• IFN-g alone leads to aplastic anemia by disrupting the generation of common myeloid progenitors and lineage differentiation.• The inhibitory effect of IFN-g on hematopoiesis is intrinsic to hematopoietic stem/ progenitor cells.Aplastic anemia (AA) is characterized by hypocellular marrow and peripheral pancytopenia. Because interferon gamma (IFN-g) can be detected in peripheral blood mononuclear cells of AA patients, it has been hypothesized that autoreactive T lymphocytes may be involved in destroying the hematopoietic stem cells. We have observed AA-like symptoms in our IFN-g adenylate-uridylate-rich element (ARE)-deleted (del) mice, which constitutively express a low level of IFN-g under normal physiologic conditions. Because no T-cell autoimmunity was observed, we hypothesized that IFN-g may be directly involved in the pathophysiology of AA. In these mice, we did not detect infiltration of T cells in bone marrow (BM), and the existing T cells seemed to be hyporesponsive. We observed inhibition in myeloid progenitor differentiation despite an increase in serum levels of cytokines involved in hematopoietic differentiation and maturation. Furthermore, there was a disruption in erythropoiesis and B-cell differentiation. The same phenomena were also observed in wild-type recipients of IFN-g ARE-del BM. The data suggest that AA occurs when IFN-g inhibits the generation of myeloid progenitors and prevents lineage differentiation, as opposed to infiltration of activated T cells. These results may be useful in improving treatment as well as maintaining a disease-free status. (Blood. 2014;124(25):3699-3708)

Differential HLA-C levels influence several human diseases, but the mechanisms responsible are incompletely characterized. Using a validated prediction algorithm, we imputed HLA-C cell surface levels in 228 individuals from the 1000 Genomes dataset. We tested 68,726 SNPs within the MHC for association with HLA-C level. The HLA-C promoter region variant, rs2395471, 800 bp upstream of the transcription start site, gave the most significant association with HLA-C levels (p = 4.2 × 10). This imputed expression quantitative trait locus, termed impeQTL, was also shown to associate with HLA-C expression in a genome-wide association study of 273 donors in which HLA-C mRNA expression levels were determined by quantitative PCR (qPCR) (p = 1.8 × 10) and in two cohorts where HLA-C cell surface levels were determined directly by flow cytometry (n = 369 combined, p < 10). rs2395471 is located in an Oct1 transcription factor consensus binding site motif where the A allele is predicted to have higher affinity for Oct1 than the G allele. Mobility shift electrophoresis demonstrated that Oct1 binds to both alleles in vitro, but decreased HLA-C promoter activity was observed in a luciferase reporter assay for rs2395471_G relative to rs2395471_A on a fixed promoter background. The rs2395471 variant accounts for up to 36% of the explained variation of HLA-C level. These data strengthen our understanding of HLA-C transcriptional regulation and provide a basis for understanding the potential consequences of manipulating HLA-C levels therapeutically.

Background & Aims Liver inflammatory diseases associated with cancer promoting somatic oncogene mutations are increasing in frequency. Preclinical cancer models that allow for the study of early tumor progression are often protracted, which limits the experimental study parameters due to time and expense. Here we report a robust inexpensive approach using Sleeping Beauty Transposition (SBT) delivery of oncogenes along with Gaussia Luciferase expression vector G.luc, to assess de novo liver tumor progression, as well as the detection of immune responses or responses induced by therapeutic intervention. Methods Tracking de novo liver tumor progression with G.luc was demonstrated in models of hepatocellular carcinoma (HCC) or adenoma (HCA) initiated by hydrodynamic delivery of SBT oncogenes. Results Rising serum luciferase levels correlated directly with increasing liver tumor burden and eventual morbidity. Early detection of hepatocyte apoptosis from mice with MET+CAT transfected hepatocytes was associated with a transient delay in HCC growth mediated by a CD8+ T-cell response against transformed hepatocytes. Furthermore, mice that lack B cells or macrophages had an increase in TUNEL+ hepatocytes following liver MET transfection demonstrating these cells provided protection from MET-induced hepatocyte apoptosis. Treatment of mice bearing established HCC with IL-18+IL-12 decreased tumor burden that was associated with decreased levels of serum luciferase. Conclusions Hydrodynamic delivery of the SBT vector G.luc to hepatocytes serves as a simple blood-based approach for real-time tracking of pathologically distinct types of liver cancer, which revealed tumor-induced immunologic responses and was beneficial in monitoring the efficacy of therapeutic interventions.