Introduction: Transplantation is a widely used effective treatment in clinical therapy, where post-operative immune-mediated rejection leading to graft loss remains a significant challenge. Chemokine CCL28 has multiple functions in both innate and adaptive immune. It acts as an effective antibacterial factor in the physiological environment, as well as a chemokine to attract cells expressing CCR10 and/or CCR3, thereby promoting or inhibiting local immune responses. This study established engineered MSCs capable of self-regulation and secretion of CCL28, aimed at reducing the side effects of immunosuppressants and prolonging the survival of transplants.
Methods: In vitro, MSCs wewe infected with CCl28 and inducible caspase-9 overexpression lentivirus to construct engineered MSCs which can release the chemokine CCL28 and express suicide genes. Next, we established an allogeneic mouse skin transplantation model and treated it with engineered MSCs through tail vein injection. After treatment, the survival status of the mouse grafts were recorded, the morphology of the graft tissues were examined by HE staining, the proportions of various immune cells and cytokines in peripheral blood and local graft were detected by flow cytometry, immunofluorescence and ELISA. Additionally, we outlined the changes in the local immune microenvironment of the grafts and identified key regulatory factors and immune cell populations associated with the anti-rejection response of the transplantation through multi-omics techniques.
Results: The results of the graft survival curve showed that the survival of CCL28 group was extended by 4 days compared to the control group in the allogeneic mouse skin transplant model. The HE staining results showed that CCL28 group had a more complete tissue morphology and less inflammatory cell infiltration. The flow cytometry and immunofluorescence results showed that CCL28 group was able to recruit more Tregs and reduce the number of CD4+ T cells, thereby altering the local immune microenvironment of the graft. In vivo and in vitro, flow cytometry and qPCR showed that the key transcription factors (FOXP3 and HELIOS) expression of Treg cells were up-regulated, while the effector molecules (TNF and IFN-γ) expression of CD4+ T cells were down regulated. Furthermore, CCL28 receptor blockade experiment revealed that CCL28 exerted its effects through CCR10, enhancing the number and function of inhibitory immune cells, thereby reducing the immune rejection response. In vitro and in vivo, ELISA results showed that engineered MSCs successfully and continuously released chemokine CCL28 and the release could be terminated using chemical inducer of dimerizatio.
Conclusion: The release of chemokine CCL28 by engineered MSCs remodels the immune microenvironment, suppresses the rejection, and prolongs the survival of skin grafts. Our study enriches the understanding of chemokines' roles and regulatory mechanisms in transplantation and provides new insights into reducing immune rejection-mediated graft loss.