In the human disease AA, proliferation of oligoclonal pathogenic T cells and up-regulation in Fas expression on BM hematopoietic cells accompanies profound BM destruction [33
]. This pathology was reproduced in experimental models of BM failure in which expansion of oligoclonal T cells and the activation of Th1 immune response and Fas-mediated cell apoptosis led to massive BM cell destruction [9
In the current study, we observed MHC class II up-regulation and co-expression/co-localization with Fas in both B6
C.B10 and B6
CByB6F1 LN cell infusion models. We also observed an association of MHC class II+
BM cells with CD4+
T cells, implicating CD4 cell-mediated cytotoxicity in the destruction of MHC class II+
BM cells. The infusion of allogeneic LN cells caused significant declines in BM cells (marrow hypoplasia) and mature blood cells (pancytopenia). We did not specifically measure changes in the numbers of CD150+
hematopoietic cells in our current study, as we have previously reported that infusion of 5 × 106
B6 LN cells to 5 Gy-irradiated C.B10 or CByB6F1 recipients caused significant declines in radiation-protective hematopoietic cells [19
], in Lin−
hematopoietic stem and progenitor cells [7
], and in Lin−
hematopoietic cells [8
], along with development of marrow hypoplasia and peripheral blood pancytopenia. We specifically examined MHC class II expression and MHC class II-Fas co-localization on residual BM cells by excluding CD4+
mature cells that do not contain hematopoietic stem or progenitor cells but do include MHC class II-positive antigen presenting cells. Thus, we focused on those less mature hematopoietic cells that are the likely main targets for cell destruction in BM failure.
The co-localization of MHC class II and the apoptotic receptor Fas on residual BM cells, as observed by Amnis ImageStream imaging flow cytometry analysis, provided fresh evidence of an association between these two types of molecules and their potential role in cell destruction. MHC class II molecules have short cytoplasmic tails with no known signaling motifs [36
]. Physical co-localization with Fas would provide a possibility for MHC class II to transmit signals through Fas to its downstream molecular components to initiate cell destruction. This mechanism of cell killing is consistent with previous reports indicating that Fas-mediated cell apoptosis is the major pathway of cell destruction in AA patients [33
] as well as in mouse models of immune-mediated BM failure [10
]. Ideally we would analyze MHC class II-Fas co-expression and co-localization on residual BM cells since the destruction of residual BM cells is most relevant to immune-mediated BM destruction. However, residual BM cells are based on negative selection of CD4−
cells. Thus, we examined MHC class II and Fas co-localization on CD150+
cells because these two antibodies provide positive identification of cell populations that are enriched in hematopoietic stem and progenitor cells.
Confocal microscopy revealed a close association between CD4+
T cells and MHC class II+
BM cells in the BM cavity of mice that had developed BM failure (). Our observation is concordant with previous reports showing CD4 T cell-mediated cell destruction [20
]. In a particular AA patient, a specific CD4 T cell clone was isolated and was found to have cytotoxic activity toward autologous and allogeneic cell targets [11
]. It has also been reported that AA patients show oligo-clonal CD4 T cell expansion in blood and BM [14
]. Thus, the infusion of allogeneic LN cells may cause initial T cell expansion and IFN-γ up-regulation in the BM of affected animals. Elevated IFN-γ then up-regulates MHC class II and Fas co-expression/co-localization on residual BM cells, including hemaopoietic progenitor and stem cells, that may attract activated CD4 and CD8 T cells. Engagement of CD4 and MHC class II between effector and target cells transduces signals through the co-localized Fas receptor to trigger a chain of molecular responses leading to target cell destruction. Indeed, the majority of MHC class II+
residual BM cells in LN-cell infused animals were Annexin V+
, indicating cell apoptosis, and only a very small fraction of MHC class II−
residual BM cells were Annexin V+
(). Incubation of CD4 and CD8 T cells caused more cell apoptosis in MHC class II+
residual BM cells than in MHC class II−
residual BM cells, further indicating that the expression of MHC class II and Fas facilitated target cell apoptosis. While overall the CD4 T cell effect was less than for CD8 T cells in the induction of target cell death in vitro
, CD4 T cells did express slightly higher cytotoxicity toward MHC class II+
residual BM cells than did CD8 T cells (); further supporting an MHC class II engagement with CD4 T cells in immune-mediated cell destruction of hematopoietic targets.
Since IFN-γ is a key cytokine up-regulated during inflammation, we speculate that the IFN-γ-driven MHC class II up-regulation, and the associated elevation in MHC class II/Fas co-localization on target cells and T-cell-mediated target cell apoptosis, might represent a general mechanism for cell destruction in BM failure as well as in other inflammatory conditions. Indeed, we found that IFN-γ-deficient LN cells failed to cause BM failure (). Lack of IFN-γ may have abrogated BM destruction through (at least) two mechanisms: 1) by preventing expansion and activation of cytotoxic T cells as immune effectors; 2) by reducing IFN-γ-mediated bystander destruction of BM cells.
Data from our cell culture studies in vitro supported the hypothesis that IFN-γ was an active regulator of MHC class II and Fas, since culturing normal BM cells with recombinant IFN-γ resulted in increased MHC class II and Fas co-expression and co-localization. In direct comparison, cytokines that promote hematopoiesis, such as IL3 and SCF, lacked these effects (). Notably, IFN-γ-induced MHC class II and Fas co-expression/co-localization in vitro () was less than seen in residual BM cells in the animal models ( & ), suggesting that the interaction of IFN-γ with other cellular components in the in vivo settings, such as CD4 and CD8 T cells, are important in the induction of BM failure. It is possible that other inflammatory cytokines, such as IL-5, IL-15 and IL-17, might also be able to induce MHC class II-Fas co-expression and co-localization on target cells during inflammation. Future studies are certainly needed to reveal the roles of each specific cytokine.