Ikaros Deficiency Enhances Notch Signaling in Host Antigen Presenting Cells and Differentially Regulates Gvhd and GVL Responses After Experimental BMT.

Abstract 690

Host antigen presenting cells (APCs) are necessary for induction of graft-versus-host (GVH) responses. But the APC autonomous molecular mechanisms that are critical for modulation of GVH are not known. Ikaros (Ik) is a zinc-finger DNA binding protein that is essential for the development of lymphoid cells and hematological malignancies. Because Ik is known to negatively regulate certain dendritic cell (DC), the most potent APCs, functions (Wu et al, Immunity, 1997) we hypothesized that its deficiency in host APCs will reduce GVHD. We generated [B6→B6] and [Ik^-/-B6→B6] chimeras and utilized them as recipients in well characterized MHC matched, minor mismatched C3H.SW B6 model of acute GVHD. [B6→B6] and [Ik^/-B6 → B6] animals received 9 Gy and were transplanted with 3 × 10⁶ T cells and 5 × 10⁶ BM from either syngeneic B6 or allogeneic C3H.SW donors. Contrary to our hypothesis, [Ik^-/-B6 < 0.001). The increase in mortality was associated with a significant increase in GVHD specific clinical severity, histopathological damage of the liver and GI tract, donor T cell expansion, serum levels TNF-αa, IFNγ and IL-17 on days +7 and +14 after BMT. Similar results were obtained in a second, BALB/c→B6 model of GVHD and also in recipient chimeras generated from dominant negative Ikaros (IkDN) mice, demonstrating a role for Ik in a strain independent manner. We next performed systematic analyses of DCs to uncover the mechanisms underpinning our surprising results. Phenotypic analysis of spleen and BM derived DCs demonstrated similar numbers of CD8⁺ and CD8^-CD11c⁺DCs but a reduced number of pDCs were present in Ik^-/- animals when compared with WT animals. Ik-/-DCs demonstrated an immature phenotype as determined by the expression of CD40/80. By contrast, functional analyses of Ik^-/- DCs, when stimulated with LPS showed significantly greater secretion of proinflammatory cytokines TNF-αa and IL-6 and greater stimulation of allo-T cells in MLRs when compared with WT B6 DCs. CFSE, annexin and CD107a labeling studies demonstrated that Ik^-/-DCs caused greater proliferation without altering the rate of apoptosis or the cytotoxic function of the allo-T cells. To characterize the molecular mechanisms we evaluated the role of putative molecular targets of Ikaros, the Notch signaling pathway because Ikaros and Notch antagonistically regulate target genes (Chari et al. J Immunol 2008 and Bugeon, et al. J Immunol 2008). Both Ik^-/- and IkDN DCs, at steady state, showed an increase in the expression of several Notch target genes such as Hes-1, Hex-1, Deltex1, Notch3, Jagged-1 and Jagged-2. Blockade of Notch signaling with γ-secretase inhibitor (DAPT) in MLR mitigated the enhanced allo-stimulatory capacity of the Ik^-/- DCs, thus demonstrating a functional relevance for enhanced notch signaling in the absence of Ikaros. We next hypothesized that given the enhanced GVHD response that was associated with increased proliferation and preserved cytotoxicity of allo-T cells, the GVL response will also be enhanced in the recipients with Ik^-/- APCs. Unexpectedly, the [B6→ B6] and [Ik^-/-B6 → B6] chimeras when transplanted with allogeneic T cells from C3H.SW cells along with two different doses of syngeneic 0.5 or 1×10⁴ MBL-2 tumor cells demonstrated equivalent GVL responses despite greater severity of GVHD. Together our data demonstrate (a) for the first time, to our knowledge, suggest differential regulation of GVHD and GVL at the level of host APCs and (b) show a role for a novel molecular pathway, the Ik-Notch axis, in the host APCs as an important modulator of GVH responses.