above) were incubated with lymphocytes at 37°C and cultures were treated with Golgi Stop (2 µmol/L; BD Biosciences, San Diego, CA) after 1 h. Five hours later, nonadherent cells were stained for CD4 and intracellular IL-2 using a kit (BD Biosciences). Samples were analyzed by CyAn ADP cytometer (DakoCytomation, Fort Collins, CO) and data were analyzed using FlowJo software (Ashland, OR).

DNA sequencing. DNA sequencing was done by The Wistar Institute's DNA sequencing facility. DNA and deduced amino acid sequence (ExPASy)8 comparisons were done with the BLAST program.9

Peptide design. Potentially DRB1*070101 binding epitopes were determined from the deduced amino acid sequence by using the Rammensee epitope prediction model10 and were limited to epitopes with a binding score of >20. Selected peptides were synthesized and high-pressure liquid chromatography purified by Invitrogen. The following peptides were used: VGLIAARRTGRLRGT, with a score of 24 [peptide 1, ribosomal protein L8 (RPL8) position 235–249]; TGRLRGTKTVQEKEN, with a score of 24 (peptide 2, RPL8 position 243–257); and RPGLLGASVLGLDDI, with a score of 22 (control peptide, telomerase reverse transcriptase).

Full-length RPL8 cloning. The GeneRacer kit (Invitrogen) and oligonucleotides based on the cDNA sequence of the phage that stimulated Th35-1A cell proliferation were used to determine the 5' and 3' end of RPL8 mRNA in WM35 cells. Both fragments (5' and 3' end) were sequenced and oligonucleotides were designed to clone full-length RPL8 cDNA by reverse transcription-PCR (RT-PCR; SuperScript III One-Step RT-PCR with Platinum Taq; Invitrogen).

Northern blot analysis. Northern blot analysis was done according to standard procedures. RNA levels were compared using a Storm PhosphorImager system (GE Healthcare, Piscataway, NJ).

Real-time PCR. Single-strand cDNA derived from tumor cells was subjected to real-time PCR using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin, or RPL8-specific primers on an ABI Prism 7000 Sequencing Detection System (Applied Biosystems). The data were analyzed using 7000 System SDS software (Applied Biosystems).

Statistical analyses. Differences between experimental and control values were analyzed for significance by Student's two-sided t test or nonparametric Wilcoxon test using Proc NPAR1WAY in SAS/STAT software (SAS Institute, Inc., Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Library screening and clone identification. A single phage clone of the cDNA library from WM35 melanoma cells induced proliferation and IFN-{gamma} release in Th35-1A cells. The clone had an insert of 189 bp, encoding an open reading frame of 58 amino acids, and represented the COOH-terminal part of RPL8 (18). The nucleotide sequence of full-length RPL8 subsequently cloned from WM35 melanoma cells was 100% identical with the published RPL8 sequence (Genbank GI:15431305; data not shown). Thus, the WM35 antigen recognized by Th35-1A cells was not mutated, which is in contrast to the other cloned mutated Th antigens or Th antigens using alternative reading frames (12–16).

Epitope determination. To confirm that RPL8 was recognized by Th35-1A, we determined the peptide epitope recognized by Th35-1A. This epitope was predicted to associate with HLA-DR7 (7). The deduced amino acid sequence of the cloned cDNA contains two potential DR7 (DRB1*070101) binding sites (see Materials and Methods). Only peptide 2 (TGRLRGTKTVQEKEN) was recognized by Th35-1A after presentation by autologous monocytes, and peptide recognition was HLA class II dependent (Fig. 2A ). Th35-1A proliferation was peptide concentration dependent (Fig. 2B). Allogeneic DR7+ monocytes presented peptide 2 to Th35-1A cells (Fig. 2C), whereas DR7– monocytes did not (Fig. 2D).


Figure 2
View larger version (15K):
[in this window]
[in a new window]

  		Figure 2. Recognition of RPL8 peptide 2 by Th35-1A cells is HLA-DR7 and peptide concentration dependent. A, Th35-1A cells were stimulated with peptide (25 µmol/L)–pulsed autologous DR7+ monocytes in the absence of antibody or presence of either control mouse immunoglobulin (Ig) or anti-HLA class II antibody (both at 10 µg/mL). B to D, Th35-1A cells were stimulated with peptide (between 3.1 and 50 µmol/L)–pulsed autologous monocytes (B), DR7+ allogeneic monocytes (C), or DR7– allogeneic monocytes (D). Proliferation of Th35-1A cells was measured by [3H]Tdr incorporation assay. Values with identical symbols (* and #) differ significantly (P < 0.01) from each other (A). *, P < 0.01, experimental values that differ significantly from the corresponding control values (B and C).

 
RPL8 protein is a component of the 60S subunit of ribosomes and is involved in protein synthesis (18). It is expressed by all normal cell.11 RPL8 RNA is overexpressed in metastatic versus primary carcinomas (19). In light of the ubiquitous expression of RPL8 RNA (Table 1 ), it was surprising that some, but not all, tumor cell lysates derived from different patients stimulated proliferation of Th35-1A (Table 1). Because an antibody to RPL8 is not available, RPL8 protein levels and possible post-translational modifications in tumors of various tissue origins could not be investigated.


View this table:
[in this window]
[in a new window]

  		Table 1. RPL8 RNA levels in normal and tumor cells relative to WM35 cells

 
Recognition of peptide 2 by lymphocytes of HLA-DR7+ melanoma patients. PBMC from two of four HLA-DR7+ melanoma patients tested significantly proliferated after stimulation with RPL8 peptide 2 (Table 2 ). PBMC from four of seven HLA-DR7+ patients tested showed peptide reactivity as determined by intracellular staining for IL-2 (Table 2). Not all nine HLA-DR7+ patients' PBMC could be tested in both assays because we lacked sufficient numbers of PBMC. However, two patients (patients 35 and 3533) tested in both assays showed lymphocyte responses in both assays. Lymphocytes from two HLA-DR7– patients and four healthy donors were nonresponsive in both assays (Table 2). Tumor RPL8 RNA levels were similar in all patients tested (Table 2).


View this table:
[in this window]
[in a new window]

  		Table 2. Summary of patients' clinical status, immune responses, and tumor mRNA level

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have successfully cloned the antigen, RPL8, recognized by a CD4+ HLA class II–restricted Th cell clone (Th35-1A) directed against melanoma. Until now, only the Ii-cDNA library fusion approach developed by Wang et al. (15) has been available for the molecular cloning of antigens recognized by MHC class II–restricted Th cells. The major limitation of the cDNA library-Ii fusion approach is the need for prior knowledge and isolation of the MHC class II restriction element used by the Th cell. We have developed a novel approach to the cloning of MHC class II–restricted antigens. This strategy does not require prior knowledge of the MHC class II restriction element used by Th cells for antigen recognition as phage-expressed cDNA libraries are presented to Th cells by MHC class II–positive autologous EBV-transformed B cells. Natural processing of the antigen by APC is another potential advantage of the phage protein fusion approach. Instead of the lysogenic filamentous phages commonly used in phage display libraries, we used the lytic phage T7. The use of T7 phage has two major advantages: (a) the cDNA is located at the 3' end of protein 10B, whereas, in filamentous phage, the cDNA is located in the middle of pIII, thus requiring two in-frame fusions, and (b) the lytic life cycle of T7 phage avoids negative selection of proteins during protein transport through the bacterial membrane, which is necessary for assembling filamentous phage.

Antigen phage display is a novel and powerful approach to the cloning of MHC class II–dependent antigens recognized by CD4+ MHC class II–restricted T cells. Using this approach, we have cloned RPL8, which is shared by melanomas (this study and ref. 7), gliomas (7), colorectal carcinomas (19), and ovarian carcinomas (20).

Antigen recognition by Th35 is HLA-DR7 restricted (7), and a putatively HLA-DR7 binding peptide of RPL8 stimulated Th35 cells in an HLA class II–restricted manner. Moreover, RPL8 peptide-specific lymphocytes could be detected in four of nine HLA-DR7+ melanoma patients but not in HLA-DR7– patients or healthy donors. There was no statistically significant correlation between melanoma status (stable versus progressive disease) and lymphocyte responses or RPL8 RNA levels of tumor cells and lymphocyte responses. Furthermore, recognition of allogeneic tumor cells by Th35 was independent of tumor RNA level. Interestingly, RPL8 may contain additional epitopes presented by a broad range of HLA class I/II alleles (i.e. A1, A2, A3, A24, B7, B44, B51, DR1, DR4, and DR11), based on preliminary algorithm screens. This suggests that this antigen may represent an important vaccine target in patients bearing RPL8+ tumors.


   Acknowledgments
 
Grant support: NIH grants CA60975, CA88193, CA25874, and CA10815 and the Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Jeffrey Faust and Matthew Farabaugh for flow cytometry analysis and Andrea Raymond for her help with real-time PCR.


   Footnotes
 
Note: R.K. Swoboda and R. Somasundaram contributed equally to this work.

8 http://us.expasy.org/tools/dna.html Back

9 http://www.ncbi.nlm.nih.gov/blast Back

10 http://www.syfpeithi.de Back

11 http://genome-www5.stanford.edu/cgi-bin/source/sourceResult Back

Received 7/26/06. Revised 2/23/07. Accepted 3/ 1/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gerloni M, Zanetti M. CD4 T cells in tumor immunity. Springer Semin Immunopathol 2005;27:37–48.[CrossRef][Medline]
  2. Yu P, Fu YX. Tumor-infiltrating T lymphocytes: friends or foes? Lab Invest 2006;86:231–45.[CrossRef][Medline]
  3. Radrizzani M, Benedetti B, Castelli C, et al. Human allogeneic melanoma-reactive T-helper lymphocyte clones: functional analysis of lymphocyte-melanoma interactions. Int J Cancer 1991;49:823–30.[CrossRef][Medline]
  4. Takahashi T, Chapman PB, Yang SY, Hara I, Vijayasaradhi S, Houghton AN. Reactivity of autologous CD4+ T lymphocytes against human melanoma. Evidence for a shared melanoma antigen presented by HLA-DR15. J Immunol 1995;154:772–9.[Abstract]
  5. Topalian SL, Rivoltini L, Mancini M, et al. Human CD4+ T cells specifically recognize a shared melanoma-associated antigen encoded by the tyrosinase gene. Proc Natl Acad Sci U S A 1994;91:9461–5.[Abstract/Free Full Text]
  6. Wang RF. The role of MHC class II-restricted tumor antigens and CD4+ T cells in antitumor immunity. Trends Immunol 2001;22:269–76.[CrossRef][Medline]
  7. Somasundaram R, Swoboda R, Caputo L, et al. A CD4+, HLA-DR7-restricted T-helper lymphocyte clone recognizes an antigen shared by human malignant melanoma and glioma. Int J Cancer 2003;104:362–8.[CrossRef][Medline]
  8. Novellino L, Castelli C, Parmiani G. A listing of human tumor antigens recognized by T cells: March 2004 update. Cancer Immunol Immunother 2005;54:187–207.[CrossRef][Medline]
  9. Weber J. Peptide vaccines for cancer. Cancer Invest 2002;20:208–21.[CrossRef][Medline]
  10. Maruyama H, Zaloudik J, Li W, et al. Cancer vaccines: single-epitope anti-idiotype vaccine versus multiple-epitope antigen vaccine. Cancer Immunol Immunother 2000;49:123–32.[CrossRef][Medline]
  11. Maccalli C, Li YF, El-Gamil M, Rosenberg SA, Robbins PF. Identification of a colorectal tumor-associated antigen (COA-1) recognized by CD4(+) T lymphocytes. Cancer Res 2003;63:6735–43.[Abstract/Free Full Text]
  12. Voo KS, Zeng G, Mu JB, Zhou J, Su XZ, Wang RF. CD4+ T-cell response to mitochondrial cytochrome B in human melanoma. Cancer Res 2006;66:5919–26.[Abstract/Free Full Text]
  13. Wang HY, Peng G, Guo Z, Shevach EM, Wang RF. Recognition of a new ARTC1 peptide ligand uniquely expressed in tumor cells by antigen-specific CD4+ regulatory T cells. J Immunol 2005;174:2661–70.[Abstract/Free Full Text]
  14. Wang HY, Zhou J, Zhu K, Riker AI, Marincola FM, Wang RF. Identification of a mutated fibronectin as a tumor antigen recognized by CD4+ T cells: its role in extracellular matrix formation and tumor metastasis. J Exp Med 2002;195:1397–406.[Abstract/Free Full Text]
  15. Wang RF, Wang X, Atwood AC, Topalian SL, Rosenberg SA. Cloning genes encoding MHC class II-restricted antigens: mutated CDC27 as a tumor antigen. Science 1999;284:1351–4.[Abstract/Free Full Text]
  16. Wang RF, Wang X, Rosenberg SA. Identification of a novel major histocompatibility complex class II-restricted tumor antigen resulting from a chromosomal rearrangement recognized by CD4(+) T cells. J Exp Med 1999;189:1659–68.[Abstract/Free Full Text]
  17. Somasundaram R, Zaloudik J, Jacob L, et al. Induction of antigen-specific T and B cell immunity in colon carcinoma patients by anti-idiotypic antibody. J Immunol 1995;155:3253–61.[Abstract]
  18. Hanes J, Klaudiny J, von der Kammer H, Scheit KH. Characterization by cDNA cloning of the mRNA of human ribosomal protein L8. Biochem Biophys Res Commun 1993;197:1223–8.[CrossRef][Medline]
  19. Futschik M, Jeffs A, Pattison S, et al. Gene expression profiling of metastatic and nonmetastatic colorectal cancer cell lines. Genome Letters 2002;1:26–34.[CrossRef]
  20. Luo LY, Herrera I, Soosaipillai A, Diamandis EP. Identification of heat shock protein 90 and other proteins as tumour antigens by serological screening of an ovarian carcinoma expression library. Br J Cancer 2002;87:339–43.[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Swoboda, R. K.
Right arrow Articles by Herlyn, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Swoboda, R. K.
Right arrow Articles by Herlyn, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online