Increased frequency of myeloid-derived suppressor cells facilitating skin allograft survival in aged mice

Background More and more aged people have organ transplantation recently. Aging process may have an inuence on immunity, which conducts an adjustment of immunosuppressive agents to prevent adverse effects. Understanding of aging effects on immunity will be helpful for post-transplant care of aged recipients. Results A mouse model using C3H mice as donors and aged/young C57BL/10J mice as recipients was employed to study the aging effects on immunity. The frequency of CD4 + , CD8 + and native CD4 + foxp3 + regulatory T-cells in the spleen were not different between aged and young mice. However, the frequency of CD11b + Gr-1 + myeloid-derived suppressor cells (MDSC) was higher in aged mice (4.4 ± 1.4% versus 1.6 ± 1.1%, p=0.026). To measure cytokines in the serum, the level of TGF-β was higher in aged mice than in young mice (21.04 ± 3.91ng/ml versus 15.26 ± 5.01ng/ml, p = 0.026). In vitro, enriched T-cells from aged mice had lower proliferation capacity (0.350±0.003 O.D. versus 0.430±0.017 O.D. at responders/stimulatory cells = 100/1) and lower Ag-specic cytotoxic ability (21.2 ± 3.0% versus 39.3 ± 4.8% at target cell/effector cells = 1/100, p=0.003) than T-cells from young mice. In vivo, the skin allografts survived on aged recipients was 19.7 ± 5.2 days, compared 11.9 ± 4.1 days on young mice (p = 0.005). When entinostat was applied to aged mice to block MDSC, the survival of skin allografts was shorten to 13.5 ± 4.7 days which was not different from the survival on young mice (p = 0.359). Conclusion The allogeneic immunity was lower in aged than in young mice evidenced by a higher frequency of MDSC, higher serum level of TGF-β, decreased function of


Background
Organ transplantation is widely accepted as the nal treatment for the patients with a speci c organ failure or even multi-organ failure. In solid organ transplantation, graft/patient survival is much improved recently because of the advances in immunosuppressive management, surgical techniques and perioperative care. Among these advances, immunosuppressive management is most important because it is not only employed to prevent acute rejection in the short-term but also contribute to long-term graft/patient survival. However, heavy loading of immunosuppressants may be complicated with the adverse effects including neurotoxicity, nephrotoxicity, hypertension, hyperglycemia, bone marrow suppression, gastrointestinal disturbance, and so on. (1)(2)(3) Heavy immunosuppressants may also inhibit hosts' defensive immunity, increase the risk of opportunistic infection and induce the development of neoplasms.(4-6) Therefore, suitable levels of immunosuppressive agents are essential to keep allografts with good function and prevent the adverse effects of organ transplantation. (7) Currently, aging people is increasing. Many and many aged people may have organ transplantation. In OPTN/SRTR report in 2015, liver transplant recipients above aged 65 years were increased from 9.2% in literature, a narrower repertoire of T-cells and senescent T-cells to decrease the defending abilities to viral infection were reported. (10,11) Therefore, the dosage of immunosuppressive agents should be minimized in aged people to avoid over-immunosuppression-related morbidities (12). However, what are the basic reasons beyond T-cells that immunosuppressive agents can be minimized for aged people is not clearly de ned.
In this study, we designed a mouse model using aged and young C57BL/10J (B10) mice as recipients to de ne the aging effects on immunity. The understanding of aging effects on immunity is helpful for posttransplant care of aged recipients in the future.

Results
Immune cell population in aged mice. To determine the alteration of immune cell population in aged mice, the spleens were obtained from aged and young mice to yield spleen cells. The spleen cells were stained by a panel of monoclonal antibodies and analyzed by ow cytometry. The results showed that the frequency of CD4 + T-cells in aged mice (n = 7) was 14.6 ± 2.9% compared to 16.5 ± 1.2% in young mice (p = 0.185) and CD8 + T-cells in aged mice was 8.4 ± 1.6% compared to 8.9 ± 0.7% in young mice (p = 0.324). The frequency of T-cells was not different between aged and young mice. The frequency of native regulatory T-cells (CD4 + foxp3 + ) in aged and young mice was not different, either (0.4 ± 0.6% versus 1.0 ± 0.8%, p = 0.160). However, the frequency of myeloid-derived suppressor cells (MDSC; CD11b + Gr-1 + ) in aged mice was 4.4 ± 1.4% which was higher than 1.6 ± 1.1% in young mice (p = 0.026, Fig. 1).
Cytokine measurements in serum. To determine whether the common cytokines in serum were different in aged and young mice, IL-12, IL-10, IFN-γ and TGF-β were measured. The result showed that the level of TGF-β was higher in aged mice than young mice (21.04 ± 3.91 ng/ml versus 15.26 ± 5.01 ng/ml, p = 0.026). The levels of IL-12, IL-10 and IFN-γ were not different between aged and young mice.
Immunological reactions of direct antigen presentation in aged recipients. Bone marrow-derived C3H dendritic cells (DC) was employed as donor antigen-presenting cells and enriched T-cells from aged and young B10 mice were employed as recipients' cells to perform mixed lymphocyte reaction (MLR). The C3H DC cultured in GM-CSF and IL-4 expressed mature phenotype with high levels of CD40, CD80. CD86 and I-A k (Fig. 2a). When this mature C3H DC was applied to activate enriched T-cells derived from aged or young B10 mice, MLR showed that proliferation capacity of T-cells from aged mice was lower than those from young mice (0.350 ± 0.003 optic density (O.D.) versus 0.430 ± 0.017 O.D. at responders/stimulatory cells = 100/1, p = 0.001) (Fig. 2b).
Cytotoxic abilities of T-cells derived from aged mice. T-cells derived from aged and young B10 mice were activated by C3H DC for 3 days and employed as effector cells to perform antigen (Ag)-speci c and nonspeci c cytotoxic abilities. R1.1 cells (H-2 k , allogeneic), p815 cells (H-2 d , third party) and Yac-1 cells (non-Ag-speci c, nature killer-sensitive) were used as target cells. The results showed that T-cells derived from aged mice had a lower Ag-speci c cytotoxic ability than T-cells derived from young mice (21.2 ± 3.0% versus 39.3 ± 4.8% at target cell/effector cells = 1/100, p = 0.003) (Fig. 3a). Even for the third party targets, T-cells derived from aged mice also had a lower cytotoxic ability than T-cells derived from young mice (6.0 ± 0.6% versus 11.0 ± 2.7% at target cell/effector cells = 1/100, p = 0.017, Fig. 3b). For non-speci c targets, the cytotoxic ability of T-cells derived from aged or young mice were not different. (28.6 ± 0.6% versus 32.8 ± 3.5% at target cell/effector cells = 1/100, p = 0.100, Fig. 3c), Inducible regulatory T-cells in aged mice. To determine whether regulatory T-cells could be induced in vitro, enriched T-cells derived from aged and young B10 mice were activated by C3H DC for 3 days and CD4 + foxp3 + cells were examined. The results showed that a higher frequency of CD4 + foxp3 + regulatory cells was induced in T-cells derived from aged mice than young mice (7.87 ± 3.42% versus 5.04 ± 2.71%, p = 0.023) (Fig. 4). Survival of skin allograft survival. To determine whether allograft survival was different in aged and young recipients, skin grafts from C3H mice were transplanted to aged (n = 20) and young (n = 11) B10 mice and survival time was recorded. To determine whether MDSC played an important role of immunosuppression in aged mice, a half of aged mice was fed with entinostat (50 mg/kg/week, Selleck Chemicals, Houston, Tx) after skin transplantation. The results showed that the skin allograft survival on young B10 mice was 11.9 ± 5.2 days and was prolonged to 19.7 ± 5.2 days on aged mice (p = 0.005).
When the aged mice was fed with entinostat to inhibit MDSC, the survival of skin grafts was shorten to 13.5 ± 4.7 days, which was signi cantly shorter than the survival on aged mice without entinostat feeding (p = 0.042) and not different from the survival on young mice (p = 0.359, Fig. 6).

Discussion
The different frequency of immune cells in aged and young mice was mainly in MDSC. By analyzing the immune cells in the spleen, the frequency of CD4 + T-cell, CD8 + T-cells and regulatory T-cells were not different between aged and young mice. However, the frequency of MDSC in aged mice was higher than in young mice. MDSC is a well-known immune-suppressor cell which is associated with cancer, sepsis, chronic in ammation, trauma, etc. (13,14) While the frequency of MDSC increases, the immunity of the hosts is suppressed. Hurez et al in their study described that MDSC was increased in aged mice and depletion of MDSC by anti-Gr-1 antibody improved the anti-tumor immunity in aged mice. (15) In this study, the higher frequency of MDSC in aged mice con rmed the frequency of MDSC was increased along with aging. While the function of MDSC was neutralized by entinostat (a class I-speci c histone deacetylase inhibitor)(16), the survival of allogeneic skin grafts on aged mice was shortened and similar to the survival on young mice. This re ected the immunologic suppression of MDSC in aged mice.
The proliferating capacities of T-cells were decreased in aged mice. According to the results in this study, the proliferation of T-cells from aged mice was lower than those from young mice when the T-cells were stimulated by allogeneic dendritic cells. In Shen's study, they adoptively transferred young naïve T-cells to aged and young mice and found that the expansion of T-cells was reduced in the microenvironment of aged mice, and T-cell response to allo-transplantation in vivo was also impaired.(9) They concluded that T-cell immunity was impaired by intrinsic and extrinsic factors. Yager et al reported that the repertoire diversity in T-cells was declined in aged mice, which impaired CD8 + T-cells response to known immunedominant epitopes.(11) Thus, proliferating ability of T-cells of aged mice was reduced and the reduction of T-cells was related to T-cells themselves and environmental factors.
Cytokine pro le is one of the environmental factors in aged and young mice. Thus, a panel of cytokines in the serum were measured for aged and young mice. The results showed that the levels of TGF-β in the serum of aged mice were signi cantly higher than that in young mice. TGF-β is a multi-functional cytokine with the property of immunosuppression. In the microenvironment with a high level of TGF-β, dendritic cells express low levels of co-stimulatory molecules and decrease the stimulatory capacity on Tcells. (17,18) Naïve T-cells exposed in a high level of TGF-β will be induced to become regulatory T-cells. (19,20) In this study, we also found that T-cell proliferation capacity were lower in indirect antigenpresenting pathway when they were stimulated by dendritic cells coming from aged mice compared to young mice. All these results implied that immune cells were suppressed in aged mice which had high serum levels of TGF-β.
Although the phenotypic population of CD4 + and CD8 + T-cells were not altered in aged mice, the cytotoxic abilities of T-cell coming from aged mice were lower than those coming from young mice. In vitro study, the results clearly showed that antigen-speci c cytotoxicity of aged T-cells was signi cant lower than young T-cells. In vivo study, skin allografts survived longer in aged recipients than in young recipients. In a model of mice implanted with tumors, Grizzle et al demonstrated that T-cell cytotoxicity to tumor cells was decreased in aged mice and the tumor implanted in aged mice grew more rapidly than in young mice. (21) They also found that the decline of T-cell cytotoxicity was correlated to accumulation of myeloid-derived suppressor cells. Clearly, T-cell immunity declined along with aging and leaded to prolong skin graft survival in aged mice.
Regulatory T-cells could be induced in aged mice although native Treg was not different between aged and young mice. Treg is an important cells to modulate immune reactions in the hosts. (22) In organ transplantation, Treg is believed to induce infectious tolerance. (23,24) Currently, ex vivo expansion of Treg has been applied to induce tolerance in clinical trials. (25) In this study, Treg was easier to be induced from T-cells derived from aged mice than from young mice. The true mechanism was not known. However, the higher serum level of TGF-β in aged mice may prime the property of Treg and contributed to following induction. It was reported that TGF-β played a critical role of Treg development in thymus, but also could induce Treg in peripheral. (19) The higher level of TGF-β implied that T-cell-mediated immunity could be suppressed easier in aged mice than in young mice and the grafts survival was prolonged.

Conclusion
The allogeneic immunity was lower in aged than in young mice evidenced by a higher frequency of MDSC, higher serum level of TGF-β, decreased function of T-cells, and easy-to-induced regulatory T-cells in aged mice. Blocking the function of MDSC by entinostat reversed the low immunity in aged mice and cause skin allograft rejection similar to young recipients. Taking together, the cellular immunity is weaker in aged mice than in young mice and MDSC plays the important role. Based on these experimental results, the clinical regimen of immunosuppression induction after transplantation for aged recipients should be adjusted to prevent or decrease adverse effects. Enriched T-cells. Enriched T-cells were obtained from splenocytes passing through nylon wool column. Nylon wool column was prepared by packing 0.5 gram nylon wool in a 10-c.c. syringe. The column was equilibrated by running cultural medium and incubated at 37 o C for an hour before splenocytes were loaded into nylon wool column. Splenocyte-loaded nylon wool column was incubated at 37 o C for one hour. Then, the non-adherent cells, enriched T-cells, were collected. Quantitative T-cell proliferation. The allogeneic stimulatory capacities of DC were determined via an oneway mixed lymphocyte reaction (MLR) employing colorimetric tetrazolium (MTT) assay. (27,28) Enriched T-cells were stimulated by irradiated DC triplicatedly in 96-well plates for 3 days. In the last 4 hours of the procedure, sterilized MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (5 mg/ml, Sigma, St Louis, MO) was added to each well (10 ul/well) to produce blue formazan. Upon termination of MLR, acid-isopropanol (0.04N HCL-isopropanol, 100 ul/well) was added to each well and mixed thoroughly to dissolve the blue crystals. After 10 minutes at room temperature to ensure that all crystals were dissolved, the plates were read on a Dynatech MR580, microelisa reader, employing a test wavelength of 570 nm to measure the optical densities (OD) of formazan formation.

Materials And Methods
Cell-mediated cytotoxicity. T-cells activated by DC for three days were applied as effector cells. P815 (H-2 d ), R1.1 (H-2 k ), and YAC-1 (natural killer sensitivity) cells were applied as targets. Cell-mediated cytotoxicity was performed at various effector-to-target ratio triplicatedly in 96-well plates, and assessed by ow cytometry. To assess the cell-mediated cytotoxicity by ow cytometry, the targets were labeled with PKH-26 and 5-(and-6)-carboxy uorescein diacetate succinimidyl ester (CFSE) according to the Cytokine measurement. The serum of young and old mice was collected. The product of cytokines was measured by enzyme-linked immunoabsorbent assay. The procedure was conducted as the instructions of producers. (PharMingen, San Diego, CA).
Skin transplantation. Under adequate anesthesia, an incision was made on the ank of B10 mice. A 1 × 1 cm skin ap taken from C3H mice was attached to the ank of B10 mice and xed to adjacent skin with 3 − 0 Dexon sutures. The skin ap was protected by circulated gauge for 3 days. The skin ap were observed very 2-3 days. Rejection was diagnosed when the skin grafts were fully detached. Statistical analysis. Unpaired Student's t-test was used to analyze continuous variables. Categorical variables were analyzed by either Chi-square test or Fisher's exact test. All pairwise multiple comparisons were done by Holm-Sidak method. The survival rates were calculated using the Kaplan-Meier method. The statistical analyses were all performed with SigmaPlot 12.3 for Window software (Systat Software, Inc., San Jose, CA, USA). P < 0.05 was considered statistically signi cant.

Declarations
Ethics approval: Experimental use of these mice was approved by Animal Care Committee of Chang-Gung Memorial Hospital (No. 105-0293C).
Consent for publication: not applicable.
Availability of data and materials: All data generated or analysed during this study are included in this published article Competing interests: There is no con ict of interest among the authors.    third party) were used as target cells, T-cells derived from aged mice had a lower cytotoxic ability than Tcells derived from young mice (6.0±0.6% versus 11.0±2.7% at target cell/effector cells = 1/100, p=0.017). (c) When Yac-1 cells (non-Ag-speci c, nature killer-sensitive) were used as target cells, the cytotoxic ability of T-cells derived from aged or young mice were not different (28.6±0.6% versus 32.8±3.5% at target cell/effector cells = 1/100, p=0.100).

Figure 4
A representative of induced regulatory T-cells. Enriched T-cells derived from aged and young B10 mice were activated by C3H DC for 3 days, a higher frequency of CD4+foxp3+ regulatory cells was induced in the T-cells derived from aged mice than young mice (7.87±3.42% versus 5.04±2.71%, p=0.023).  Kaplan-Meier survival curve of skin allografts. Skin grafts from C3H mice were transplanted to young (n=11) B10 mice, aged B10 mice (n=10) and aged B10 mice (n=10) fed with entinostate. The results showed that the skin allograft survival on young B10 mice was 11.9 ± 5.2 days and was prolonged to 19.7 ± 5.2 days on aged mice (p = 0.005). When the aged mice was fed with entinostat to inhibit MDSC, the survival of skin grafts was shorten to 13.5 ± 4.7 days, which was not different from the survival on young mice (p = 0.359).