小野寺 淳

BACKGROUND: Like its parent base 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) is a direct epigenetic modification of cytosines in the context of CpG dinucleotides. 5hmC is the most abundant oxidized form of 5mC, generated through the action of TET dioxygenases at gene bodies of actively-transcribed genes and at active or lineage-specific enhancers. Although such enrichments are reported for 5hmC, to date, predictive models of gene expression state or putative regulatory regions for genes using 5hmC have not been developed. RESULTS: Here, by using only 5hmC enrichment in genic regions and their vicinity, we develop neural network models that predict gene expression state across 49 cell types. We show that our deep neural network models distinguish high vs low expression state utilizing only 5hmC levels and these predictive models generalize to unseen cell types. Further, in order to leverage 5hmC signal in distal enhancers for expression prediction, we employ an Activity-by-Contact model and also develop a graph convolutional neural network model with both utilizing Hi-C data and 5hmC enrichment to prioritize enhancer-promoter links. These approaches identify known and novel putative enhancers for key genes in multiple immune cell subsets. CONCLUSIONS: Our work highlights the importance of 5hmC in gene regulation through proximal and distal mechanisms and provides a framework to link it to genome function. With the recent advances in 6-letter DNA sequencing by short and long-read techniques, profiling of 5mC and 5hmC may be done routinely in the near future, hence, providing a broad range of applications for the methods developed here.

Abstract Purpose Auto-antibodies (auto-abs) to type I interferons (IFNs) have been identified in patients with life-threatening coronavirus disease 2019 (COVID-19), suggesting that the presence of auto-abs may be a risk factor for disease severity. We therefore investigated the mechanism underlying COVID-19 exacerbation induced by auto-abs to type I IFNs. Methods We evaluated plasma from 123 patients with COVID-19 to measure auto-abs to type I IFNs. We performed single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells from the patients with auto-abs and conducted epitope mapping of the auto-abs. Results Three of 19 severe and 4 of 42 critical COVID-19 patients had neutralizing auto-abs to type I IFNs. Patients with auto-abs to type I IFNs showed no characteristic clinical features. scRNA-seq from 38 patients with COVID-19 revealed that IFN signaling in conventional dendritic cells and canonical monocytes was attenuated, and SARS-CoV-2-specific BCR repertoires were decreased in patients with auto-abs. Furthermore, auto-abs to IFN-α2 from COVID-19 patients with auto-abs recognized characteristic epitopes of IFN-α2, which binds to the receptor. Conclusion Auto-abs to type I IFN found in COVID-19 patients inhibited IFN signaling in dendritic cells and monocytes by blocking the binding of type I IFN to its receptor. The failure to properly induce production of an antibody to SARS-CoV-2 may be a causative factor of COVID-19 severity.

UNLABELLED: Invariant natural killer T (iNKT) cells play an essential role in antitumor immunity by exerting cytotoxicity and producing massive amounts of cytokines. iNKT cells express invariant T-cell receptors (TCR) to recognize their cognate glycolipid antigens such as α-galactosylceramide (α-GalCer) presented on CD1d. We recently reported that iNKT cells recognize CD1d-negative leukemia cell line K562 in a TCR-dependent manner. However, it remains controversial how iNKT cells use TCRs to recognize and exhibit cytotoxic activity toward CD1d-negative tumors cells without CD1d restriction. Here, we report that iNKT cells exerted cytotoxicity toward K562 cells via a carried over anti-Vα24 TCR mAb from positive selection by magnetic bead sorting. We found that addition of the anti-Vα24Jα18 TCR mAb (6B11 mAb) rendered iNKT cells cytotoxic to K562 cells in an FcγRII (CD32)-dependent manner. Moreover, iNKT cells treated with 6B11 mAb became cytotoxic to other CD32+ cell lines (U937 and Daudi). In addition, iNKT cells treated with 6B11 mAb suppressed K562 cell growth in a murine xenograft model in vivo. These data suggest that anti-iNKT TCR mAb treatment of iNKT cells can be applied as a therapeutic strategy to treat CD32+ cancers such as leukemia, lymphoma, and lung cancer. SIGNIFICANCE: Our findings unveiled that iNKT cells recognize and kill CD1d-negative target tumors via the anti-iNKT TCR mAb bound to CD32 at the tumor site, thereby bridging iNKT cells and CD1d-negative tumors. These findings shed light on the therapeutic potential of anti-iNKT TCR mAbs in NKT cell-based immunotherapy to treat CD1d-negative CD32+ cancers.

Tumor-specific CD8+ T cells play a pivotal role in anti-tumor immunity and are a key target of immunotherapeutic approaches. Intratumoral CD8+ T cells are heterogeneous; Tcf1+ stem-like CD8+ T cells give rise to their cytotoxic progeny - Tim-3+ terminally differentiated CD8+ T cells. However, where and how this differentiation process occurs has not been elucidated. We herein show that terminally differentiated CD8+ T cells can be generated within tumor-draining lymph nodes (TDLNs) and that CD69 expression on tumor-specific CD8+ T cells controls its differentiation process through regulating the expression of the transcription factor TOX. In TDLNs, CD69 deficiency diminished TOX expression in tumor-specific CD8+ T cells, and consequently promoted generation of functional terminally differentiated CD8+ T cells. Anti-CD69 administration promoted the generation of terminally differentiated CD8+ T cells, and the combined use of anti-CD69 and anti-PD-1 showed an efficient anti-tumor effect. Thus, CD69 is an attractive target for cancer immunotherapy that synergizes with immune checkpoint blockade.