Yingying Lin

iPSC-MSCs Combined with Low-Dose Rapamycin Induced Islet Allograft Tolerance Through Suppressing Th1 and Enhancing Regulatory T-Cell Differentiation

Mesenchymal stem cell (MSC) differentiation is dramatically reduced after long-term in vitro culture, which limits their application. MSCs derived from induced pluripotent stem cells (iPSCs-MSCs) represent a novel source of MSCs. In this study, we investigated the therapeutic effect of iPSC-MSCs on diabetic mice. Streptozocin-induced diabetic mice transplanted with 400 islets alone or with 1×10(6) iPSC-MSCs were examined following rapamycin injection (0.1 mg/kg/day, i.p., from days 0 to 9) after transplantation. Our results showed that iPSC-MSCs combined with rapamycin significantly prolonged islet allograft survival in the diabetic mice; 50% of recipients exhibited long-term survival (>100 days). Histopathological analysis revealed that iPSC-MSCs combined with rapamycin preserved the graft effectively, inhibited inflammatory cell infiltration, and resulted in substantial release of insulin. Flow cytometry results showed that the proportion of CD4(+) and CD8(+) T cells was significantly reduced, and the number of T regulatory cells increased in the spleen and lymph nodes in the iPSC-MSCs combined with the rapamycin group compared with the rapamycin-alone group. Production of the Th1 proinflammatory cytokines interleukin-2 (IL-2) and interferon-γ was reduced, and secretion of the anti-inflammatory cytokines IL-10 and transforming growth factor-β was enhanced compared with the rapamycin group, as determined using enzyme-linked immunosorbent assays. Transwell separation significantly weakened the immunosuppressive effects of iPSC-MSCs on the proliferation of Con A-treated splenic T cells, which indicated that the combined treatment exerted immunosuppressive effects through cell-cell contact and regulation of cytokine production. Taken together, these findings highlight the potential application of iPSC-MSCs in islet transplantation.

Arsenic trioxide is a novel agent for combination therapy to prolong heart allograft survival in allo-primed T cells transferred mice.

Alloreactive memory T cells are major barriers to transplantation acceptance due to their capacity to accelerate rejection. Here, we investigated the effects of combined treatment with arsenic trioxide (As(2)O(3)) and blocking monoclonal antibodies (mAb) against CD154 and LFA-1 (anti-CD154/LFA-1) on graft survival as well as changes in pathology and immunological responses in mice with adoptively transferred allo-primed T cells. The mean survival time (MST) for the cardiac allografts in recipient mice receiving the combination of As(2)O(3) and anti-CD154/LFA-1 was significantly longer (>113.7days) compared to those receiving anti-CD154/LFA-1 (23.2days), As(2)O(3) (12.5days) alone or no treatment (5.5days). This combined strategy distinctly inhibited lymphocyte infiltration in grafts, proliferation of splenic T cells and the generation of memory T cells in spleens. Moreover, the combined treatment caused the significant down-regulation of IL-2 and IFN-γ accompanied by increased expression of TGF-β and regulatory T cells (Tregs) in spleens, which led to long-term cardiac allograft survival in recipient mice. These results highlight the potential application of As(2)O(3) and its contribution in combination therapy with antibody blockade to delay rejection by memory T cells.

Overexpression of Jagged-1 combined with blockade of CD40 pathway prolongs allograft survival

Dendritic cells (DCs) have the tolerogenic potential to regulate adaptive immunity and induce allografts acceptance. Here we investigated whether blockade of the CD40 pathway could enhance the immune tolerance induced by DC2.4 cells modified to express Jagged‐1 (JAG1‐DC) in heart transplantation. Results showed that JAG1‐DC treatment combined with anti‐CD40L monoclonal antibody (mAb) administration significantly prolonged cardiac allograft survival in mice, with long‐term survival (>110 days) of 50% of the allografts in the recipients. The therapy specifically inhibited the immune response, induced alloantigen‐specific T‐cell hyporesponsiveness, upregulated transforming growth factor‐β synthesis and increased the population of regulatory T cells (Tregs) driven by Jagged‐1‐Notch activation. These results highlight the potential application of gene therapy to induce alloantigen‐specific Tregs effectively by providing the Jagged‐1 stimulation.

Inhibition of accelerated rejection mediated by alloreactive CD4(+) memory T cells and prolonged allograft survival by arsenic trioxide

The aim of this study was to evaluate and determine the potential mechanisms of As2O3 in accelerated rejection mediated by alloreactive CD4+ memory T cells. Vascularized heterotopic cardiac transplantation from C57BL/6 mice to nude mice (pre-transferred CD4+ memory T cells) was performed on Day 0, and As2O3 was administered to recipient mice from Day 0 to 10. As a result, As2O3 could reduce the proliferation of allo-primed CD4+ memory T cells in vitro in MLR and the baseline rate of proliferation was restored by the addition of exogenous IL-2. In vivo, compared with the control[+] group, the mean survival time of cardiac allografts in the As2O3 group was prolonged from 5.8 ± 0.7 to 14.2 ± 2.5 days. Five days after transplantation, the relative gene expression of IL-2, IFN-γ and Foxp3 was reduced in the grafts by As2O3 treatment, but the expression of IL-10 and TGF-β was increased. Correspondingly, the proportions of CD4+ T cells, CD4+ memory T cells and regulatory T cells (Tregs), both in recipient spleens and lymph nodes, were lowered. These results indicate the potential of As2O3 as a novel immunosuppressant targeting CD4+ memory T cells.

Blockade of CD27/CD70 pathway to reduce the generation of memory T cells and markedly prolong the survival of heart allografts in presensitized mice

BACKGROUND Alloreactive memory T cells are a major obstacle to transplantation acceptance due to their capacity for accelerated rejection. METHODS C57BL/6 mice that had rejected BALB/c skin grafts 4 weeks earlier were used as recipients. The recipient mice were treated with anti-CD154/LFA-1 with or without anti-CD70 during the primary skin transplantation and anti-CD154/LFA-1 or not during the secondary transplantation of BALB/c heart. We evaluated the impact of combinations of antibody-mediated blockade on the generation of memory T cells and graft survival after fully MHC-mismatched transplantations. RESULTS One month after the primary skin transplantation, the proportions of CD4(+) memory T cells/CD4(+) T cells and CD8(+)memory T cells/CD8(+) T cells in the anti-CD154/LFA-1 combination group were 47.32±4.28% and 23.18±2.77%, respectively. In the group that included anti-CD70 treatment, the proportions were reduced to 34.10±2.71% and 12.19±3.52% (P<0.05 when comparing the proportion of memory T cells between the two groups). The addition of anti-CD70 to the treatment regimen prolonged the mean survival time following secondary heart transplantation from 10days to more than 90days (P<0.001). Furthermore, allogenic proliferation of recipient splenic T cells and graft-infiltrating lymphocytes were significantly decreased. Meanwhile, the proportion of regulatory T cells was increased to 9.46±1.48% on day 100 post-transplantation (P<0.05). CONCLUSIONS The addition of anti-CD70 to the anti-CD154/LFA-1 combination given during the primary transplantation reduced the generation of memory T cells. This therapy regimen provided a potential means to alleviate the accelerated rejection mediated by memory T cells during secondary heart transplantation and markedly prolong the survival of heart allografts.

Arsenic Trioxide Induces T Cell Apoptosis and Prolongs Islet Allograft Survival in Mice.

Background T cell–mediated immune rejection is a key barrier to islet transplantation. Preliminary studies have shown that arsenic trioxide (As2O3) can inhibit T cell responses and prolong heart allograft survival. Here, we sought to investigate the possibility of using As2O3 to prolong islet allograft survival in an acute rejection model of Balb/c to C57B/6 mice. Methods Recipient mice were treated with As2O3 and/or rapamycin after islet allograft transplantation. At day 10 after transplantation, the graft, spleen, lymph nodes, and blood of the recipient mice were recovered for analysis. In vitro, to further examine the mechanism underlying As2O3 protection of islet allografts against T cell–mediated rejection, mixed lymphocyte reaction and apoptosis analyses of T cells were performed. The phosphorylation levels of I&kgr;B&agr; and p38 were also evaluated to confirm the proliferation and apoptosis of As2O3-treated T cells. Results We found that As2O3 prolonged islet allograft survival by reducing inflammatory reactions, influencing cytokine synthesis and secretion and T-cell subset proportions, and inhibiting T-cell responses. Furthermore, As2O3 and rapamycin showed a synergistic effect in suppressing islet allotransplant rejection. Conclusions Arsenic trioxide may prevent allograft rejection by inhibiting T-cell proliferation and inducing T-cell apoptosis.

Immunosuppressive Effect of Compound K on Islet Transplantation in an STZ-Induced Diabetic Mouse Model

Islet transplantation is a therapeutic option for type 1 diabetes, but its long-term success is limited by islet allograft survival. Many factors imperil islet survival, especially the adverse effects and toxicity due to clinical immunosuppressants. Compound (Cpd) K is a synthesized analog of highly unsaturated fatty acids from Isatis tinctoria L. (Cruciferae). Here we investigated the therapeutic effect of Cpd K in diabetic mice and found that it significantly prolonged islet allograft survival with minimal adverse effects after 10 days. Furthermore, it reduced the proportion of CD4+ and CD8+ T cells in spleen and lymph nodes, inhibited inflammatory cell infiltration in allografts, suppressed serum interleukin-2 and interferon-γ secretion, and increased transforming growth factor-β and Foxp3 mRNA expression. Surprisingly, Cpd K and rapamycin had a synergistic effect. Cpd K suppressed proliferation of naïve T cells by inducing T-cell anergy and promoting the generation of regulatory T cells. In addition, nuclear factor-κB signaling was also blocked. Taken together, these findings indicate that Cpd K may have a potential immunosuppressant effect on islet transplantation.

Arsenic trioxide inhibits accelerated allograft rejection mediated by alloreactive CD8(+) memory T cells and prolongs allograft survival time.

CD8(+) memory T (Tm) cells are a significant barrier to transplant tolerance induction in alloantigen-primed recipients, and are insensitive to existing clinical immunosuppressants. Here, we studied the inhibition of CD8(+) Tm cells by arsenic trioxide (As2O3) for the first time. Alloantigen-primed CD8(+) Tm cells were transferred to T cell immunodeficient nude mice. The mice were subjected to heart allotransplantation, and treated with As2O3. The transplant survival time was determined, and the inhibitory effects of As2O3 on CD8(+) Tm cell-mediated immune rejection were assessed through serological studies and inspection of the transplanted heart and lymphoid organs. We found that As2O3 treatment prolonged the mean survival time of the graft and reduced the number of CD8(+) Tm cells in the spleen and lymph nodes. The expression of the genes encoding interleukin (IL)-2, and IFN-γ was reduced, while expression of IL-10 and transforming growth factor-β was increased in the transplant. Our findings show that As2O3 treatment inhibits allograft rejection mediated by alloreactive CD8(+) Tm cells in the mouse heart transplantation model.

Inhibiting accelerated rejection mediated by alloreactive CD4+ memory T cells and prolonging allograft survival by 1α,25-dihydroxyvitamin D3 in nude mice

Donor-reactive memory T cells are major barriers to long-term survival of transplanted organs due to their capacity to accelerate rejection. In this study we investigated the ability of 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] to inhibit accelerated rejection mediated by alloreactive CD4(+) memory T cells and to prolong cardiac allograft survival in an adoptive T cell memory/heart transplant model of nude mice. In vitro, the proliferation of CD4(+) memory T cells was significantly inhibited by 1,25(OH)(2)D(3) and was restored following addition of exogenous IL-2. Compared with the control group, the mean survival time of cardiac allografts in the 1,25(OH)(2)D(3) group was prolonged from 6.5±0.3 to 20.2±0.8 days in vivo. Five days after transplantation, the levels of IL-2 and IFN-γ were reduced in the grafts and the recipient sera by 1,25(OH)(2)D(3) treatment, while that of IL-10 increased. The proportions of CD4(+) memory T cells and CD4(+)Foxp3(+) T cells, both in recipient spleen and lymph nodes, were lowered by 1,25(OH)(2)D(3) treatment when compared with the control group. Our data suggests that 1,25(OH)(2)D(3) inhibits expansion of CD4(+) memory T cells, possibly by inducing clonal anergy and/or clonal deletion, resulting in prolongation of cardiac allograft survival in nude mice. These results may provide a rational basis for exploiting 1,25(OH)(2)D(3) as a novel immunosuppressant targeting CD4(+) memory T cells.

CD44/CD70 blockade and anti-CD154/LFA-1 treatment synergistically suppress accelerated rejection and prolong cardiac allograft survival in mice.

Current treatments that are efficient in controlling effector T cell responses to allografts have limited efficacy on the accelerated rejection mediated by memory T cells. Effective targeting of alloreactive memory T cells may therefore be explored to improve therapeutic approaches towards solving this problem. In this study, we investigated the synergistic effect of CD44/CD70 blockade and anti‐CD154/LFA‐1 treatment on the accelerated rejection mediated by memory T cells. While CD44/CD70 blockade had limited effects on the alloresponses of effector T cells in vivo, it diminished the expansion of both CD4+ and CD8+ memory T cells in recipients adoptively transferred with donor‐sensitized T cells. In combination with anti‐CD154/LFA‐1 treatment, CD44/CD70 blockade significantly prolonged cardiac allograft survival in adoptive transfer recipients. We demonstrated that treatment with the combination of all four antibodies (anti‐CD154/LFA‐1/CD44/CD70) inhibited accelerated rejection by markedly suppressing the alloresponses of effector and memory T cells and reducing the number of graft‐infiltration lymphocytes in adoptive transfer recipients. Meanwhile, CD44/CD70 blockade and anti‐CD154/LFA‐1 treatment synergically enhanced regulatory T cells (Tregs) by increasing the proportion of splenic Tregs and the expression of IL‐10 in these recipients. Our findings contribute to the potential design of therapies for accelerated allograft rejection.