CDCP1+ mesenchymal stem cells preferentially inhibit acute graft-versus-host disease(aGVHD) Free

Object The main complication after hematopoietic stem cells is acute graft-versus-host disease(aGVHD), which affects the success of transplant and survival of patients. It is effective and low-toxicity for mesenchymal stem cells (MSCs) in preventing and treating aGVHD. However, the high heterogeneity of MSCs is unsolved, which leads to instability of clinical outcome and limits the extensive use. Therefore, it is urgent to find a subset of MSCs with advantages in the prevention and treatment of aGVHD while clarifying their roles and mechanisms in regulating aGVHD to solve the bottleneck problem of clinical treatment. This study aims to explore the dominant role and molecular mechanism of CDCP1⁺MSCs in inducing the differentiation of T cells into immunosuppressive cells to control aGVHD, laying a theoretical and experimental foundation for the precise prevention and treatment of aGVHD and promoting the cell therapy of MSCs in clinical practice.

Methods Single-cell sequencing and analysis identified the CDCP1⁺MSCs ; Isolate and culture MSCs from umbilical cord tissues; CDCP1⁺MSCs were sorted out by flow cytometry. The migration ability of MSCs was detected by Transwell. The proliferation of T cells was detected by CCK-8 and CSFE proliferation experiments; The changes in the proportion of T cell subsets were detected by flow cytometry; The release of cytokines was detected by ELISA. The expression levels of chemokines were detected by qPCR and Western blot. shCDCP1-MSCs were constructed using lentiviruses that knocked down CDCP1; The gene changes after knockdown of CDCP1 were detected by transcriptome sequencing. Data were analyzed using t test or one-way analysis of variance.

Results Through single-cell sequencing and bioinformatics analysis, we found that the CDCP1⁺MSCs derived from the umbilical cord has a stronger potential for immune negative regulation. Through co-culture of CDCP1⁺MSCs and T cells, we found that the CDCP1⁺MSCs group inhibited T cell proliferation compared with the CDCP1 knockdown group. At the same time, it was found that the number of Treg cells increased in the CDCP1⁺MSCs group, while the number of Th2, Th17, Tc1, Tc2, and Tc17 cells decreased. We also found that in the CDCP1⁺MSCs group, the release of cytokines TNF-α, IL-4, IL-6, and IL-21 decreased, while the expression of chemokine CCR2 increased. We found through Transwell detection that CDCP1⁺MSCs have a stronger migration ability. However, after knocking down CDCP1, the migration ability of MSCs decreased significantly. Through RNA-Seq detection, we found that the down-regulation of the CDKN2B gene was the most significant after knockdown of CDCP1, and it might be the key gene affecting the differentiation of T cells into immunosuppressive cells.. Furthermore, through the allo-HSCT aGVHD model mice, we found that the clinical score of aGVHD in mice infused with CDCP1⁺MSCs was lower.

Conclusion This study indicates that CDCP1⁺MSCs derived from the umbilical cord have a stronger immune negative regulatory ability, and they may control aGVHD by inducing T cells to differentiate into immunosuppressive cells through the CDKN2B signaling pathway. The CDCP1⁺MSCs derived from umbilical cord may be a more effective and stable cell for the prevention and treatment of aGVHD.

Keywords Mesenchymal stem cell; Acute graft-versus-host disease; CDCP1