Germline pathogenic variants in DNMT3A were recently described in patients with overgrowth, obesity, behavioral, and learning difficulties (DNMT3A Overgrowth Syndrome/DOS). Somatic mutations in the DNMT3A gene are also the most common cause of clonal hematopoiesis, and can initiate acute myeloid leukemia (AML). Using whole genome bisulfite sequencing, we studied DNA methylation in peripheral blood cells of 11 DOS patients and found a focal, canonical hypomethylation phenotype, which is most severe with the dominant negative DNMT3AR882H mutation. A germline mouse model expressing the homologous Dnmt3aR878H mutation phenocopies most aspects of the human DOS syndrome, including the methylation phenotype and an increased incidence of spontaneous hematopoietic malignancies, suggesting that all aspects of this syndrome are caused by this mutation.
© 2021. The Author(s).

Pluripotent stem cells (PSCs) have the potential to provide homogeneous cell populations of T cells that can be grown at a clinical scale and genetically engineered to meet specific clinical needs. OP9-DLL4, a stromal line ectopically expressing the Notch ligand Delta-like 4 (DLL4) is used to support differentiation of PSCs to T-lymphocytes. This article outlines several protocols related to generation of T cells from human and non-human primate (NHP) PSCs, including initial hematopoietic differentiation of PSC on OP9 feeders or defined conditions, followed by coculture of the OP9-DLL4 cells with the PSC-derived hematopoietic progenitors (HPs), leading to efficient differentiation to T lymphocytes. In addition, we describe a protocol for robust T cell generation from hPSCs conditionally expressing ETS1. The presented protocols provide a platform for T cell production for disease modeling and evaluating their use for immunotherapy in large animal models.
Copyright © 2020 The Authors; exclusive licensee Bio-protocol LLC.

HLA-DPB1 antigens are mismatched in about 80% of allogeneic hematopoietic stem cell transplantations from HLA 10/10 matched unrelated donors and were shown to be associated with a decreased risk of leukemia relapse. We recently developed a reliable in vitro method to generate HLA-DPB1 mismatch-reactive CD4 T-cell clones from allogeneic donors. Here, we isolated HLA-DPB1 specific T cell receptors (TCR DP) and used them either as wild-type or genetically optimized receptors to analyze in detail the reactivity of transduced CD4 and CD8 T cells toward primary AML blasts. While both CD4 and CD8 T cells showed strong AML reactivity in vitro, only CD4 T cells were able to effectively eliminate leukemia blasts in AML engrafted NOD/SCID/IL2Rγc-/- (NSG) mice. Further analysis showed that optimized TCR DP and under some conditions wild-type TCR DP also mediated reactivity to non-hematopoietic cells like fibroblasts or tumor cell lines after HLA-DP upregulation. In conclusion, T cells engineered with selected allo-HLA-DPB1 specific TCRs might be powerful off-the-shelf reagents in allogeneic T-cell therapy of leukemia. However, because of frequent (common) cross-reactivity to non-hematopoietic cells with optimized TCR DP T cells, safety mechanisms are mandatory.

Human mononuclear phagocytes comprise phenotypically and functionally overlapping subsets of dendritic cells (DCs) and monocytes, but the extent of their heterogeneity and distinct markers for subset identification remains elusive. By integrating high-dimensional single-cell protein and RNA expression data, we identified distinct markers to delineate monocytes from conventional DC2 (cDC2s). Using CD88 and CD89 for monocytes and HLA-DQ and FcεRIα for cDC2s allowed for their specific identification in blood and tissues. We also showed that cDC2s could be subdivided into phenotypically and functionally distinct subsets based on CD5, CD163, and CD14 expression, including a distinct subset of circulating inflammatory CD5-CD163+CD14+ cells related to previously defined DC3s. These inflammatory DC3s were expanded in systemic lupus erythematosus patients and correlated with disease activity. These findings further unravel the heterogeneity of DC subpopulations in health and disease and may pave the way for the identification of specific DC subset-targeting therapies.
Copyright © 2019 Elsevier Inc. All rights reserved.

Cancer immunotherapies provide survival benefits in responding patients, but many patients fail to respond. Identifying the biology of treatment response and resistance are a priority to optimize drug selection and improve patient outcomes. We performed transcriptomic and immune profiling on 158 tumor biopsies from melanoma patients treated with anti-PD-1 monotherapy (n = 63) or combined anti-PD-1 and anti-CTLA-4 (n = 57). These data identified activated T cell signatures and T cell populations in responders to both treatments. Further mass cytometry analysis identified an EOMES+CD69+CD45RO+ effector memory T cell phenotype that was significantly more abundant in responders to combined immunotherapy compared with non-responders (n = 18). The gene expression profile of this population was associated with longer progression-free survival in patients treated with single agent and greater tumor shrinkage in both treatments.
Copyright © 2019 Elsevier Inc. All rights reserved.