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The use of genetically engineered tools, including combinations of Cre-LoxP and Flp-FRT systems, enable the interrogation of complex biology. Steroidogenic factor-1 (SF-1) is expressed in the ventromedial hypothalamic nucleus (VMH). Development of genetic tools, such as mice expressing Flp recombinase (Flp) in SF-1 neurons (Sf-1-Flp), will be useful for future studies that unravel the complex physiology regulated by the VMH. Here, we developed and characterized Sf-1-Flp mice and demonstrated its utility. Flp sequence was inserted into Sf-1 locus with P2A. This insertion did not affect Sf-1 mRNA expression levels and Sf-1-Flp mice do not have any visible phenotypes. They are fertile and metabolically comparable to wild-type littermate mice. Optogenetic stimulation using adeno-associated virus (AAV)-bearing Flp-dependent channelrhodopsin-2 (ChR2) increased blood glucose and skeletal muscle PGC-1α in Sf-1-Flp mice. This was similar to SF-1 neuronal activation using Sf-1-BAC-Cre and AAV-bearing Cre-dependent ChR2. Finally, we generated Sf-1-Flp mice that lack β2-adrenergic receptors (Adrβ2) only in skeletal muscle with a combination of Cre/LoxP technology (Sf-1-Flp::SKM∆Adrβ2). Optogenetic stimulation of SF-1 neurons failed to increase skeletal muscle PGC-1α in Sf-1-Flp::SKM∆Adrβ2 mice, suggesting that Adrβ2 in skeletal muscle is required for augmented skeletal muscle PGC-1α by SF-1 neuronal activation. Our data demonstrate that Sf-1-Flp mice are useful for interrogating complex physiology.

Chlamydia trachomatis (CT) is the most common bacterial sexually transmitted infection globally. Understanding natural immunity to CT will inform vaccine design. This study aimed to profile immune cells and associated functional features in CT-infected women, and determine immune profiles associated with reduced risk of ascended endometrial CT infection and CT reinfection. PBMCs from CT-exposed women were profiled by mass cytometry and random forest models identified key features that distinguish outcomes. CT+ participants exhibited higher frequencies of CD4+ Th2, Th17, and Th17 DN CD4 T effector memory (TEM) cells than uninfected participants with decreased expression of T cell activation and differentiation markers. Minimal differences were detected between women with or without endometrial CT infection. Participants who remained follow-up negative (FU-) showed higher frequencies of CD4 T central memory (TCM) Th1, Th17, Th1/17, and Th17 DN but reduced CD4 TEM Th2 cells than FU+ participants. Expression of markers associated with central memory and Th17 lineage were increased on T cell subsets among FU- participants. These data indicate that peripheral T cells exhibit distinct features associated with resistance to CT reinfection. The highly plastic Th17 lineage appears to contribute to protection. Addressing these immune nuances could promote efficacy of CT vaccines.

While immune checkpoint inhibition (CPI) has reshaped cancer treatment, the majority of cancer patients do not benefit from this approach, which can also cause immune-related adverse events. Induction of IFNγ responses is thought be necessary for anti-tumor immunity, but growing evidence also implicates IFNγ as a tumor-intrinsic mediator of CPI resistance. CPI-induced IFNγ mediates activation-induced cell death in T cells as an immune-intrinsic mechanism of resistance. In this study, we show that transient block of IFNγ signaling through administration of the JAK1 inhibitor ABT-317 enhances anti-tumor T cell responses with CPI in pre-clinical models. Importantly, sequential but not concomitant ABT-317 treatment led to significantly reduced toxicity and improved tumor efficacy. Sequential treatment reduced activation-induced T cell death and enhanced expansion of tumor-reactive T cell subsets with increased effector function in vivo and ex vivo. Only CPI in combination with ABT-317 also enhanced memory responses by protecting mice from tumor rechallenge. These results demonstrate that JAK inhibition within a discrete time window following CPI addresses an immune-intrinsic mechanism of therapeutic resistance.

Type 1 diabetes (T1D) is precipitated by the autoimmune destruction of the insulin-producing β-cells in the pancreatic islets of Langerhans. Chemokines have been identified as major conductors of islet infiltration by autoaggressive leukocytes, including antigen-presenting cells and islet autoantigen-specific T cells. We have previously generated a roadmap of gene expression in the islet microenvironment during T1D in a mouse model and found that most of the chemokine axes are chronically upregulated during T1D. The XCL1/XCR1 chemokine axis is of particular interest, since XCR1 is exclusively expressed on conventional dendritic cells type 1 (cDC1) that excel by their high capacity for T cell activation. Here we demonstrate cDC1 expressing XCR1 are present in and around the islets of patients with T1D and of islet-autoantibody positive individuals. Further, we show that XCL1 plays an important role in the attraction of highly potent dendritic cells expressing XCR1 to the islets in an inducible mouse model for T1D. XCL1-deficient mice display a diminished infiltration of XCR1+ cDC1 and subsequently a reduced magnitude and activity of islet autoantigen-specific T cells resulting in a profound decrease in T1D incidence. Interference with the XCL1/XCR1 chemokine axis might constitute a novel therapy for T1D.

Metabolic dysfunction-associated steatotic liver disease (MASLD)-characterized by excess accumulation of fat in the liver-now affects one third of the world's population. As MASLD progresses, extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis, a major determinant of disease severity and mortality. To identify transcriptional regulators of fibrosis, we computationally inferred the activity of transcription factors (TFs) relevant to fibrosis by profiling the matched transcriptomes and epigenomes of 108 human liver biopsies from a deeply characterized cohort of patients spanning the full histopathologic spectrum of MASLD. CRISPR-based genetic knockout of the top 100 TFs identified ZNF469 as a regulator of collagen expression in primary human hepatic stellate cells (HSCs). Gain- and loss-of-function studies established that ZNF469 regulates collagen genes and genes involved in matrix homeostasis through direct binding to gene bodies and regulatory elements. By integrating multiomic large-scale profiling of human biopsies with extensive experimental validation we demonstrate that ZNF469 is a transcriptional regulator of collagen in HSCs. Overall, these data nominate ZNF469 as a previously unrecognized determinant of MASLD-associated liver fibrosis.

The role of mesenchymal cells during respiratory infection is not well defined, including whether, which, and how the different types of mesenchymal cells respond. We collected all mesenchymal cells from lung single-cell suspensions of mice that were naïve (after receiving only saline vehicle), pneumonic (after intratracheal instillation of pneumococcus 24 hours previously), or resolved from infection (after non-lethal pneumococcal infections 6 weeks previously) and performed single-cell RNA sequencing. Cells clustered into five well-separated groups based on their transcriptomes: matrix fibroblasts, myofibroblasts, pericytes, smooth muscle cells, and mesothelial cells. Fibroblasts were the most abundant and could be further segregated into Pdgfra+Npnt+Ces1d+Col13a1+ alveolar fibroblasts and Cd9+Pi16+Sca1+Col14a1+ adventitial fibroblasts. The cells from naïve and resolved groups overlapped in dimension reduction plots, suggesting the mesenchymal cells returned to baseline transcriptomes after resolution. During pneumonia, all mesenchymal cells responded with altered transcriptomes, revealing a core response that had been conserved across cell types as well as distinct mesenchymal cell type-specific responses. The different subsets of fibroblasts induced similar gene sets, but the alveolar fibroblasts responded more strongly than the adventitial fibroblasts. These data demonstrated diverse and specialized immune activities of lung mesenchymal cells during pneumonia.

The functional plasticity of tumor-infiltrating B (TIL-B) cells spans from anti-tumor responses to non-canonical immune suppression. Yet, how tumor microenvironment (TME) influences TIL-B development is still underappreciated. Our current study integrated single cell transcriptomics and BCR (B cell receptor) sequencing to profile TIL-B phenotypes and clonalities in hepatocellular carcinoma (HCC). Using trajectory and gene regulatory network analysis, we were able to characterize plasma cells, memory and naïve B cells within the HCC TME and further revealed a downregulation of BCR-signaling genes in plasma cells and a subset of inflammatory TNF+ memory B cells. Within the TME, non-switch memory B cell subset acquires an age-associated B cell phenotype (TBET+, CD11c+) and expressed higher levels of PD-L1, CD25 and granzyme B. We further demonstrated that the presence of HCC tumor cells could confer suppressive functions on peripheral blood B cells which in turn, dampen T cell co-stimulation. To the best of our knowledge, these findings represent novel mechanisms of non-canonical immune suppression in HCC. While previous studies identified atypical memory B cells in chronic hepatitis and across several solid cancer types, we further highlighted their potential role as regulatory B cells (Bregs) within both the TME and peripheral blood of HCC patients.

Lower urinary tract symptoms (LUTS) affect approximately 50% of the population over 40 years of age and are strongly associated with obesity and metabolic syndrome. Adipose tissue plays a key role in obesity/metabolic syndrome by releasing adipokines that regulate systemic energy/lipid metabolism, insulin resistance, and inflammation. Adiponectin (ADPN), the most abundant adipokine, modulates energy/metabolism homeostasis through its insulin-sensitizing and antiinflammatory effects. Human plasma ADPN levels are inversely associated with obesity and diabetes. To the best of our knowledge, the role of adipokines such as ADPN in the LUTS associated with obesity/metabolic syndrome remains unknown. We have tested such a possible role in a global ADPN-knockout (Adpn-/-) mouse model. Adpn-/- mice exhibited increased voiding frequency, small voids, and reduced bladder smooth muscle (BSM) contractility, with absence of purinergic contraction. Molecular examination indicated significantly altered metabolic and purinergic pathways. The ADPN receptor agonist AdipoRon was found to abolish acute BSM contraction. Intriguingly, both AMPK activators and inhibitors also abolished BSM purinergic contraction. These data indicate the important contribution of what we believe is a novel ADPN signaling pathway to the regulation of BSM contractility. Dysregulation of this ADPN signaling pathway might be an important mechanism leading to LUTS associated with obesity/metabolic syndrome.

Access to the brain for treating neurological sequalae requires a craniotomy, which can be complicated by infection. Staphylococcus aureus accounts for half of craniotomy infections, increasing morbidity in a medically fragile patient population. T cells preferentially traffic to the brain during craniotomy infection; however, their functional importance is unknown. Using a mouse model of S. aureus craniotomy infection, CD4+ T cells were critical for bacterial containment, as treatment of WT animals with anti-CD4 exacerbated infection that was similar to phenotypes in Rag1-/- mice. Single-cell RNA-Seq (scRNA-Seq) revealed transcriptional heterogeneity in brain CD3+ infiltrates, with CD4+ cells most prominent that displayed Th1- and Th17-like characteristics, and adoptive transfer of either subset in Rag1-/- animals during early infection prevented S. aureus outgrowth. scRNA-Seq identified a robust IFN signature in several innate immune clusters, and examination of cell-to-cell interactions revealed extensive T cell crosstalk with monocytes/macrophages that was also observed in human craniotomy infection. A cooperative role for Th1 and Th17 responses was demonstrated by treatment of Ifng-/- mice with IL-17A neutralizing antibody that recapitulated phenotypes in Rag1-/- animals. Collectively, these findings implicate Th1- and Th17-mediated proinflammatory responses in shaping the innate immune landscape for S. aureus containment during craniotomy infection.

Chronic lung allograft dysfunction (CLAD) substantially limits long-term survival following lung transplantation. To identify potential targets for CLAD prevention, T cells from explanted CLAD lungs and lung-draining lymph nodes, as well as diseased and nondiseased controls were isolated and single-cell RNA sequencing and TCR sequencing were performed. TCR sequencing revealed a clonally expanded population of CD8+ tissue-resident memory T cells (TRMs) with high cytotoxic potential, including upregulation of KLRK1, encoding the co-receptor NKG2D. These cytotoxic CD8+ TRMs accumulated around the CLAD airways and had a 100-fold increase in clonal overlap with lung-draining lymph nodes when compared with non-CLAD lungs. Using a murine model of orthotopic lung transplantation, we confirmed that cytotoxic CD8+ TRM accumulation was due to chronic rejection and not transplantation alone. Furthermore, blocking NKG2D in vivo attenuated the airway remodeling following transplantation and diminished airway accumulation of CD8+ T cells. Our findings support NKG2D as a potential therapeutic target for CLAD, affecting cytotoxic CD8+ TRM accumulation.