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Objectives: DROSHA and DICER have central roles in the biogenesis of microRNAs (miRNAs). However, we previously showed that in the murine system, DROSHA has an alternate function where it directly recognises and cleaves protein-coding messenger (m)RNAs and this is critical for safeguarding the pluripotency of haematopoietic stem cells (HSCs). Maintenance of murine HSC function is dependent on DROSHA-mediated cleavage of two mRNAs, Myl9 and Todr1 . The goal of this study is to determine whether this pathway is conserved in human HSCs., Methods: DROSHA and DICER were knocked down in human cord blood CD34 + HSCs with short hairpin RNAs. The function of HSCs was analysed in vitro and in humanised mice. Analysis of mRNA cleavage was performed by capture of 5' phosphorylated RNAs., Results: Consistent with murine HSCs, DROSHA knockdown impaired the differentiation of human HSCs in vitro and engraftment into humanised mice, whereas DICER knockdown had no impact. DROSHA cleaves the MYL9 mRNA in human HSCs and DROSHA deficiency resulted in the accumulation of the mRNA. However, ectopic expression of MYL9 did not impair human HSC function. We were unable to identify a human homolog of Todr1., Conclusion: A miRNA-independent function of DROSHA is critical for the function of human HSCs. DROSHA directly recognises and degrades mRNAs in humans HSCs. However, unlike in murine HSCs, the degradation of the MYL9 mRNA alone is not critical for human HSC function. Therefore, DROSHA must be inhibiting other targets and/or has another miRNA-independent function that is essential for safeguarding the pluripotency of human HSCs., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Objectives: Vaccines that prime Wilms' tumor 1 (WT1)-specific CD8 + T cells are attractive cancer immunotherapies. However, immunogenicity and clinical response rates may be enhanced by delivering WT1 to CD141 + dendritic cells (DCs). The C-type lectin-like receptor CLEC9A is expressed exclusively by CD141 + DCs and regulates CD8 + T-cell responses. We developed a new vaccine comprising a human anti-CLEC9A antibody fused to WT1 and investigated its capacity to target human CD141 + DCs and activate naïve and memory WT1-specific CD8 + T cells., Methods: WT1 was genetically fused to antibodies specific for human CLEC9A, DEC-205 or β-galactosidase (untargeted control). Activation of WT1-specific CD8 + T-cell lines following cross-presentation by CD141 + DCs was quantified by IFNγ ELISPOT. Humanised mice reconstituted with human immune cell subsets, including a repertoire of naïve WT1-specific CD8 + T cells, were used to investigate naïve WT1-specific CD8 + T-cell priming., Results: The CLEC9A-WT1 vaccine promoted cross-presentation of WT1 epitopes to CD8 + T cells and mediated priming of naïve CD8 + T cells more effectively than the DEC-205-WT1 and untargeted control-WT1 vaccines., Conclusions: Delivery of WT1 to CD141 + DCs via CLEC9A stimulates CD8 + T cells more potently than either untargeted delivery or widespread delivery to all Ag-presenting cells via DEC-205, suggesting that cross-presentation by CD141 + DCs is sufficient for effective CD8 + T-cell priming in humans. The CLEC9A-WT1 vaccine is a promising candidate immunotherapy for malignancies that express WT1., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)