Regulation of Acute Graft-Versus-Host Disease by MicroRNA-155

Abstract 245

Acute Graft-versus-host disease (aGVHD) is a frequent and lethal complication of allogeneic hematopoietic stem cell transplantation (HSCT) in which donor T cells destroy HLA mismatched host tissues by secreting soluble inflammatory cytokines (TNF-α, IFN-γ) and/or inducing direct cytotoxic cellular responses. Despite recent advances, GVHD still remains a major clinical problem, underscoring the need to elucidate further its mechanisms to then develop novel therapeutic strategies. Recent studies indicate that microRNAs (miR) play critical roles in the development and function of the immune system. In particular, miR-155 is required for normal function of B and T lymphocytes. MiR-155 has been shown to be up-regulated upon B and T cell activation and mice deficient for miR-155 are immunodeficient, exhibiting T cells with attenuated INF-γ and TNF-α release in response to antigen stimulation. Based on these observations, we hypothesized that miR-155 is up-regulated in donor T cells during aGVHD and is involved in the modulation of this process. To test this hypothesis, we initially measured miR-155 expression from activated CD8 T cells obtained from an experimental animal model of aGVHD. For this, a major histocompatibility complex (MHC) mismatched HSCT model was used in which spleen cells and BM from C57BL/6 (B6) donors were transferred i.v. into lethally irradiated B6D2F1 recipient mice (n=4). Two additional groups were included as controls with one group receiving no cell infusion (radiation only, n=4); and a second group receiving T cell depleted bone marrow only (t-BM) (5×10⁶) (n=4). GVHD scores were performed daily according to Cooke et al, Blood 1996;8:3230-9. Recipient mice were sacrificed and tissues harvested when GVHD scores were ≥7 or at the end of the experiment. After an average of 3–4 weeks, mice receiving donor spleen cells but not BM alone developed severe GVHD (scores >7) which was confirmed by histology. Activated CD8 T cells from the spleen were isolated using CD8+/CD44+ antibodies and after obtaining total RNA, miR-155 expression was measured by RT-PCR. Mice receiving donor BM plus spleen cells developed severe aGVHD and exhibited increased miR-155 expression with respect to the BM only group (fold change 5.2, t-test p<0.001). To confirm that a causal relationship exists between miR-155 and aGVHD severity, we repeated the above MHC mismatched murine experiment using BL/6 mice deficient for miR-155 expression as donors (Rodriguez et al, Science 2007;316;608-611). The groups were as follows; radiation alone (n=4), t-BM (n=8), BM + wild type (WT) spleen cells (n=8), and t-BM+ miR-155 KO spleen cells (n=8). We found that mice receiving donor spleen cells from miR-155 KO mice exhibited dramatically lower mean GVHD scores (3 Vs. 5.5, t-test p=0.0007) and improved survival compared to those receiving WT spleen cells(87% vs. 13% of mice alive at 70 days, respectively; log-rank test p<0.001). Our results showed that recipients from miR-155 KO spleen cells did not exhibit high GVHD histological scores (III-IV) in the spleen, liver or gut, while 80%, 60% and 40% of the WT did. Overall survival, GVHD scores and histological GHVD findings were similar between miR-155 KO and WT t-BM only group. Since high levels of soluble TNF-α is characteristic for aGVHD, we measured serum TNF-α by ELISA assay in mice at the time of harvest. Mice receiving miR-155 KO spleen cells had significantly lower TNF-α levels (mean 14 pg/ml) than WT controls (mean 48 pg/ml, t-test, p=0.005). Finally, to establish the relevance of this finding to the human system, we measured miR-155 expression using Locked Nucleic Acid in situ hybridization from histologically confirmed gut biopsies of aGVHD patients (n=5), and healthy controls (n=3). We found a strong up-regulation of miR-155 expression in all patients with gut aGVHD while miR-155 expression was absent in normal gut. In summary, we have shown that miR-155 expression is up-regulated in CD8 T cells from mice with aGVHD and showed that mice receiving donor lymphocyte cells deficient for miR-155 exhibited less GVHD and improved survival as compared to mice receiving WT donor lymphocytes. Finally, up-regulation of miR-155 was also found in clinical specimens from patients with gut aGVHD. Altogether our data indicate a role for miR-155 in the modulation of aGVHD, and thus point to miR-155 as a novel target for therapeutic intervention in aGVHD.