| Background and object Vascularized composite allotransplantation (VCA) is an encouraging reconstructive option for patients with devastating tissue loss. However, acute rejection after VCA is the most common complication. Immunosuppressive agents aimed to prevent acute rejection are needed, and thereby VCA is hampered by severe side effects secondery to high-dose and lifelong agents. MSCs, proven to have immunomodulatory capacity in vitro, represent a promising approach to modulate T cell responses associated with allograft acute rejection. MSCs were demonstrated to suppress T cells activation and proliferation with the mechanism of paracrine secretion and cell-to-cell direct contact.Among different sources of MSCs, adipose-derived stem cells (ASCs) have superior immunomodulatory potency as compared with other counterparts. Normally, T cells reside within SLOs, such as spleen and lymph node (LN), in which adaptive immune response are initiated. Despite of the effective suppressive effect in vitro, the suppressive function are not that dramatic as expected when MSCs delivered systematically. Previous researches showed that up-regulation of CCR7 expression on murine bone marrow-derived stem cells by lentiviral transduction enabled an efficient migration to SLOs together with an alleviation of graft-versus-host disease. Therefore, we aim to manipulate human ASCs(hASCs) to express rat CCR7 for an advanced examination of immunomodulatory effects in rat VCA model, which may provide new guidances for acute rejection treatment in VCA.Method 1.Cell preparation.? hASCs were isolated from subcutaneous adipose tissue; ? CCR7 espression on hASCs cultured with or without inflammatory cytokins was tested;? hASCs were transfected into rCCR7-GFP-hASCs (C-A) and GFP-hASCs (G-A)by lentivirus encoded with CCR7-GFP gene or GFP gene alone, and the latter one were served as control; ?hASCs, C-A and G-A were performed phenotype, differentiation and cell proliferation analysis. 2. Cell migration assays. In vitro, C-A and G-A were placed in transwell upper chamber with or without CCL21 in the lower chamber. In vivo, GFP+ cell proportion in spleen and LNs was tested by FCM following cell injection; Next, frozen sections of LNs and spleen were performed immunofluorescent staining to examine the GFP+ cells and their distribution. 3. In vitro, hASCs, C-A and G-A were tested for suppressive function using mixed lymphocyte reactivity assays. T cell proliferation and cytokin levels in supernatant was examined by FCM; in vivo, C-A and G-A injection rats were performed vascularized abdominal flap allotransplantation. Allografts were evaluated daily and exerted histological examination 3d, 7d and 14d post operation; meanwhile, cell suspension of LN and spleen was collected for T cell proliferation examination and plasma was harvested for cytokins level test.Results 1. hASCs were successfully isolated and qualified as MSCs, and no CCR7 expression was detected in hASCs, even co-cultured with inflammatory cytokins. High CCR7 expression on hASCs was detected following CCR7 gene encoded letiviral transducion. In addition, WPRE level are significantly higher in C-A and G-A than in hASCs (P< 0.001). Moreover, hASCs, C-A and G-A shared the similar characteristics in cell phenotype, cell differentiation and proliferation. 2. The number of cells that migrated toward CCL21 was much higher of C-A group than G-A group (P <0.001). Furthermore,the GFP+ cells proportion in LNs and spleen was higher in C-A injection group than in G-A group (P<0.01). Likewise, more GFP+ cells in frozen sections were observed in C-A injection group and these GFP+ cells distributed within the red fluorescent T cell rich areas.3. A decreased cell clusters in hASCs groups was observed when hASCs were co-cultured with mixed lymphocyte reaction, together with a decreased proportion of CD4+ cells and a decreased ratio of Thl/ Th2 but an inceased ratio of Treg/ Th17. Moreover, a significant reduction of cytokines level of IFN-?, IL-2, IL-17 and IL-6 was observed whereas IL-4 and IL-10 were induced in the hASCs groups. Besides, hASC exhibited dose-dependent suppressive effect in vitro. 4. In the rat model, the presence of C-A prolonged the allograft survival and postponed the acute rejection. In the histological examination, much severer damage was shown in G-A injection group than in C-A group at the same time post operation. In addition, a delayed soaring Th1/ Th2 ration was observed in C-A group and the similar trend can be seen in proinflammatory cytokins level of IFN-y, IL-2, IL-17 and IL-6, which then declined rapidly; whereas the Treg/ Th17 ratio kept increasing from the beginning as well as the anti-inflammatory cytokins level of IL-4 and IL-10.Conclusion Upregulation of CCR7 enables migration of hASCs to SLOs and accumulation within T cell rich zone, which leads to an effective modulation of Thl/ Th2 and Treg/ Th17 balance in rat VC A model. CCR7-hASCs, that have been proven to alleviate acute rejection after VCA, may be an promising way in the advanced researches in immunomodulatory field of VCA. |
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