Role of regulatory T cells in CD47/donor specific transfusioninduced immune tolerance in skin heart transplantation mice

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1 Received: 31 May 2018 Revised: 10 August 2018 Accepted: 21 August 2018 DOI: /tid ORIGINAL ARTICLE Role of regulatory T in CD47/donor specific transfusioninduced immune tolerance in skin heart transplantation mice Yu Hu 1 Honglan Zhou 2 Baoshan Gao 2 Gang Wang 2 Yuantao Wang 2 1 Department of Pathology, China Japan Union Hospital, Jilin University, Changchun, China 2 Department of Urology, The First Hospital of Jilin University, Changchun, China Correspondence Yuantao Wang, Department of Urology, The First Hospital of Jilin University, Changchun, China. wyt160802@163.com Funding information The Work is funded by National Natural Science Foundation of China (No , and ). Abstract Objectives: To explore the role of regulatory T (T reg ) in the establishment of immune tolerance induced by donor specific transfusion (DST) in mice with skin heart transplantation. Methods: C57BL/6 mice received DST of splenocytes from CD47 +/+ or CD47 / H 2 bm1 mice or no DST 7 days before skin heart transplantation from major histocompatibility complex class I mismatched H 2 bm1 donors. The number and proportion in graft and lymphoid organs were measured by flow cytometry (FACS) and immunohistochemistry (IHC). The inhibitory function and anti donor T cell responses were assessed by mixed lymphocyte reaction. Results: We observed that mean survival time (MST) of skin or heart graft was significantly longer in C57BL/6 mice which received DST from CD47 +/+ H 2 bm1 mice than from CD47 / H 2 bm1 mice. By FACS, we found that the number in spleen was increased significantly in mice which received CD47 / DST compared to mice which received CD47 +/+ DST. However, the percentages in total splenocytes and lymph node were significantly higher in mice that received CD47 +/+ DST than mice which received CD47 / DST. Immunohistochemistry showed an increased heart grafts infiltration in the recipients with CD47 / DST, but not CD47 +/+ DST. Supporting this, we found that donor T cell proliferation was significantly suppressed in mice which received CD47 +/+ DST compared to mice which received CD47 / DST. There was no difference of inhibitory function between these two groups. Conclusion: Our results indicated that CD47 expression on DST plays an important role in the induction of immune tolerance in mice with skin heart transplantation. Increased percentage may contribute to immune tolerance induced by CD47 +/+ DST. KEYWORDS co transplantation of skin and heart, DST, immune tolerance, Treg 1 INTRODUCTION Organ transplantation is the ultimate treatment option for end stage organ failure. Successful transplantation requires the establishment of immune Yu Hu and Honglan Zhou authors are co first authors. tolerance to eliminate graft rejection. While there are variety of ways to eliminate graft rejection, donor specific transfusion (DST) has been utilized to induce tolerance in animal model of transplantation. It has been shown that DST facilitates tolerance and prolongs allograft survival. 1-3 Different animal models of transplantation have been utilized for transplantation immunology research, with each being unique. Transpl Infect Dis. 2019;21:e wileyonlinelibrary.com/journal/tid 2018 John Wiley & Sons A/S. 1 of 8 Published by John Wiley & Sons Ltd

2 2 of 8 Mouse skin transplantation is one of the most common animal models for studying graft rejection 4 because skin graft rejection is often rapid, intensive, and visible. However, once rejection occurs, skin graft shrinks, scabs and becomes necrotic, which makes it difficult to measure cell infiltration in the graft. The mouse model of abdominal heterotopic heart transplantation was established by Corry in Although it is technically difficult to operate, the model has an advantage over the skin model that cell infiltration in the graft at the time of rejection can be easily measured because of the large organ size. We have previously shown that the graft rejection occurs in major histocompatibility complex (MHC) I mismatched skin transplantation model. 6 However, graft rejection does not occur in MHC I mismatched heart transplantation models. From our preliminary data, we observed that with co transplantation of skin and heart, skin graft induced rejection of homologous heart grafts. Therefore, using the mouse model of co transplantation of skin and heart, we are able to measure survival time of both skin and heart grafts, determine cell infiltration in the graft, and explore underlying mechanisms by which tolerance or rejection is induced. CD47, known as integrin associated protein, is a member of the immunoglobulin superfamily and the receptor for signal regulatory protein alpha (SIRPα). CD47 is a self marker and interaction between CD47 SIRPa delivers a signal of don t eat me to macrophages that prevent macrophage from phagocytozing of self. 7,8 It has been shown that CD47 gene knockdown to interrupt interaction between donor CD47 and recipient SIRPα induces macrophage tolerance and rapid rejection of xenogeneic hematopoietic. 9,10 Regulatory T (T reg ) are a subset of CD4 + T with the property to suppress effector T. T reg are crucially involved in a variety of physiological and pathophysiological conditions such as autoimmunity and tumor immunity. 11,12 Foxp3 is a master transcriptional factor and a marker for T reg. Foxp3 expressing T reg possess a strong ability to inhibit the proliferation of effector T in vivo. 13 In transplantation, T reg play an important role in DST induced immune tolerance. 14,15 Our previous studies showed that C57BL/6 mice rejected skin grafts but not heart grafts from MHC mismatched bm1 mice and the rejection was reversed in C57BL/6 mice receiving DST, suggesting DST induces immune tolerance in C57BL mice compared to MHC mismatched skin grafts. 16 In the present study, we established a MHC mismatched mouse model of skin heart transplantation to be able to easily assess the infiltration in grafts. We then investigated the role in the induction of immune tolerance by different CD47 genotype DSTs in the transplant mouse model. 2 MATERIALS AND METHODS 2.1 Mice C57BL/6 (H 2 b ), MHC I mismatched bm1 (CD47 +/+ H 2 bm1 ), and CD47 KO bm1 (CD47 / H 2 bm1 ) mice were purchased from the animal center of Medical College of Jilin University. All mice were female and aged about 6 8 weeks old with a body weight of around 20 g. Mice were maintained at the animal facility under pathogenfree conditions. The study was approved by the animal care committee at Medical College of Jilin University. 2.2 Donor specific transfusion In the present study, C57BL/6 mice were used as recipients and H 2 bm1 mice were used as donors to establish a mouse model of skin heart transplantation. Splenocytes from CD47 +/+ and CD47 / H 2 bm1 mice were depleted of erythrocytes using ammonium chloride potassium (ACK) lysing buffer (Cambrex Bio Science Walkersville, Walkarsville, MD, USA) and intravenously injected into C57BL/6 mice ( per mouse) 7 days prior to skin heart transplantation. Mice were divided into three groups. (a) Non DST group: C57BL/6 mice had skin heart transplantation without other pretreatment. (b) CD47 / DST group: C57BL/6 mice were given DST from CD47 / H 2 bm1 mice 7 days before surgery of skin heart transplantation. (c) CD47 +/+ DST group: C57BL/6 mice were given DST from CD47 +/+ H 2 bm1 mice 7 days before surgery of skin heart transplantation. 2.3 Surgical instruments and reagents Surgical microscope was purchased from Leica (Panasonic Corporation, Osaka, Japan) and microsurgical instruments were purchased from the Roboz Surgical Instrument Co (Gaithersburg, MD, USA). Vascular sutures 10 0 were purchased from Ethicon (Somerville, NJ, USA). LLC and other silk sutures were purchased from Shanghai Medical Suture Factory (Minhang, China). Vaseline oil sand gauzes were purchased from Gauze (Huanggang Huangzhou Xianghui Textiles Co., Ltd., Huanggang, China). Band Aid bandages were purchased from Johnson & Johnson (New Brunswick, NJ, USA). Chloral hydrate and heparin were provided by the College of Translational Medicine, Jilin University Surgical procedure Donor heart preparation Anesthesia was given by intraperitoneal injection of 4% chloral hydrate (10 12 μl/g). The abdominal cavity of H 2 bm1 mice was open by longitudinal incision from the xiphoid down to the pubic symphyseal. The ribs and diaphragm were cut to expose heart. The inferior vena cava was ligated with a 6 0 silk suture and the right lung was removed after bundled ligation of the right portal hilum. Pulmonary artery was horizontally cut at bifurcation and heart was laced aged with 10 ml ice saline through ascending aorta. After cutting off the aorta at brachiocephalic trunk bifurcation and bundled ligation of the remaining vessels, the heart was removed and placed in ice saline at 4 C until use. Heart transplantation C57BL/6 mice were anesthetized and fixed on plates. The abdomen was shaved, disinfected, and opened through the abdominal incision. The intestine was pushed rightward and covered with wet saline

3 3 of 8 gauze to fully expose the renal veins, the inferior vena cava, and inferior abdominal vena cava. After blocking blood flow using the vascular clamps, longitudinal incisions were made along the abdominal aorta and inferior vena cava, respectively. The length of the incision was the same as the width of aorta and pulmonary arteries from the donor. The donor s aorta and the recipient s abdominal aorta, and the donor s pulmonary artery and the recipient s inferior vena cava were anastomosed to end to side, respectively. After the anastomosis was completed, the vascular clamps were slowly loosened and the heart was gently touched to promote heart resuscitation. The abdomen was then closed by continuous suturing. Skin graft surgery The skin graft was performed after the heart transplantation. The tail skin of donor H 2 bm1 mice was collected and cut into small pieces of 1 1 cm 2 in size. After anesthetization, the back of C57BL/6 mice was shaved, and an area of full thick skin equal to the size of the graft was cut off. Skin graft was fixed by 5 0 silk thread to suture four corners of graft to the back skin of C57BL/6 mice. The skin graft was covered with oil gauze and the Bondi bandage. The mice were kept warm under the lamp without restriction of water after surgery. 2.4 Graft function monitoring For heart graft, an abdominal palpation for transplanted heart was performed on day 3 after surgery. The functional status of heart graft was given a level of 4 0 according to the strength of heart beat from strong to weak and recorded daily. The rejection date was measured at the time when the heartbeat of the graft heart was unable to be palpated. For skin graft, the band aid was removed and the skin graft survival was monitored 3 days after transplantation. Ulceration and necrosis were developed on the transplanted skin in the early stage of skin graft rejection with some of the skin fused into pieces. Subsequently, necrotic skin became dry and formed scab. When the area of healthy graft skin was <20% of the original skin graft, the transplantation was considered as rejection. 2.5 Immunohistochemistry The graft tissue specimens were fixed in 10% formalin solution. The specimens were embedded, sectioned, dewaxed, and dehydrated in gradient alcohol. The sections were sealed after stained with hematoxylin eosin. The sections were then stained with primary antibody (anti Foxp3 Ab, 1:50; Invitrogen; Thermo Fisher Scientific Co., Ltd., Shanghai, China) and secondary antibody (Goat anti r abbit IgG, 1:500; Invitrogen). The infiltration of inflammatory and T reg in the grafts were assessed under light microscope. 2.6 Flow cytometry Mononuclear from spleen and lymph node were isolated by gradient density centrifugation. Cells were stained with fluorochrome conjugated anti mouse CD4 and Foxp3 antibodies (BD Biosciences). Rabbit anti mouse Fc γ mab (2.4G2; BD Biosciences, Shanghai, China) was used to block non specific staining. Cells stained with mouse IGg2a and rabbit IgG were served as homologous controls. After staining and washing, were analyzed using a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) MLR assay and T reg cell inhibition detection Single cell suspensions were prepared from spleen and lymph nodes of recipient mice. Total T lymphocytes were isolated using pan T immunomagnetic beads and then flow sorted to remove Foxp3 +. Remaining were used for in vitro (mixed lymphocyte reaction) MLR. Whole spleen from H 2 bm1 mice were γ irradiated at 30 Gy and used as stimulating. About responding were mixed with stimulating at 1:1 ratio in 96 well plate and incubated in a 37 C incubator with a humidity 95% and 5% CO 2 for 3 days. One microliter of 3 H TdR was added to each well 16 hours before were collected on a glass fiber membrane. After drying, were counted on a liquid scintillation counter. T cell proliferation was measured as counts per minute (cpm) and expressed as stimulation index ([cpm of responder in well with stimulator added cpm of blank well]/[cpm of the same responder in well containing responder only cpm of blank well]). For detection cell inhibition function, we isolated effector from total T in mice received DST (CD47 / or CD47 +/+ ). Before isolation, GFP Foxp3 + T (T reg ) were presorted from the mice. We stimulated T using bm1 spleen. Isolated T reg were mixed with responding at a ratio of 1:2, 1:4, 1:8, or 1:16 for 3 days. Proliferation of responding was measured by a liquid scintillation counter. T cell proliferation of responding was measured as counts per minute (cpm) and expressed as suppression index ([cpm of responder in well with T reg added cpm of blank well]/[cpm of the same responder in well containing responder only cpm of blank well]). 2.7 Statistical analysis The software SPSS 11.0 (SAS Institute, Cary, NC, USA) was used for statistical analysis. Survival time of transplanted heart was analyzed using the Kaplan Meier survival curve and plotted using GraphPad Prism (La Jolla, CA, USA). The log rank test was used to analyze the difference of survival time between different groups. The measurement data were expressed as mean ± standard deviation and compared using paired t test. P < 0.05 was considered statistically significant. 3 RESULTS 3.1 Effect of different CD47 genotype DST on graft survival We first determined graft survival time of C57BL/6 mice (H 2 b ) that received skin heart transplantation from MHC I mismatched H 2 bm1

4 4 of 8 FIGURE 1 Effect of donor specific transfusion (DST) of different CD47 gene types on graft survival. Graphs showing graft survival of skin (A) or heart (B) in recipient C57BL/6 mice which received DST from H 2 bm1 mice of CD47 KO (CD47 / DST), CD47 WT (CD47 +/+ DST). C57BL/6 mice which did not receive DST (Non DST) served as control. Graft survival time was measured from the day of transplantation surgery to the day when the graft was rejected mice. One group of C57BL/6 mice were given DST from CD47 KO (CD47 / DST) H 2 bm1 mice and another group of C57BL/6 mice received DST from CD47 WT (CD47 +/+ DST) H 2 bm1 mice 7 days before surgery. C57BL/6 mice which received DST (Non DST) served as control. Graft survival time was measured from the day of transplantation surgery to the day when the graft was rejected. As shown in Figure 1A, mean survival time (MST) of skin graft in C57BL/6 mice that did not receive DST was 17.5 days and MST of skin graft in mice that received DST from CD47 / H 2 bm1 mice was 20 days. There was no statistically significant difference of survival time between these two groups (P > 0.05). However, MST of skin grafts in C57BL/6 mice that received DST from CD47 +/+ H 2 bm1 mice was significantly prolonged compared to other two groups (MST: 46.5 days, P < 0.005). Similarly, there was no significant difference in survival time of cardiac graft between the CD47 / DST group (MST: 17 days) and the Non DST group (MST: 15.5 days). However, MST of cardiac grafts in the CD47 +/+ DST group was significantly longer (MST: 42.5 days, P < 0.005) than both the CD47 / DST group and the Non DST group (Figure 1B). These results suggest that while DST from CD47 WT H 2 bm1 mice induces immune tolerance and prolongs graft survival, DST from CD47 KO bm1 mice fails to trigger the tolerance to skin/ heart grafts in C57BL/6 mice. 3.2 Effect of different CD47 genotype DST on the numbers To explore the underlying mechanism by which CD47 / DST suppressed CD47 +/+ DST mediated long graft survival, we measured numbers in spleen and lymph node from C57BL/6 mice transplanted with skin heart from MHC I mismatched H 2 bm1 mice. The spleen and lymph nodes of C57BL/6 mice were harvested 18 days after transplantation and absolute numbers and percentages were measured by flow cytometry. As shown in Figure 2A, C57BL/6 mice without transplantation (naive) had lower number in spleen than C57BL/6 mice with transplantation. The transplanted mice without receiving DST (No DST) had highest number in spleen. Among mice with transplantation, the number in spleen of mice received CD47 / DST increased significantly (P < 0.01) when compared to mice that received CD47 +/+ DST (Figure 2A). We then determined the percentages in total splenocytes, we, however, found that percentage in total splenocytes was significant higher in mice that received CD47 +/+ DST than mice which received CD47 / DST (P < 0.05; Figure 2B). In lymph nodes, the number was lower in mice without transplantation than mice received transplantation without difference among groups (Figure 2C). Percentage in total LN was also lower in mice without transplantation than in mice which received transplantation (Figure 2D). Mice received CD47 +/+ DST had increased percentage in total LN when compared to mice received CD47 / DST (Figure 2D). We also measured the percentage in CD4 + T from spleen or LN and found that percentage in CD4 + T was not different between mice which received CD47 +/+ DST and CD47 / DST (Figure 2E,F). 3.3 Effect of different CD47 genotype DST on infiltration in heart graft To further assess the role in CD47 DST induced tolerance, we next measured infiltration in heart graft. The graft was collected and Foxp3 staining was performed 18 days after transplantation. As shown in Figure 3, we observed that infiltration was more prevalent in mice which received CD47 / DST than mice that received CD47 +/+ DST. It appeared that the number of Foxp3 stained was higher in heart graft from mice which received CD47 / DST group (Figure 3A) when compared to heart graft from mice which received CD47 +/+ DST (Figure 3B) Effect of different CD47 genotype DST on donor T cell proliferation Given differentiated effect of CD47 DST on development and infiltration T reg in recipient mice, we detected effect of DST of different CD47 gene types on donor T cell proliferation. Using MLR assay, we found that donor T cell proliferation significantly increased in mice which received CD47 / DST compared to mice that received CD47 +/+

5 5 of 8 FIGURE 2 Effect of different CD47 genotype donor specific transfusion (DST) on the numbers of regulatory T (T reg ). A,C, Graphs showing numbers in spleen (A) and lymph node (C) from skin heart transplanted C57BL/6 received CD47 /, CD47 +/+ DST, or No DST. C57BL/6 mice without transplantation (Naive) were used as a control. B,D, Graphs showing percentage in total splenocytes (A) or in total lymph node (C) from skin heart transplanted C57BL/6 received CD47 /, CD47 +/+ DST or No DST. C57BL/6 mice without transplantation (Naive) were used as control. E,F, Graphs showing percentage in CD4 + splenocytes (E) or in CD4 + from lymph node (F) from skinheart transplanted C57BL/6 that received CD47 /, CD47 +/+ DST, or No DST. *P < 0.05, **P < 0.01, and ***P < C57BL/6 mice without transplantation (Naive) were used as control (A) (B) FIGURE 3 Effect of different CD47 genotype donor specific transfusion (DST) on infiltration of regulatory T (T reg ) in heart graft. Immunohistochemical images showing infiltration of Foxp3 + T reg in heart graft of C57BL/6 mice which received CD47 / (A) or CD47 +/+ (B) DST

6 6 of 8 FIGURE 4 Effect of different CD47 genotype donor specific transfusion (DST) on donor T cell proliferation. Graph showing donor stimulation (proliferation) index of T in C57BL/6 mice which received CD47 / or CD47 +/+ DST or Non DST. *P < T cell proliferation index was measured as counts per minute (cpm) and expressed as stimulation index (cpm of responder in wells with stimulator added/cpm of the same responders in wells containing responder only) DST (P < 0.05). There was no difference of stimulation index in mice received CD47 / DST when comparing Non DST mice (P > 0.05; Figure 4). These results suggest that donor T cell proliferation in CD47 +/+ DST group was significantly inhibited. 3.4 Effect of different CD47 genotype DST on inhibition function of Treg We next determined inhibitory function isolated from skin heart transplanted C57BL/6 mice received different CD47 genotype DST. To do this, we co cultured T reg with effector T at a ratio of 1:2, 1:4, 1:8, or 1:16 for 3 days and proliferation of effector was measured by a liquid scintillation counter using cpm and expressed as suppression index. As shown in of CD47 expressing induces durable immune tolerance and failure of graft rejection. In contrast, DST of CD47 / activates dendritic in recipient mice, resulting in transplant rejection. 6,16 Our study found that CD47 +/+ DST induced immune tolerance and prolonged survival of skin and heart grafts in C57BL/6 mouse while CD47 / DST failed to induce immune tolerance, which is consistent with previous studies. Regulatory T are a subset of T with immunomodulatory functions. It has been shown that changes in both number and function play a critical role in the development of autoimmune diseases. 14 Previous studies have shown that the proportion was significantly higher in grafts or lymphoid organs from tolerant mice than rejected mice. 17,18 However, subsequent studies found that the number increased during graft rejection in liver and heart transplantation. 19,20 Therefore, whether there is an association between the number in lymphoid organs and grafts and immune tolerance remains controversial. In the present study, we found that the number of spleen T reg was significantly higher in mice received CD47 / DST (rejection group) than mice received CD47 +/+ DST (tolerance group). However, the proportion in splenocytes was significantly lower in mice of rejection group than in mice of tolerance group. An explanation is that more T reg developed in order to inhibit the excessive proliferation of effector in spleen from mice of rejection, but this increase in T reg was still not enough to suppress the proliferation of effector. Supporting this, the proportion significantly increased in lymph nodes of mice which received CD47 +/+ DST (tolerance group). Similar findings were observed in the grafts. The infiltration and effector in heart graft was significantly higher in CD47 / DST group (rejection group) than in CD47 +/+ DST group (tolerance group). However, the infiltration was more significant by effector than by T reg. These results suggest that the increase of the proportion Figure 5, while suppression index decreased as the ratio /effector went down, we found that there was no significant difference of suppression index in T reg from C57BL/6 mice with or without DST. Furthermore, suppression index of T reg was not significantly different in C57BL/6 mice which received CD47 +/+ DST when compared to mice that received CD47 / DST (Figure 5). 4 DISCUSSION Donor specific transfusion before transplantation has been used to induce graft tolerance in animal graft models. 1 Our previous studies showed that DST induces immune tolerance in full MHC mismatched mouse model of heart transplantation and the MHC I mismatch mouse model of skin transplantation. It has been shown that CD47 expression on donor is critical to induce immune tolerance. DST FIGURE 5 Effect of different CD47 genotype donor specific transfusion (DST) on regulatory T (T reg ) cell inhibition function. Graph showing suppression index on the effector T in C57BL/6 mice which received CD47 / or CD47 +/+ DST or Non DST at different ratios

7 7 of 8 in lymphoid organs or grafts often indicates the occurrence of immune tolerance and the increase in the number reflects significant proliferation of effector, which indicates the occurrence of rejection. Regulatory T inhibit the proliferation of effector T through various mechanisms that involve cell contact and soluble proteins. 21,22 In the present study, we isolated T reg from mice that received CD47 / DST or CD47 +/+ DST and co cultured with effector T. We found that there was no significant difference in inhibitory capacity isolated from these two groups. Given that T reg cell proportion in mice received CD47 / DST is different from CD47 +/+ DST mice, it is believed that whether to induce immune tolerance or not by DST depends more on the proportion of T reg in the graft and lymphoid organs rather than the number or inhibitory ability. While T reg play an important role in transplanting immune tolerance, other mechanisms were also involved in the induction of immune tolerance. We found that CD47 +/+ DST induces immune tolerance, which is associated with increased proportion in lymph node. CD47 / DST cannot induce immune tolerance with increased number. Because the proliferation of effector is more pronounced, the proportion in lymphocytes decreases. Given that there was no significant difference in the inhibition function between CD47 +/+ and CD47 / DST, we believe that the proportion in lymphocytes has a greater effect on the outcome of immune response (tolerance or rejection). As for the effect of DST on DC, it has been shown 6,16 that the mouse DC is in a mature state after CD47 / DST. Matured DCs more effectively present the antigen to the effector, which leads to an immune response toward graft rejection. In contrast, DC is in an immature state after CD47 +/+ DST, which promotes the development, thereby making the immune response towards graft tolerance. In summary, we observed that mice received CD47 +/+ DST had longer graft survival than mice received CD47 / DST, suggesting the immune tolerance induced by CD47 +/+ DST. We found that the proportion in grafts and lymphoid organs was significantly higher in mice which received CD47 +/+ DST than mice that received CD47 / DST, indicating that T reg play an important role in the development of immune tolerance in mice with skinheart transplantation. To the best of our knowledge, this is the first study to investigate the role in the mouse model of co transplantation. CONFLICTS OF INTEREST None. AUTHORS CONTRIBUTIONS Yu Hu and Honglan Zhou performed the experiments, Baoshan Gao prepared the manuscript, Gang Wang polished the manuscript, and Yuantao Wang designed the study. 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