Voriconazole-induced periostitis is a rare adverse effect in patients on long-term therapy, characterised by periosteal inflammation and associated bony pain. The accompanying lab abnormalities (elevated serum alkaline phosphatase and fluoride) and characteristic imaging findings (uptake of radionuclide tracer on nuclear bone scan) are critical for diagnosis. The disease process is thought to be secondary to excess fluoride from voriconazole which stimulates bone formation and decreases osteoclast bone resorption. Management includes stopping voriconazole and switching to another agent.

After resolution of infection, T cells differentiate into long-lived memory cells that recirculate through secondary lymphoid organs or establish residence in tissues. In contrast to CD8 + tissue-resident memory T cells (T RM ), the developmental origins and transcriptional regulation of CD4 + T RM remain largely undefined. Here, we investigated the phenotypic, functional, and transcriptional profiles of CD4 + T RM in the small intestine (SI) responding to acute viral infection, revealing a shared gene expression program and chromatin accessibility profile with circulating T H 1 and the progressive acquisition of a mature T RM program. Single-cell RNA sequencing identified heterogeneity among established CD4 + T RM , which were predominantly located in the lamina propria, and revealed a population of cells that coexpressed both effector- and memory-associated genes, including the transcriptional regulators Blimp1, Id2, and Bcl6. T H 1-associated Blimp1 and Id2 and T FH -associated Bcl6 were required for early T RM formation and development of a mature T RM population in the SI. These results demonstrate a developmental relationship between T H 1 effector cells and the establishment of early T RM , as well as highlighted differences in CD4 + versus CD8 + T RM populations, providing insights into the mechanisms underlying the origins, differentiation, and persistence of CD4 + T RM in response to viral infection.

Memory CD4 + T cells play a pivotal role in mediating long-term protective immunity, positioning them as an important target in vaccine development. However, multiple functionally distinct helper CD4 + T-cell subsets can arise in response to a single invading pathogen, complicating the identification of rare populations of memory precursor cells during the effector phase of infection and memory CD4 + T cells following pathogen clearance and the contraction phase of infection. Furthermore, current literature remains unclear regarding whether a single CD4 + memory T-cell lineage gives rise to secondary CD4 + T helper subsets or if there are unique memory precursor cells within each helper lineage. A majority of T follicular helper (Tfh) cells, which have established memory potential, express Id3, an inhibitor of E protein transcription factors, following acute viral infection. We show that expression of Id3 definitively identified a subset of cells within both the CD4 + Tfh and T helper 1 (Th1) lineages at memory time points that exhibited memory potential, with the capacity for significant re-expansion in response to secondary infection. Notably, we demonstrate that a subset of Th1 cells that survive into the memory phase were marked by Id3 expression and possessed the potential for enhanced expansion and generation of both Th1 and Tfh secondary effector cell populations in a secondary response to pathogen. Additionally, these cells exhibited enrichment of key molecules associated with memory potential when compared with Id3 lo Th1 cells. Therefore, we propose that Id3 expression serves as an important marker to indicate multipotent potential in memory CD4 + T cells.

Tissue macrophages self-renew during homeostasis and produce inflammatory mediators upon microbial infection. We examined the relationship between proliferative and inflammatory properties of tissue macrophages by defining the impact of the Wnt/β-catenin pathway, a central regulator of self-renewal, in alveolar macrophages (AMs). Activation of β-catenin by Wnt ligand inhibited AM proliferation and stemness, but promoted inflammatory activity. In a murine influenza viral pneumonia model, β-catenin-mediated AM inflammatory activity promoted acute host morbidity; in contrast, AM proliferation enabled repopulation of reparative AMs and tissue recovery following viral clearance. Mechanistically, Wnt treatment promoted β-catenin-HIF-1α interaction and glycolysis-dependent inflammation while suppressing mitochondrial metabolism and thereby, AM proliferation. Differential HIF-1α activities distinguished proliferative and inflammatory AMs in vivo. This β-catenin-HIF-1α axis was conserved in human AMs and enhanced HIF-1α expression associated with macrophage inflammation in COVID-19 patients. Thus, inflammatory and reparative activities of lung macrophages are regulated by β-catenin-HIF-1α signaling, with implications for the treatment of severe respiratory diseases.

Unremitting defense against diverse pathogens and malignancies requires a dynamic and durable immune response. Tissue-resident memory CD8 + T cells (T rm ) afford robust protection against infection and cancer progression through continuous surveillance of non-lymphoid tissues. Here, we provide insight into how T rm confer potent and persistent immunity through partitioning of distinct cellular subsets differing in longevity, effector function, and multipotency. Antigen-specific CD8 + T cells localized to the epithelium of the small intestine are primarily comprised of a shorter-lived effector population most prominent early following both acute viral and bacterial infections, and a longer-lived Id3 hi T rm population that subsequently accumulates at later memory timepoints. We define regulatory gene-programs driving these distinct T rm states, and further clarify roles for Blimp1, T-bet, Id2, and Id3 in supporting and maintaining intestinal T rm heterogeneity during infection. Further, through single-cell RNAseq analysis we demonstrate that tumor-infiltrating lymphocytes broadly differentiate into discrete populations of short-lived and long-lived T rm -like subsets, which share qualities with terminally-exhausted and progenitor-exhausted cells, respectively. As the clinical relevance of T rm continues to widen from acute infections to settings of chronic inflammation and malignancy, clarification of the spectrum of phenotypic and functional states exhibited by CD8 + T cells that reside in non-lymphoid tissues will provide a framework for understanding their regulation and identity in diverse pathophysiological contexts.

Tissue-resident memory CD8+ T cells (Trm) provide host protection through continuous surveillance of non-lymphoid tissues. Using single-cell RNA-sequencing (scRNA-seq) and genetic reporter mice, we identified discrete lineages of intestinal antigen-specific CD8+ T cells, including a Blimp1hiId3lo tissue-resident effector cell population most prominent in the early phase of acute viral and bacterial infections and a molecularly distinct Blimp1loId3hi tissue-resident memory population that subsequently accumulated at later infection time points. These Trm populations exhibited distinct cytokine production, secondary memory potential, and transcriptional programs including differential roles for transcriptional regulators Blimp1, T-bet, Id2, and Id3 in supporting and maintaining intestinal Trm. Extending our analysis to malignant tissue, we also identified discrete populations of effector-like and memory-like CD8+ T cell populations with tissue-resident gene-expression signatures that shared features of terminally exhausted and progenitor-exhausted T cells, respectively. Our findings provide insight into the development and functional heterogeneity of Trm cells, which has implications for enhancing vaccination and immunotherapy approaches.

Fetal-derived tissue-resident macrophages exhibit stem cell-like features of self-renewal in adulthood to maintain macrophage population during homeostasis and/or various insults. However, little is known about the cellular and molecular mechanisms modulating proliferative and inflammatory fate decisions of tissue-resident macrophages in vivo. Here, we show that WNT-β-catenin signaling inhibited lung-resident alveolar macrophage (AM) self-renewal, while simultaneously promoted AM inflammatory activities in vitro and in vivo during influenza virus infection. Mechanistically, WNT engagement facilitated the binding of β-catenin with HIF-1α over its conventional binding partner TCF-4. Such a binding choice led to the elevated macrophage inflammation in a glycolysis-dependent manner, while inhibited AM self-renewal by causing mitochondrial damage and impairing oxidative phosphorylation. Thus, AM self-renewal and inflammatory activity are uncoupled by WNT-β-catenin signaling through HIF-1α-mediated cellular metabolic choice. Importantly, we showed that AM with high HIF-1α activity had limited proliferative capacity and produced inflammatory cytokines, while AM with low HIF-1α activity were highly proliferative and expressed genes associated with tissue repair function in vivo during influenza virus infection. In accordance, we demonstrated that AM proliferation and repopulation were needed for optimal lung repair following the clearance of influenza virus in the respiratory tract. Our results have revealed key mechanisms modulating macrophage fate choice between progeny production versus inflammatory effector activity, and subsequent effects on tissue inflammation and repair.

Generation of T-cell memory is crucial in conferring vaccine-induced immunity, particularly against pathogens where neutralizing antibodies alone are insufficient at providing long-term protection. While great advances have been made in understanding the generation and maintenance of memory CD8+ T cells and B cells, mechanisms underlying the generation of memory CD4+ T cells have remained relatively elusive. This limitation is in part due to the multi-potency and lineage plasticity exhibited by CD4+ T helper (TH) cells. Using the LCMV viral infection model, we show that the T follicular helper (TFH) subset are the predominant CD4+ memory T cell type based on its relative abundance following pathogen elimination as well as its multi-potent potential upon antigen re-challenge. This multi-potency of TFH memory cells during secondary challenge has also been observed in influenza infection and acute bacterial infection, suggesting that the TFH memory subset is most capable of providing a comprehensive and robust secondary response. From advancements in CD8+ memory T cell studies, it has become clear that transcription factors (TF) serve as crucial arbiters for the cell-fate decisions between short-lived effector and memory. To investigate the transcriptional mechanisms underlying TFH formation, we employed a novel bioinformatics analysis utilizing the PageRank algorithm that combines both RNA-seq and ATAC-seq and have identified putative transcriptional regulators of memory TFH differentiation. Further understanding of TFH memory formation will undoubtedly unveil new insights into CD4+ T cell memory in hopes of improving vaccine-based immunity.

In response to infection, naive CD4+ T-cells proliferate and differentiate into several possible effector subsets, including conventional T helper effector cells (TH 1, TH 2, TH 17), T regulatory cells (Treg ) and T follicular helper cells (TFH ). Once infection is cleared, a small population of long-lived memory cells remains that mediate immune defenses against reinfection. Memory T lymphocytes have classically been categorized into central memory cell (TCM ) and effector memory cell (TEM ) subsets, both of which circulate between blood, secondary lymphoid organs and in some cases non-lymphoid tissues. A third subset of memory cells, referred to as tissue-resident memory cells (TRM ), resides in tissues without recirculation, serving as 'first line' of defense at barrier sites, such as skin, lung and intestinal mucosa, and augmenting innate immunity in the earliest phases of reinfection and recruiting circulating CD4+ and CD8+ T-cells. The presence of multiple CD4+ T helper subsets has complicated studies of CD4+ memory T-cell differentiation, and the mediators required to support their function. In this review, we summarize recent investigations into the origins of CD4+ memory T-cell populations and discuss studies addressing CD4+  TRM differentiation in barrier tissues.

In response to acute infection, naive T cells proliferate and differentiate into effector cells, which aid in clearing pathogens, and long-lived memory cells, which provide protection from reinfection. CD4+ T cells play a key role in this response because they mediate the activity of innate cell, B cell, and CD8+ T cell immunity. Memory T cells have classically been categorized into central memory cells (TCM), which circulate between blood and secondary lymphoid organs, or effector memory cells (TEM), which can enter non-lymphoid tissues. Recently, evidence for a third subset of memory cells has emerged, called tissue-resident memory T cells (TRM), which populate barrier surfaces such as skin and intestinal mucosa. TRM serve as sentinels at the sites of pathogen exposure, coordinating the initial response and providing a significant boost to immunity. The ontogeny and regulation of CD8+ TRM differentiation have been characterized in recent years, but less is known about CD4+TRM within the context of antiviral immunity. Using bulk and single-cell RNA-seq and ATAC-seq, we characterize the transcriptional and epigenetic profiles of viral-specific CD4+ T cells in the small intestine. We demonstrate that antigen-specific CD4+ TRM have a transcriptional profile distinct from circulating populations. CD4+ TRM can also be distinguished from circulating memory CD4+ T cells by expression of CD69, Ly6C, and CD27 cell-surface receptors. Additionally, we examine the role of transcriptional regulators, including Bcl6, Prdm1, and Id2/Id3, in CD4+ TRM development. A better understanding of CD4+ TRM will allow us to harness the protective capacity of this memory population and modulate activity in infection or inflammatory diseases.

498 Objectives: The lesion-to-cerebellum uptake ratio (L/C) appears to be a valuable semiquantitative index for the evaluation of malignancy in pulmonary nodules with 18F-FDG PET, since it does not require blood sampling, accurate dose and body weight measurements. Furthermore, L/C demonstrates a significantly higher sensitivity than the standardized uptake value (SUV) for nodules that are faintly positive on visual estimation. However, it is not known if the L/C is superior to the lesion-to-organ uptake ratios (L/O) of other organs, which similarly would not require accurate dose and body weight measurements for the evaluation of lung nodules. Therefore, this study compares the receiver operating characteristic (ROC) curves of the L/C and L/O for several organs, including the lungs, liver, spleen and bone marrow, for differentiating malignant from benign lung nodules. Methods: 40 patients underwent whole body PET imaging after an IV injection of 543 ± 69 MBq (14.7 ± 1.9 mCi) of 18F-FDG. Regions of interest (ROIs) were overlaid onto each attenuation corrected axial image in the areas of radiographically known lung densities, and mean activities were obtained. Mean activities were also acquired for ROIs of the cerebellar cortex, lungs, spleen, bone marrow in the lumbar spine and for the liver in the mid-section of each organ. The L/C and the L/O were calculated for each nodule and ROC curves were analyzed using the ROCKIT 0.9B software package. Results: Surgical pathology and serial CT scans (follow-up > 24 months) revealed 21 malignant and 28 benign lung lesions less than 3 cm in size. The mean L/C was 0.73 ± 0.40 for malignant nodules and 0.22 ± 0.11 for benign nodules (P Conclusions: The L/C method demonstrated significantly more favorable ROC characteristics than the L/O-marrow method for evaluation of malignancy in pulmonary nodules. Furthermore, since the Az for L/C data was greater than the Az for L/O-spleen, L/O-liver and L/O-lung data, the L/C method may also demonstrate more favorable ROC characteristics than the L/O method for these organs. However, since the data for these organs did not show a statistically significant difference between the L/C method and the L/O method, studies with a larger patient population are needed to confirm these preliminary results.