International Immunopharmacology Manuscript Draft Manuscript Number: INTIMP-D-11-00299 Title: Characterization of MT-2 cells as a human regulatory T cell-like cell

line Article Type: Preliminary Study/Report Keywords: MT-2 cells, HTLV-1, regulatory T cells, phenotype, immunesuppression Abstract: The paucity of naturally occurring CD4+FoxP3+ regulatory T cells (Tregs) and difficulties in the identification and isolation of such cells are major obstacles in the studies of human Tregs. The availability of human Treg-like cell lines is likely to facilitate a better understanding of the molecular basis of Treg function and to discover pharmacological reagents to regulate Treg activity in disease state. In this study, we examined the Treg phenotype and the immune suppressive effect of a human Tcell leukemia virus type 1 (HTLV-1) infected cell line MT-2. The results showed that MT-2 cells express typical human Treg markers CD4, CD25, FoxP3, CTLA-4, GITR, CCR4, TNFR2 and HLA DR, but not for CD127. Importantly, MT-2 cells were potent suppressors of the polyclonal proliferation of both normal human peripheral blood (PB) CD4 cells and mouse peripheral CD4 cells. In addition, MT-2 cells suppressed the production of IL-17 by co-cultured human CD4 cells and INF production by cocultured mouse CD4 cells. In contrast, another HTLV-1-infected cell line MS-9 did not express FoxP3 and CTLA-4 and did not show any detectable suppressive activity. Our data clearly demonstrate that MT-2 cell line has the phenotypic and functional characteristics of human Tregs and is a useful tool to study human Tregs.

Highlights

Highlights

MT-2 cells express typical human Treg markers MT-2 cells inhibit activation of both human and mouse responder cells Immunosuppressive activity of MT-2 cells is not based on HTLV-1 infection Thus, MT-2 is a human Treg-like cell line which may be a useful tool to study Tregs

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R. Hamano et al. MT-2 cells are human Treg-like cells. 7/29/2011 Characterization of MT-2 cells as a human regulatory T cell-like cell line Ryoko Hamanoa, d, Xueqiang Wua, Christine Lai a, Kathryn S. Jones b, O.M. Zack Howard a, Francis W. Ruscetti c, Joost J. Oppenheim a and Xin Chen b From a Laboratory of Molecular Immunoregulation, Cancer Inflammation Program, Center for Cancer Research, NCI-Frederick, USA; b Basic Science Program, SAICFrederick, Inc., NCI-Frederick, USA; c Laboratory of Experimental Immunology, Cancer Inflammation Program, Center for Cancer Research, NCI-Frederick, USA; d Division of Rheumatology, Kanazawa University, Kanazawa, Ishikawa, Japan The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the U.S. Government. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract HHSN261200800001E. This Research was supported [in part] by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. Correspondence to Dr. Xin Chen: BSP, SAIC-Frederick, Inc., Lab of Molecular Immunoregulation, CIP, CCR, NCI-Frederick. P.O.Box B, Bldg 560, Rm 31-19, Maryland 21702-1201, USA. Tel.: +001-301-846-1347; Fax.: +001-301-846-6752, Email address: chenxin@mail.nih.gov Abbreviations: HTLV-1: human T-cell leukemia virus type 1; Tregs: regulatory T cells; PB: peripheral blood; PBMC: Peripheral blood mononuclear cell.

R. Hamano et al. MT-2 cells are human Treg-like cells. 7/29/2011 Abstract The paucity of naturally occurring CD4+FoxP3+ regulatory T cells (Tregs) and difficulties in the identification and isolation of such cells are major obstacles in the studies of human Tregs. The availability of human Treg-like cell lines is likely to facilitate a better understanding of the molecular basis of Treg function and to discover pharmacological reagents to regulate Treg activity in disease state. In this study, we examined the Treg phenotype and the immune suppressive effect of a human T-cell leukemia virus type 1 (HTLV-1) infected cell line MT-2. The results showed that MT-2 cells express typical human Treg markers CD4, CD25, FoxP3, CTLA-4, GITR, CCR4, TNFR2 and HLA DR, but not for CD127. Importantly, MT-2 cells were potent suppressors of the polyclonal proliferation of both normal human peripheral blood (PB) CD4 cells and mouse peripheral CD4 cells. In addition, MT-2 cells suppressed the production of IL-17 by cocultured human CD4 cells and INF production by co-cultured mouse CD4 cells. In contrast, another HTLV-1-infected cell line MS-9 did not express FoxP3 and CTLA-4 and did not show any detectable suppressive activity. Our data clearly demonstrate that MT-2 cell line has the phenotypic and functional characteristics of human Tregs and is a useful tool to study human Tregs. Key words: MT-2 cells, HTLV-1, regulatory T cells, phenotype, immunesuppression

R. Hamano et al. MT-2 cells are human Treg-like cells. 7/29/2011 1. Introduction CD4+CD25+ regulatory T cells (Tregs), representing a minor fraction (~10%) of peripheral CD4+ T cells and expressing the X chromosome-encoded forkhead transcription factor, FoxP3, play an essential role in the maintenance of immunological homeostasis and prevention of autoimmune responses [1]. Despite substantial progress over the past decade, the fundamental mechanism underlying the immunosuppressive activity of Tregs remains poorly understood. A major obstacle of Treg study is the difficulty in isolating sizable numbers of naturally occurring Tregs, and the lack of specific surface marker(s) for the identification of human Tregs. A human Treg-like cell line which readily provides abundant source of cells may prove useful for the study of immunobiology and immunopharmacology of Tregs. The MT-2 cell line was derived from normal human cord leukocytes of a healthy donor by co-cultivation with leukemic cells from an ATL patient [2]. It has been shown that MT-2 cells expressed FoxP3, and when mixed at a ratio of 1:2 (suppressors: responders), MT-2 cells inhibited the proliferative response of purified human PB CD4+CD25- cells to stimulation with dendritic cells and anti-CD3 Ab [3]. Thus, MT-2 cells could represent a human Treg-like cell line. However, the Treg phenotypic markers and relative suppressive activity of MT-2 cells on both the proliferation and cytokine production of co-cultured responder cells need to be further evaluated. Tregs express a panel of markers associated with their suppressive function. For example, it has been shown that CD25 and TNFR2 expressed by Tregs transduced the signaling of IL-2 and TNF in the activation of Tregs [4-5] and expression of CTLA-4 reportedly

R. Hamano et al. MT-2 cells are human Treg-like cells. 7/29/2011 contributed to the immunosuppressive function of Tregs [1]. Thus, establishing the similarity of MT-2 cells to human Tregs is crucial for the utilization of this cell line to study Tregs.

R. Hamano et al. MT-2 cells are human Treg-like cells. 7/29/2011 2. 2.1. Material and Method Cells and reagents

Human peripheral blood enriched in mononuclear cells was obtained from normal donors by leukapheresis (Transfusion Medicine Department, Clinical Center, National Institutes of Health, Bethesda, MD, with an approved human subjects agreement). The blood was centrifuged through Ficoll-Hypaque (Sigma), and PBMCs collected at the interface were washed with PBS and centrifuged through isoosmotic Percoll (Pharmacia, Uppsala, Sweden) gradient. MT-2 cells were from ATCC. MS-9 cells were a gift from Dr. Gisela Heidecker at NCI-Frederick. Female wild type C57BL/6 mice were provided by the Animal Production Area of the National Cancer Institute (NCI) (Frederick, MD). NCIFrederick is accredited by American Association for the Accreditation of Laboratory Animal Care International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the Procedures outlined in the Guide for Care and Use of Laboratory Animals published by the National Research Council (National Research Council, National Academy of Sciences. (1996) Guide for Care and Use of Laboratory Animals. National Academy Press, Washington D.C. Anti-human CD4-FITC, CD25-PE-Cy5, CD152 (CTLA-4)-APC, CD45RO-PE-Cy5 and CD120b (TNFR2)-PE were purchased from BD Pharmingen (San Diego, CA). Antihuman CD45RA-PerCP-Cy5.5, CD127-PerCP-Cy5.5, HLA-DR-PerCP-Cy5.5, CD45ROAPC, GITR-APC, FoxP3-APC staining set, and both anti-human and anti-mouse

R. Hamano et al. MT-2 cells are human Treg-like cells. 7/29/2011 functional grade purified anti-CD3 Abs were from eBioscience (San Diego, CA). CFDA SE Cell trace Kit was purchased from Molecular Probes. 2.2. In vitro cell culture and proliferation assay

Human CD4+ cells were purified from freshly isolated human PBMCs with anti-human CD4 microbeads and LS column (Miltenyi Biotec Inc., Auburn, CA). CD4 depleted PBMCs were used as APCs. Human APCs were irradiated with 4,000 R. Mouse lymphocytes were harvested from mouse spleen, axillary lymph node, inguinal lymph node and mesenteric lymph nodes. CD4+ cells were purified from lymphocytes with mouse CD4 (L3T4) MicroBeads and LS column (Miltenyi Biotec). T-depleted spleen cells were used as APCs and were prepared by depletion of CD90+ cells with anti-mouse CD90 MicroBeads and LD column (Miltenyi Biotec). Mouse APCs were irradiated with 3,000 R. CFSE-labelled (2 M, 8 min at room temperature) human or mouse CD4 cells (2104 cells/well) were seeded in a U-bottom 96-well plate in medium [For culture of human CD4 cells: RPMI 1640 with 10% fetal bovine serum (FBS, Hyclone, Logan, UT) containing 2 mM glutamine, 100 IU/ml penicillin, and 100 g/ml streptomycin, 10 mM HEPES. Additional 1 mM sodium pyruvate and 50 M 2-ME were added for culture of mouse CD4 cells]. The cells were co-cultured with 2105 cells/well of human or mouse APCs and 1 g/ml of soluble anti-human or anti-mouse CD3 Ab, respectively. MT-2 cells or MS-9 cells were added to the wells at a desired ratio to CD4+ cells. After 48~72 hr, CFSE dilution was determined with FACS. In some experiment, cytokine levels in the supernatants of cultures were determined by SearchLight Human or mouse Cytokine Array (Aushon Biosystems, Billerica, MA).

R. Hamano et al. MT-2 cells are human Treg-like cells. 7/29/2011 2.3. Flow Cytometry

After blocking FcR, cells were incubated with appropriately diluted antibodies. Data were acquired on a FACSort (BD Biosciences, Mountain View, CA) and analysis was conducted using CellQuest software (BD Biosciences). 2.4. Statistical analysis

Comparisons of data were analyzed by two-tailed Students t test using Graphpad Prism 4.0.

R. Hamano et al. MT-2 cells are human Treg-like cells. 7/29/2011 3. 3.1. Results and discussion Human Treg-like phenotypic markers expressed by MT-2 cells

First, we examined the expression of Treg phenotypic markers on MT-2 cells. As shown in Figure 1, MT-2 cells expressed CD4, the human Treg lineage maker. However, unlike human PB CD4 T cells, MT-2 cells did not express CD3. MT-2 cells expressed a high level of CD25, the chain of IL-2 receptor which is a key surface marker for the identification of mouse Tregs [6]. CD25 expressed by human PB CD4 cells is relatively dim as compared with mouse CD4 cells and only highly expressing CD25 (CD25high) cells had consistent suppressive function [7]. Nevertheless, all MT-2 cells expressed high levels of CD25 and thus were CD25high cells. We confirmed the previous observation [3] that MT-2 cells expressed FoxP3, the most specific marker for Tregs to date. Thus, MT-2 cells had the essential CD4+CD25hiFoxP3+ characteristic human Treg phenotype. Human Tregs express low levels of CD127, the IL-7 receptor chain, and the absence of this molecule therefore can be used with CD25 to define human Tregs [8]. Similar to human Tregs, MT-2 cells did not express CD127. We and others reported that TNFR2 was preferentially expressed by ~40% of mouse Tregs [4], and by a majority of human PB CD4+FoxP3+ Tregs [9]. Only a small fraction of MT-2 cells expressed low levels of TNFR2, which is similar to TNFR2 expression pattern of mouse Tregs, but not human Tregs. All MT-2 cells express high levels of GITR on the surface, and majority of them expressed CTLA-4, a potential effector molecule of Tregs. It has been shown that approximately one-third of human Tregs expressed MHC DR, and MHC DR+ Tregs represented a distinct functional subset of Tregs which were responsible for contact-

R. Hamano et al. MT-2 cells are human Treg-like cells. 7/29/2011 dependent suppression [7]. Interestingly, more than half of MT-2 cells expressed MHC DR. In addition, MT-2 cells expressed high levels of CCR4, a chemokine receptor recently found to enhance viability and maintain the high frequency of Tregs in patients with HTLV-1 infection [10], in addition to directing the traffic of Tregs to the tumor environment. MT-2 cells were negative for CD45RA and expressed high levels of CD45RO, suggesting that they may be an effector/memory type of human Treg-like cells. 3.2. Suppressive activity of MT-2 cells on primary human PB CD4 cells

and mouse CD4 cells The in vitro functional hallmark of Tregs is that they markedly suppress the activation of co-cultured T responder cells. We therefore examined the number of MT-2 cells required to inhibit co-cultured human PB CD4 cells. As shown in Figure 2A, the proliferative responses of PB CD4 cells to TCR stimulation, as measured by CFSE-dilution, were suppressed by 9.7%, 31.6%, 63.3% and 91.2% after addition of MT-2 cells at ratios of 10:1, 10:2, 10:5 and 10:10 (PB CD4 cells: MT-2 cells). Since MT-2 cells by themselves alone produced high levels of IFN and TNF, but not IL-17A (data not shown), we therefore used IL-17A as an indicator to examine the suppressive effect of MT-2 cells on the cytokine production of co-cultured human primary CD4 cells. As shown in Figure 2B, MT-2 cells potently inhibited IL-17A production over a ratio of 10:1~10:10 (CD4: MT-2 cells, p<0.05). Mouse CD4 cells are more readily obtainable than human PB CD4 cells. Thus, we investigated whether it is possible to use mouse CD4 cells as responder cells to examine

R. Hamano et al. MT-2 cells are human Treg-like cells. 7/29/2011 suppressive effect of MT-2 cells. As shown in Figure 2C, at a ratio of 10:5 to 10: 10 (CD4: MT-2 cells), MT-2 cells inhibited the proliferation of co-cultured mouse CD4 cells by 8% and 51.9% respectively. At 10:1 ratio, however, MT-2 cells stimulated to a limited extent the proliferation of co-cultured mouse CD4 cells (p>0.05), which was probably caused by an allostimulatory effect of MT-2 cells. Similarly, IFN production by mouse CD4 cells was also markedly inhibited by MT-2 cells when they were co-cultured at a ratio of 10:5 and 10:10 (CD4: MT-2, p<0.05~0.01), but not at a ratio of 10:1 (p>0.05). Although MT-2 cells also suppressed TNF and IL-17 production by mouse CD4 cells (data not shown), they did not suppress IL-2 production (data not shown). 3.3. HTLV-1 infection is not sufficient to confer Treg phenotype and

function of MT-2 cells It has been shown that, similar to human Tregs, MT-2 cells inhibited proliferation of CD4+CD25- T cells in a cell-to-cell contact manner which can be abrogated by separating MT-2 cells from responder cells [3]. Thus, MT-2 cells are unlikely to exert their suppressive activity by transferring HTLV-1 virus to responder cells. For further clarification, we compared MT-2 cells with HTLV-1-infected MS-9 cells, since both of them can infect or transform other T cells in vitro [11-12]. As shown in Fig 3A-B, MT-2 cells consistently showed suppressive activity, while MS-9 cells did not suppress proliferation of co-cultured primary human CD4 cells at all. Furthermore, unlike MT-2 cells, MS-9 cells did not express the Treg-fingerprint molecule FoxP3 and CTLA-4 (Fig 3C), although they expressed CD25 (data not shown). These data indicate that the infection by HTLV-1 is not sufficient to confer phenotypic and functional attributes of Tregs and that the suppressive activity of MT-2 cells is not based on its release of 10

R. Hamano et al. MT-2 cells are human Treg-like cells. 7/29/2011 infectious HTLV-1. It has been shown previously that the suppressive activity of MT-2 cells could not be abrogated by anti-CTLA-4 Ab [3]. However, the Ab may not efficiently block the short-distance interaction in the cell-to-cell contact environment. Since CTLA-4 is expressed by MT-2 cells but not by MS-9 cells, its role in mediating the suppressive activity of MT-2 cells should be further studied. The suppressive activity of MT-2 cells is also unlikely based on the consumption of growth factors in the co-cultures, since Jurkat cells, which grow faster than MT-2 cells, did not show suppression at all (data not shown). Taken together, our data suggested that MT-2 cells had the phenotypic and functional characteristics of human Tregs and therefore may be used as a human Treg-like cell line for Treg studies. Similar to human Tregs, MT-2 cells were CD4+CD25hiFoxP3+, and expressed CTLA-4, GITR, CCR4, MHC DR, CD45RO and TNFR2, but not CD127. The expression levels of these molecules on MT-2 cells are readily manipulated by molecular biological techniques, and this approach may help reveal their roles in the biological function of Tregs. The suppressive activity of MT-2 cells can be conveniently monitored with CFSE-dilution based proliferation assays and cytokine levels by using normal human PB CD4 cells or with normal mouse CD4 cells as responders. Our data also revealed some differences between MT-2 cells and primary human Tregs. One example is that MT-2 cells did not express CD3. In addition, special safety measures and the potential impact of HTLV-1 produced by MT-2 cells on co-cultured responder cells have to be considered in the study by using MT-2 cells as Treg-like cell line. These differences should be taken into account when using MT-2 cells as a cellular model to study human Tregs. Nevertheless, MT-2 cells can be used as positive control for human Treg studies

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R. Hamano et al. MT-2 cells are human Treg-like cells. 7/29/2011 and for biochemical studies of Tregs which require larger numbers of cells. Patients with HTLV-1 infection often have a high frequency of FoxP3+ Tregs in the periphery and the underlying mechanism remains to be fully understood [10]. MT-2 cells immortalized by HTLV-1 represent phenotypically and functionally typical Treg-like cells, thus providing promising a model to study the molecular pathway by which HTLV-1-infection promotes the generation of Tregs in human patients. Acknowledgement The authors thank Dr. Ji Ming Wang for discussion and critical review of the manuscript.

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R. Hamano et al. MT-2 cells are human Treg-like cells. 7/29/2011 Reference: [1] Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune tolerance. Cell. 2008;133:775-87. [2] Miyoshi I, Kubonishi I, Yoshimoto S, Akagi T, Ohtsuki Y, Shiraishi Y, et al. Type C virus particles in a cord T-cell line derived by co-cultivating normal human cord leukocytes and human leukaemic T cells. Nature. 1981;294:770-1. [3] Chen S, Ishii N, Ine S, Ikeda S, Fujimura T, Ndhlovu LC, et al. Regulatory T cell-like activity of Foxp3+ adult T cell leukemia cells. Int Immunol. 2006;18:269-77. [4] Chen X, Baumel M, Mannel DN, Howard OM, Oppenheim JJ. Interaction of TNF with TNF Receptor Type 2 Promotes Expansion and Function of Mouse CD4+CD25+ T Regulatory Cells. J Immunol. 2007;179:154-61. [5] Oppenheim JJ. IL-2: more than a T cell growth factor. J Immunol. 2007;179:1413-4. [6] Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol. 1995;155:1151-64. [7] Costantino CM, Baecher-Allan CM, Hafler DA. Human regulatory T cells and autoimmunity. Eur J Immunol. 2008;38:921-4. [8] Liu W, Putnam AL, Xu-Yu Z, Szot GL, Lee MR, Zhu S, et al. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J Exp Med. 2006;203:1701-11. [9] Chen X, Subleski JJ, Hamano R, Howard OM, Wiltrout RH, Oppenheim JJ. Coexpression of TNFR2 and CD25 identifies more of the functional CD4+FOXP3+ regulatory T cells in human peripheral blood. Eur J Immunol. 2010;40:1099-106. [10] Toulza F, Nosaka K, Tanaka Y, Schioppa T, Balkwill F, Taylor GP, et al. Human Tlymphotropic virus type 1-induced CC chemokine ligand 22 maintains a high frequency of functional FoxP3(+) regulatory T cells. J Immunol. 2010;185:183-9. [11] Hill SA, Shuh M, Derse D. Comparisons of defective HTLV-I proviruses predict the mode of origin and coding potential of internally deleted genomes. Virology. 1999;263:273-81. [12] Jones KS, Petrow-Sadowski C, Huang YK, Bertolette DC, Ruscetti FW. Cell-free HTLV-1 infects dendritic cells leading to transmission and transformation of CD4(+) T cells. Nat Med. 2008;14:429-36.

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R. Hamano et al. MT-2 cells are human Treg-like cells. 7/29/2011

Figure caption Figure 1. FACS analysis of Treg-like phenotype of MT-2 cells. MT-2 cells were stained with indicated anti human Abs and then analyzed with FACS. CTLA-4 and FoxP3 were stained intracellularly. Other antibodies stained the cell surface. Grey histogram shows antibody staining. Isotype controls are shown by line histogram. Data shown are representatives of at least three separate experiments with similar results. Figure 2. Suppressive effect of MT-2 cells on primary human PB CD4 cells and primary mouse CD4 cells. MACS-purified PB human CD4 cells or mouse CD4 cells were stained with CFSE and were co-cultured with MT-2 cells at the indicated ratios. The cells were stimulated with APCs and anti-CD3 Ab for 3 days. The proliferation of PB human CD4 cells (A) or mouse CD4 cells (C) were measured by CFSE dilution with FACS. The percentage of CFSE-diluted cells is shown in the histograms. Production of IL-17 by human CD4 cells (B) and IFN by mouse CD4 cells (D) in the supernatant were determined and data shown (meanSD, N=3~5) were summarized from three separate experiments. Compared with CD4 cells alone: * p<0.05, ** p<0.01. Figure 3. The phenotypic and functional properties of HTLV-1-infected MS-9 cells. CFSE-labeled human PB CD4 cells were co-cultured with MT-2 cells or MS-9 cells at a desired ratio. The cells were stimulated with APCs and anti-CD3 for 3 days. The proliferation of human PB CD4 cells was measured by CFSE dilution with FACS. (A) Typical FACS analysis. Grey histogram: human PB CD4 cells cultured alone (without MT-2 or MS-9 cells); solid line histogram: human PB CD4 cells cultured with MT-2 cells (left) or MS-9 cells (right) at a ratio of 10:10. (B) CFSE expression (MFI) by human PB 14

R. Hamano et al. MT-2 cells are human Treg-like cells. 7/29/2011 CD4 cells when cultured alone, co-cultured with MT-2 cells (open bar) or with MS-9 cells (black bar). *p<0.05 (meanSD, N=3). (C) Intracellular expression of FoxP3 and CTLA-4 by MS-9 cells was analyzed by FACS. Grey histogram: anti FoxP3 Ab or anti CTLA-4 Ab staining; dashed line histogram: isotype control staining. The data shown are representatives of at least three separate experiments with similar results.

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Figure 1 Click here to download high resolution image

Figure 2 Click here to download high resolution image

Figure 3 Click here to download high resolution image