Bone Marrow Transplantation (2013) 48, 124–128 & 2013 Macmillan Publishers Limited All rights reserved 0268-3369/13 www.nature.com/bmt

ORIGINAL ARTICLE Effects of ICOSLG expressed in mouse hematological neoplasm cell lines in the GVL reaction

B Wang1,2,NMa3, H Cheng1, H Zhou1, H Qiu1, J Yang1 and J Wang1

As a member of the B7 family, inducible co-stimulator ligand (ICOSLG) expressed on tumor cell has been reported to have an important role in tumor immunity. In this study, we sought to determine whether the expression of ICOSLG in mouse hematological malignancy cells influences GVL reaction after mouse allogeneic BMT. In our study, we analyzed the expression of ICOSLG in six mice hematological malignancy cell lines for the first time, and found that FBL3, A20 and P388 cells expressed high levels of ICOSLG. Then, we chose A20 cells as targets to construct a GVL model and study the effects on the GVL reaction by silencing the ICOSLG gene. The survival was analyzed. We found that in GVL model, mortality of interference groups was significantly delayed compared with the control group (P ¼ 0.0005). Our results indicate that knockdown of ICOSLG of mouse leukemic cells may significantly enhance GVL effect after allogeneic BMT.

Bone Marrow Transplantation (2013) 48, 124–128; doi:10.1038/bmt.2012.103; published online 25 June 2012 Keywords: mice; leukemia; ICOSLG; GVL; BMT

INTRODUCTION shorter survival time. Another study showed that human myeloma Allogeneic hematopoietic SCT (allo-HSCT) is a potentially curative cell lines commonly expressed B7.2 and ICOSLG molecules. Plasma therapy for hematological malignancies and GVL activity by cells expressing ICOSLG were observed in myeloma patients alloreactive donor T cells has an important role in the prevention alone, while not in patients with monoclonal gammopathy of of post-transplantation tumor relapse. However, alloreactive donor unknown significance or hematologically normal individuals and T cells are also responsible for GVHD, a major cause of post- that expression of these molecules was associated with a growth transplantation morbidity and mortality. Thus, a major challenge advantage in vitro.20 Furthermore, this study provided evidence for allo-HSCT is to inhibit GVHD while maintaining the beneficial that these molecules were involved in the pathophysiology of effects of T-cell-mediated GVL activity. multiple myeloma. Optimal activation of T cells requires two signals: the first signal ICOSLG is a positive co-stimulatory molecule that has a primary is delivered by the engagement of the T-cell receptor with the role in Th2 responses.21 Furthermore, ICOS blockade reduces the peptide–MHC complex on APC, and the second signal is a co- production of Th2 cytokines but not type 1T helper cell stimulatory signal. Inducible co-stimulator (ICOS) is a relatively cytokines.22 Therefore, the ICOSLG-ICOS signal most likely favors recently characterized member of the B7 family of immune- the induction of Th2-type responses, and thus may be involved in regulatory ligands, which is involved in the co-stimulation of T the negative regulation of cell-mediated immune responses cells and is expressed only on activated T cells.1–3 against tumors. The role of ICOSLG expressed on hematological The first member of the B7 family, B7-1, was identified over 20 neoplasms in tumor immunity is not clear now. Although it have years ago and revealed to have co-stimulatory function when revealed that a variety of human hematological cell lines, leukemic ligated to the CD28 receptor on T cells.4–7 Several new members of cells from acute myeloid leukemia patients, lymphoma cells and the B7 family, such as B7-H1, ICOS ligand (LG), B7-H3 and B7-H4, plasma cells express ICOSLG,19,23 it has not been reported whether have been identified since then.8,9 As the counter ligand of ICOS, a ICOSLG molecules are expressed in cell lines of mouse CD28-related molecule, ICOSLG is expressed on professional APC hematological malignancies. (such as DCs and B cells) and on some tumor cells (such as glioma In our previous study,24 we choose mouse hematological cells and gastric carcinoma cells).10–13 ICOSLG is the only B7 family malignancies as objects and used flow cytometry to analyze the member that preferentially co-stimulates type 2T helper cell (Th2) expression of ICOSLG in six mouse hematological neoplasm cell responses, and interestingly, this unique molecule has a critical role lines. We found that FBL3, A20 and P388 cells expressed high in antitumor immunity.14–16 ICOSLG expression on solid tumors levels of ICOSLG, whereas L6565, YAC-1 and EL4 cells showed little (implanted-transfected tumors) aids in both NK mediated and to no expression of ICOSLG. Based upon ICOSLG expression, we CD8 þ CTL activation and killing.16 Several studies using ICOSLG- chose one erythroid malignancy cell line(FBL3) and one transfected solid tumor cell lines found that ICOSLG induced lymphocytic malignancy cell line(A20) as target cells to CD8 þ cytotoxic lymphocyte-mediated tumor regression.17,18 determine whether the blockade of ICOSLG influences Conversely, Tamura et al.19 reported that the expression of cytotoxicity in allogeneic mixed lymphocyte—hematological ICOSLG in acute myeloid leukemic cells was associated with a neoplasm cell reaction. We directly blocked the ICOSLG molecule

1Department of Hematology, Changhai Hospital, The Second Military Medical University, Shanghai, China; 2Department of Oncology, Changhai Hospital, The Second Military Medical University, Shanghai, China and 3 Clinical Laboratory, Shanghai, 85th Hospital PLA, China. Correspondence: Professor J Wang, Department of Hematology, Changhai Hospital, The Second Military Medical University, 168 Changhai Road, Shanghai 200433, China. E-mail: [email protected] Received 12 January 2012; revised 26 April 2012; accepted 1 May 2012; published online 25 June 2012 Effects of ICOSLG in the GVL reaction B Wang et al 125 with anti-mouse ICOSLG antibody and demonstrated that 50-TTCAAGACG-30. A non-silencing siRNA was used as a negative control. blocking ICOSLG was sufficient to enhance the cytotoxicity of The complementary sequences of this control siRNA were as follows: alloreactive T cells against mouse hematological neoplasm cells. (50-TTCTCCGAACGTGTCACGT-30)50-TTCAAGAGA-30 (50-ACGTGACACGT TCG 0 30 Our data also suggest that when ICOSLG was blocked, production GAGAA-3 ). of the two Th2 cytokines (IL-4 and IL-10) noticeably decreased, but the blockade did not significantly influence the production of the Real-time reverse transcription-PCR two type 1T helper cell (IFN-g and IL-2) cytokines. We used the RNAiso Plus kit for RNA isolation, the PrimeScript RT reagent Previous studies showed that ICOS blockade may be able to kit for reverse transcription and the SYBR Premix Ex Taq kit for real-time inhibit or treat GVHD while GVL activity is slightly diminished.25–27 PCR (Takara Bio Inc., Shiga, Japan). All procedures were carried out However, as far as the ICOSLG molecule that expressed on according to the manufacturer’s instructions. The real-time PCR was performed on cDNA from all six cell lines using an ABI Prism 7700 leukemic cells is concerned, which role it has on the GVL reaction Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The after allogeneic BMT has not yet been reported. A study has primers used to amplify a 165-bp product of B7-H2 were 50-TGG AAG AGG revealed that the administration of soluble ICOSLG-Fc may delay TGG TCA GGC-30 (forward) and 50-TTA GGC TAT TGT CCG TTG-30 (reverse). tumor growth in some mouse hematological neoplasms in B7-H2 mRNA expression was normalized to that of b-actin, and the primers syngeneic hosts.28 In this study, we analyzed the expression of used to amplify a 266-bp product of b-actin were as follows: 50-GTC CCT ICOSLG in FBL3, A20 and P388 cell lines again, and chose A20 as CAC CCT CCC AAA AG-30 (forward) and 50-GCT GCC TCA ACA CCT CAA C target cells to construct a GVL model. Then, we introduced shRNA CC-30 (reverse). into A20 cells to knockdown the ICOSLG gene to represent its effect on the GVL reaction after allogeneic BMT for the first time. IHA20 sort, in vitro transfection and G418 selection ICOSLG-positive A20 cells were sorted using a FACS Vantage cell sorter according to the manufacturer’s instructions (BD Biosciences). Those cells MATERIALS AND METHODS were diluted for a limiting value. The cells that express ICOSLG highly were Cell lines and cell culture selected by inverted microscope and cultured for days. We called these cells IHA20 (ICOSLG highly expressed in A20 cells). IHA20 cells were plated Three mouse cell lines derived from hematological malignancies were on 24-well culture plates (at a density of 7 Â 105 cells/well). After an used. A20, FBL3 and P388 were all obtained from the Cell Bank of Type overnight incubation, transfection of pGSci or pGSci-shRNA was performed Culture Collection, Shanghai Institute of Cell Biology, Chinese Academy of using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) at a final Sciences (Shanghai, China). These cells were cultured in RPMI 1640 concentration of 2 mg pDNA/mL according to the manufacturer’s instruc- medium with 10% FCS, 100 units/mL penicillin, 100 mg/mL streptomycin tions. In brief, 1 mg ICOSLG-specific shRNA expressing plasmids pGSci- and 2 mML-glutamine. shRNA, or negative control pGSci-DNA, was mixed with 3 mg Lipofectamine 2000 (Invitrogen), in 500uL serum-free medium per well. At 8 h after Mice and allo-BMT transfection, the medium was replaced by fresh RPMI-1640 medium with C57BL/6 (B6) and BALB/c mice were obtained from the Shanghai SLAC 10% FBS and cultured for 48 h. Those cells were selected using 800 ug/mL Laboratory Animal Co., Ltd (Shanghai, China). Mice used in allo-HSCT of G418 for 14–20 days to obtain a clone expressing the highest EGFP and experiments were between 6 and 8 weeks of age. Referring to the neomycin-resistant activity. experimental method of our colleagues Tao et al.,29 BM cells were removed aseptically from femurs and tibias of donor mice. Splenocytes were The proliferation of selected cells was measured by cell count obtained by centrifugation in lymphocyte-M. T cells were calculated in in vitro 6 5 splenocytes. Cells (5 Â 10 BM with or without 4.5 Â 10 splenic T In all, 1.0 Â 104 cells were plated onto a 24-well multiwell plate in 1 mL of lymphocytes) were resuspended in PBS medium and transplanted by tail medium consisted of RPMI 1640 supplemented with 10% FCS. The number vein infusion (0.25 mL total volume) into lethally irradiated recipients on 60 of the cells of the three samples was calculated with counting plate at the day 0. Before transplantation, on day 0, recipients received 9.0 Gy TBI ( Co same time everyday for 6 days. The proliferation curves of the three source). Mice were housed in sterilized microisolator cages and received different cell populations IHA20, IHA20-control shRNA and IHA20-shRNA normal chow and autoclaved hyperchlorinated drinking water. They were were drawn. maintained under standard conditions and cared for according to the Second Military Medical University guidelines for animal care. All animal experiments were approved by that institution’s Committee on the Use of Tumor induction and the erect of GVL model Live Animals in Teaching and Research. To assess the effects of ICOSLG gene knockdown on GVL activity of alloreactive T cells, we constructed the GVL model: C57B6-BALB/c with IHA20. In this model, animals received 8 Â 104 leukemia cells intravenously Flow cytometry in a separate injection on day 0 of allo-HSCT, and a low dose of donor To analyze the expression of cell surface proteins, we incubated our cells splenic T-lymphocytes cells (4.5 Â 105) was used to decrease GVHD with phycoerythrin-conjugated anti-mouse ICOSLG (BD Pharmingen, mortality and allow for a better measurement of GVL activity. Survivals San Diego, CA, USA). Isotype-matched negative controls were used in all were monitored daily. The cause of death was determined by necropsy assays. Cells were analyzed on a FACScan cytometer according to the and histopathology. Briefly, all animals with macroscopic liver/spleen manufacturer’s instructions (BD Biosciences, San Diego, CA, USA). The metastasis at autopsy were recorded as tumor deaths. The spleens and CellQuest software ver.3.2 (BD Bioscience) was used to analyze the results. livers from animals that did not exhibit any gross evidence of tumor were The enhanced green fluorescent protein (EGFP) was also analyzed by flow analyzed by histopathology for evidence of microscopic tumor, and cause cytometry. of death was subsequently determined. If no evidence of tumor was detected and the animals displayed signs of GVHD, the cause of death was Construction of expression plasmids established as GVHD. If tissues were lysed at autopsy the cause of death was labeled as not analyzed. In addition, mice splenic lymphocytes of ICOSL-specific siRNAs were designed with BLOCK-IT RNAi Designer (at control groups and interference groups were obtained at day 24. The https://rnaidesigner.invitrogen.com/rnaiexpress/), and synthesized using percentage of EGFP-positive cells in the two groups was calculated. the chemical synthesis method by GeneChem Co. (Shanghai, China). Plasmids expressing siRNA were under the control of the human U6 promoter in pGSci/U6 vector (GeneChem Co.). The pGSci vector contains Statistical analysis the H1 promoter driving the siRNA cassette together with a CMV-driven Comparison between groups was done by analysis of variance. The coral EGFP (EGFP) cDNA and neomycin-resistant gene. The complementary statistical significance of differences was determined by Student’s two- sequences of siRNA were as follows: 50-CACTCAGCTTCACAACATCAATT tailed t-test in two groups and one-way analysis of variance in multiple CAAGACGTTGATGTTGTGAAGCTGAGTG-30, which forms stem-loop struc- groups with the SAS 9.1.3 software (Shanghai, China). The Mantel–Cox log- tured siRNA, targeted to Mus ICOS ligand (ICOSLG) mRNA (targeted rank test was used for survival data. P-values of o0.05 were considered to sequence: 50-CTCAGCTTCACAACATCAA-30), with loop sequences of be statistically significant.

& 2013 Macmillan Publishers Limited Bone Marrow Transplantation (2013) 124 – 128 Effects of ICOSLG in the GVL reaction B Wang et al 126 RESULTS IHA20, IHA20-control shRNA and IHA20-shRNA groups (P40.05; ICOSLG is highly expressed in three mouse leukemic cell lines Figure 3). Using flow cytometry, we found that FBL3, A20 and P388 cells expressed high levels of ICOSLG. The rates of ICOSLG þ cells were ± ± Knock-down of ICOSLG gene of mice A20 cell may significantly as follows: 85.22 0.24% of FBL3 cells, 47.13 0.44% of A20 delayed mortality enhance in our GVL model and 21.56±0.56% of P388 (Figure 1a). These results have been confirmed by real-time reverse transcription-PCR (Figure 1b). In this GVL model, we observed that all the mice in the group for GVHD that received alloHSCs and donor splenic lymphocytes cells were still alive at the end of our observation period (Figure 4a). We Silencing of ICOSLG by shRNA also observed that no mortality without tumor challenge in mice After sorting with a FACS Vantage cell sorter, the cells that express that received IHA20-control shRNA or IHA20-shRNA cells (data not ICOSLG highly were selected and cultured for 14 days. The positive shown). rate of IHA20 was steady at 85.22±2.04%. After transfection and Mortality of interference groups for GVL that received alloHSCs G418 selection, the ICOSLG-positive rates of IHA20-control shRNA with donor splenic lymphocytes and IHA20-shRNA cells was and IHA20-shRNA was 84.33±4.11 and 16.01±2.14%, respec- significantly delayed compared with control groups for GVL that tively, (Po0.01; Figures 2a–c), ICOSLG mRNA in shRNA-transfected received alloHSCs with donor splenic lymphocytes and IHA20- IHA20 cells reduced (95.78±0.18)% compared with that in the control shRNA cells, while the mortality of the latter one was control (Po0.01; Figure 2d). The proliferation was calculated also significantly delayed compared with the tumor challenge group by cell counting and there were no significant differences among that received alloHSCs and IHA20-control shRNA cells (Figure 4a).

FBL3 A20 P388 30 200 200 300 25 240 160 160 M1 20 85.22% M1 120 120 (0.24) 180 47.13% M1 15 120 (0.44) 80 21.56% 80 Counts Counts Counts (0.56) 10 40 60 40 0 0 0 5 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Ratio of ICOSL /action (%) 0 ICOSL PE ICOSL PE ICOSL PE FBL3 A20 P388 Figure 1. ICOSLG expression in six mouse hematological neoplasms cell lines. (a) Histograms of flow cytometric analysis. The black line represents the negative isotypic control group; the gray line represents the ICOSLG þ group. M1: ICOSLG þ cells. (b) The number of transcript copies of ICOSLG per nanogram of input RNA, as determined by real-time reverse transcription-PCR (mean±s.d.; n ¼ 3).

A20 IHA20 678 116 300 400 1 15 1 82 4 4 240 320 10 10 M1 103 103 180 M1 240 85.21% 120 47.13% 160 (2.02) 102 102 (0.44) 1 1

Counts 60 80 10 10

0 ICOSLG-PE 100 100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

ICOSL-PE EGFP

** ** 140 140 * * 120 120 100 100 80 80 60 60 actin (%) actin (%) 40 40 20 20 Ratio of ICOSLG mRNA/ Ratio of ICOSLG mRNA/ 0 0 IHA20-control IHA20 IHA20- IHA20-control IHA20 IHA20- shRNA shRNA shRNA shRNA Figure 2. Inhibition of ICOSLG expression. (a, b) Flow cytometry analysis the ICOSLG-positive rate in A20, IHA20, IHA20-control shRNA and IHA20-shRNA. (c) Statistical analysis showed that the ICOSLG expression had no difference between IHA20 and IHA20-control shRNA cells. (*P ¼ 0.1804, n ¼ 3). The degree of reduction in ICOSLG expression in IHA20-shRNA was (80.92±1.63)% compared with that in IHA20-control shRNA (**Po0.01, n ¼ 3). (d) Silencing of ICOSLG detected by Real-time PCR. The mRNA level was examined by real-time reverse transcription- PCR. No Statistical difference in the mRNA level between IHA20 and control IHA20-pGSci cells. (*P ¼ 0.4236, n ¼ 3). The degree of reduction in ICOSLG mRNA with pGSci-shRNA was (85.01±0.41)% compared with the control plasmid pGSci. (**Po0.00005, n ¼ 3).

Bone Marrow Transplantation (2013) 124 – 128 & 2013 Macmillan Publishers Limited Effects of ICOSLG in the GVL reaction B Wang et al 127 Knockdown of ICOSLG gene of mice A20 cell may significantly embedded, sectioned, and hematoxylin and eosin stained. delay tumor progression in our GVL model Figure 4d is the representative photograph of infiltration of the Spleens of interference groups and control groups were removed liver by leukemia cells in interference groups compared with that and lymphocytes were obtained by centrifugation in lymphocyte- in control groups. M at day 24. EGFP-positive cells (leukemia cells) were analyzed on a FACScan cytometer. The percentage of EGFP-positive cells in the two groups is (17.65±0.07)% and (2.10±0.03), respectively, DISCUSSION (Figures 4b and c). Mouse liver and spleen were paraffin We introduced ICOSLG-specific shRNA to knockdown the ICOSLG gene of IHA20 cell. The ICOSLG-positive rates reduced from 84.33±4.11 to 16.01±2.14% and ICOSLG mRNA reduced (95.78±0.18)% than before. In our GVL model, mortality of 600 interference groups for GVL was significantly delayed compared with control groups for GVL. We also observed that mortality was IHA20-shRNA 500 no difference between the group received alloHSCs with IHA20- IHA20-control shRNA shRNA and the group received alloHSCs with IHA20-control shRNA cells. These indicate that it is not the difference of the cell IHA20 ) 400 between IHA20-control shRNA and IHA20-shRNA but the GVL 4 effect that made mortality difference between interference groups for GVL and control groups for GVL. 300 We all know that recognition by donor T cells of leukemia- restricted antigens would require either direct antigen processing and presentation by the leukemia, or presentation of leukemia Counts (1x10 200 antigens by hostor donor-derived professional APC. The naive T cells were primed to the Th2 cells after direct antigen presentation by the leukemia and conjunction of ICOS and ICOSLG. It is 100 favorable for leukemia cells to grow with high concentration of Th2 cytokine around them. We blocked the direct antigen 0 presentation path by silencing of its ICOSLG gene to inhibit the 123456 growth of leukemia and so enhance the GVL effect. 27 À / À Time (day) Hubbard et al. reported that alloreactive ICOS donor T cells induce less GVHD while GVL activity is slightly diminished. In Figure 3. The effect of ICOSLG-shRNA transfection on cell prolifera- 4 this study, not only direct antigen presentation path but also tion. In all, 1.0 Â 10 cells were plated onto a 24-well multiwell plate indirect antigen presentation path was blocked. The GVL effect in 1 mL of medium. The number of the different kinds of cells was calculated with counting plate at the same time everyday for 6 days. through presentation of leukemia antigens by APC cannot achieve The graph shows the average of three separate experiments. We and this partly covered the enhancement to GVL through direct found that no significant influence to cell proliferation was observed path. 31 À / À in IHA20-shRNA cells and IHA20-control shRNA cells (P40.05). There Yu et al. found that recipients of ICOS CD4 þ T cells was no significant influence between IHA20-shRNA cells and IHA20 exhibited significantly less GVHD morbidity and delayed mortality cells (P40.05). Cell growth curve was plotted against time. while recipients of ICOS À / À CD8 þ T cells exhibited significantly

20 B6 BALB/C 18 100 16 BM+IHA20-control shRNA 14 80 BM+IHA20-shRNA P =0.0001 12 BM+SC+IHA20-control shRNA 60 BM+SC+IHA20-shRNA 10 40 BM+SC 8 6 % survival 20 4 % EGFP positive cells 0 2 0 01020 30 40 50 Control groups Interference groups Days post BMT for GVL for GVL

Control groups Interference groups for GVL for GVL 4 4 10 64 1810 42 2 3 3 10 18 0010 51 102 102 101 101 ICOSLG-PE 100 100 100 101 102 103 104 100 101 102 103 104 IHA20-shRNA IHA20-control shRNA EGFP Figure 4. Knockdown of ICOSLG gene of mouse A20 cell significantly enhanced GVL effect. (a) B6 into BALB/c and challenge with A20. B6 mice were lethally irradiated (900 cGy) and received transplants according to the methods described in section 2 (n ¼ 10). Statistical analyses: ~ versus ., P ¼ 0.64; m versus ~, P ¼ 0.0004; and ’ versus m, P ¼ 0.0005. (b, c) Mouse splenic lymphocytes of control groups and interference groups were obtained at day 24. The percentage of EGFP-positive cells in the two groups is (17.65±0.07) and (2.10±0.03)%. The percentage of EGFP-positive cells in the former than the latter (P ¼ 0.0001). (d) Liver histopathology in recipients infused HBA20-shRNA (left panel) and infused HBA20-control shRNA (right panel). Arrowheads denote leukemia cells. Note the tumor cells. Original magnification  100.

& 2013 Macmillan Publishers Limited Bone Marrow Transplantation (2013) 124 – 128 Effects of ICOSLG in the GVL reaction B Wang et al 128 enhanced GVHD morbidity and accelerated mortality. ICOS þ 11 Schreiner B, Wischhusen J, Mitsdoerffer M, Schneider D, Bornemann A, Melms A CD4 þ T cells might have a more important role in GVHD. et al. Expression of the B7-related molecule ICOSL by human glioma cells in vitro 32 In recent years, Strauss et al. found that CD4 þ CD25 þ Treg in and in vivo. Glia 2003; 44: 296–301. tumor-infiltrating lymphocytes expressed ICOS, while Treg in 12 Wang S, Zhu G, Chapoval AI, Dong H, Tamada K, Ni J et al. Costimulation of PBMC had low ICOS expression. ICOShigh Treg upregulated Treg T cells by B7-H2, a B7-like molecule that binds ICOS. Blood 2000; 96: markers and mediated stronger suppression relative to ICOSlow 2808–2813. Treg partly mediated via IL-10 and transforming growth factor-b. 13 Yoshinaga SK, Whoriskey JS, Khare SD, Sarmiento U, Guo J, Horan T et al. T-cell co- We all know that the cytokine environment in vivo is very stimulation through B7RP-1 and ICOS. Nature 1999; 402: 827–832. 14 Liang L, Sha WC. The right place at the right time: novel B7 family members complex, and the change of cytokines when ICOS/ICOSLG blocked 27 regulate effector T cell responses. Curr Opin Immunol 2002; 14: 384–390. still remains controversial in different disease models. Also, the 15 Flies DB, Chen L. Modulation of immune response by B7 family molecules in GVL reaction does not only depend on cytokines. Why knockdown tumor microenvironments. Immunol Invest 2006; 35: 395–418. of ICOSLG gene of mice leukemic cell significantly enhances GVL 16 Flies DB, Chen L. The new B7s: playing a pivotal role in tumor immunity. effect after allogeneic BMT perhaps needs to be studied further. J Immunother 2007; 30: 251–260. 17 Liu X, Bai XF, Wen J, Gao JX, Liu J, Lu P et al. B7H costimulates clonal expansion of, and cognate destruction of tumor cells by, CD8 ( þ ) T lymphocytes in vivo. J Exp CONCLUSIONS Med 2001; 194: 1339–1348. In summary, our results indicate that knockdown of ICOSLG gene 18 Wallin JJ, Liang L, Bakardjiev A, Sha WC. Enhancement of CD8 þ T cell responses by ICOS/B7h costimulation. J Immunol 2001; 167: 132–139. of mice leukemic cell may significantly enhance GVL effect after 19 Tamura H, Dan K, Tamada K, Nakamura K, Shioi Y, Hyodo H et al. Expression allogeneic BMT. In our study, it is the ICOSLG molecule expressed of functional B7-H2 and B7.2 costimulatory molecules and their prognostic on leukemic cell that we focused on. Just as previous studies implications in de novo acute myeloid leukemia. Clin Cancer Res 2005; 11: showed that ICOS blockade may be able to inhibit or treat 5708–5717. 26,33 GVHD, our study indicates that ICOSLG blockade may provide 20 Yamashita T, Tamura H, Satoh C, Shinya E, Takahashi H, Chen L et al. Functional a new strategy to enhance the GVL effect in allogeneic PBSC B7.2 and B7-H2 molecules on myeloma cells are associated with a growth transplantation. advantage. Clin Cancer Res 2009; 15: 770–777. 21 Vieira PL, Wassink L, Smith LM, Nam S, Kingsbury GA, Gutierrez-Ramos JC et al. ICOS-mediated signaling regulates cytokine production by human T cells and CONFLICT OF INTEREST provides a unique signal to selectively control the clonal expansion of Th2 helper cells. Eur J Immunol 2004; 34: 1282–1290. The authors declare no conflict of interests. 22 Coyle AJ, Lehar S, Lloyd C, Tian J, Delaney T, Manning S et al. The CD28-related molecule ICOS is required for effective T cell-dependent immune responses. Immunity 2000; 13: 95–105. ACKNOWLEDGEMENTS 23 Wang F, Zhu W, Liu T, Sun Z, Ju S, Ju S et al. The expression analysis of ICOS-L on This work was supported in part by grants from the National Natural Science activated T cells and immature dendritic cells as well as malignant B cells and Foundation of China (NSFC No. 30871100, 81090110) to JMW, NSFC No. C140402 Grave’s-disease-derived thyroid tissues by two novel mAbs against human ICOS-L. to HYQ. Tissue Antigens 2007; 69: 62–72. 24 Wang B, Cheng H, Wang L, Zhou H, Wang J. Expression of ICOSLG on mouse hematologic neoplasm cell lines and their influence on cytotoxicity in allogeneic REFERENCES mixed lymphocyte reactions. Leuk Lymphoma 2012; 53: 674–680. 1 Her M, Kim D, Oh M, Jeong H, Choi I. Increased expression of soluble inducible 25 Chen BJ, Cui X, Liu C, Chao NJ. Prevention of graft-versus-host disease costimulator ligand (ICOSL) in patients with systemic lupus erythematosus. Lupus while preserving graft-versus-leukemia effect after selective depletion of 2009; 18: 501–507. host-reactive T cells by photodynamic cell purging process. Blood 2002; 99: 2 Hutloff A, Dittrich AM, Beier KC, Eljaschewitsch B, Kraft R, Anagnostopoulos I et al. 3083–3088. ICOS is an inducible T-cell co-stimulator structurally and functionally related to 26 Taylor PA, Panoskaltsis-Mortari A, Freeman GJ, Sharpe AH, Noelle RJ, Rudensky AY CD28. Nature 1999; 397: 263–266. et al. Targeting of inducible costimulator (ICOS) expressed on alloreactive T cells 3 Ling V, Wu PW, Finnerty HF, Bean KM, Spaulding V, Fouser LA, Leonard JP et al. down-regulates graft-versus-host disease (GVHD) and facilitates engraftment of Cutting edge: identification of GL50, a novel B7-like protein that functionally allogeneic bone marrow (BM). Blood 2005; 105: 3372–3380. binds to ICOS receptor. J. Immunol 2000; 164: 1653–1657. 27 Hubbard VM, Eng JM, Ramirez-Montagut T, Tjoe KH, Muriglan SJ, Kochman AA 4 Freedman AS, Freeman GJ, Rhynhart K, Nadler LM. Selective induction of B7/BB-1 et al. Absence of inducible costimulator on alloreactive T cells reduces graft on interferon-gamma stimulated monocytes: a potential mechanism for amplifi- versus host disease and induces Th2 deviation. Blood 2005; 106: 3285–3292. cation of T cell activation through the CD28 pathway. Cell Immunol 1991; 137: 28 Ara G, Baher A, Storm N, Horan T, Baikalov C, Brisan E et al. Potent activity of 429–437. soluble B7RP-1-Fc in therapy of murine tumors in syngeneic hosts. Int J Cancer 5 Linsley PS, Clark EA, Ledbetter JA. T-cell antigen CD28 mediates adhesion with B 2003; 103: 501–507. cells by interacting with activation antigen B7/BB-1. Proc Natl Acad Sci USA 1990; 29 Tao Y, Zhang W, Fang Y, Yang D, Wang L, Zhou H et al. Bortezomib attenuates 87: 5031–5035. acute graft-vs.-host disease through interfering with host immature dendritic 6 Yokochi T, Holly RD, Clark EA. B lymphoblast antigen (BB-1) expressed on epstein- cells. Exp Hematol 2011; 39: 710–720. barr virus-activated B cell blasts, B lymphoblastoid cell lines, and burkitt’s 30 Bektas M, Payne SG, Liu H, Goparaju S, Milstien S, Spiegel S. A novel acylglycerol lymphomas. J Immunol 1982; 128: 823–827. kinase that produces lysophosphatidic acid modulates cross talk with EGFR in 7 Bretscher P, Cohn M. A theory of self-nonself discrimination. Science 1970; 169: prostate cancer cells. J Cell Biol 2005; 169: 801–811. 1042–1049. 31 Yu XZ, Liang Y, Nurieva RI, Guo F, Anasetti C, Dong C. Opposing effects of ICOS on 8 Subudhi SK, Zhou P, Yerian LM, Chin RK, Lo JC, Anders RA et al. Local expression of graft-versus-host disease mediated by CD4 and CD8 T cells. J Immunol 2006; 176: B7-H1 promotes organ-specific autoimmunity and transplant rejection. J Clin 7394–7401. Invest 2004; 113: 694–700. 32 Strauss L, Bergmann C, Szczepanski MJ, Lang S, Kirkwood JM, Whiteside TL. 9 Swallow MM, Wallin JJ, Sha WC. B7h, a novel costimulatory homolog of B7.1 and Expression of ICOS on human melanoma-infiltrating CD4 þ CD25highFoxp3 þ T B7.2, is induced by TNFalpha. Immunity 1999; 11: 423–432. regulatory cells: implications and impact on tumor-mediated immune suppres- 10 Chen XL, Cao XD, Kang AJ, Wang KM, Su BS, Wang YL. In situ expression and sion. J Immunol 2008; 180: 2967–2980. significance of B7 costimulatory molecules within tissues of human gastric car- 33 Frauwirth KA, Thompson CB. Activation and inhibition of lymphocytes by costi- cinoma. World J Gastroenterol 2003; 9: 1370–1373. mulation. J Clin Invest 2002; 109: 295–299.

Bone Marrow Transplantation (2013) 124 – 128 & 2013 Macmillan Publishers Limited