Sox9 links biliary maturation to branching morphogenesis.

Branching morphogenesis couples cellular differentiation with development of tissue architecture. Intrahepatic bile duct (IHBD) morphogenesis is initiated with biliary epithelial cell (BEC) specification and eventually forms a heterogeneous network of large ducts and small ductules. Here, we show that Sox9 is required for developmental establishment of small ductules. IHBDs emerge as a webbed structure by E15.5 and undergo morphological maturation through 2 weeks of age. Developmental knockout of Sox9 leads to decreased postnatal branching morphogenesis, manifesting as loss of ductules in adult livers. In the absence of Sox9, BECs fail to mature and exhibit elevated TGF-ß signaling and Activin A. Activin A induces developmental gene expression and morphological defects in BEC organoids and represses ductule formation in postnatal livers. Our data demonstrate that adult IHBD morphology and BEC maturation is regulated by the Sox9-dependent formation of precursors to ductules during development, mediated in part by downregulation of Activin A.

LENS: Landscape of Effective Neoantigens Software.

MOTIVATION: Elimination of cancer cells by T cells is a critical mechanism of anti-tumor immunity and cancer immunotherapy response. T cells recognize cancer cells by engagement of T cell receptors with peptide epitopes presented by major histocompatibility complex molecules on the cancer cell surface. Peptide epitopes can be derived from antigen proteins coded for by multiple genomic sources. Bioinformatics tools used to identify tumor-specific epitopes via analysis of DNA and RNA-sequencing data have largely focused on epitopes derived from somatic variants, though a smaller number have evaluated potential antigens from other genomic sources. RESULTS: We report here an open-source workflow utilizing the Nextflow DSL2 workflow manager, Landscape of Effective Neoantigens Software (LENS), which predicts tumor-specific and tumor-associated antigens from single nucleotide variants, insertions and deletions, fusion events, splice variants, cancer-testis antigens, overexpressed self-antigens, viruses, and endogenous retroviruses. The primary advantage of LENS is that it expands the breadth of genomic sources of discoverable tumor antigens using genomics data. Other advantages include modularity, extensibility, ease of use, and harmonization of relative expression level and immunogenicity prediction across multiple genomic sources. We present an analysis of 115 acute myeloid leukemia samples to demonstrate the utility of LENS. We expect LENS will be a valuable platform and resource for T cell epitope discovery bioinformatics, especially in cancers with few somatic variants where tumor-specific epitopes from alternative genomic sources are an elevated priority. AVAILABILITY AND IMPLEMENTATION: More information about LENS, including code, workflow documentation, and instructions, can be found at (https://gitlab.com/landscape-of-effective-neoantigens-software).

Associations of minor histocompatibility antigens with outcomes following allogeneic hematopoietic cell transplantation.

The role of minor histocompatibility antigens (mHAs) in mediating graft versus leukemia and graft versus host disease (GvHD) following allogeneic hematopoietic cell transplantation (alloHCT) is recognized but not well-characterized. By implementing improved methods for mHA prediction in two large patient cohorts, this study aimed to comprehensively explore the role of mHAs in alloHCT by analyzing whether (1) the number of predicted mHAs, or (2) individual mHAs are associated with clinical outcomes. The study population consisted of 2249 donor-recipient pairs treated for acute myeloid leukemia and myelodysplastic syndrome with alloHCT. A Cox proportional hazard model showed that patients with a class I mHA count greater than the population median had an increased hazard of GvHD mortality (hazard ratio [HR] = 1.39, 95% confidence interval [CI] = 1.01, 1.77, p = .046). Competing risk analyses identified the class I mHAs DLRCKYISL (GSTP), WEHGPTSLL (CRISPLD2), and STSPTTNVL (SERPINF2) were associated with increased GVHD mortality (HR = 2.84, 95% CI = 1.52, 5.31, p = .01), decreased leukemia-free survival (LFS) (HR = 1.94, 95% CI = 1.27, 2.95, p = .044), and increased disease-related mortality (DRM) (HR = 2.32, 95% CI = 1.5, 3.6, p = .008), respectively. One class II mHA YQEIAAIPSAGRERQ (TACC2) was associated with increased risk of treatment-related mortality (TRM) (HR = 3.05, 95% CI = 1.75, 5.31, p = .02). WEHGPTSLL and STSPTTNVL were both present within HLA haplotype B*40:01-C*03:04 and showed a positive dose-response relationship with increased all-cause mortality and DRM and decreased LFS, indicating these two mHAs contribute to the risk of mortality in an additive manner. Our study reports the first large-scale investigation of the associations of predicted mHA peptides with clinical outcomes following alloHCT.

Shared graft-versus-leukemia minor histocompatibility antigens in DISCOVeRY-BMT.

T-cell responses to minor histocompatibility antigens (mHAs) mediate graft-versus-leukemia (GVL) effects and graft-versus-host disease (GVHD) in allogeneic hematopoietic cell transplantation. Therapies that boost T-cell responses improve allogeneic hematopoietic cell transplant (alloHCT) efficacy but are limited by concurrent increases in the incidence and severity of GVHD. mHAs with expression restricted to hematopoietic tissue (GVL mHAs) are attractive targets for driving GVL without causing GVHD. Prior work to identify mHAs has focused on a small set of mHAs or population-level single-nucleotide polymorphism-association studies. We report the discovery of a large set of novel GVL mHAs based on predicted immunogenicity, tissue expression, and degree of sharing among donor-recipient pairs (DRPs) in the DISCOVeRY-BMT data set of 3231 alloHCT DRPs. The total number of predicted mHAs varied by HLA allele, and the total number and number of each class of mHA significantly differed by recipient genomic ancestry group. From the pool of predicted mHAs, we identified the smallest sets of GVL mHAs needed to cover 100% of DRPs with a given HLA allele. We used mass spectrometry to search for high-population frequency mHAs for 3 common HLA alleles. We validated 24 predicted novel GVL mHAs that are found cumulatively within 98.8%, 60.7%, and 78.9% of DRPs within DISCOVeRY-BMT that express HLA-A∗02:01, HLA-B∗35:01, and HLA-C∗07:02, respectively. We confirmed the immunogenicity of an example novel mHA via T-cell coculture with peptide-pulsed dendritic cells. This work demonstrates that the identification of shared mHAs is a feasible and promising technique for expanding mHA-targeting immunotherapeutics.

Non-Hodgkin Lymphomas: Malignancies Arising from Mature B Cells.

Non-Hodgkin lymphomas (NHLs) are a diverse group of entities, both clinically and molecularly. Here, we review the evolution of classification schemes in B-cell lymphoma, noting the now standard WHO classification system that is based on immune cell-of-origin and molecular phenotypes. We review how lymphomas arise throughout the B-cell development process as well as the molecular and clinical features of prominent B-cell lymphomas. We provide an overview of the major progress that has occurred over the past decade in terms of our molecular understanding of these diseases. We discuss treatment options available and focus on a number of the diverse research tools that have been employed to improve our understanding of these diseases. We discuss the problem of heterogeneity in lymphomas and anticipate that the near future will bring significant advances that provide a measurable impact on NHL outcomes.

Identifying Key Genetic Regions for Cell Sheet Morphogenesis on Chromosome 2L Using a Drosophila Deficiency Screen in Dorsal Closure.

Cell sheet morphogenesis is essential for metazoan development and homeostasis of animal form - it contributes to developmental milestones including gastrulation, neural tube closure, heart and palate formation and to tissue maintenance during wound healing. Dorsal closure, a well-characterized stage in Drosophila embryogenesis and a model for cell sheet morphogenesis, is a remarkably robust process during which coordination of conserved gene expression patterns and signaling cascades regulate the cellular shape changes and movements. New 'dorsal closure genes' continue to be discovered due to advances in imaging and genetics. Here, we extend our previous study of the right arm of the 2nd chromosome to the left arm of the 2nd chromosome using the Bloomington deficiency kit's set of large deletions, which collectively remove 98.9% of the genes on the left arm of chromosome two (2L) to identify 'dorsal closure deficiencies'. We successfully screened 87.2% of the genes and identified diverse dorsal closure defects in embryos homozygous for 49 deficiencies, 27 of which delete no known dorsal closure gene. These homozygous deficiencies cause defects in cell shape, canthus formation and tissue dynamics. Within these deficiencies, we have identified pimples, odd-skipped, paired, and sloppy-paired 1 as dorsal closure genes on 2L that affect lateral epidermal cells. We will continue to identify novel 'dorsal closure genes' with further analysis. These forward genetic screens are expected to identify new processes and pathways that contribute to closure and links between pathways and structures already known to coordinate various aspects of closure.

Cas9-AAV6-engineered human mesenchymal stromal cells improved cutaneous wound healing in diabetic mice.

Human mesenchymal stromal cells (hMSCs) are a promising source for engineered cell-based therapies in which genetic engineering could enhance therapeutic efficacy and install novel cellular functions. Here, we describe an optimized Cas9-AAV6-based genome editing tool platform for site-specific mutagenesis and integration of up to more than 3 kilobases of exogenous DNA in the genome of hMSCs derived from the bone marrow, adipose tissue, and umbilical cord blood without altering their ex vivo characteristics. We generate safe harbor-integrated lines of engineered hMSCs and show that engineered luciferase-expressing hMSCs are transiently active in vivo in wound beds of db/db mice. Moreover, we generate PDGF-BB- and VEGFA-hypersecreting hMSC lines as short-term, local wound healing agents with superior therapeutic efficacy over wildtype hMSCs in the diabetic mouse model without replacing resident cells long-term. This study establishes a precise genetic engineering platform for genetic studies of hMSCs and development of engineered hMSC-based therapies.