CD4⁺ CTLs Act as a Key Effector Population for Allograft Rejection of MSCs in a Donor MHC-II Dependent Manner in Injured Liver

Mesenchymal stromal/stem cells (MSCs) have been considered an attractive source of cytotherapy due to their promising effects on treating various diseases. Allogeneic MSCs (allo-MSCs) are extensively used in clinical trials due to their convenient preparation and credible performance. Traditionally, allo-MSCs are considered immunoprivileged with minimal immunogenicity and potent immunomodulatory capacity. However, growing evidence has suggested that allo-MSCs also induce immune response and cause rejection after transplantation, but the underlying cellular and molecular mechanisms remain to be elucidated. Here, we demonstrated that allografted MSCs upregulated MHC-II upon stimulation of IFN-γ in hepatic inflammatory environment by using mouse model of CCl₄-induced liver injury. MHC-II upregulation enhanced the immunogenicity of allo-MSCs, leading to the activation of alloreactive T cells and rejection of allo-MSCs. However, MHC-II deficiency impaired the allogenic reactivity, thereby rescuing the loss of allo-MSCs. Mechanistically, CD4⁺ cytotoxic T lymphocytes (CTLs), rather than CD8⁺ CTLs, acted as the major effector for allo-MSC rejection. Under liver injury condition, the transplanted allo-MSCs upregulated CD80 and PD-L1, and CD8⁺ CTLs highly expressed CTLA-4 and PD-1, thereby inducing immune tolerance of CD8⁺ T cells to allo-MSCs. On the contrary, CD4⁺ CTLs minimally expressed CTLA-4 and PD-1; thus, they remain cytotoxic to allo-MSCs. Consequently, transplantation of MHC-II-deficient allo-MSCs substantially promoted their therapeutic effects in treating liver injury. This study revealed a novel mechanism of MSC allograft rejection mediated by CD4⁺ CTLs in injured liver, which provided new strategies for improving clinical performance of allo-MSCs in benefiting hepatic injury repair.