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Resident memory T cells (TRMs) play a vital role in regional immune defense. Although laboratory rodents have been extensively used to study fundamental TRM biology, poor isolation efficiency and low cell survival rates have limited the implementation of TRM-focused high-throughput assays. Here, we engineer a murine vaginal epithelial organoid (VEO)-CD8 T cell co-culture system that supports CD8 TRM differentiation. These in - vitro -generated TRMs are phenotypically and transcriptionally similar to in vivo TRMs. Pharmacological and genetic approaches showed that transforming growth factor β (TGF-β) signaling plays a crucial role in their differentiation. The VEOs in our model are susceptible to viral infections and the CD8 T cells are amenable to genetic manipulation, both of which will allow a detailed interrogation of antiviral CD8 T cell biology. Altogether we have established a robust in vitro TRM differentiation system that is scalable and can be subjected to high-throughput assays that will rapidly add to our understanding of TRMs. [Display omitted] • Vaginal epithelial stem cells form organoids mimicking in vivo vaginal epithelium • CD8 T cells co-cultured with organoids differentiate into resident memory CD8 T cells • In vitro CD8 TRMs phenotypically and transcriptionally resemble in vivo epithelial TRMs • VEO-CD8 co-culture model can be interrogated to reveal fundamental TRM biology Technical issues with resident memory T cell (TRM) isolation and survival have hindered detailed inquiries into TRM biology. Ulibarri et al. establish a vaginal epithelial organoid (VEO)-CD8 T cell co-culture system that enables epithelial TRM differentiation in vitro. This co-culture model will expand fundamental and translational mucosal TRM research. [ABSTRACT FROM AUTHOR]

Tissue-resident memory CD8 + T cells (T RM ) are localized in non-lymphoid tissues throughout the body where they mediate long-lived protective immunity at common sites of pathogen exposure. As the signals controlling T RM differentiation are uncovered, it is becoming apparent that the dynamic activities of numerous transcription factors are intricately involved in T RM formation. Here, we highlight known transcriptional regulators of T RM differentiation and discuss how understanding the transcriptional programming of CD8 + T cell residency in non-lymphoid tissues can be leveraged to prevent or treat disease. [ABSTRACT FROM AUTHOR]

Objective A novel approach to regulate obesity-associated adipose inflammation may be through metabolic reprogramming of macrophages (MΦs). Broadly speaking, MΦs dependent on glucose are pro-inflammatory, classically activated MΦs (CAM), which contribute to adipose inflammation and insulin resistance. In contrast, MΦs that primarily metabolize fatty acids are alternatively activated MΦs (AAM) and maintain tissue insulin sensitivity. In actuality, there is much flexibility and overlap in the CAM-AAM spectrum in vivo dependent upon various stimuli in the microenvironment. We hypothesized that specific lipid trafficking proteins, e.g. fatty acid transport protein 1 (FATP1), would direct MΦ fatty acid transport and metabolism to limit inflammation and contribute to the maintenance of adipose tissue homeostasis. Methods Bone marrow derived MΦs (BMDMs) from Fatp1 −/− and Fatp1 +/+ mice were used to investigate FATP1-dependent substrate metabolism, bioenergetics, metabolomics, and inflammatory responses. We also generated C57BL/6J chimeric mice by bone marrow transplant specifically lacking hematopoetic FATP1 ( Fatp1 B −/− ) and controls Fatp1 B +/+ . Mice were challenged by high fat diet (HFD) or low fat diet (LFD) and analyses including MRI, glucose and insulin tolerance tests, flow cytometric, histologic, and protein quantification assays were conducted. Finally, an FATP1-overexpressing RAW 264.7 MΦ cell line (FATP1-OE) and empty vector control (FATP1-EV) were developed as a gain of function model to test effects on substrate metabolism, bioenergetics, metabolomics, and inflammatory responses. Results Fatp1 is downregulated with pro-inflammatory stimulation of MΦs. Fatp1 −/− BMDMs and FATP1-OE RAW 264.7 MΦs demonstrated that FATP1 reciprocally controled metabolic flexibility, i.e. lipid and glucose metabolism, which was associated with inflammatory response. Supporting our previous work demonstrating the positive relationship between glucose metabolism and inflammation, loss of FATP1 enhanced glucose metabolism and exaggerated the pro-inflammatory CAM phenotype. Fatp1 B−/− chimeras fed a HFD gained more epididymal white adipose mass, which was inflamed and oxidatively stressed, compared to HFD-fed Fatp1 B+/+ controls. Adipose tissue macrophages displayed a CAM-like phenotype in the absence of Fatp1 . Conversely, functional overexpression of FATP1 decreased many aspects of glucose metabolism and diminished CAM-stimulated inflammation in vitro . FATP1 displayed acyl-CoA synthetase activity for long chain fatty acids in MΦs and modulated lipid mediator metabolism in MΦs. Conclusion Our findings provide evidence that FATP1 is a novel regulator of MΦ activation through control of substrate metabolism. Absence of FATP1 exacerbated pro-inflammatory activation in vitro and increased local and systemic components of the metabolic syndrome in HFD-fed Fatp1 B−/− mice. In contrast, gain of FATP1 activity in MΦs suggested that Fatp1 -mediated activation of fatty acids, substrate switch to glucose, oxidative stress, and lipid mediator synthesis are potential mechanisms. We demonstrate for the first time that FATP1 provides a unique mechanism by which the inflammatory tone of adipose and systemic metabolism may be regulated. [ABSTRACT FROM AUTHOR]

Glucose is a critical component in the proinflammatory response of macrophages (MΦs). However, the contribution of glucose transporters (GLUTs) and the mechanisms regulating subsequent glucose metabolism in the inflammatory response are not well understood. Because MΦs contribute to obesity-induced inflammation, it is important to understand how substrate metabolism may alter inflammatory function. We report that GLUT1 (SLC2A1) is the primary rate-limiting glucose transporter on proinflammatory-polarized MΦs. Furthermore, in high fat diet-fed rodents, MΦs in crown-like structures and inflammatory loci in adipose and liver, respectively, stain positively for GLUT1. We hypothesized that metabolic reprogramming via increased glucose availability could modulate the MΦs inflammatory response. To increase glucose uptake, we stably overexpressed the GLUT1 transporter in RAW264.7 MΦs (GLUT1-OE MΦs). Cellular bioenergetics analysis, metabolomics, and radiotracer studies demonstrated that GLUT1 overexpression resulted in elevated glucose uptake and metabolism, increased pentose phosphate pathway intermediates, with a complimentary reduction in cellular oxygen consumption rates. Gene expression and proteome profiling analysis revealed that GLUT1-OE MΦs demonstrated a hyperinflammatory state characterized by elevated secretion of inflammatory mediators and that this effect could be blunted by pharmacologic inhibition of glycolysis. Finally, reactive oxygen species production and evidence of oxidative stress were significantly enhanced in GLUT1-OE MΦs; antioxidant treatment blunted the expression of inflammatory mediators such as PAI-1 (plasminogen activator inhibitor 1), suggesting that glucose-mediated oxidative stress was driving the proinflammatory response. Our results indicate that increased utilization of glucose induced a ROS-driven proinflammatory phenotype in MΦs, which may play an integral role in the promotion of obesity-associated insulin resistance. [ABSTRACT FROM AUTHOR]