TRIM-00960; No of Pages 8 Transplant Immunology xxx (2015) xxx–xxx

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Anti-OX40L monoclonal antibody prolongs secondary heart allograft survival based on CD40/CD40L and LFA-1/ICAM-1 blockade Helong Dai a,1, Fenghua Peng a,2, Minjie Lin b,3, Junjie Xia c,4, Shaojie Yu a,5, Gongbin Lan a,6, Yu Wang a,7, Xubiao Xie a,8, Chunhua Fang a,9, Matthias Corbascio d,10, Zhongquan Qi c,⁎,11, Longkai Peng a,⁎⁎,12 a

Department of Urological Organ Transplantation, Center of Organ Transplantation, Second Xiangya Hospital, Central South University, Hunan Province, PR China Department of Cardiology, Second Xiangya Hospital, Central South University, Hunan Province, PR China Organ Transplantation Institute, Xiamen University, Fujian Province, PR China d Malmö Hospital of Lund University, Malmö, Sweden b c

a r t i c l e

i n f o

Article history: Received 1 October 2014 Received in revised form 11 January 2015 Accepted 12 January 2015 Available online xxxx Keywords: Anti-OX40L mAb Memory T cell Regulatory T cell Heart transplantation

a b s t r a c t Background: Memory T cells (Tms) form a barrier against long-term allograft survival; however, CD4+Foxp3+ regulatory T cells (Tregs) can suppress allograft rejection. The OX40/OX40L pathway is critical to the generation of Tms and turns off Treg suppressor function. Methods: B6 mice that rejected BALB/c skin grafts after 4 weeks were used as the secondary heart transplant recipients. The skin recipient mice, termed S0, S2 and S3, were treated with the isotype antibodies, anti-CD40L/LFA1 or anti-OX40L combined with anti-CD40L/LFA-1 mAbs, respectively. The secondary heart recipients, termed H0 and H2, received anti-CD40L/LFA-1 mAbs or not, respectively (Fig. 1). Results: Four weeks after primary skin transplantation, the Tms in the S3 group that received anti-OX40L with anti-CD40L/LFA-1 mAbs were reduced compared to those in the S2 group (CD4+ Tm: 32.61 ± 2.20% in S2 vs. 25.36 ± 1.16% in S3; CD8+ Tm: 27.76 ± 1.96% in S2 vs. 20.95 ± 1.30% in S3; P b 0.01). Meanwhile, the proportions of Tregs in S3 increased compared to those in S2 (P b 0.05). The anti-OX40L with anti-CD40L/LFA-1 mAbs group (S3H2) prolonged the mean survival time (MST) following secondary heart transplantation from 9.5 days to 21 days (P b 0.001). Furthermore, allogeneic proliferation of recipient splenic T cells and graft-infiltrating lymphocytes were significantly inhibited in the S3H2 group. Additionally, a higher level of IL-10 was detected in sera and allografts. Conclusions: Anti-OX40L mAb could prolong secondary heart allograft survival based on CD40/CD40L and LFA-1/ ICAM-1 blockade. The mechanism of protecting allografts using anti-OX40L mAb involved impairing the generation of Tm and up-regulating IL-10 producing Tregs, inhibiting the function of T cells. © 2015 Elsevier B.V. All rights reserved.

⁎ Correspondence to: Z. Qi, Organ Transplantation Institute, Xiamen University, Fujian Province 361005, PR China. Tel.: +86 731 85295141. ⁎⁎ Correspondence to: L. Peng, Department of Urological Organ Transplantation, Center of Organ Transplantation, Second Xiangya Hospital, Central South University, Hunan Province 410011, PR China. E-mail address: [email protected] (L. Peng). 1 Helong Dai, performed research and wrote the paper. 2 Fenghua Peng, performed heart transplantation and modified the paper. 3 Minjie Lin, modified the paper. 4 Junjie Xia, performed flow cytometry. 5 Shaojie Yu, performed heart transplantation. 6 Gongbin Lan, performed mixed lymphocyte reaction (MLR) assays. 7 Yu Wang, performed qRT-PCR. 8 Xubiao Xie, performed skin transplantation. 9 Chunhua Fang, performed ELISAs. 10 Matthias Corbascio, contributed important reagents. 11 Zhongquan Qi, contributed supporting funds. 12 Longkai Peng, contributed important reagents and supporting funds.

1 . Introduction Many studies have shown that transplant recipients can develop alloreactive memory T cells (Tms) after exposure to alloantigen during previous transplantations, pregnancies, blood transfusions, as well as due to continuous exposure to bacteria or viruses [1,2]. The two main subsets of Tms are CD4+ Tm and CD8+ Tm, which express cell surface markers such as CD44, CCR7 and CD62L that distinguish them from naive counterparts [3]. Tms have enhanced functions and mediate accelerated graft rejection, which makes them the most difficult obstacle to extend allograft survival time for both primary and secondary transplants [4]. However, Tregs show great potential in alleviating transplant rejection and protecting the allografts effectively [5,6]. Various strategies have been investigated to prevent rejection, including effective usage of agents to block co-stimulatory molecules to

http://dx.doi.org/10.1016/j.trim.2015.01.001 0966-3274/© 2015 Elsevier B.V. All rights reserved.

Please cite this article as: Dai H, et al, Anti-OX40L monoclonal antibody prolongs secondary heart allograft survival based on CD40/CD40L and LFA-1/ICAM-1 blockade, Transpl Immunol (2015), http://dx.doi.org/10.1016/j.trim.2015.01.001

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induce long-term survival [7,8]. In many murine transplant models, blockade of CD40/CD40L and LFA-1/ICAM-1 has proven to be remarkably effective in preventing acute allograft rejection [9–12]. The OX40/OX40L pathway belongs to members of the TNFR and TNF superfamilies similar to CD40/CD40L pathway [13,14]. The ligand for OX40 (OX40L) is expressed on dendritic cells, B cells, and inflamed vascular endothelial cells which acts to mediate the survival, proliferation, and effector differentiation of activated T cells, as well as the generation of Tms [14–21]. What's more, the OX40/OX40L pathway has been identified as a key negative regulator of Tregs [22,23]. Targeting the OX40/OX40L pathway could be therapeutically important for organ transplantation. However, most studies have focused on treatments when the Tms have been produced. Little is known about the application of anti-OX40L mAb for the prevention of Tm generation for extending the secondary transplantation survival. Here, we use blocking anti-OX40L mAb combined with anti-CD40L/LFA-1 mAbs during primary skin transplantation to reduce the generation of Tms and enhance Tregs, using anti-CD40L/LFA-1 mAbs to inhibit the function of T cells during the secondary heart transplantation. This regimen markedly prolongs the heart allograft survival. Additionally, we explore the mechanism of prolonged survival of secondary allografts. 2 . Materials and methods 2.1 . Animals Female C57BL/6 (B6, H-2b) and BALB/c (H-2d) mice (8–12 weeks old) were purchased from Slac Laboratory Animal Co. Ltd. (Shanghai, China) and used as graft recipients and donors, respectively. All animals were maintained and bred in a pathogen-free facility, and all procedures were performed according to the Institutional Animal Care and Use Committee (IACUC) guidelines. 2.2 . Antibodies All antibodies administered in vivo were produced by Bioexpress (West Lebanon, NH, USA), including blocking monoclonal antibodies (mAbs) anti-OX40L (RM134L), anti-CD40L (MR-1), anti-LFA-1(M17/ 4), and their respective isotype controls. Antibodies used for flow cytometric analysis, including FITC-anti-CD4 (GK1.5), FITC-anti-CD8 (53–6.7), PE-anti-CD44 (IM7), PECy5-anti-CD62L (MEL14), and their isotype controls were purchased from Biolegend (San Diego, CA, USA). CD4+Foxp3+ regulatory T cells (Tregs) were detected using a Mouse Regulatory T cell Staining Kit from eBioscience (San Diego, CA, USA). 2.3 . Primary skin transplantation Full-thickness BALB⁄c trunk skin tissues (circular pieces, diameter = 1–1.2 cm) were engrafted onto the lumbar region of B6 mice. After the fully MHC-mismatched transplantation, the B6 mice received a 2antibody treatment regimen consisting of 0.25 mg of anti-CD40L mAb and 0.1 mg of anti-LFA-1 mAb, or a 3-antibody treatment regimen consisting of 0.25 mg of anti-CD40L mAb, 0.1 mg of anti-LFA-1 mAb,

Table 1 Various abbreviations standing for different treatments used in our study. Abbreviation treatment S0 S2 S3 H0 H2

Skin transplantation + isotype antibodies Skin transplantation + anti-CD40L/LFA-1 Skin transplantation + anti-CD40L/LFA-1/OX40L Heart transplantation + isotype antibodies Heart transplantation + anti-CD40L/LFA-1

Days post-transplantation At days 0 and 2 At days 0 and 2 At days 0 and 2 At days 0, 2, 4 and 6 At days 0, 2, 4 and 6

Table legend: mAbs were pooled and administered i.p. after the transplantation procedure as follows: 0.25 mg/dose anti-CD40L, 0.1 mg/dose anti-LFA-1, and 0.25 mg/dose antiOX40L. Heart transplantations were performed four weeks after primary skin transplantation.

and 0.25 mg of anti-OX40L mAb. Control group mice were treated with isotype antibodies (Table 1, Fig. 1). The mAbs were intraperitoneally (i.p.) administered on days 0 and 2 post-transplantation.

2.4 . Alloantigen-primed heart transplantation model Four weeks after primary skin grafting, the B6 mice were defined as alloantigen-primed mice. Vascularized heterotopic heart transplantations from BALB/c donors to the alloantigen-primed B6 recipients were performed with anastomosis to the vessels of the neck using a non-suture cuff technique as described previously [24]. This method is known as the alloantigen-primed model. The mice were treated with 0.25 mg anti-CD40L mAb and 0.1 mg of anti-LFA-1 mAb or isotype controls on days 0, 2, 4, and 6 after the secondary heart transplantation (Table 1, Fig. 1). Graft survival was monitored by daily palpation. Rejection was defined as the complete loss of a palpable heart beat.

2.5 . Flow cytometry Spleens and heart allografts were isolated from mice at four weeks after skin transplantation and six days after heart transplantation, respectively. T lymphocytes were isolated from spleens of B6 mice using nylon wool columns (Wako, Osaka, Japan). Approximately 1 × 106 T lymphocytes from recipient spleens and heart grafts were stained using fluorescent antibodies according to the manufacturer's instructions. At last, all of the positively stained cells were analyzed with a flow cytometer (Partec Co, Munster, Germany). Data were analyzed with Flow Jo 7.5 software (Tree Star Inc., Ashland, OR, USA).

2.6 . Enzyme-linked immunosorbent assay (ELISA) ELISAs were performed using commercially available kits (Neo Bioscience Technology Limited Company, Shenzhen, China) to detect concentrations of IL-2, IFN-γ, IL-10, and TGF-β in the sera according to the manufacturer's instructions. A standard curve was generated using known amounts of purified recombinant murine cytokines.

2.7 . Mixed lymphocyte reactions (MLRs) T lymphocytes were isolated from spleens of B6 mice using nylon wool columns (Wako, Osaka, Japan) and were used as responder cells. Donor spleen cells were used as stimulator cells and treated with mitomycin (40 μg/ml, Amresco, Solon, OH, USA) before their use in the MLR assay. For proliferation assays, 1 × 105 stimulator cells were cultured with 5 × 105 responder cells in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin in 96-well plates. Three separate wells were dedicated to each responder–stimulator combination and each experiment was repeated three times. Cells were incubated for 72 h at 37 °C in 95% humidified air mixed with 5% carbon dioxide. After 72 h of culture, cell proliferation was quantified using a BrdU kit (Roche Applied Science, Mannheim, Germany).

2.8 . Pathological examination of heart allografts The heart allografts were resected from the recipient mice on day sixth post-transplantation. Tissues were fixed in 10% buffered formalin solution, embedded in paraffin, cut into 5 μm sections and stained with hematoxylin and eosin (H&E). Graft rejection was graded on the extent of infiltration and the anatomical localization of inflammatory cells according to the International Society of Heart and Lung Transplantation (ISHLT) standard [25].

Please cite this article as: Dai H, et al, Anti-OX40L monoclonal antibody prolongs secondary heart allograft survival based on CD40/CD40L and LFA-1/ICAM-1 blockade, Transpl Immunol (2015), http://dx.doi.org/10.1016/j.trim.2015.01.001

H. Dai et al. / Transplant Immunology xxx (2015) xxx–xxx

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Fig. 1. Flow chart of the experiment. Full-thickness skin grafts were transplanted from BALB/c mice to B6 mice. Four weeks after skin grafting, the B6 mice were defined as alloantigenprimed mice which were divided into 3 groups (S0, S2 and S3). Subsequently, they were used in the next three steps. Firstly, spleens were isolated for analyzing the proportion of Tms and Tregs. (n = 3 mice/group). Secondly, alloantigen-primed B6 mice were performed the secondary heart transplantation from BALB/c donor and recorded the survival time. Recipients were divided into 2 stages (H0 and H2), so there were 6 groups (S0H0, S2H0, S3H0; S0H2, S2H2 and S3H2, n = 6 mice/group). Thirdly, explored the mechanism of prolonged survival in the S0H2, S2H2 and S3H2 groups (n = 3 mice/group).

2.9 . Quantitative real-time PCR (qRT-PCR)

2.11 . Statistical analyses

RNA was isolated from heart allografts using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Reverse transcription and qRT-PCR were performed using commercially available reagents (TOYOBO, Osaka, Japan) with the StepOne Real-Time PCR System (ABI, Foster, UK). Syber Green I was used to detect amplification and β-actin was used as a normalizing control. Calculations were performed using the 2−ΔΔCT method. Each reaction was carried out in triplicate. The primer sequences used for the qRT-PCR were listed in Table 2.

The MST of the six groups was analyzed by the Kaplan–Meier method and Log-rank test. All other data were analyzed by one-way analysis of variance (ANOVA). Because multiple comparisons were made during the analysis, a Bonferroni correction was calculated and applied. A P value less than 0.05 was considered to represent a statistically significant difference; P b 0.01 and P b 0.001 indicated highly significant differences. All analyses were performed using GraphPad Prism® software (GraphPad, Inc., La Jolla, CA, USA).

3 . Results

2.10 . Extraction of lymphocytes from heart allografts The harvested heart allografts were minced with a sterile blade and incubated in 10 ml buffered saline with 2% bovine serum albumin (BSA) and 2 mg/ml collagenase at 37 °C for 2 h. Cells were strained through a 70 μm nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ, USA). Lymphocytes were isolated from these cells using EZ-SepTM Mouse lymphocyte separation medium (Dakewe Biotech Company, Shenzhen, China) with centrifugation for 20 min at 1600 rpm. After washed twice in RPMI 1640, lymphocytes were resuspended in phosphate buffered saline (PBS) with 10% fetal bovine serum for analysis by flow cytometry.

Table 2 Primer sequences used for qRT-PCR. Primer name

Sequences

β-actin

forward 5′-CATCCGTAAAGACCTCTATGCCAAC-3′, reverse 5′-ATGGAGCCACCGATCCACA-3′ forward 5′-GGAGCAGCTGTTGATGGACCTAC-3′, reverse 5′-AATCCAGAACATGCCGCAGAG-3′ forward 5′-CGGCACAGTCATTGAAAGCCTA-3′, reverse 5′-GTTGCTGATGGCCTGATTGTC-3′ forward 5′-GACCAGCTGGACAACATACTGCTAA-3′, reverse 5′-GATAAGGCTTGGCAACCCAAGTAA-3′ forward 5′-TGACGTCACTGGAGTTGTACGG-3′, reverse 5′-GGTTCATGTCATGGATGGTGC-3′ forward 5′-CAGCTCTGCTGGCGAAAGTG-3′, reverse 5′-TCG.TCTGAAGGCAGAGTCAGGA-3′

IL-2 IFN-γ IL-10 TGF-β Foxp3

3.1 . Anti-OX40L mAb combined with anti-CD40L/LFA-1 mAbs reduced Tms but increased Tregs during primary skin transplantation According to previous studies, we hypothesized that anti-OX40L mAb treatment would affect the generation of alloreactive effector Tm (CD44highCD62Llow) and the proportions of CD4 +Foxp3+ Tregs. The abbreviations of S 0, S 2, and S 3 represented mice post skin transplantation that received isotype antibodies, anti-CD40L/LFA-1 mAbs, or anti-OX40L mAb combined with anti-CD40L/LFA-1 mAbs treatment, respectively (Fig. 1). We isolated B6 mouse splenic T lymphocytes at four weeks posttransplantation and analyzed CD4, CD8, CD44, CD62L, and Foxp3 expression by flow cytometry (Fig. 2A). Serum concentrations of IL-2, IFN-γ, IL-10, and TGF-β were tested by ELISAs (Fig. 2B). Compared to the S0 group, the proportions of CD4+ Tm/CD4+ T cells in both the S2 and S3 groups were significantly reduced (39.76 ± 3.17% in the S0 group vs. 32.61 ± 2.20% in the S2 group [P b 0.05] and 25.36 ± 1.16% in the S3 group [P b 0.01]), and the proportions of CD8+ Tm/CD8+ T cells were also significantly reduced (33.32 ± 2.84% in the S0 group vs. 27.76 ± 1.96% in the S2 group [P b 0.05] and 20.95 ± 1.30% in the S3 group [P b 0.01]). By contrast, the proportions of Tregs among splenic T cells increased in the S2 group (1.85 ± 0.15%) and S3 group (2.42 ± 0.19%; P b 0.05 vs. the S2 group) compared to the S0 group (1.18 ± 0.30%; P b 0.05 vs. the S2 group). There were no statistical differences in IL-2 concentrations detected among the S0, S2, and S3 groups. The concentrations of IFN-γ were reduced in the S2 and S3 groups (Fig. 2B, P b 0.001). The levels of IL-10 increased in the S2 and S3 groups compared to the S0 group (Fig. 2B, P b 0.05). However, we detected no significant differences in TGF-β concentrations between the S2 and S3 groups.

3.2 . Anti-OX40L mAbs markedly prolonged secondary heart allograft survival based on CD40/ CD40L and LFA-1/ICAM-1 blockade during primary and secondary transplantation To investigate the effect of the reduced Tms and increased Tregs observed in the S3 group in secondary transplantation, we used B6 mice which came from the former three groups (S 0, S 2, and S3) as recipients for heart transplantation. Mice from all groups were treated with isotype control antibodies (S0H0, S2H0, and S3H0) or antiCD40L/LFA-1 mAbs (S0H2, S2H2, and S3H2) during the secondary heart transplantation (Fig. 1).

Please cite this article as: Dai H, et al, Anti-OX40L monoclonal antibody prolongs secondary heart allograft survival based on CD40/CD40L and LFA-1/ICAM-1 blockade, Transpl Immunol (2015), http://dx.doi.org/10.1016/j.trim.2015.01.001

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Fig. 2. The proportion of Tms and CD4+Foxp3+ Tregs in recipient splenic lymphocytes cells and levels of various cytokines in sera of recipient mice following skin transplantation. Spleens and sera were isolated from mice at 4 weeks post-transplantation. (A) The proportions of CD4+ Tm/CD4+ T cells (top), CD8+ Tm/CD8+ T cells (middle), and CD4+Foxp3+ Tregs (bottom) were analyzed by flow cytometry; Tregs are shown as the proportion of splenic lymphocytes. Naïve B6 mice were used as negative controls (n = 3 mice/group). (B) ELISAs were used to measure the concentrations of IL-2, IFN-γ, IL-10, and TGF-β in the sera (n = 3 mice/group; *P b 0.05; ***P b 0.001; ns, non-significant). Each reaction was carried out in triplicate. Data are all representative of three separate experiments.

The MST following heart transplantation was 3.25 days for the S0H0 control group, 4.5 days for the S2H0 treated group (P b 0.01 vs. either group), and 5.5 days for the S3H0 treated group (P b 0.001 vs. the S0H0 group; Fig. 3A). Unfortunately, survival was not very long in any of the three groups listed above. Therefore, we also used anti-CD40L/LFA-1 mAbs during the secondary heart transplantation. The MST was 6 days for the S0H2 group, 9.5 days for the S2H2 group, and 21 days for the S3H2 treated group (P b 0.001 for each between group comparisons; Fig. 3B). The MST in S3H2 group was more than twice that of the S2H2 group. This finding indicated that antiOX40L mAbs could markedly prolong heart allograft survival based on CD40/CD40L and LFA-1/ICAM-1 blockade during primary and secondary transplantation.

3.3 . The proportions of Tms and functions of lymphocytes were inhibited, meanwhile, the Tregs were enhanced during secondary heart transplantation To explore the mechanism of prolonged allograft survival induced by treatments including anti-OX40L mAbs, we extracted spleens and sera from recipient mice at day 6 post-transplantation for analysis (Fig. 1). The Tms and Tregs were analyzed by flow cytometry (Fig. 4A). We used MLR to test the proliferative responses of recipient splenic T cells to donor BALB/c cells (Fig. 4B). The concentrations of rejection/tolerance associated with cytokines, such as IL-2, IFN-γ, IL-10, and TGF-β in the sera were detected by ELISAs (Fig. 4C).

Compared to the S0H2 group, the proportions of CD4+ Tm/CD4+ T cells in both the S2H2 group and S3H2 groups were obviously reduced (25.46 ± 1.56% in S0H2 group vs. 19.98 ± 1.35% in S2H2 group and 15.23 ± 2.47% in S3H2 group). The proportions of CD8+ Tm/CD8+ T cells were also significantly reduced (24.31 ± 1.31% in S0H2 group vs. 19.78 ± 1.10% in S2H2 group and 15.36 ± 1.15% in S3H2 group). P b 0.05 when comparing the proportion of CD4+ and CD8+ Tms between the S0H2 group and S2H2 group. When comparing the proportion of CD4+ and CD8+ Tms between S2H2 groups to S3H2 group, the P value was less than 0.05 and 0.01, respectively. Additionally, the proportions of Tregs in splenic T cells increased in the S2H2 group (2.79 ± 0.33%) and S3H2 group (4.21 ± 0.59%) compared to the S0H2 group (2.12 ± 0.24%). P b 0.05 when comparing the proportion of Tregs in S2H2 group to S0H2 group and S3H2 group. P b 0.01 when comparing the proportion of Tregs in S3H2 group to S0H2 (Fig. 4A). The MLR results showed that cell proliferation in the S2H2 group was reduced more than in the S0H2 group. Furthermore, mice that received anti-OX40L mAb treatment based on CD40/CD40L and LFA-1/ICAM-1 blockade (S3H2 group) had lower cell proliferative responses than mice that did not receive anti-OX40L mAbs (S2H2 group) (Fig. 4B; P b 0.05).We found that the concentration of IFN-γ in sera was significantly reduced from the S0H2 group to the S3H2 group (Fig. 4C, S0H2 group vs. S2H2 group, P b 0.001; S2H2 group vs. S3H2 group, P b 0.05). Furthermore, the levels of IL-10 were increased in the S3H2 group compared to the S2H2 group (Fig. 4C, P b 0.05). Similar to primary skin transplantation, we did not detect statistically significant differences in TGF-β concentrations between the S3H2 and S2H2 groups (Fig. 4C).

Fig. 3. Survival time of secondary heart allografts in each group. We performed the secondary heart transplantation at 4 weeks post primary skin grafting as described previously. Allograft survival times are shown by the Kaplan–Meier method and were compared by the Log-rank test. Groups that received isotype control antibodies (A) or anti-CD40L/LFA-1 mAbs (B) during the secondary heart transplantation were analyzed respective (n = 6 mice/group).

Please cite this article as: Dai H, et al, Anti-OX40L monoclonal antibody prolongs secondary heart allograft survival based on CD40/CD40L and LFA-1/ICAM-1 blockade, Transpl Immunol (2015), http://dx.doi.org/10.1016/j.trim.2015.01.001

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Fig. 4. The proportion of Tms and CD4+Foxp3+ Tregs among splenic lymphocyte cells and the function of splenic lymphocyte in recipients treated with anti-CD40L/LFA-1 mAbs during the secondary heart transplantation. Spleens and sera were harvested from recipient mice at day 6 post-transplantation for analysis. (A) The proportions of CD4+ Tm/CD4+ T cells (top), CD8+ Tm/CD8+ T cells (middle), and CD4+Foxp3+ Tregs (bottom) were analyzed by flow cytometry, Tregs are shown as the proportion of splenic lymphocytes (n = 3 mice/group). (B) MLR assays were used to test the proliferative responses of recipient splenic T cells to donor BALB/c cells. The mean OD values were compared among the groups (n = 3 mice/group; * P b 0.05). Three separate wells were dedicated to each responder–stimulator combination and each reaction was carried out in triplicate. Splenic T cells alone served as a negative control. (C) The concentrations of IL-2, IFN-γ, IL-10, and TGF-β in the sera were measured by ELISAs (n = 3 mice/group; *P b 0.05; **P b 0.01; ***P b 0.001; ns, non-significant). Each reaction was carried out in triplicate. Data are all representative of three separate experiments.

Fig. 5. The relative mRNA expression of key cytokines in allografts, the detection of graft-infiltrating lymphocytes and the pathological analysis from mice treated with anti-CD40L/LFA-1 mAbs during the secondary heart transplantation. Allografts were removed from recipient mice at day 6 post-transplantation. (A) The mRNA levels of IL-2, IFN-γ, IL-10, TGF-β, and Foxp3 were measured by qRT-PCR (n = 3 mice/group; *P b 0.05; **P b 0.01; ***P b 0.001; ns, non-significant). Each reaction was performed in triplicate. (B) Flow cytometric analysis of graftinfiltrating lymphocytes (n = 3 mice/group). A no FITC-anti-CD8 stain served as a negative control. (C) H&E staining of allografts and the ISHLT scores. The point represents the score from one biopsy (n = 3 mice/group, and 3 biopsies for each mouse for a total of 9 data points, **P b 0.01; *** P b 0.001). The line represents the mean scores. Data are all representative of three separate experiments.

Please cite this article as: Dai H, et al, Anti-OX40L monoclonal antibody prolongs secondary heart allograft survival based on CD40/CD40L and LFA-1/ICAM-1 blockade, Transpl Immunol (2015), http://dx.doi.org/10.1016/j.trim.2015.01.001

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To summarize the results of the MLRs and ELISAs, we found that the functions of lymphocytes were clearly inhibited and that IL-10 concentrations were increased when mice received anti-OX40L mAbs treatment based on CD40/CD40L and LFA-1/ICAM-1 blockade. 3.4 . Heart allografts showed higher levels of Tregs and IL-10 Allografts were removed from recipient mice at day 6 post-transplantation to measure the rejection/tolerance associated with mRNA levels by qRT-PCR, including IL-2, IFN-γ, IL-10, TGF-β, and Foxp3 (Fig. 5A). Compared to the S2H2 group, the inflammatory factor IFN-γ in the S3H2 group was significantly reduced (Fig. 5A; P b 0.001). Treatment with anti-OX40L mAbs induced higher levels of Foxp3 gene expression (Fig. 5A; P b 0.05). The expression of IL-10 was enhanced in the S0H2 group compared to the S2H2 and S3H2 groups (Fig. 5A; S0H2 vs. S2H2, P b 0.001; S2H2 vs. S3H2, P b 0.01). However, there was no difference in TGF-β expression levels between the S2H2 and S3H2 groups (Fig. 5A). 3.5 . Fewer graft-infiltrating lymphocytes and intact myocardial structure were detected in the S3H2 group The proportions of CD8+ T cells/graft-infiltrated cells in the S0H2 and S2H2 groups were 33.58 ± 2.41% and 22.11 ± 2.85%, respectively (Fig. 5B; P b 0.01). By contrast, the proportion was reduced to 13.57 ± 2.12% in the S3H2 group mice that had received antiOX40L mAb treatment (P b 0.001 vs. the S0H2 group; Fig. 5B). These data are accordant to the pathological examination of heart allografts which tissues from mice in the S3H2 group showed lower levels of inflammatory infiltration and fewer changes in myocardial structural integrity compared to the S2H2 group (Fig. 5C; P b 0.01).

4 . Discussion Memory T cells (Tms) possess the ability to quickly and robustly respond to alloantigen, which sets a major barrier to successful transplantation, especially for the secondary transplantation [26–28]. Nevertheless, more and more findings clarify that subsets of Tregs are crucial in prolonging the transplant survival [29,30]. Several different types of Tregs have been identified to play a pivotal role in the control of long-term graft survival in rodents, such as innate regulatory NKT cells, γδT cells, and induced Tregs including IL-10-secreting Tr1 CD4+ T cells or TGF-β-producing Th3 CD4+ T cells [31–33]. The paradoxical roles of Tms and Tregs need more exploration in transplantation. Researchers have found that combination blockade of the CD40/ CD40L and LFA-1/ICAM-1 pathways can effectively protect the primary islet, heart, skin and bone marrow transplantations [9–12,34–37]. Those results are validated during the primary transplant, however, a reliable method to target Tms has remained challenging [38]. Another pathway that belongs to members of the TNFR and TNF superfamilies similar to the CD40/CD40L pathway is the OX40-OX40L pathway which has been reported to have a diametric role in the response of Tms and Tregs to allografts [13,14,21]. Using blocking OX40L costimulation alone or in combination with other reagents could decrease the generation of Tms [15,16,18–20,39]. By contrast, the OX40/OX40L pathway has been identified as a key negative regulator of Tregs [22,40,41]. But the exact role of anti-OX40L mAb in the secondary transplantation remains incompletely defined. As we previously reported, we focused on the prevention to reduce the generation of Tms during primary transplantation to prolong the survival of secondary allografts [42]. In this study, we used a blocking anti-OX40L mAb combined with anti-CD40L/LFA-1 to inhibit the generation of Tms and enhance Tregs. Indeed, we found that both the CD4+ Tm and CD8+ Tm in the S3 group (anti-OX40L plus anti-CD40L/LFA-1) were significantly reduced compared to the S2 group that received anti-CD40L/LFA-1 alone (Fig. 2A). This finding demonstrated that anti-OX40L could reduce the proportion of Tm, which is consistent with the finding of Min Diem Vu and colleagues [15,16]. Meanwhile, the concentrations of IFN-γ in sera reduced in the additive usage anti-OX40L group (Fig. 2B; P b 0.001). These data are consistent with the findings reported in others' studies [43–45]. Interestingly, the proportions of Tregs among splenic T cells increased in the S2 group and S3 group compared to the S0 group

(Fig. 2A). A study using blocking and stimulating anti-OX40L also explained the Treg-mediated long-term survival in CD40L deficient settings [13]. The levels of IL-10 increased in the S3 group compared to the S2 and S0 groups (Fig. 2B, P b 0.05). However, we didn't detect any significant differences in TGF-β concentrations between the S2 and S3 groups (Fig. 2B). To investigate the effect of the reduced Tms and increased Tregs observed in the S3 group upon secondary transplantation, we performed the heart transplantations. The MST following heart transplantation was prolonged from 3.25 to 5.5 days (Fig. 3A). Unfortunately, this period of survival remained very brief among all of the treatment groups. Accordingly, we used anti-CD40L/LFA-1 mAbs during the secondary heart transplantation to inhibit the function of effector T cells. The MST in the S3H2 group reached 21 days, which was more than twice as long as that of the S2H2 group (Fig. 3B). This finding indicated that anti-OX40L mAbs could markedly prolong heart allograft survival based on CD40/CD40L and LFA-1/ICAM-1 blockade during the primary and secondary transplantations. We attempted to explore the mechanism of prolonged heart allograft survival induced by the treatment that included antiOX40L mAbs. Our flow cytometry results once again showed that the addition of anti-OX40L mAbs based on CD40/CD40L and LFA1/ICAM-1 blockade could maintain the low levels of CD4 + Tm and CD8 + Tm, which was beneficial for secondary allografts (Fig. 4A) [46]. Moreover, the proportions of Tregs in splenic T cells increased in the S3H2 group (Fig. 4A). The treatment with anti-OX40L mAbs also induced higher Foxp3 gene expression in the grafts (Fig. 5A, P b 0.05). All of these results indicated that the anti-OX40L mAb did protect the allografts by promoting Tregs, which has also been observed in other animal models [13,22,40,47]. The levels of IL-10 were enhanced in the S3H2 group compared to the S2H2 group (Fig. 4C, 5A). However, there was no significant difference in TGF-β expression levels between the S2H2 and S3H2 groups (Fig. 4C, 5A). Based on the levels of IL-10 and TGF-β during primary skin transplantation and secondary heart transplantation, we speculated that the protective Tregs population might be IL-10-secreting Tr1 cells [22,33,48]. The MLR results demonstrated that cell proliferation in the S3H2 group mice that received anti-OX40L mAb treatment had lower cell proliferative responses than did the S2H2 group (Fig. 4B, P b 0.05). IFN-γ has been found to play a pivotal role in maximizing the function of T cells, while simultaneously limiting the expansion of Tregs [40,43]. Our findings showed that the rejection associated with cytokines IFN-γ in sera was significantly reduced in the S3H2 group compared to the S2H2 group (Fig. 4C, P b 0.05). To summarize the results of cell proliferation and IFN-γ secretion, we found that the function of T lymphocytes was obviously inhibited when mice received anti-OX40L mAb treatment based on CD40/CD40L and LFA-1/ICAM-1 blockade. Heart allograft tissues from mice in the S3H2 group had lower levels of inflammatory infiltration and fewer damage of myocardial structure compared to the S2H2 groups (Fig. 5C, P b 0.01). Results showed that anti-OX40L mAb treatment could reduce the infiltration of CD8+ T cells into the allografts (Fig. 5B, P b 0.05). We detected fewer graft-infiltrating CD4+ T cells (data are not shown), suggesting that CD4+ T cells may play a key role in secondary lymphoid organs. This finding was consistent with previous results reported by Yalai et al. [49]. Unfortunately, our treatment regimen did not achieve a distinguished prolonged survival. We speculate that B cells that produce antibodies specific for donor antigens, and memory B cells that could be activated, proliferated, or converted to alloantibody-secreting plasma cells in the secondary immune response, would have the potential ability to prevent allograft acceptance [50–55]. Therefore, further research will be required to determine whether memory B cells, donor specific antibodies, infiltrating macrophage, or natural killer cells impair the long-term acceptance of allografts [56–58].

Please cite this article as: Dai H, et al, Anti-OX40L monoclonal antibody prolongs secondary heart allograft survival based on CD40/CD40L and LFA-1/ICAM-1 blockade, Transpl Immunol (2015), http://dx.doi.org/10.1016/j.trim.2015.01.001

H. Dai et al. / Transplant Immunology xxx (2015) xxx–xxx

In summary, anti-OX40L mAb could prolong secondary allograft survival based on CD40/CD40L and LFA-1/ICAM-1 blockade during primary skin transplantation and secondary heart transplantation. The mechanism of allograft protection might include impaired generation of CD4+ Tm and CD8+ Tm and up-regulated IL-10-producing Tregs, inhibited function of T cells, and suppressed inflammatory cells infiltrating into allografts. Although this treatment program did not achieve long-term allograft survival, it did demonstrate that anti-OX40L was very effective in inhibiting alloreactive Tms and enhancing the IL-10secreting Tr1 which is beneficial to the secondary transplantation. We expect that all of these basic findings could promote the inclusion of anti-OX40L mAb in clinical transplant therapy, especially for preventing the generation of Tms.

Acknowledgments This work was supported by the Fundamental Research Funds for the Central Universities of Central South University (No.2014zzts083) (to D.H.L).

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Please cite this article as: Dai H, et al, Anti-OX40L monoclonal antibody prolongs secondary heart allograft survival based on CD40/CD40L and LFA-1/ICAM-1 blockade, Transpl Immunol (2015), http://dx.doi.org/10.1016/j.trim.2015.01.001