Allospecific CD8 T suppressor cells induced by multiple MLC stimulation or priming in the presence of ILT3.Fc have similar gene expression profiles

Human Immunology 75 (2014) 190–196

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Allospecific CD8 T suppressor cells induced by multiple MLC stimulation or priming in the presence of ILT3.Fc have similar gene expression profiles q Ling Chen a,b, Zheng Xu a, Chris Chang a, Sophey Ho a, Zhuoru Liu a, George Vlad a, Raffaello Cortesini a, Raphael A. Clynes a, Yun Luo b, Nicole Suciu-Foca a,⇑ a b

Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, United States Department of Cardiology, The First People’s Hospital of Jiujiang, Jiujiang Affiliated Hospital, Nanchang University, Jiujiang, Jiangxi 332000, China

a r t i c l e

i n f o

Article history: Received 19 August 2013 Accepted 23 October 2013 Available online 9 November 2013

a b s t r a c t Alloantigen specific CD8 T suppressor cells can be generated in vitro either by multiple stimulations of CD3 T cells with allogeneic APC or by single stimulation in primary MLC containing recombinant ILT3.Fc protein. The aim of the present study was to determine whether multiple MLC stimulation induced in CD8+ CD28 T suppressor cells molecular changes that are similar to those observed in CD8 T suppressor cells from primary MLC containing ILT3.Fc protein. Our study demonstrates that the characteristic signatures of CD8 T suppressor cells, generated by either of these methods are the same consisting of up-regulation of the BCL6 transcriptional repressor and down-regulation of inflammatory microRNAs, miR-21, miR-30b, miR-146a, and miR-155 expression. In conclusion microRNAs which are increased under inflammatory conditions in activated CD4 and CD8 T cells with helper or cytotoxic function show low levels of expression in CD8 T cells which have acquired antigen-specific suppressor activity. Ó 2014 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.

1. Introduction Immune regulation has gained great attention since the discovery of CD4 regulatory T cells (CD4 Tregs) [1,2]. Several types of CD4 Tregs such as natural CD4+CD25+ FOXP3+ Tregs and lymphokine induced CD4+ Tr1 have been described [1–3]. CD4 Tregs exercise their suppressive function either by cell-to-cell contact or by secreting suppressive cytokines, such as IL-10, IL-4 and TGF-b [1– 5]. CD4 Treg based therapies have been tested in both animal models and clinical trials [6–9]. Although CD8 T suppressor cells (CD8 Ts, also called CD8 Tregs) were first described decades ago, their study has attracted less attention [10]. Like CD4 Tregs, CD8 Tregs are natural components of the immune system [11–14] or can be induced by various methods [15–19]. Different phenotypic markers of CD8 Tregs have been reported in various systems such as CD28, CD25+,CD56+, CD57+,

Abbreviations: Ts, T suppressor cells; Treg, T regulatory cells; BCL6, B cell CLL/ lymphoma 6; miR, microRNA. q This work was funded by Juvenile Diabetes Research Foundation (1-2008-550) and the International Organ Transplantation Consortium (Roma, Italy). ⇑ Corresponding author. Address: Department of Pathology and Cell Biology, Columbia University, 630 West 168th Street, VC15-204, New York, NY 10032, United States. E-mail address: [email protected] (N. Suciu-Foca).

CD103+, CD122+, CTLA4+, CXCR3+, and CD25+Foxp3+[12–19]. CD8 Tregs exert their inhibitory function through cell-to-cell interactions or by secreting suppressive cytokines. Evidence has been presented that CD8 Tregs play an important role in maintenance of self [11,13] and allogeneic tolerance [15,20], and in prevention of autoimmune disorders [18,21]. Our group has reported for the first time the generation of human allo-specific CD8+CD28 Ts by repeated in vitro stimulation of CD3 T cells with allogeneic APCs in MLC [15]. CD8+CD28 Ts did not suppress the activation of CD4+ T cells by T–T interaction or secretion of soluble cytokines, but rather by tolerizing the priming APCs [15,22] in which they induced down-regulation of the costimulatory molecules, CD80, CD86, CD40, and up-regulation of the expression of the inhibitory molecules ILT3 and ILT4 [23]. Tolerized APCs interacted with CD4 T cells with cognate specificity inducing T cell anergy [15,22]. CD8+CD28 Tregs have been described in both autoimmune patients [24] and in vivo experimental systems [25]. Since the discovery of the suppressive effect of the ILT3 molecule induced in APC by CD8+CD28 Ts, our research has focused on the potential of ILT3 to promote immune tolerance. We engineered a recombinant ILT3.Fc protein and showed it to be able to modulate the immune response by anergizing CD4 T cells and inducing the differentiation of CD8 Ts [26]. The immunomodula-

0198-8859/$36.00 - see front matter Ó 2014 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics. http://dx.doi.org/10.1016/j.humimm.2013.10.004

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tory activity of ILT3.Fc was further tested in vivo using a humanized NOD/SCID mouse model [25,27]. In this model, mice were humanized by injection of fresh PBMC from a blood donor and transplanted with normal or tumoral allogeneic human tissue. Treatment with ILT3.Fc prevented rejection or reverted its progression when administered after the onset of rejection [28]. To understand the mechanism underlying the generation of CD8 Ts, we analyzed their gene profiles. There was a striking increase of a zinc finger transcriptional repressor BCL6 in CD8 Ts [28]. By transfecting unprimed CD8 T cells with BCL6 or knocking it down from CD8 Ts, we proved that BCL6 is essential to the differentiation of CD8 Ts. The finding that human CD8 Ts cells harvested from ILT3.Fc treated NOD/SCID mice bearing islet allografts, displayed high levels of BCL6 and provided further proof that BCL6 is crucial to the function of human CD8 Ts. More recently we have shown that inflammatory microRNAs such as miR-21, miR-30b, miR-146a and miR-155 are down-regulated by ILT3.Fc leading to increased expression of their target genes including BCL6, DUSP10, CXCR4, and SOCS1 [29]. The aim of this study was to determine whether CD8+CD28 Ts and ILT3.Fc induced CD8 Ts share molecular characteristics. We analyzed molecularly CD8+CD28 Ts generated by multiple MLC stimulations and demonstrated that the expression of the BCL6 gene is increased while that of inflammatory microRNAs is decreased in CD8+CD28 Ts, similar to the events occurring in ILT3.Fc induced CD8 Ts. 2. Materials and methods

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by LKB 1250 betaplate counter (PerkinElmer). Mean cpm of the triplicate cultures and SD to the mean were calculated. Percent of suppression was calculated as [1  (cpmofculturesofCD4 Tcells + APCs + CD8+CD28+ or CD8+CD28 Tcells)/(cpmofculturesofCD4 Tcells + APCs)]. 2.3. Real-time PCR of protein coding genes Total RNA was prepared with Absolutely RNAR miniprep kit (Agilent Technologies, La Jolla, CA) from CD8+CD28+ and CD8+CD28 T cells unprimed, primed one or two times as described above. cDNA was synthesized using the first strand cDNA synthesis kit for RT-PCR (Roche Diagnostics, Basel, Switzerland). Real time PCR was performed using proprietary gene expression probes provided by Applied Biosystems (Foster City, CA). 7300 SDS 1.3.1 software (Applied Biosystems) was used for data collection and analysis. Formula 2ddCt was used for calculation of the gene expression relative to the housekeeping gene GAPDH. ddCt = (Ct [gene]  Ct [GAPDH]). Ct is the crossing threshold value returned by the PCR instrument for every gene amplified. 2.4. Real-time PCR of microRNA Total RNA was isolated from CD8+CD28+ or CD8+CD28 T cells with TRIzol (Invitrogen). The RNA was annealed to oligonucleotide primers and hybridized to oligonucleotide probes. Real-time PCR detection of microRNA was performed with TagMan small RNA assay (Applied Biosystems). Relative expression of microRNA was normalized by U6 microRNA.

2.1. T cell isolation and in vitro stimulation PBMCs were separated by gradient centrifugation from buffy coats purchased from the New York Blood Center (Long Island City, NY). CD3+ T cells were negatively isolated using T cell isolation kits (Miltenyi Biotech, Auburn CA). CD4+ T cells and CD8+ T cells were further purified by negative selection depleting CD8+ or CD4+ T cells with CD8 or CD4 T cells kits (Miltenyi Biotech). CD8+CD28+ and CD8+CD28 T cells were separated from CD8+ T cells using CD28 kits (Miltenyi Biotech). CD2-depleted PBMCs, used as APCs, were prepared from PBMCs using CD2 beads from Invitrogen (Grand Island, NY) for depletion of CD2+ cells. All the procedures were performed according to the manufacturer’s instructions. CD3+ T cells (106/ml) were primed with irradiated (3000 rad) allogeneic APCs (5  105/ml) for 7 days. Primed T cells were washed and cultured (106/ml) with irradiated APCs (5  105/ml) from the same donor for secondary 7 day MLC stimulation. IL-2 (R&D systems, Minneapolis, MN) was added at10 u/ml on day 3 [15]. All cell cultures were incubated at 37 °C in a 5% CO2 atmosphere and were performed in complete medium (RPMI1640 supplemented with 10% FCS, 2 mM L-glutamine and 50 lg/ml gentamycin from Mediatech, Manassas, VA). 2.2. Suppression assays CD8+CD28+ or CD8+CD28 T cells were isolated from unprimed T cells, or from cultures of T cells primed or restimulated with allogeneic APCs as described above. 5  104 responding CD4+ T cells/ well and 2.5  104/well irradiated allogeneic stimulating APCs were cultured in the presence or absence of 2.5  104 CD8+CD28+ or CD8+CD28 T cells/well. The responders and stimulators used for assaying the putative Ts were from the same blood donors whose cells were used for generating Ts by multiple MLC stimulations. The cultures were harvested on day 6. 1 lCi of 3[H] thymidine (Perkin Elmer, Melville, NY) was added to each well 18 h before harvesting and 3[H] thymidine incorporation was measured

3. Results 3.1. Acquisition of suppressor function by CD8+CD28 T cells was accompanied by significantly higher expression of BCL6 compared to CD8+CD28+ T cells CD3+ T cells were stimulated with allogeneic APCs for 7 days in primary MLC. T cells were then re-stimulated in secondary MLC with APCs from the same allogeneic donor for another 7 days. CD8+CD28+ or CD8+CD28 T cells were isolated from unprimed T cells and T cells primed in primary and secondary MLC. The CD28 positive and negative populations were tested for their ability to suppress the proliferation of CD4+ T cells from the same responder against the stimulator used for primary and secondary MLC. As shown in Fig. 1, neither CD8+CD28+ nor CD8+CD28 T cells isolated from unprimed T cells or T cells at the end of primary MLC showed suppressive activity. CD8+CD28 T cells, but not CD8+CD28+ T cells, acquired suppressor function after the secondary stimulation in MLC. The results were consistent with our previous findings that repeated in vitro allostimulations result in CD8+CD28 Ts cells [15]. RNAs were extracted from each population of CD8+CD28+or CD8+CD28 T cells used in the suppression assays. Real-time PCRs were performed for BCL6 expression. There were no significant differences between BCL6 expression in CD8+CD28+ and CD8+CD28 T cells that were either unprimed or primed only once. However, BCL6 expression became significantly higher in CD8+CD28 T cells than in CD8+CD28+ T cells after two stimulations, consistent with the difference between the inhibitory activities they displayed when tested in the suppressor assay. This result suggested that the BCL6 gene plays the same role in multiply stimulated CD8+CD28 Ts as it does in CD8 Ts generated in primary MLC in the presence of ILT3.Fc protein. We then performed a time-point analysis of BCL6 gene expression in CD8+CD28+ and CD8+CD28 T cells in order to determine the time after stimulation when changes occur. BCL6 gene expression levels in both CD8+CD28+ and CD8+CD28 T cells decreased

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Fig. 1. Differences in suppressive activity are associated with differences in BCL6 expression in CD8+CD28+ and CD8+CD28 T cells from secondary MLC. Panel A. Comparison of suppressive function of unprimed, primed and restimulated CD8+CD28+ and CD8+CD28 T cells. Panel B. Differences in BCL6 expression between CD8+CD28+ and CD8+CD28 T cells unprimed, or primed by allogeneic APCs in primary or secondary MLC N = 4, ⁄⁄⁄P < 0.001.

dramatically during the first 3 days of primary MLC, then gradually increased within day 5 and 7 to a level which was still 7–8 times lower than that seen in unprimed cells (Fig. 2). Upon re-stimulation BCL6 expression remained low in CD8+CD28+ T cells (Fig. 2). In contrast, BCL6 expression in CD8+CD28 T cells decreased on day 1 yet increased gradually during the next 6 days to a level that was 6–7 times higher than that detected in CD8+CD28+ T cells (Fig. 2). The progressive increase of BCL6 expression in CD8+CD28 T cells during secondary MLC stimulation was similar to that seen in ILT3.Fc-induced CD8 Ts during 7 days of primary MLC stimulation in medium containing ILT3.Fc. Acquisition of suppressor function by CD8+CD28 T cells was accompanied by high level of BCL6 expression.

To further investigate the changes of BCL6 expression in CD8+CD28 Ts cells, we stimulated CD8+CD28+ and CD8+CD28 T cells for the third time. There was little change of BCL6 expression in CD8+CD28 Ts cells after the third stimulation, as it remained at the same high level as seen at the end of secondary MLC (Fig. 3). 3.2. Expression levels of genes affected by BCL6 in CD8+CD28 Ts cells BCL6 promotes the expression of CXCR4 [30], and represses a group of genes such as IFNc [30], granzyme B [31]. We analyzed the genes affected by BCL6 in both CD8+CD28+ and CD8+CD28 T cells after two stimulations. Fig. 4 shows that CXCR4 expression was significantly higher in CD8+CD28 T cells than in CD8+CD28+ T cells, while IFNc, granzyme B and IL-2 genes were much lower in CD8+CD28 T cells than in CD8+CD28+ T cells.

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3.3. Expression of pro-inflammatory microRNAs and their targeted genes in CD8+CD28 Ts cells In previous studies, we demonstrated that in ILT3.Fc-induced CD8 Ts a number of microRNAs which target BCL6 and other genes with immune-inhibitory functions were down-regulated [29]. We analyzed the expression of miR-21, miR-30b, miR-146a, and miR-155 in CD8+CD28+ and CD8+CD28 T cells after two MLC stimulations. Fig. 5 shows that all four microRNAs were greatly downregulated in CD8+CD28 T cells compared to levels of expression in CD8+CD28+ T cells. BCL6 and CXCR4 are targeted by miR-30b and miR-146a, respectively [29]. Decreased expression of miR-30b and miR-146a correlates with increased expression of BCL6 and CXCR4 in CD8+CD28 Tregs (Figs. 1 and 4). The 30 -UTR site of SOCS1 gene, a negative regulator of cytokine signaling through STAT1 [32], contains potential binding sites for miR-30b and miR-155 [29,33]. MiR-21 targets DUSP10, known as inhibitor of the MAPK pathway and cytokine production [34]. We studied the expression of SOCS1 and DUSP10 expression in CD8+CD28 Ts. Just as in ILT3.Fc induced CD8+ Ts, the expression of both genes was significantly up-regulated in CD8+CD28 Ts, compared to their CD8+CD28+ T cell counterpart (Fig. 6). 4. Discussion Fifteen years ago, our group demonstrated for the first time that antigen-specific MHC class I allorestricted CD8+CD28 T suppressor cells can be generated by multiple T cell stimulation in MLC

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Fig. 3. RT-PCR showing the BCL6 expression dynamics in CD8+CD28+T cells and CD8+CD28 Ts cells during allo-stimulation. (A) Down-regulation of BCL6 expression is observed in both CD8+CD28+ and CD8+CD28 cells one day after priming in primary MLC. (B) Down-regulation of BCL6 expression is observed in CD8+CD28+ but not in CD8+CD28 cells one day after priming in tertiary MLC. n = 3, ⁄P < 0.05, ⁄⁄P < 0.01.

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[15]. We discovered that CD8+CD28 Ts did not suppress CD4+ T helper cells by direct T cell to T cell interaction or cytokine secretion, but rather by acting directly on priming APCs [15,22]. CD8+CD28 Ts induced down-regulation of co-stimulatory molecules [15,22] and up-regulation of inhibitory receptors, such as ILT3 and ILT4 on APCs [23]. We showed that CD4+ or CD8+ T cells which interact with tolerogenic APCs become anergic and develop regulatory function [26].

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At the same time several other groups described a ‘‘temporal bridging’’ model in which CD4+ T helper cells conditioned or licensed the APC to become immunogenic, expressing high levels of co-stimulatory molecules which enabled them to trigger the activation of CD8+ CTL with cognate antigen specificity [35–37]. Our data complemented the finding showing that the APC bridge is the key to switching on or off the immune response according the prevalence of stimulatory or inhibitory membrane molecules.

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Fig. 5. Decreased expression of inflammatory microRNAs in CD8 CD28 Ts cells. Expression of miR-21, miR-30b, miR-146a and miR-155 was significantly lower in CD8+CD28 Ts cells than in their CD8+CD28+ T cell counterpart. N = 3, ⁄P < 0.05, P < 0.01.

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Fig. 6. The inhibitory genes targeted by inflammatory microRNAs are increased in CD8+CD28 Ts cells. SOCS1 targeted by miR-30b and miR-155 and DUSP10 targeted by miR-21 are increased in CD8+CD28 Ts as compared with CD8+CD28+ T cells. N = 3, ⁄⁄P < 0.01, P < 0.001.

Next, we demonstrated that not only membrane but also soluble ILT3 engineered as a recombinant ILT3.Fc molecule induced the differentiation of CD8 Ts during primary MLC. CD8 Ts also have the capacity of inducing tolerogenic APC [26]. In ILT3.Fc induced allospecific CD8 Ts the expression of genes with inhibitory function such as BCL6, CXCR4, SOCS1, and DUSP10 is increased, while that of inflammatory microRNA which target these genes is decreased [28,29]. In this study, we compared the molecular profile of CD8+CD28 Ts with the one of ILT3 induced CD8 Ts. Before priming CD8+CD28+ and CD8+CD28 T cells had no suppressor function, and expressed similar level of BCL6. After priming, the level of BCL6 expression decreased sharply in both populations of T cells. Neither of them

acquired suppressor function. However, after secondary MLC stimulation CD8+CD28 T cells but not CD8+CD28+ T cells from the same culture became Ts and displayed high levels of BCL6. The suppressive function correlated with increased expression of BCL6. The increased level of BCL6 preceded acquisition of Ts function by CD8+CD28 T cells as indicated by the time course analysis of CD8 T cells restimulated in the secondary MLC. Of notice, unprimed CD8+CD28+ and CD8+CD28 T cells had higher levels of BCL6 compared to those seen after primary MLC yet displayed no suppressive function. The BCL6 level was significantly reduced one day after stimulation in primary MLC. However after tertiary stimulation in MLC, BCL6 level remained high in CD8+CD28 Ts cells, as seen at the end of secondary MLC, but decreased further in the CD8+CD28+ subset of the same culture. This indicates that CD8+CD28 Ts cells have to maintain high expression of BCL6 during stimulation for their suppressive function. Evidence has been accumulating that microRNAs, small noncoding RNAs, serve as important regulators of coding genes, which control the differentiation of T cells with diverse effector or regulatory function [38–40]. The role of microRNAs in development, differentiation and function of CD4+ Tregs has been documented [39–42]. Our study on CD8 Ts generated by priming in the presence of ILT3.Fc or by multiple MLC stimulation in medium without ILT3.Fc has demonstrated that expression of certain microRNAs such as miR-21, 30b, miR-146a and miR-155 is down-regulated. Although there is no other literature concerning the role of these microRNAs in CD8 Tregs, their involvement in T cell responses and regulation has been reported. MiR-155 promotes the development of Th17 and Th1 cells in autoimmune inflammation [43]. The lack of miR-155 impaired cytotoxic CD8+ T responses to viral and bacterial infections [44]. Up-regulation of miR-30b was shown to induce down-regulation of BCL6 in B lymphocytes and B lymphoma [45]. The function of miR-146a and miR-21 is not completely understood. MiR-146a was shown to be indispensable for suppression mediated by CD4 Tregs in vivo although miR146a deficient Tregs were as suppressive as miR-146a sufficient Tregs in vitro [42]. Expression of miR-21 in human CD4+CD25+ Tregs was reported to be higher than CD4+CD25 T cells [41]. On the other hand, silencing of miR-21 reverses the activation type

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of T cells in patients with systemic lupus erythematosus [46], and ameliorates autoimmune splenomegaly in a mouse model [47]. MiR-21, miR-146a and miR-155 expression were all significantly increased in both CD4+ and CD8+ T cells from lupus mice as compared to their expression levels in T cells from control mice [47]. The reported function of those microRNAs are mostly consistent with our discovery that down-regulation of miR-21, miR-30b, miR-146 and miR-155 results in the up-regulation of inhibitory genes essential to the generation and function of CD8 Ts cells. In this study, we demonstrated that the molecular profile of CD8+CD28 Ts induced by repetitive in vitro stimulations is similar to CD8 Ts produced by priming in the presence of ILT3.Fc. In the presence of ILT3.Fc CD4+ T cells were anergized and failed to activate APCs. In previous studies we showed that in the absence of CD4+ T cell’ help, CD8+ T cell cannot develop into cytotoxic T cells [26]. Furthermore, other studies showed that less inflammation and IL-2 lead to higher expression of BCL6, and lower expression of granzyme B and perforin in CD8+ T cells [48]. These results are consistent with our discovery that in the presence of ILT3.Fc, CD4+ T cell become anergic, and CD8+ T cells acquire high expression of BCL6 and suppressive rather than CTL activity. The development of CD8+CD28 Ts is more complicated since CD8+CD28+ and CD8+CD28 T cells were cultured under the same conditions in both primary and secondary MLC. We speculate that these two subsets of CD8+ T cells may acquire different sensitivities to inflammatory stimuli from the micro-environments leading to the differentiation of either CD8+CD28+ CTL or CD8+CD28 Ts. A strong parallel can be drawn to other author’s CD4 Treg studies. Unlike CD8 T cells, the CD4 T cell compartment does not contain a significant fraction of CD28 cells. However, CD28 costimulation blockade during priming, in conjunction with an inhibitory receptor signaling (CTLA4 in that case), leads to poor CD4 Th activation and increased development of CD4+CD25+ Treg [49], emphasizing the importance of costimulation through CD28 as a checkpoint for activation versus acquisition of regulatory function. The common molecular profile of ILT3-induced CD8 Ts and CD8+CD28 Ts suggests that by changing the expression of Ts signature genes the immune responses could be manipulated. It has been demonstrated that exosomes contain mRNA and microRNA, which can be delivered to another cells [50]. Other methods, such as LNA (locked nucleic acid)-modified ant-microRNA, have been described to treat diseases in animal systems [47,51]. The existence of CD8 Ts, CD8+CD28 Ts and soluble ILT3 protein in circulation, and their association with disease status have been documented in transplantation, autoimmune disease, and cancer of both human and animal systems by us [25,27,28,52,53] and other groups [13,18–20,24]. Delivering specific microRNAs and mRNA to CD8+T cells with exosome or LNA-modified microRNA to increase or decrease CD8 Ts or CD8+CD28 Ts may become an effective therapeutic approach. We postulate that the level of expression of the CD28 costimulatory receptor on CD8 T cells at certain times during stimulation may control or reflect its susceptibility to inflammatory or inhibitory factors within the micromilieu in which they are primed. Manipulating BCL6 or microRNA expression, and propagating these cells in vitro may provide a tool for alloantigen specific suppression in clinical transplantation.

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