Single-cell RNA sequencing distinguishes dendritic cell subsets in the rat, allowing advanced characterization of the effects of FMS-like tyrosine kinase 3 ligand.

Tissue-resident dendritic cells (DCs) are essential for immunological homeostasis and hold promise for a variety of therapeutic interventions. The rare nature of tissue-resident DCs and their suboptimal description in the lab rat model has limited their characterization. To address this limitation, FMS-like tyrosine kinase 3 ligand (FLT3L) has been utilized to expand these population in vitro and in vivo for investigative or therapeutic purposes. However, conflicting reports have suggested that FLT3L can either promote immune tolerance or enhance immunogenicity, necessitating clarification of the effects of FLT3L on DC phenotype and functionality. We first paired single-cell RNA sequencing with multicolour spectral flow cytometry to provide an updated strategy for the identification of tissue-resident classical and plasmacytoid DCs in the rat model. We then administered FLT3L to Lewis rats in vivo to investigate its effect on tissue-resident DC enumeration and phenotype in the liver, spleen, and mesenteric lymph nodes. We found that FLT3L expands classical DCs (cDCs) 1 and 2 in a dose-dependent manner and that cDC1 and cDC2 in secondary lymphoid organs had altered MHC I, MHC II, CD40, CD80, CD86, and PD-L1 cell-surface expression levels following FLT3L administration. These changes were accompanied by an increase in gene expression levels of toll-like receptors 2, 4, 7, and 9 as well as inflammatory cytokines IL-6 and TNF-α. In conclusion, FLT3L administration in vivo increases cDC enumeration in the liver, spleen, and mesenteric lymph nodes accompanied by a tissue-restricted alteration in expression of antigen presentation machinery and inflammatory mediators.

Interleukin-10 and Transforming Growth Factor-ß Cytokines Decrease Immune Activation During Normothermic Ex Vivo Machine Perfusion of the Rat Liver.

Normothermic ex vivo liver perfusion (NEVLP) is a novel system for organ preservation that may improve over static cold storage clinically and offers the chance for graft modification prior to transplantation. Although recent studies have shown the presence of inflammatory molecules during perfusion, none have yet shown the effects of NEVLP on liver-resident immune cell activation. We investigated the effects of NEVLP on liver-resident immune cell activation and assessed the ability of anti-inflammatory cytokines interleukin 10 (IL10) and transforming growth factor ß (TGF-ß) to improve organ function and reduce immune activation during perfusion. Rat livers were perfused for 4 hours at 37°C with or without the addition of 20 ng/mL of each IL10 and TGF-ß (n = 7). Naïve and cold storage (4 hours at 4°C) livers served as controls (n = 4). Following preservation, gene expression profiles were assessed through single-cell RNA sequencing; dendritic cell and macrophage activation was measured by flow cytometry; and cytokine production was assessed by enzyme-linked immunosorbent assay. NEVLP induced a global inflammatory gene expression signature, most notably in liver-resident macrophages and dendritic cells, which was accompanied by an increase in cell-surface levels of major histocompatibility complex (MHC) II, CD40, and CD86. Immune activation was partially ameliorated by IL10 and TGF-ß treatment, but no changes were observed in inflammatory cytokine production. Overall levels of liver damage and cellular apoptosis from perfusion were low, and liver function was improved with IL10 and TGF-ß treatment. This is the first study to demonstrate that liver-resident immune cells gain an activated phenotype during NEVLP on both the gene and protein level and that this activation can be reduced through therapeutic intervention with IL10 and TGF-ß.

The Immunological Effect of Oxygen Carriers on Normothermic Ex Vivo Liver Perfusion.

Introduction: Normothermic ex vivo liver perfusion (NEVLP) is an organ preservation method that allows liver graft functional assessment prior to transplantation. One key component of normothermic perfusion solution is an oxygen carrier to provide oxygen to the liver to sustain metabolic activities. Oxygen carriers such as red blood cells (RBCs) or hemoglobin-based oxygen carriers have an unknown effect on the liver-resident immune cells during NEVLP. In this study, we assessed the effects of different oxygen carriers on the phenotype and function of liver-resident immune cells. Methods: Adult Lewis rat livers underwent NEVLP using three different oxygen carriers: human packed RBCs (pRBCs), rat pRBCs, or Oxyglobin (a synthetic hemoglobin-based oxygen carrier). Hourly perfusate samples were collected for downstream analysis, and livers were digested to isolate immune cells. The concentration of common cytokines was measured in the perfusate, and the immune cells underwent phenotypic characterization with flow cytometry and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The stimulatory function of the liver-resident immune cells was assessed using mixed lymphocyte reactions. Results: There were no differences in liver function, liver damage, or histology between the three oxygen carriers. qRT-PCR revealed that the gene expression of nuclear factor κ light chain enhancer of activated B cells (NF-kB), Interleukin (IL-1ß), C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine ligand 7 (CCL7), and CD14 was significantly upregulated in the human pRBC group compared with that in the naive, whereas the rat pRBC and Oxyglobin groups were not different from that of naive. Flow cytometry demonstrated that the cell surface expression of the immune co-stimulatory protein, CD86, was significantly higher on liver-resident macrophages and plasmacytoid dendritic cells perfused with human pRBC compared to Oxyglobin. Mixed lymphocyte reactions revealed increased allogeneic T-cell proliferation in the human and rat pRBC groups compared to that in the Oxyglobin group. Conclusions: Liver-resident immune cells are important mediators of rejection after transplantation. In this study, we show that the oxygen carrier used in NEVLP solutions can affect the phenotype of these liver-resident immune cells. The synthetic hemoglobin-based oxygen carrier, Oxyglobin, showed the least amount of liver-resident immune cell activation and the least amount of allogeneic proliferation when compared to human or rat pRBCs. To mitigate liver-resident immune cell activation during NEVLP (and subsequent transplantation), Oxyglobin may be an optimal oxygen carrier.

Reversible epigenetic alterations regulate class I HLA loss in prostate cancer.

Downregulation of HLA class I (HLA-I) impairs immune recognition and surveillance in prostate cancer and may underlie the ineffectiveness of checkpoint blockade. However, the molecular mechanisms regulating HLA-I loss in prostate cancer have not been fully explored. Here, we conducted a comprehensive analysis of HLA-I genomic, epigenomic and gene expression alterations in primary and metastatic human prostate cancer. Loss of HLA-I gene expression was associated with repressive chromatin states including DNA methylation, histone H3 tri-methylation at lysine 27, and reduced chromatin accessibility. Pharmacological DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibition decreased DNA methylation and increased H3 lysine 27 acetylation and resulted in re-expression of HLA-I on the surface of tumor cells. Re-expression of HLA-I on LNCaP cells by DNMT and HDAC inhibition increased activation of co-cultured prostate specific membrane antigen (PSMA)27-38-specific CD8+ T-cells. HLA-I expression is epigenetically regulated by functionally reversible DNA methylation and chromatin modifications in human prostate cancer. Methylated HLA-I was detected in HLA-Ilow circulating tumor cells (CTCs), which may serve as a minimally invasive biomarker for identifying patients who would benefit from epigenetic targeted therapies.

Live cell molecular analysis of primary prostate cancer organoids identifies persistent androgen receptor signaling.

Prostate Cancer (PC) is a disease with remarkable tumor heterogeneity that often manifests in significant intra-patient variability with regards to clinical outcomes and treatment response. Commonly available PC cell lines do not accurately reflect the complexity of this disease and there is critical need for development of new models to recapitulate the intricate hierarchy of tumor pathogenesis. In current study, we established ex vivo primary patient-derived cancer organoid (PDCO) cultures from prostatectomy specimens of patients with locally advanced PC. We then performed a comprehensive multi-parameter characterization of the cellular composition utilizing a novel approach for live-cell staining and direct imaging in the integrated microfluidic Stacks device. Using orthogonal flow cytometry analysis, we demonstrate that primary PDCOs maintain distinct subsets of epithelial cells throughout culture and that these cells conserve expression of androgen receptor (AR)-related elements. Furthermore, to confirm the tumor-origin of the PDCOs we have analyzed the expression of PC-associated epigenetic biomarkers including promoter methylation of the GSTP1, RASSF1 and APC and RARb genes by employing a novel microfluidic rare-event screening protocol. These results demonstrate that this ex vivo PDCO model recapitulates the complexity of the epithelial tumor microenvironment of multifocal PC using orthogonal analyses. Furthermore, we propose to leverage the Stacks microfluidic device as a high-throughput, translational platform to interrogate phenotypic and molecular endpoints with the capacity to incorporate a complex tumor microenvironment.