Nuclear Receptor Subfamily 4 Group A Member 1 Overexpression Prolongs Free Flap Allotransplant Graft Survival by Inducing
T-Cell Anergy in the Rat

Bo Xiaoa,*, Yanqun Zhangb, Shiqiang Liua, Jianke Dinga, Zhou Yua, Tong Wanga, and Baoqiang Songa,*
aDepartment of Plastic Surgery, Xijing Hospital, Air Force Medical University, Xi’an, Shanxi Province, China; and bDepartment of Emergency, Xijing Hospital, Air Force Medical University, Xi’an, Shanxi Province, China

ABSTRACT
Background. New strategies to inhibit acute rejection are needed for further applications of composite tissue allotransplantation. The nuclear receptor subfamily 4 group A member 1 (NR4A1) is considered a key controller of maintaining tolerance homeostasis. However, the effect of NR4A1 in suppressing rejection responses after allotransplantation remains unknown.

Methods. Brown Norway rat groin flaps were transplanted into Lewis rat recipients. The recipients were administrated cytosporone B, an NR4A1 activator. NR4A1 expression and graft survival time were assessed. T helper type 1 and regulatory T cell populations in the second lymphoid organ were detected by flow cytometry. Furthermore, a retrovirus con- taining NR4A1 was constructed and transfected to T cells in vitro. After stimulation, interleukin 2 and interferon gamma secretions were detected in the T cells.

Results. Administration of cytosporone B activated NR4A1 expression in allotransplant recipients and was associated with prolonged survival time of the vascularized free flap allograft. T helper type 1 cells in the recipient secondary lymphoid organs were decreased, whereas the population of regulatory T cells did not change. Interleukin 2 and interferon gamma were suppressed in vitro in the T cells overexpressing NR4A1.

Conclusions. We demonstrated that the overexpressed NR4A1 is associated with sup- pressed graft rejection response. The suppression effect may attribute to induction of T-cell anergy and blockade of key immunologic cytokines.

A LLOGRAFT transplantation of facial tissue is emerging as a potential treatment for complex facial defects caused by trauma, tumor resection, or congenital abnormalities. At present, more than 40 facial trans- plantations have been performed worldwide [1]. However, the large doses and the lifelong administration of immu- nosuppressant drugs required after transplantations may lead to complications such as opportunistic infections, organ impairment, and malignancies. These complications caused by immunosuppressants remain the main obstacle in facial transplantation therapies. Thus, there is a need for new strategies to inhibit the immune response to the trans- planted tissue and minimize the need for
immunosuppressants.

The activation and differentiation of T cells after stimu- lation by alloantigens is a key step in graft rejection response.
ª 2020 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
Normally, naive CD4þ T cells exit from quiescence to un- dergo clonal expansion and secrete cytokines such as inter- leukin 2 (IL-2) and interferon gamma (IFN-g) after stimulation by alloantigens. In some circumstances, T cells are unable to react to stimulation by alloantigens; thisBo Xiao and Yanqun Zhang contributed equally to this work.

*Address correspondence to Bo Xiao, MD, PhD, Department of Plastic Surgery, Xijing Hospital, Air Force Medical University, No. 127 Changle West Road, Xi’an, Shanxi 710032, China. Tel: ( 86) 29 84775301; Fax: ( 86) 29 84775301. E-mail: xiaobo@fmmu.
edu.cn; and Baoqiang Song, MD, PhD. Department of Plastic Surgery, Xijing Hospital, Air Force Medical University, No. 127 Changle West Road, Xi’an, Shanxi 710032, China. E-mail: [email protected]

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https://doi.org/10.1016/j.transproceed.2020.08.023

Transplantation Proceedings, XX, 1e7 (2020) 1

2 XIAO, ZHANG, LIU ET AL
unresponsive state is called T-cell anergy [2]. Induction of T-cell anergy is a potential strategy to inhibit the graft rejection response.
The transcription factor nuclear receptor subfamily 4 group A member 1 (NR4A1), also named Nur77, has emerged as an important player in the immune response through its contribution to maintaining tolerance and its capacity to inhibit T-cell expansion [3]. For example, over- expression of NR4A1 inhibits the effector T-cell differenti- ation [4], and deletion of NR4A1 overcomes T-cell tolerance and enhances the CD4þ and the CD8þ T-cell activation [4,5]. Furthermore, deletion of NR4A1 enhances immunity against tumors [6] and chronic viruses [7]. However, the effect of NR4A1 in allotransplantation immunity remains to be investigated.
Although no endogenous ligands of NR4A1 have yet been identified, cytosporone B (Csn-B) is a pharmacologic agent that affects NR4A1 activity [8]. In this study, we tested whether administration of Csn-B could induce a sustained NR4A1 expression and suppress the graft rejection response in a rat free flap allotransplantation model. Furthermore, T-cell activation and differentiation in the secondary lymphoid organs in vivo as well as the immunologic cytokine production in vitro were investigated to explore the effects of NR4A1 on immune inhibition.

MATERIALS AND METHODS
Animals
Adult male Brown Norway (RT.1n) and Lewis (RT.11) rats (Vital River, Beijing, China) weighing 200 to 250 g were used. Animal experiments were performed with permission of the local author- ities (The Fourth Military Medical University, Xi’an, China). Ani- mals used were housed in the animal facility of The Fourth Military Medical University according to animal care and use guidelines.

Free Flap Preparation and Transplantation
Superficial groin free flaps removed from donor Brown Norway rats were transplanted into recipient Lewis rats. Briefly, rats were anesthetized and the groin and leg areas were prepared and draped in a sterile fashion. The flaps were harvested and the flap bed was perfused with 5 mL of phosphate-buffered saline. Then the native vessels were reanastomosed using interrupted 11-0 nylon sutures. All of these manipulations were done at room temperature (20◦C).
The operative area was securely dressed, and the rat was placed in a biohazard level 2 animal care rooms. The allografts were evaluated daily for evidence of rejection. The endpoint of the study was first sign of epidermolysis.

In Vivo Experimental DesignLewis recipients were classified into 3 groups: group I, allo- transplantation group; group II, Csn-B perioperative treatment (allotransplantation Csn-B); group III, DMSO perioperative treatment (allotransplantation DMSO). Csn-B was purchased from Sigma-Aldrich (Shanghai, China) and reconstituted with DMSO. Drug was freshly dissolved in saline (0.9% wt/vol) prior to daily treatment of recipients from the day of and days 1 to 6 after allotransplantation (10 mg/kg i.p.). DMSO were injected as placebo (0.9% saline, 3.5% DMSO vol/vol). Naive Lewis rats composed the naive group and naive Lewis rats receiving the same Csn-B protocol as Group II composed the Csn-B group.

Flow Cytometry
At 7 days after transplantation, spleens from each group were harvested. Single-cell suspensions were prepared by grinding the tissues with the plunger of a 5 mL disposable syringe and then suspending them in 1640 medium. Splenocytes were treated with a hemolysis buffer to remove red blood cells. For IFN-g staining, splenocytes were incubated with FITC-labeled anti-IFN-g (BD Biosciences, San Diego, Calif, United States). For regulatory T (Treg) cell staining, splenocytes were incubated with fluorescein isothiocyanate–labeled anti-CD4 (BD Biosciences) and
phycoerythrin-labeled anti-CD25 monoclonal antibodies (BD Bio-
sciences) for 30 minutes at 4◦C in the dark. After washing, these cells were then fixed and stained with allophycocyanin-labeled anti- rat Foxp3 monoclonal antibody, according to the manufacturer’s instructions (eBioscience, San Diego, Calif, United States). Cells
were then washed 3 times and resuspended in flow cytometry buffer; 106 cells were assayed using a FACSCalibur flow cytometer (Becton Dickinson, San Diego, Calif, United States), and data were analyzed using WinMDI 2.9 software (Scripps Research Institute, La Jolla, Calif, United States).

Cell Culture and Retrovirus Transfection
Spleens from adult male C56BL/6 (Vital River) were harvested. Single-cell suspensions were prepared by grinding the tissues with the plunger of a 5 mL disposable syringe and then suspending them in 1640 medium. Splenocytes were treated with a hemolysis buffer to remove red blood cells. The suspension was centrifuged at 250g for 5 minutes and the pellet was washed in MACS buffer. Then 30
Survival data for allotransplanta- tion group (n 6), allotransplantation DMSO group (n 6), and allotransplantation cytosporone B group (n 7). The criterion for limb allo-
graft loss was the first sign of epidermol- ysis. Statistical significance of P < .05 was calculated using log-rank test.

þNR4A1 OVEREXPRESSION PROLONGS GRAFT SURVIVAL 3Image
Fig 2. Representative histologic finding on postoperative day 7. (left) Histologic finding of the allotransplantation cytosporone B group is normal (histologic grade 0). The epidermis is intact and there is little lymphocyte infiltration in the dermis. (right) The skin in the allotransplantation þ DMSO group demonstrates intense and diffuse dermal inflammatory infiltrate composed of mononuclear cells.
mL of CD4þ T-cell beads (Miltenyi Biotec, Darmstadt, Germany) was added to each suspension and incubated at 4◦C for 15 minutes. Cell suspensions were then topped up with 5 mL MACS buffer and centrifuged at 200g, and the pellet was resuspended in 1 mL MACS
buffer. During this time, a large separation column was placed on the magnet as per the manufacturers’ instructions. Then the cell suspension passed through the column by gravity. The column was removed from the magnetic field and 2 mL MACS buffer was added to the column to wash the cells into a fresh 15 mL tube. The cells
were then checked for purity by FACS and then resuspended in culture media for further applications.

Human full-length NR4A1 was constructed in a pcDNA3.1 plasmid vector. The plasmid was packaged into a retrovirus to enhance T-cell transfection efficiency. An empty vector (RV-vec- tor) served as a negative control. All of the plasmids and retrovirus were synthesized and packaged by GenePharma (Shanghai, China). CD4þ T cells were activated with plate-bound anti-CD3e (BD Biosciences) and anti-CD28 (BD Biosciences) under neutral
conditions. Thirty-six hours after activation, cells were infected with retrovirus RV-NR4A1 or the control empty vector (RV-vector).
Quantitative Real-Time Polymerase Chain Reaction

The expression of NR4A1 mRNA both in recipient spleens and acti- vated T cells was analyzed by real-time polymerase chain reaction (RT- PCR). Strand DNA was synthesized with the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Shanghai, China) as per the manufacturer’s instructions. Quantitative RT-PCR was performed on an iQ5 multicolor RT-PCR detection system using FastStart Universal
SYBR Green Master (Thermo Scientific) according to the manufac- turer’s instructions. The primers for the amplification of NR4A1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed and synthesized by Shenggong (Shanghai, China). The primer se- quences were as follows: NR4A1 (human) sense: 50-ATA- CACCCGTGACCTCAACCA-30, antisense: 50-TTCTGCAC
TGTGCGCTTGAA-30; NR4A1 (rat) sense: 50-CGTGCCTTTA

Cytosporone B administration induces NR4A1 expression in vivo. An induction of NR4A1 mRNA (left) and protein expression (right) was observed after NR4A1 stimulation. NR4A1 mRNA expression was detected by quantitative real-time polymerase chain re- action, and NR4A1 protein expression was determined by Western blotting analysis after recipient spleen tissue were harvested on day 7. Data are presented as means SE of 3 independent experiments performed in triplicate. *P < .05 vs naive; #P < .05 vs allo-
transplantation or cytosporone B (unpaired t test).

4 XIAO, ZHANG, LIU ET AL
Image
Fig 4. T helper type 1 (Th1) cell ratio but not T regulatory (Treg) cell ratio was reduced by cytosporone B administration in recipient spleens. Recipient spleens were harvested and examined for Th1 and Treg cell characteristics by flow cytometry for interferon gamma and Foxp3 expression on day 7 in each group. The Th1 cell ratio was significantly lower in the allotransplantation cytosporone B group compared to the allotransplantation DMSO group (P < .05, unpaired t test). There was no difference in Treg cell ratio betweenthe 2 groups (P > .05, unpaired t test).
AGCCCATAGC-30, antisense: 50-TCTGGAATGAGGAGATACAT CAGTCT-30; GAPDH (human) sense: 50-CCACCCATGGCA AATTCCATGGCA-30, antisense: 50-TCTAGACGGCAGGTC AGGTCCACC-30; and GAPDH (rat): sense: 50-CGTATCG- GACGCCTGGTTA-30, antisense: 50-GACTGTGCC
GTTGAACTTGC-30. After 5 minutes of initial denaturation at 95◦C, PCR was carried out for 40 cycles at 95◦C for 10 seconds and 60◦C for
30 seconds. GAPDH was used as the housekeeping gene. The
threshold cycle (Ct) value was measured, and the comparative gene expression was calculated by the 2—DDC1 method as described before.
Enzyme-Linked Immunosorbent Assay
IL-2 and IFN-g concentrations in cell culture supernatants were determined using a Quantikine ELISA (enzyme-linked immuno- sorbent assay) kit (R&D Systems, Shanghai, China), following the manufacturer’s protocol.

NR4A1 OVEREXPRESSION PROLONGS GRAFT SURVIVAL 5
Image
Fig 5. NR4A1 was overexpressed after RV-NR4A1 transduction in T cells after stimulation. NR4A1 mRNA expression in the vector transduction group and the RV-NR4A1 transduction group was detected by quantitative real-time polymerase chain reaction for the indicated times. Data are presented as means SE of 3 independent experiments. *P < .05 (unpaired t test).

RESULTS
Csn-B Administration Activates NR4A1 Expression and Is Associated With Prolonged Allograft Survival Time
Csn-B administration from day 0 to day 6 is associated with the prolonged survival time of the rat free flap allografts (Fig 1). On day 7, flaps in the group of rats treated with Csn- B had a normal appearance, and skin biopsy specimens showed few infiltrated inflammatory cells. Flaps in the
group of rats treated with DMSO showed extraordinary swelling, and skin biopsy specimens showed high inflam- matory cell infiltration, which was consistent with Banff grade IV rejection (Fig 2). On day 7, spleens from each group were harvested and RT-PCR) and Western blotting were performed to assess NR4A1 expression. NR4A1 mRNA and NR4A1 protein expression trends in each group were similar (Fig 3), and NR4A1 expression was lowest in

NR4A1 overexpression inhibited interleukin 2 (IL-2) and interferon gamma (IFN-g) secretion in activated T cells. IL-2 and IFN-g levels were determined by enzyme-linked immunosorbent assay at the indicated times. Data are presented as means SE of 3 independent exper- iments. Except for IL-2 at 1 hour, the levels of IL-2 and IFN-g in the RV-
NR4A1 group were significantly lower than those of the vector group (P < .05, unpaired t test).

6 XIAO, ZHANG, LIU ET AL

the naïve rats. In both allotransplantation groups regardless of treatment with Csn-B or DMSO injections, the expres- sion of NR4A1 was significantly elevated compared to that in the naive group, which shows that allotransplantation alone can stimulate NR4A1 expression. Additionally, in the group receiving the allotransplantation with Csn-B admin- istration, the expression of NR4A1 was significantly higher than that in the groups receiving only Csn-B treatment or only receiving an allotransplantation.
Csn-B Administration Reduced T Helper Type 1 Ratio in Recipient Spleens

To investigate the mechanism underlying the survival of the transplanted grafts, the recipient spleens were har- vested and examined for T helper type 1 (Th1) and Treg cell characteristics by flow cytometry on day 7 in each group (Fig 4). The Th1 cell population was significantly smaller in the group with allotransplantation and treat- ment with Csn-B than in the group with allo- transplantation and treatment with DMSO (3.9%0.42% vs 10.2% 0.87%, P < .05), showing that Csn-B administration and NR4A1 overexpression both severely
impaired Th1 cell differentiation. There was no significant difference in the population of Treg cells between the 2 groups (1.9% 0.21% vs 2.1% 0.17%, P > .05),showing that Csn-B administration and NR4A1 over-
expression do not affect Treg cell proliferation.NR4A1 Overexpression Reduces IL-2 and IFN-g Levels in CD4þ T Cells
þWe overexpressed NR4A1 in CD4 T cells by transfecting the cells with retrovirus RV containing NR4A1. We then tested the mRNA expression of NR4A1. We found that NR4A1 mRNA expression was higher than in the control group, which showed successful RV infection and NR4A1 overexpression (Fig 5). We next tested the protein expres- sion of IL-2 and IFN-g at different times after stimulation in each group by enzyme-linked immunosorbent assay. We found that both IL-2 and IFN-g expressions peaked at 3 to 6 hours after stimulation in the vector-transduced group. The expressions of IL-2 and IFN-g were both much lower than that in the control group at each time point, showing that protein secretion was also strongly suppressed in the NR4A1 overexpression cells (Fig 6).

DISCUSSION
More than 150 vascularized composite allotransplantation procedures have been performed worldwide, including procedures on the hand, face, penis, and lower extremities, since the first successful hand transplant was performed in 1999 [9]. Complications of anti-rejection drugs remain a cause of graft in tolerance and death of recipients. Toler-
ance induction continues to be considered the “holy grail” of allotransplantation. However, traditional anti-rejection
drugs such as tacrolimus can suppress transplant rejection response while failing to induce tolerance.
NR4A1 is considered a key regulator of transplant toler- ance. High levels of NR4A1 expression have been reported in several tolerance models [4,10].
In our free flap allo- transplantation model, NR4A1 stimulation was able to suppress the graft rejection response and was associated with prolonged graft survival time. The inhibitive immuno- logic status was in accordance with the high level of NR4A1 expression. However, owing to a lack of induction of persistently high-level expression of NR4A1, grafts were rejected after NR4A1 expression returned to normal levels. The survival of the grafts was in accordance with the
decreased pool of Th1 cells in the secondary lymphoid organs. Because rejection response belongs to the Th1 cell– mediated cell immunity, the inhibition of differentiation of Th1 cells due to NR4A1 stimulation has a special role in
suppressing graft rejection response. This result is in line with that of a previous study that reported that an enforced NR4A1 expression severely impaired Th1 cell differentia- tion in vitro [6]. Although Treg cells were demonstrated to be effective in suppressing graft rejection and inducing tolerance, our study showed that the Treg cell pool did not change after NR4A1 stimulation, which demonstrated that the inhibition of rejection was not caused by Treg expansion in our model. This result confirmed the results of the pre- vious study that reported that NR4A1 expression did not affect the differentiation of Treg cells in vitro [4].

IL-2 is mainly secreted by the antigen-activated T cells. IL-2 has a wide range of activities, including the ability to boost the cytolytic activity of natural killer cells or lymphokine-activated killer cells, to increase the cytolytic activity of tumor-infiltrating lymphocytes, to augment immunoglobulin production by activated B cells, and to modulate effector T-cell differentiation [11]. Shortage of IL-2 in the microenvironment leads to anergy of the acti- vated T-cell expansion [12]. IFN-g is the main cytokine that induces Th1 differentiation and is also an important factor that mediates graft rejection. In the overexpressed NR4A1 T-cell model, the levels of IL-2 and IFN-g were both decreased after stimulation. These results indicate that high persistent expression of NR4A1 suppresses the graft rejec- tion response by blocking the production of key immuno- logic cytokines. In other experiments, the regulation of NR4A1 expression has also been reported to influence cytokine production. Hamers et al reported a higher neutrophil influx and enhanced IL-6, monocyte chemotactic protein 1, and neutrophil attracting chemokine production in the NR4A1-deficient colons in a colitis animal model [7]. McEvoy et al reported that altered NR4A1 activity leads to the suppression of lipopolysaccharide induced monocyte chemotactic protein 1 mRNA and protein expressions in human and murine myeloid cells [13].

In conclusion, we demonstrated that administration of Csn-B activated the expression of NR4A1 in allotransplant recipients and was associated with prolonged survival time of vascularized free flap allografts. Our study found that suppression of the immune response was in accordance with lower pools of Th1 cells in the recipient secondary lymphoidNR4A1 OVEREXPRESSION PROLONGS GRAFT SURVIVAorgans. Our in vitro study found that IL-2 and IFN-g levels were suppressed in T cells with NR4A1 overexpression, which suggests that overexpressed NR4A1 may block downstream cytokine expression and induce T-cell anergy to suppress the graft rejection response.

FUNDING
This study was supported by the Natural Science Founda- tion of China (NSFC 81401615).

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