Chronic Opioid Treatment Arrests Neurodevelopment and Alters Synaptic Activity in Human Midbrain Organoids.

Understanding the impact of long-term opioid exposure on the embryonic brain is critical due to the surging number of pregnant mothers with opioid dependency. However, this has been limited by human brain inaccessibility and cross-species differences in animal models. Here, a human midbrain model is established that uses hiPSC-derived midbrain organoids to assess cell-type-specific responses to acute and chronic fentanyl treatment and fentanyl withdrawal. Single-cell mRNA sequencing of 25,510 cells from organoids in different treatment groups reveals that chronic fentanyl treatment arrests neuronal subtype specification during early midbrain development and alters synaptic activity and neuron projection. In contrast, acute fentanyl treatment increases dopamine release but does not significantly alter gene expression related to cell lineage development. These results provide the first examination of the effects of opioid exposure on human midbrain development at the single-cell level.

Tff2 defines transit-amplifying pancreatic acinar progenitors that lack regenerative potential and are protective against Kras-driven carcinogenesis.

While adult pancreatic stem cells are thought not to exist, it is now appreciated that the acinar compartment harbors progenitors, including tissue-repairing facultative progenitors (FPs). Here, we study a pancreatic acinar population marked by trefoil factor 2 (Tff2) expression. Long-term lineage tracing and single-cell RNA sequencing (scRNA-seq) analysis of Tff2-DTR-CreERT2-targeted cells defines a transit-amplifying progenitor (TAP) population that contributes to normal homeostasis. Following acute and chronic injury, Tff2+ cells, distinct from FPs, undergo depopulation but are eventually replenished. At baseline, oncogenic KrasG12D-targeted Tff2+ cells are resistant to PDAC initiation. However, KrasG12D activation in Tff2+ cells leads to survival and clonal expansion following pancreatitis and a cancer stem/progenitor cell-like state. Selective ablation of Tff2+ cells prior to KrasG12D activation in Mist1+ acinar or Dclk1+ FP cells results in enhanced tumorigenesis, which can be partially rescued by adenoviral Tff2 treatment. Together, Tff2 defines a pancreatic TAP population that protects against Kras-driven carcinogenesis.

Tbc1d10c is a selective, constitutive suppressor of the CD8 T-cell anti-tumor response.

Cancer immunotherapy approaches target signaling pathways that are highly synonymous between CD4 and CD8 T-cell subsets and, therefore, often stimulate nonspecific lymphocyte activation, resulting in cytotoxicity to otherwise healthy tissue. The goal of our study was to identify intrinsic modulators of basic T lymphocyte activation pathways that could discriminately bolster CD8 anti-tumor effector responses. Using a Tbc1d10c null mouse, we observed marked resistance to a range of tumor types conferred by Tbc1d10c deficiency. Moreover, tumor-bearing Tbc1d10c null mice receiving PD-1 or CTLA-4 monotherapy exhibited a 33% or 90% cure rate, respectively. While Tbc1d10c was not expressed in solid tumor cells, Tbc1d10c disruption selectively augmented CD8 T-cell activation and cytotoxic effector responses and adoptive transfer of CD8 T cells alone was sufficient to recapitulate Tbc1d10c null tumor resistance. Mechanistically, Tbc1d10c suppressed CD8 T-cell activation and anti-tumor function by intersecting canonical NF-κB pathway activation via regulation of Map3k3-mediated IKKß phosphorylation. Strikingly, none of these cellular or molecular perturbations in the NF-κB pathway were featured in Tbc1d10c null CD4 T cells. Our findings identify a Tbc1d10c-Map3k3-NF-κB signaling axis as a viable therapeutic target to promote CD8 T-cell anti-tumor immunity while circumventing CD4 T cell-associated cytotoxicity and NF-κB activation in tumor cells.

Widespread Traces of Lytic Kaposi Sarcoma-Associated Herpesvirus in Primary Effusion Lymphoma at Single-Cell Resolution.

Cancer cells of primary effusion lymphoma (PEL) often contain both Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). We measured the interplay of human, KSHV, and EBV transcription in a cell culture model of PEL using single-cell RNA sequencing. The data detect widespread trace expression of lytic KSHV genes.

Harnessing Hematopoietic Stem Cell Low Intracellular Calcium Improves Their Maintenance In Vitro.

The specific cellular physiology of hematopoietic stem cells (HSCs) is underexplored, and their maintenance in vitro remains challenging. We discovered that culture of HSCs in low calcium increased their maintenance as determined by phenotype, function, and single-cell expression signature. HSCs are endowed with low intracellular calcium conveyed by elevated activity of glycolysis-fueled plasma membrane calcium efflux pumps and a low-bone-marrow interstitial fluid calcium concentration. Low-calcium conditions inhibited calpain proteases, which target ten-eleven translocated (TET) enzymes, of which TET2 was required for the effect of low calcium conditions on HSC maintenance in vitro. These observations reveal a physiological feature of HSCs that can be harnessed to improve their maintenance in vitro.

Generation and persistence of human tissue-resident memory T cells in lung transplantation.

Tissue-resident memory T cells (TRM) maintain immunity in diverse sites as determined in mouse models, whereas their establishment and role in human tissues have been difficult to assess. Here, we investigated human lung TRM generation, maintenance, and function in airway samples obtained longitudinally from human leukocyte antigen (HLA)-disparate lung transplant recipients, where donor and recipient T cells could be localized and tracked over time. Donor T cells persist specifically in the lungs (and not blood) of transplant recipients and express high levels of TRM signature markers including CD69, CD103, and CD49a, whereas lung-infiltrating recipient T cells gradually acquire TRM phenotypes over months in vivo. Single-cell transcriptome profiling of airway T cells reveals that donor T cells comprise two TRM-like subsets with varying levels of expression of TRM-associated genes, whereas recipient T cells comprised non-TRM and similar TRM-like subpopulations, suggesting de novo TRM generation. Transplant recipients exhibiting higher frequencies of persisting donor TRM experienced fewer adverse clinical events such as primary graft dysfunction and acute cellular rejection compared with recipients with low donor TRM persistence, suggesting that monitoring TRM dynamics could be clinically informative. Together, our results provide spatial and temporal insights into how human TRM develop, function, persist, and affect tissue integrity within the complexities of lung transplantation.