Hideki Ohdan

Whole-genome sequencing of liver cancers identifies etiological influences on mutation patterns and recurrent mutations in chromatin regulators

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. We sequenced and analyzed the whole genomes of 27 HCCs, 25 of which were associated with hepatitis B or C virus infections, including two sets of multicentric tumors. Although no common somatic mutations were identified in the multicentric tumor pairs, their whole-genome substitution patterns were similar, suggesting that these tumors developed from independent mutations, although their shared etiological backgrounds may have strongly influenced their somatic mutation patterns. Statistical and functional analyses yielded a list of recurrently mutated genes. Multiple chromatin regulators, including ARID1A, ARID1B, ARID2, MLL and MLL3, were mutated in ∼50% of the tumors. Hepatitis B virus genome integration in the TERT locus was frequently observed in a high clonal proportion. Our whole-genome sequencing analysis of HCCs identified the influence of etiological background on somatic mutation patterns and subsequent carcinogenesis, as well as recurrent mutations in chromatin regulators in HCCs.

Role for CD47-SIRPα signaling in xenograft rejection by macrophages

We have previously proven that human macrophages can phagocytose porcine cells even in the absence of Ab or complement opsonization, indicating that macrophages present a pivotal immunological obstacle to xenotransplantation. A recent report indicates that the signal regulatory protein (SIRP)α is a critical immune inhibitory receptor on macrophages, and its interaction with CD47, a ligand for SIRPα, prevents autologous phagocytosis. Considering the limited compatibility (73%) in amino acid sequences between pig and human CD47, we hypothesized that the interspecies incompatibility of CD47 may contribute to the rejection of xenogeneic cells by macrophages. In the present study, we have demonstrated that porcine CD47 does not induce SIRPα tyrosine phosphorylation in human macrophage-like cell line, and soluble human CD47-Fc fusion protein inhibits the phagocytic activity of human macrophages toward porcine cells. In addition, we have verified that manipulation of porcine cells for expression of human CD47 radically reduces the susceptibility of the cells to phagocytosis by human macrophages. These results indicate that the interspecies incompatibility of CD47 significantly contributes to the rejection of xenogeneic cells by macrophages. Genetic induction of human CD47 on porcine cells could provide inhibitory signaling to SIRPα on human macrophages, providing a novel approach to preventing macrophage-mediated xenograft rejection.

Deubiquitylation of histone H2A activates transcriptional initiation via trans-histone cross-talk with H3K4 di- and trimethylation

Transcriptional initiation is a key step in the control of mRNA synthesis and is intimately related to chromatin structure and histone modification. Here, we show that the ubiquitylation of H2A (ubH2A) correlates with silent chromatin and regulates transcriptional initiation. The levels of ubH2A vary during hepatocyte regeneration, and based on microarray expression data from regenerating liver, we identified USP21, a ubiquitin-specific protease that catalyzes the hydrolysis of ubH2A. When chromatin is assembled in vitro, ubH2A, but not H2A, specifically represses the di- and trimethylation of H3K4. USP21 relieves this ubH2A-specific repression. In addition, in vitro transcription analysis revealed that ubH2A represses transcriptional initiation, but not transcriptional elongation, by inhibiting H3K4 methylation. Notably, ubH2A-mediated repression was not observed when H3 Lys 4 was changed to arginine. Furthermore, overexpression of USP21 in the liver up-regulates a gene that is normally down-regulated during hepatocyte regeneration. Our studies revealed a novel mode of trans-histone cross-talk, in which H2A ubiquitylation controls the di- and trimethylation of H3K4, resulting in regulation of transcriptional initiation.

Whole-genome mutational landscape and characterization of noncoding and structural mutations in liver cancer

Liver cancer, which is most often associated with virus infection, is prevalent worldwide, and its underlying etiology and genomic structure are heterogeneous. Here we provide a whole-genome landscape of somatic alterations in 300 liver cancers from Japanese individuals. Our comprehensive analysis identified point mutations, structural variations (STVs), and virus integrations, in noncoding and coding regions. We discovered mutational signatures related to liver carcinogenesis and recurrently mutated coding and noncoding regions, such as long intergenic noncoding RNA genes (NEAT1 and MALAT1), promoters, CTCF-binding sites, and regulatory regions. STV analysis found a significant association with replication timing and identified known (CDKN2A, CCND1, APC, and TERT) and new (ASH1L, NCOR1, and MACROD2) cancer-related genes that were recurrently affected by STVs, leading to altered expression. These results emphasize the value of whole-genome sequencing analysis in discovering cancer driver mutations and understanding comprehensive molecular profiles of liver cancer, especially with regard to STVs and noncoding mutations.

Mixed chimerism induced without lethal conditioning prevents T cell– and anti-Galα1,3Gal–mediated graft rejection

Gal alpha 1,3Gal-reactive (Gal-reactive) antibodies are a major impediment to pig-to-human xenotransplantation. We investigated the potential to induce tolerance of anti-Gal-producing cells and prevent rejection of vascularized grafts in the combination of alpha 1,3-galactosyltransferase wild-type (GalT(+/+)) and deficient (GalT(-/-)) mice. Allogeneic (H-2 mismatched) GalT(+/+) bone marrow transplantation (BMT) to GalT(-/-) mice conditioned with a nonmyeloablative regimen, consisting of depleting CD4 and CD8 mAbs and 3 Gy whole-body irradiation and 7 Gy thymic irradiation, led to lasting multilineage H-2(bxd) GalT(+/+) + H-2(d) GalT(-/-) mixed chimerism. Induction of mixed chimerism was associated with a rapid reduction of serum anti-Gal naturally occurring antibody levels. Anti-Gal-producing cells were undetectable by 2 weeks after BMT, suggesting that anti-Gal-producing cells preexisting at the time of BMT are rapidly tolerized. Even after immunization with Gal-bearing xenogeneic cells, mixed chimeras were devoid of anti-Gal-producing cells and permanently accepted donor-type GalT(+/+) heart grafts (>150 days), whereas non-BMT control animals rejected these hearts within 1-7 days. B cells bearing receptors for Gal were completely absent from the spleens of mixed chimeras, suggesting that clonal deletion and/or receptor editing may maintain B-cell tolerance to Gal. These findings demonstrate the principle that induction of mixed hematopoietic chimerism with a potentially relevant nonmyeloablative regimen can simultaneously lead to tolerance among both T cells and Gal-reactive B cells, thus preventing vascularized xenograft rejection.

Difference in cytotoxicity against hepatocellular carcinoma between liver and periphery natural killer cells in humans.

In rodents, liver natural killer (NK) cells have been shown to mediate higher cytotoxic activity against tumor cells than do peripheral blood (PB) NK cells. However, such differences between liver and PB NK cells have not been extensively investigated in humans. The phenotypical and functional properties of NK cells extracted from liver perfusates at the time of living donor liver transplantation were investigated. The tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL), a critical molecule for NK cell–mediated anti‐tumor cell killing, was not expressed by freshly isolated PB NK cells or by liver NK cells. Stimulation with interleukin (IL)‐2, significantly up‐regulated the expression of TRAIL on liver NK cells, but this effect was barely observed on PB NK cells. Donor liver NK cells showed the most vigorous cytotoxicity against HepG2, a hepatocellular carcinoma (HCC) cell line, after IL‐2 stimulation (90.5% ± 2.2% at E: T = 10:1), compared with donor and recipient PB NK cells and recipient liver NK cells (64.8% ± 8.2%, 56.1% ± 8.9%, and 34.6% ± 7.5%, respectively). IL‐2 stimulation resulted in an increased expression of killing inhibitory receptors on liver NK cells in parallel with TRAIL expression. Consistently, the cytotoxicities of IL‐2–stimulated donor liver NK cells against self and recipient lymphoblasts were negligible. In conclusion, adoptive transfer of IL‐2–stimulated NK cells extracted from donor liver graft perfusate could mount an anti‐tumor response without causing toxicity against 1‐haplotype identical recipient intact tissues. These findings present a concept to prevent recurrence of HCC after liver transplantation. (HEPATOLOGY 2006;43:362–372.)

Peritoneal Cavity B Cells Are Precursors of Splenic IgM Natural Antibody-Producing Cells

Peritoneal cavity B-1 cells are believed to produce IgM natural Abs. We have used α1,3-galactosyltransferase-deficient (GalT−/−) mice, which, like humans, produce IgM natural Abs against the carbohydrate epitope Galα1,3Gal (Gal), to demonstrate that peritoneal cavity B-1b cells with anti-Gal receptors produce anti-Gal IgM Abs only after LPS stimulation. Likewise, peritoneal cavity cells of GalT−/− and wild-type mice do not produce IgM Abs of other specificities without LPS stimulation. Development of Ab-secreting capacity is associated with loss of CD11b/CD18 (Mac-1) expression. In contrast, there are large numbers of cells producing anti-Gal and other IgM Abs in fresh splenocyte preparations from GalT−/− and (for non-Gal specificities) wild-type mice. These cells are Mac-1− but otherwise B-1b-like in their phenotype. We therefore hypothesized a pathway wherein peritoneal cavity B cells migrate into the spleen after activation in vivo and lose Mac-1 expression to become IgM Ab-producing cells. Consistent with this possibility, splenectomy reduced anti-Gal Ab production after immunization of GalT−/− mice with Gal-positive rabbit RBC. Furthermore, splenectomized B6 GalT−/−, Ig μ-chain mutant (μ−/−) (both Gal- and B cell-deficient) mice produced less anti-Gal IgM than nonsplenectomized controls after adoptive transfer of peritoneal cavity cells from B6 GalT−/− mice. When sorted GalT−/− Mac-1+ peritoneal cavity B cells were adoptively transferred to B6 GalT−/−, μ−/− mice, IgM Abs including anti-Gal appeared, and IgM-producing and Mac1− B cells were present in the spleen 5 wk after transfer. These findings demonstrate that peritoneal cavity Mac-1+ B-1 cells are precursors of Mac-1− splenic IgM Ab-secreting cells.

Liver NK cells expressing TRAIL are toxic against self hepatocytes in mice.

Although it is known that activation of natural killer (NK) cells causes liver injury, the mechanisms underlying NK cell‐induced killing of self‐hepatocytes are not clear. We demonstrated that liver NK cells have cytotoxicity against normal syngeneic hepatocytes in mice. Polyinosinic‐polycytidylic acid (poly I:C) treatment enhanced hepatocyte toxicity of liver NK cells but not that of spleen NK cells. Unlike NK cells in other tissues, approximately 30%–40% of liver NK cells constitutively express tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL). An in vitro NK cell cytotoxic assay revealed that hepatocyte toxicity of liver NK cells from both naïve and poly I:C‐treated mice was inhibited partially by an anti‐TRAIL monoclonal antibody (mAb) alone and completely by the combination with anti‐Fas ligand (FasL) mAb and a perforin inhibitor, concanamycin A, indicating contribution of TRAIL to NK cell‐mediated hepatocyte toxicity. The majority of TRAIL+ NK cells lacked expression of Ly‐49 inhibitory receptors recognizing self‐major histocompatibility complex class I, indicating a propensity to targeting self‐hepatocytes. Poly I:C treatment significantly upregulated the expression of Ly‐49 receptors on TRAIL− NK cells. This might be a compensatory mechanism to protect self‐class I‐expressing cells from activated NK cell‐mediated killing. However, such compensatory alteration was not seen at all in the TRAIL+ NK cell fraction. Thus, liver TRAIL+ NK cells have less capacity for self‐recognition, and this might be involved in NK cell‐dependent self‐hepatocyte toxicity. In conclusion, our findings are consistent with a model in which TRAIL‐expressing NK cells play a critical role in self‐hepatocyte killing through poor recognition of MHC. (HEPATOLOGY 2004;39:1321–1331.)

Mac-1-negative B-1b phenotype of natural antibody-producing cells, including those responding to Gal alpha 1, 3Gal epitopes in alpha 1, 3-galactosyltransferase-deficient mice

Human natural Abs against Galα1-3Galβ1-4GlcNAc (Gal) epitopes are a major barrier to xenotransplantation. Studies in this report, which use combined multiparameter flow cytometric sorting and enzyme-linked immunospot assay, demonstrate that anti-Gal IgM-producing cells are found exclusively in a small B cell subpopulation (i.e., CD21−/low IgMhigh B220low CD5− Mac-1− 493− cells) in the spleens of α1,3-galactosyltransferase-deficient mice. All IgM-producing cells were detected in a similar splenic subpopulation of α1,3-galactosyltransferase-deficient and wild-type mice. A higher frequency of B cells with anti-Gal surface IgM receptors was observed in the peritoneal cavity than in the spleen, but these did not actively secrete Abs, and showed phenotypic properties of B-1b cells (CD21−/low IgMhigh CD5− CD43+ Mac-1+). However, these became Mac-1− and developed anti-Gal Ab-producing activity after in vitro culture with LPS. The splenic B cells with anti-Gal receptors consisted of both Mac-1+ B-1b cells and Mac-1− B-1b-like cells. The latter comprised most anti-Gal IgM-producing cells. Our studies indicate that anti-Gal natural IgM Abs are produced by a B1b-like, Mac-1− splenic B cell population and not by plasma cells or B-1a cells. They are consistent with a model whereby B-1b cells lose Mac-1 expression upon Ag exposure and that these, rather than plasma cells, become the major IgM Ab-producing cell population.

Conditional ablation of HMGB1 in mice reveals its protective function against endotoxemia and bacterial infection

Significance The high-mobility group box 1 (HMGB1) protein is abundantly expressed in the nucleus where it regulates chromatin function. More recently, it was found to also function in the cytoplasm and extracellular milieu for the regulation of immunity and inflammation. However, the in vivo study of HMGB1 has been hampered by the fact that HMGB1-deficient mice die soon after birth. In this study, we successfully generated Hmgb1-floxed mice to achieve conditional inactivation of the gene in a cell- and tissue-specific manner. We demonstrate that cytosolic HMGB1 in myeloid cells is critical for the protection of the host from endotoxemia and bacterial infection by inducing autophagy, a cellular response critical for maintaining cellular viability in the setting of various stresses including infection. High-mobility group box 1 (HMGB1) is a DNA-binding protein abundantly expressed in the nucleus that has gained much attention for its regulation of immunity and inflammation. Despite this, whether and how HMGB1 contributes to protective and/or pathological responses in vivo is unclear. In this study, we constructed Hmgb1-floxed (Hmgb1f/f) mice to achieve the conditional inactivation of the gene in a cell- and tissue-specific manner by crossing these mice with an appropriate Cre recombinase transgenic strain. Interestingly, although mice with HMGB1 ablation in myeloid cells apparently develop normally, they are more sensitive to endotoxin shock compared with control mice, which is accompanied by massive macrophage cell death. Furthermore, these mice also show an increased sensitivity to Listeria monocytogenes infection. We also provide evidence that the loss of HMGB1 in macrophages results in the suppression of autophagy, which is commonly induced by lipopolysaccharide stimulation or L. monocytogenes infection. Thus, intracellular HMGB1 contributes to the protection of mice from endotoxemia and bacterial infection by mediating autophagy in macrophages. These newly generated HMGB1 conditional knockout mice will serve a useful tool with which to study further the in vivo role of this protein in various pathological conditions.