This study provides direct evidence that VEXAS-specific TE govern HSC clonal dominance, thereby uncovering a regulatory axis underlying HSC biology and disease mechanisms, opening a therapeutic strategy directed towards the repetitive genome.
Germline gain-of-function (GOF) mutations in the signal transducer and activator of transcription 1 (STAT1) gene cause a dominantly inherited inborn error of immunity (IEI) characterized by chronic mucocutaneous candidiasis, autoimmunity, severe opportunistic infections and an increased risk of malignancy. Allogeneic hematopoietic stem cell (HSC) transplantation (HSCT) is curative but is associated with increased risk of morbidity and mortality in STAT1 GOF patients compared to other IEI. To develop a curative, autologous alternative to HSCT, we evaluated gene editing strategies in STAT1 GOF model cell lines, primary T cells, and patient-derived HSCs. Universal and mutation-specific strategies using CRISPR/Cas-mediated homology-directed repair (HDR) were limited by low efficacy (<25%), poor viability, and a lack of allele-specificity. In contrast, adenine base editing corrected the recurrent and highly pathogenic p.T385M mutation with upwards of 90% efficiency in patient T cells and HSCs without significant unintended on- or off-target genomic aberrations. Gene editing functionally restored total STAT1 expression (p<0.0217), STAT1 phosphorylation (p<0.0056), interferon-stimulated gene expression (OAS1; p=0.0005) and improved IL-17 production (p<0.0001). Edited HSCs retained multilineage differentiation capacity and sustained engraftment with persistence of the corrected allele at 16 weeks in humanized immunodeficient mice. These data demonstrate efficient and precise correction of STAT1 GOF mutations by base editing, with maintenance of the correction through long-term engraftment in vivo. This represents the first application of gene editing to correct a dominant gain-of-function mutation causing immunodeficiency, with potential applicability to other genetic disorders associated with heterozygous and gain-of-function mutations.
Robert Torrance, K. Orf, Nathan White et al.· Blood· 0 citations
In autoimmunity and chronic infection, both settings of persistent (self or foreign) antigen, immune responses are sustained by stem-like CD8 T cells, which self-renew and give rise to differentiated progeny. However, if and how T stemness is epigenetically encoded, which transcription factor(s) regulate the stem-T cells, and whether the stem-T cell state is disease-specific or shared across diseases, is currently not known.
We used clinically relevant models of autoimmune type 1 diabetes (T1D) and chronic infection and conducted serial T cell transplantation studies in vivo, combined with single cell paired RNA- and ATAC-sequencing on antigen-specific T cells. We developed CRISPR/Cas9-mediated gene-editing approaches in primary T cells as well as CUT&RUN studies, identifying a novel hierarchy of transcription factors regulating stem T cell identity and function.
We discovered that a small subset of stem-T cells (TSC) express lymphoid enhancer-binding factor 1 (LEF1), a member of the TCF/LEF TF family. Paired single cell transcriptomic and epigenomic analyses reveal that the LEF1+ TSC harbor a unique epigenetically encoded molecular state enriched in genes and pathways characteristic of embryonic and adult (somatic) stem cells (e.g. neural stem cells). Strikingly, we found that TSC in chronic infection harbor a LEF1+ TSC pool sharing the core stemness epigenetic and molecular program observed in autoimmune LEF1+ TSC. Loss- and gain-of-function studies in both autoimmune T1D and chronic infection confirmed the critical role of LEF1 in maintaining T cell stemness. CUT&RUN analyses provide clues as to how LEF1 instructs the epigenetically encoded program of stem-T cells.
Here we reveal novel insights into the molecular circuitries of CD8 T cell stemness and differentiation. We discover LEF1 as the master regulator defining T cell stemness and identify novel targets for therapeutic intervention.
NIH R01AI173249, JDRF SRA-2023-1410-S-B, MSKCC Basic Research Innovation Award, The Hearst Foundation
Lymphocyte Differentiation and Peripheral Maintenance (LYM)
Katrina M. Hawley, S. Miakicheva, P. Zumbo et al.· Journal of Immunology· 0 citations
The maintenance of homeostasis in hematopoietic stem and progenitor cells (HSPCs) is essential for the proper development of the entire hematopoietic system. However, the mechanisms underlying this regulatory equilibrium remain elusive. Here, we report that Prdm15 deficiency in HSPCs induces the accumulation of immature hematopoietic stem cells in mice. A series of transplantation assays shows that these cells display impaired reconstitution capacity and competitive fitness, which are associated with abnormal differentiation trajectories and transcriptional alterations identified by single-cell RNA sequencing. Mechanistically, integrated multi-omics analyses including ATAC-seq and CUT&Tag sequencing of HSPCs indicate that Prdm15 deficiency induces significant transcriptional and epigenetic alterations, particularly affecting the methyltransferase KMT2C and altering H3K4me1 and H3K27ac modifications at the promoters of hematopoietic developmental genes. Collectively, our findings establish PRDM15 as a critical epigenetic regulator of HSPCs, offering valuable insights into the molecular mechanisms underlying hematopoietic homeostasis.
Qiwen Dong, Weiwei Xiao, Junsong Huang et al.· Journal of genetics and geno...· 0 citations
Chronic graft-versus-host disease (cGVHD) is a severe complication of hematopoietic stem cell transplantation (HSCT) complication driven by immune dysregulation. A key feature is aberrant T follicular helper (Tfh) and germinal center B (GCB) cells, which promote pro-inflammatory cytokine release, pathogenic antibody production, and tissue fibrosis. Leveraging cGVHD patient T cell single-cell RNA sequencing data from the Kean Lab, we selected Id2, a transcription factor that inhibits Tfh differentiation while supporting antibody production, as a target for the treatment of cGvHD.
We used CRISPR/Cas9 to generate Id2-knockout (KO) naïve murine T cells and adoptively transferred into pre-conditioned B10.BR recipients of C57BL/6 bone marrow (BM) ± T cells to generate murine cGVHD model with bronchiolitis obliterans (BO) lung disease.
Id2-KO T cells maintained ≥90% KO efficiency until study end (d49). Mice receiving Id2-KO vs control T cells had significantly improved pulmonary function (p < 0.0001), normalizing resistance, compliance, and elastance to BM-only (no disease) levels. Consistent with cGVHD amelioration, a 2-fold reduction in lung collagen deposition (p < 0.0001) and 4-fold decrease in serum allo-reactive antibody (p = 0.0286) were noted. On D49, despite similar GCB and Tfh ratios to control, Id2-KO T cells had enhanced Tfh differentiation: Bcl6+T cells increased from 4.9% to 10.8% (p = 0.0038) with 1.8-fold higher Bcl6 MFI(p = 0.0005). Id2-KO Tfh cells were functionally impaired with a 50% reduction in IL-4 production (p = 0.0078) and a 1.9-fold decrease in SLAMF1+ Tfh cells (from 63.3% to 33%, p < 0.0001), indicating disrupted Tfh-GC B cell interactions.
Id2 restrains Tfh differentiation while supporting their functional capacity to help GCB cells produce pathogenic antibody. Genetic Id2 loss uncouples Tfh differentiation from effector activity, positioning Id2 inhibition as a novel therapeutic strategy to selectively disrupt pathogenic Tfh-GCB crosstalk in cGVHD
NIH
Transplantation Immunology (TRAN)
Yujie Zhao, Eun Ko, Cameron McDonald-Hyman et al.· Journal of Immunology· 0 citations
Single-cell RNA sequencing indicates that adulthood Rack1 deletion in type I interferon-I-responsive cells leads to aberrant lineage-geneset-scores of transcriptional HSCs and the emergence of stressed HSCs, and Mechanistically, RACK1 prevents HSC loss through maintaining the protein level of LDB1.
L. Deng, Junjie Du, Zhengqiu Xu et al.· Cell Death & Disease· 0 citations
Using mice carrying a mutation in the CBFβ2 isoform, it is demonstrated that CBFβ2 haploinsufficiency rewires transcriptional regulatory circuits in adult bone marrow progenitors, rendering them permissive for an early life-restricted Tγδ17 cells.
Michela Frascoli, Alyssa Berthelette, Joonsoo Kang et al.· Journal of Immunology· 0 citations