Yingnan Zhang

In vitro-expanded CD4+CD25highFoxp3+ regulatory T cells controls corneal allograft rejection

AIMS Natural CD4(+)CD25(+) regulatory cells (nTregs) have been implicated in maintaining peripheral immune tolerance. This study aims to test whether immunotherapy using in vitro-expanded Treg (iTregs) could suppress allograft rejection in corneal transplantation model. METHODS Natural CD4(+)CD25(+) T cells were freshly purified from naïve mice and expanded in vitro by culturing with anti-CD3/CD28-coated Dynabeads, interleukin (IL)-2 and transforming growth factor (TGF-β1). Suppression ability of iTregs was assayed by co-culturing with CD4(+)CD25(-) T cells (Teff) in vitro and by targeting corneal allograft rejection in vivo. Tracking of iTreg after adoptive transfer in vivo were examined by CFSE labeling. RESULTS Natural Treg cells were expanded by culturing with anti-CD3/CD28-coated Dynabeads in the presence of IL-2 and TGF-β1. Compared with nTregs, iTregs had similar expression of CD62L, and PD- L1, lower expression of CD69, higher levels of PD-1, CD25, and Foxp3. iTreg cells exerted stronger suppression function than natural Treg cells when cocultured with CD4(+)CD25(-) T cells in vitro and prevented fully MHC-mismatched corneal allograft rejection. Survival of iTreg cells could suppress alloimmune reaction and most prone to migrate to graft draining LNs and spleens. Moreover, maintaining CD25 expression on iTregs was indicative for preservation of allosuppression. CONCLUSION Therapeutic use of in vitro-expanded CD4(+)CD25(+) T cells may be a effective and safe tool for controlling allograft rejection and may help induce allograft tolerance.

Histone deacetylase inhibitors promote mice corneal allograft survival through alteration of CD4+ effector T cells and induction of Foxp3+ regulatory T cells

Trichostatin A (TSA) is classical Histone deacetylase inhibitors (HDACIs) II which is used in treatment of advanced cutaneous T-cells lymphoma. Our works focused on the roles of TSA on immuno-modulatory. We found that the TSA could induce resting Teff cells into apoptotic cell death and inhibit Teff cells proliferation in a dose-dependent manner. We also observed down-regulation effects of various costimulatory/adhesion molecules on Teff cells and up-regulation of Foxp3 expression on CD4+ CD25+ T cells. Treatment with TSA could improve mice corneal allograft survival by promoting the proportions and allosuppressive function of CD4+ CD25+ regulatory T cells. Our findings suggest that the use of TSA allows the beneficial pharmacological effect on CD4+ CD25- T activation in vitro and enhancement of Foxp3+ Treg cells in vivo.

CD154 blockade modulates the ratio of Treg to Th1 cells and prolongs the survival of allogeneic corneal grafts in mice

Administration of anti-CD154 monoclonal antibody (mAb) may prolong the survival of an allograft; however, the associated therapeutic mechanisms remain poorly understood. This study aimed to evaluate the effects of anti-CD154 mAb on T-cell responses in a mouse model of corneal allograft transplantation. BALB/c mice were transplanted with corneal grafts from C57BL/6 mice and treated intraperitoneally with 250 μg anti-CD154 mAb or isotype IgG on days 0, 3 and 6 post surgery. The transparency of the corneal grafts was evaluated for potential rejection signs by slit-lamp biomicroscopy and histopathology. The percentages of CD4+ T, Tim-3+CD4+ T helper (Th) 1 and CD4+CD25+Foxp3+ regulatory T cells (Tregs) in the spleen, ipsilateral draining lymph nodes and corneal grafts, and the frequency of splenic IFN-γ+ and IL-10+ expression in CD4+ T cells were determined by flow cytometry. Moreover, the ratio of Tregs to Th1 cells was calculated and the suppressive activity of splenic Tregs was measured. Anti-CD154 neutralization significantly prolonged the survival of the corneal allograft (P=0.0012) and reduced the numbers of inflammatory infiltrates in the corneal graft. In the spleen and lymph nodes, anti-CD154 treatment reduced the frequency of CD4+ T cells, Tregs and particularly Th1 cells. In the corneal allografts, anti-CD154 treatment downregulated graft-infiltrated CD4+ T cells and Th1 cells, but increased graft-infiltrated Tregs. Furthermore, anti-CD154 treatment increased the frequency of splenic IL-10+CD4+ T cells and decreased the concentration of splenic IFN-γ+CD4+ T cells. As a result, the ratio of Tregs to Th1 cells in the anti-CD154-treated recipients increased. Anti-CD154 treatment did not enhance the suppressive activity of Tregs in the recipients. The results indicate that the therapeutic effects of anti-CD154 mAb on prolonging the survival of the corneal allograft may be associated with an increased ratio of Tregs to Th1 cells in mice.

Tryptase compromises corneal epithelial barrier function

Corneal epithelial barrier dysfunction is harmful to corneal health; the pathogenesis is unclear. This study aims to elucidate the mechanism by which tryptase compromises corneal epithelial barrier function. Human corneal epithelial cell line (HCE cells) was cultured into monolayers using as a study platform. Quantitative reverse transcription polymerase chain reaction and Western blotting were employed to detect the expression of matrix metalloprotenases (MMP)9. The endosome/lysosome fusion was observed by confocal microscopy. The corneal epithelial barrier function was assessed in Transwell system. The results showed that HCE cells expressed proteinase‐activated receptor (PAR)2. Activation of PAR2 by tryptase induced expression of MMP9 in HCE cells, interfered with the fusion of endosome/lysosome, and compromised the epithelial barrier function, which could be prevented by pretreatment with MMP9 inhibitor. We conclude that tryptase can increase the expression of MMP9 in HCE cells and compromise the epithelial barrier function. Copyright

Adoptive transfer of donor corneal antigen-specific regulatory T cells can prolong mice corneal grafts survival.

Purpose: To explore the effects of adoptive transferring T regulatory cells (Treg cells) stimulated by donor corneal antigen (Ag) to prevent corneal transplantation immune rejection in mice. Methods: C57BL/6 mice were used as donors and BALB/c mice as recipients. Corneal Ag was harvested by homogenization and centrifugation. Bone marrow dendritic cells (DCs) from BALB/c mice were cultured with stimulation of granulocyte-macrophage colony-stimulating factor and interleukin-4 for 5 days. Then, donor corneal Ag was added to obtain donor corneal Ag-loaded DCs. The DCs were used to stimulate CD4+CD25+ and CD4+CD25− T cells from the recipient to yield Ag-stimulated Treg cells. Penetrating keratoplasty was performed in the mice. The recipients were randomly divided into 3 groups receiving 0.1 mL of phosphate-buffered saline, 1 × 106 naive Treg cells, and Ag-stimulated Treg cells, respectively, given by retroorbital injection at the end of surgery. The allografts were observed, and histopathological examination was performed 15 days after surgery. Results: The corneal Ag mainly comprised 2 proteins with molecular weight 54 and 42 kD, respectively. Corneal Ag-loaded DCs expressed higher levels of CD11c, CD80, and CD86 than bone marrow precursor cells. Both CD4+CD25+ and CD4+CD25− T cells showed vigorous proliferative responses to corneal Ag-loaded DCs. Mean survival time of the mice corneal allografts in phosphate-buffered saline, naive Treg cells, and Ag-stimulated groups was 8.1 ± 1.1, 14.3 ± 2.0, and 23.3 ± 2.6 days, respectively (P < 0.01 among groups). Histopathological examination revealed less inflammatory cells infiltration in Ag-stimulated than in naive mice. Conclusions: Adoptive transfer of donor corneal Ag-specific Treg cells prolonged survival time of corneal allografts in our mouse model, which might suggest a useful approach to cellular immunotherapy for corneal transplantation immune rejection.

Porcine endothelial grafts could survive for a long term without using systemic immunosuppressors: An investigation of feasibility and efficacy of xeno-Descemet's stripping automated endothelial keratoplasty from WZS-pig to rhesus monkey

In current non‐human primate models, full‐thickness porcine grafts could not achieve long‐term survival without using potent systemic immunosuppressors. Moreover, the thickness disparity in xeno‐PKP proved to be hard to manipulate and may cause several complications which also could prevent the grafts from long survival. Considering the advantages of Descemets stripping automated endothelial keratoplasty (DSAEK) derived from its ultrathin graft and lower rejection rate, we hypothesize xeno‐DSAEK may overcome these imperfections in xeno‐PKP. The aim of this study was to explore the feasibility and efficacy of xeno‐DSAEK and to investigate the possibility of long‐term survival of porcine DSAEK grafts only using local immunosuppressors.

The feasibility and efficacy of preparing porcine Descemet’s membrane endothelial keratoplasty (DMEK) grafts by two techniques: An ex-vivo investigation for future xeno-DMEK

Descemet’s membrane endothelial keratoplasty (DMEK) might be a promising technique for future xeno‐corneal transplantation due to its ultrathin graft, extremely low rejection occurrence, suture‐free graft fixation, and minimal immunosuppressive regime usage. The aim of this study is to explore the feasibility and efficacy of preparing porcine DMEK grafts by 2 techniques and investigate the graft ultrastructure.

Labial Salivary Gland Transplantation for Severe Dry Eye in a Rhesus Monkey Model

Purpose To evaluate the effectiveness of autologous labial salivary gland with labial mucous membrane graft in a rhesus monkey model with severe dry eye. Methods Eight eyes of eight rhesus monkeys with severe dry eye were included. Four eyes underwent autologous labial salivary gland and mucous membrane graft (group 1) and four eyes served as controls (group 2). The ocular surface was evaluated before and after transplantation surgery (at 1, 4, 8, 12, and 24 weeks). Conjunctival impression cytology was performed before and 24 weeks after transplantation. Finally, a histological analysis of the cornea, conjunctiva, and transplanted grafts was performed. Results At inclusion (n = 8) the mean Schirmer test was 1.31 ± 0.53 mm, the mean fluorescein score was 4.7 ± 1.65, and the mean lissamine green staining was 4.38 ± 0.48. After transplantation, a significant increase in tear secretion was observed with the mean Schirmer test results in group 2 significantly higher than those observed for group 1 at all time points (P < 0.05). Similarly, fluorescein and lissamine green scores were significantly lower in group 2 than in group 1 at all time points after transplantation (P < 0.05). Impression cytology specimens showed severe conjunctival squamous metaplasia without goblet cells in both groups. Under light microscopy, no significant difference was observed between the cornea and the conjunctiva of the two groups. Conclusions Labial salivary gland transplantation provided a basal secretion of tears and improved ocular surface staining scores during the first 3 months in a severe rhesus monkey model of dry eye. However, this was not accompanied by major improvement of ocular surface tissues. Chinese Abstract.

Specific demographic factors could predict deceased potential cornea donors: A retrospective study from Beijing Tongren Hospital Eye Bank

Abstract Compared with evident cornea donors (ECDs), deceased potential cornea donors (DPCDs) have no obvious donor identifications to reference, which causes many eligible cornea tissues to be wasted. The demographic characteristics of DPCDs might be different from those of ECDs owing to the following different features: donation consent provided by relatives and willingness to donate before death. Thus, the aim of this study is to reveal the demographic characteristics of DPCDs by comparing DPCDs and ECDs. The demographic factors of 138 donors (both DPCDs and ECDs) were collected from the Beijing Tongren Hospital Eye Bank database and analyzed. To differentiate DPCDs from ECDs using the above-mentioned features, we interviewed the relatives of the donors by telephone. The relatives’ attitudes toward cornea donation and their suggestions for our donation service were also acquired during the interview. Two logistic regressions were performed to reveal the demographic factors influencing the 2 features and indicate DPCDs. The donors had certain demographic characteristics (elderly, secondary, or tertiary education level, central district resident), and the most frequent cause of death for the donors was a malignant tumor (n = 56, 43.1%). All the relatives had positive attitudes toward cornea donations, and they hoped to increase publicity efforts to encourage more people to donate and establish more convenient and efficient access for cornea donation. In univariate regressions, age (P = .004, >50 years: odds ratio [OR] = 6.89, 95% confidence interval [CI]: 1.82–26.05), marital status (P = .043, divorced: OR = 9.00,95% CI: 1.33–60.80) significantly influenced relative consent, whereas age (P = .001, >50 years: OR = 15.00, 95% CI: 3.00–74.98), and family address (P = .001, central district: OR = 1) were significant factors influencing the willingness to donate before death. In multivariate regression, age (P = .021, >50 years: OR = 8.14, 95% CI: 1.37–48.41) was the only significant factor influencing relative consent. Similarly, age (P = .02, >50 years: OR = 7.55, 95% CI: 1.21–47.25) was the only factor influencing willingness to donate before death. In conclusion, specific demographic factors could indicate DPCDs and might reveal directions and methods for cornea donation coordination in the future.