Mixed hematopoietic or T-cell chimerism above a minimal threshold restores perforin-dependent immune regulation in perforin-deficient mice

Abstract

Defects in perforin and related genes lead to a loss of normal immune regulation and underlie hemophagocytic lymphohistiocytosis (HLH), which requires hematopoietic cell transplantation for long-term cure. However, transplantation may be complicated by the development of mixed chimerism and uncertainty regarding the risk of HLH recurrence. To help clarify this risk and investigate how perforin influences immune activation, we studied perforin-mediated immune regulation in the context of mixed chimerism using a murine model of HLH. We found that there is a distinct threshold of ∼10% to 20% perforin expression with either mixed hematopoietic or CD8(+) T cell chimerism, above which immune regulation was reestablished. These findings demonstrate that perforin-mediated immunoregulation functions in trans and are consistent with a feedback model in which cytotoxic T cells control immune activation by killing dendritic cells. These findings also suggest rational targets for maintenance of minimal posttransplant chimerism and for therapeutic strategies involving gene correction.

Figure 1

Restoration of normal perforin expression in a fraction of hematopoietic cells restores perforin-dependent immune regulation in vivo. (A) Prf^−/− mice were transplanted with WT (GFP marked) or prf^−/− bone marrow. Twelve weeks later, they were infected with LCMV and serum IFN-γ levels were measured. N > 8 mice/point. (B) Mixed hematopoietic chimeric mice (prf^−/− mice, reconstituted with mixtures of WT and prf^−/− marrow) were challenged with LCMV, and day 8 serum IFN-γ levels were measured. (C) Six days after LCMV infection, splenic dendritic cells were sorted from groups of mixed chimeric mice (N = 3/group) and plated with LCMV-specific T cells. After overnight culture, IFN-γ production was assessed as a measure of antigen presentation. *P < .01 (comparing WT and prf curves in panel A; comparing the 2 upper curves with the lower 2 curves in panel C).

Figure 2

Restoration of normal perforin expression in CD8⁺ T cells is necessary and sufficient to restore perforin-dependent immune regulation. (A) Prf^−/− mice were reconstituted with 90% RAG/prf^−/− (DKO) marrow and 10% mixed marrow (variable amounts of WT [GFP⁺] and prf^−/−) to give animals with variable perforin expression within the T-cell compartment and perforin expression in <10% of non–T cells. After engraftment, animals were challenged with LCMV as in Figure 1, and day 8 IFN-γ levels are plotted against WT chimerism within the T-cell compartment. (B) Prf^−/− mice were reconstituted with 90% RAG^−/− marrow and 10% mixed marrow (WT [GFP⁺] and prf^−/−) to give animals with variable perforin expression within the T-cell compartment and normal perforin expression in 90% to 100% of non–T cells. IFN-γ levels and chimerism are plotted as in panel A. (C) Purified naïve WT (CD45.1⁺) or prf^−/− CD4⁺ T cells were transferred into prf^−/− mice (after conditioning with cyclophosphamide and waiting 3 weeks for recovery/stable engraftment). Animals were challenged with LCMV; IFN-γ levels and WT CD4⁺ T cell chimerism were assessed. (D) Purified naïve WT (CD45.1⁺) CD8⁺ T cells were transferred into prf^−/− mice (as in panel C). Animals were challenged with LCMV; IFN-γ levels and WT CD8⁺ T-cell chimerism were assessed. In vivo IFN-γ production by endogenous (prf^−/−) T cells was assessed on day 8 after LCMV in animals receiving CD8⁺ T-cell transfers, as previously described. *P < .01.