Mammalian genomes encode multiple transposable element (TE) silencing pathways that distinguish their targets through different molecular features, with KRAB zinc finger proteins recognizing DNA sequence, the HUSH complex sensing intronless transcripts, and the PIWI–piRNA pathway using small RNA guides. How chromatin state itself contributes to TE recognition remains less defined. Here we identify Spindlin1 (SPIN1), a three-Tudor-domain histone reader implicated in germline piRNA-directed DNA methylation, as a transcriptional repressor of evolutionarily young TEs in mouse embryonic stem cells. SPIN1 selectively binds LINE and ERV loci carrying H3K4me3 and H3K9me3, a chromatin signature enriched at young, transcription-permissive elements, and recognition of these marks by Tudor domains 1 and 2 is required for TE targeting. SPIN1 engages SPINDOC as a Tudor 1 and 3-dependent cofactor whose loss phenocopies SPIN1 depletion, and associates with the H3K9 methyltransferases SETDB1 and G9a. SPIN1 loss reduces H3K9me3 and is accompanied by increased chromatin accessibility, without altering DNA methylation. Thus, SPIN1 uses a histone-state-based mechanism to identify and repress young TEs in pluripotent cells, mechanistically distinct from its germline mode in which SPIN1 cooperates with the PIWI–piRNA pathway to promote DNA methylation, illustrating how a single histone reader engages distinct silencing machineries across cellular contexts.
Induced pluripotent stem cell (iPSC)-derived immune cells show promise for allogeneic immunotherapies. However, their clinical efficacy can be limited by early rejection of grafted iPSC-derived cells by host T cells and natural killer (NK) cells due to HLA mismatching. In theory, disrupting HLA class I expression can prevent T cell-mediated rejection, but, in its place, NK cells can eliminate “missing-self” targets due to the absence of HLA-C and HLA-E. Therefore, we devised a strategy to extend the residence time of iPSC-derived invariant natural killer T (iPSC-derived iNKT) cells in humanized mice harboring allogeneic peripheral blood mononuclear cells (PBMCs) by genetically modifying HLA class I expression. To this end, we disrupted HLA class I expression by deleting the β2-microglobulin (B2M) gene, and then recapitulated HLA-C and/or HLA-E expression in the iPSC-derived iNKT cells. Parental and modified iPSC-derived iNKT cells were assessed for their susceptibility to T- or NK cell-mediated cytotoxicity in vitro and for their residence time in allogeneic humanized mice. In vitro, dual co-expression of HLA-C and HLA-E facilitated escape from NK cell-mediated cytotoxicity, whereas single expression of either HLA-C or HLA-E provided inconsistent protection across NK cell donors. Since allogeneic HLA-C alleles can be recognized by T cells, we examined the impact of HLA-C mismatching on T cell-mediated cytotoxicity. T cell-mediated cytotoxicity against B2M-deficient iPSC-derived iNKT cells was restored when mismatched HLA-C was re-expressed. In vivo, B2M deficiency extended the residence time of iPSC-derived iNKT cells in allogeneic humanized mice. This effect was further enhanced by expression of HLA-C alleles matched to donor PBMCs, whereas expression of incompatible HLA-C reduced residence time. Taking into account the limitations of the humanized mouse model in recapitulating human NK cell function, these observations highlight the dominant role of T cell-mediated mechanisms in the rejection of allogeneic iPSC-derived iNKT cells in vivo. This study presents a promising approach for generating iPSC-derived iNKT cells tailored for a limited master cell bank, with the potential to develop universal off-the-shelf immunotherapy.
Yun‐Hsuan Chang, T. Aoki, M. Okoshi et al.· Frontiers in Cell and Develo...· 0 citations
