A Microdissection Protocol for Proteogenomic Analysis of Histological Sections to Advance Drug Development.

Combining proteogenomics with laser capture microdissection (LCM) in cancer research offers a targeted way to explore the intricate interactions between tumor cells and the different microenvironment components. This is especially important for immuno-oncology (IO) research where improvements in the predictability of IO-based drugs are sorely needed, and depends on a better understanding of the spatial relationships involving the tumor, blood supply, and immune cell interactions, in the context of their associated microenvironments. LCM is used to isolate and obtain distinct histological cell types, which may be routinely performed on complex and heterogeneous solid tumor specimens. Once cells have been captured, nucleic acids and proteins may be extracted for in-depth multimodality molecular profiling assays. Optimizing the minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, RNA-seq may be performed for gene expression and DNA sequencing performed for the discovery and analysis of actionable mutations, copy number variation, methylation profiles, etc. However, there remains a need for highly sensitive proteomic methods targeting small-sized samples. A significant part of this protocol is an enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. Included is a detailed in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Described is a proteogenomic approach leveraged from microdissected fresh frozen tissue. The protocols may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume.

Nucleosome-binding by TP53, TP63, and TP73 is determined by the composition, accessibility, and helical orientation of their binding sites.

The p53 family of transcription factors plays key roles in driving development and combating cancer by regulating gene expression. TP53, TP63, and TP73-the three members of the p53 family-regulate gene expression by binding to their DNA binding sites, many of which are situated within nucleosomes. To thoroughly examine the nucleosome-binding abilities of the p53 family, we used Pioneer-seq, a technique that assesses a transcription factor's binding affinity to its DNA binding sites at all possible positions within the nucleosome core particle. Using Pioneer-seq, we analyzed the binding affinity of TP53, TP63, and TP73 to 10 p53-family binding sites across the nucleosome core particle. We found that the affinity of TP53, TP63, and TP73 for nucleosomes was largely determined by the positioning of p53-family binding sites within nucleosomes; p53-family members bind strongly to the more accessible edges of nucleosomes but weakly to the less accessible centers of nucleosomes. We also found that the DNA-helical orientation of p53-family binding sites within nucleosomal DNA impacted the nucleosome-binding affinity of p53-family members. The composition of their binding sites also impacted each p53-family member's nucleosome-binding affinities only when the binding site was located in an accessible location. Taken together, our results show that the accessibility, composition, and helical orientation of p53-family binding sites collectively determine the nucleosome-binding affinities of TP53, TP63, and TP73. These findings help explain the rules underlying p53-family-nucleosome binding and thus provide requisite insight into how we may better control gene-expression changes involved in development and tumor suppression.

Vitamin D regulates microbiome-dependent cancer immunity.

A role for vitamin D in immune modulation and in cancer has been suggested. In this work, we report that mice with increased availability of vitamin D display greater immune-dependent resistance to transplantable cancers and augmented responses to checkpoint blockade immunotherapies. Similarly, in humans, vitamin D-induced genes correlate with improved responses to immune checkpoint inhibitor treatment as well as with immunity to cancer and increased overall survival. In mice, resistance is attributable to the activity of vitamin D on intestinal epithelial cells, which alters microbiome composition in favor of Bacteroides fragilis, which positively regulates cancer immunity. Our findings indicate a previously unappreciated connection between vitamin D, microbial commensal communities, and immune responses to cancer. Collectively, they highlight vitamin D levels as a potential determinant of cancer immunity and immunotherapy success.

Defining Porphyromonas gingivalis strains associated with periodontal disease.

Porphyromonas gingivalis, a Gram-negative anaerobic bacterium commonly found in human subgingival plaque, is a major etiologic agent for periodontitis and has been associated with multiple systemic pathologies. Many P. gingivalis strains have been identified and different strains possess different virulence factors. Current oral microbiome approaches (16S or shotgun) have been unable to differentiate P. gingivalis strains. This study presents a new approach that aims to improve the accuracy of strain identification, using a detection method based on sequencing of the intergenic spacer region (ISR) which is variable between P. gingivalis strains. Our approach uses two-step PCR to amplify only the P. gingivalis ISR region. Samples are then sequenced with an Illumina sequencer and mapped to specific strains. Our approach was validated by examining subgingival plaque from 153 participants with and without periodontal disease. We identified the avirulent strain ATCC33277/381 as the most abundant strain across all sample types. The W83/W50 strain was significantly enriched in periodontitis, with 13% of participants harboring that strain. Overall, this approach can have significant implications not only for the diagnosis and treatment of periodontal disease but also for other diseases where P. gingivalis or its toxins have been implicated, such as Alzheimer's disease.

Alcohol Consumption and the Diversity of the Oral Microbiome in Postmenopausal Women.

BACKGROUND: Alcohol reduces neutrophil function and decreases salivary flow, which could affect the composition of the oral microbiome. OBJECTIVE: We hypothesized that the α- and ß-diversity of the oral microbiome and the relative abundance of bacterial taxa would differ by frequency and type of alcohol consumption. METHODS: We used a food frequency questionnaire to assess the frequency of consumption of beer, wine, and liquor (drinks/week) in a sample of 1179 postmenopausal women in the Osteoporosis and Periodontal Disease Study. Women were categorized as nondrinkers, drinking <1 drink/wk, ≥1 to <7 drinks/wk, or ≥7 drinks/wk for total alcohol consumption and for beer, wine, and liquor consumption. The composition and diversity of the oral microbiome was assessed from subgingival plaque samples using 16S ribosomal RNA amplicon sequencing. Permutational multivariate analysis of variance (PERMANOVA) was used to examine ß-diversity (between-sample diversity) in the microbiome between alcohol consumption categories. Analysis of covariance was used to examine the mean α-diversity (within-sample diversity), assessed by the Shannon index (species evenness), Chao1 index (species richness), and observed operational taxonomic unit (OTU) count and the mean relative abundance of 245 bacterial taxa across alcohol consumption categories. RESULTS: Over half of the participants (67%) consumed alcohol, with 14% reporting ≥1 drink/d. The ß-diversity across categories of total alcohol consumption, but not categories of alcohol type, was statistically significantly different (P for PERMANOVA = 0.016). Mean α-diversity measures were statistically significantly higher (P < 0.05) in the highest category of total alcohol and wine consumption compared to nondrinkers; no significant associations were found for beer or liquor consumption. The relative abundance of 1 OTU, Selenomonassp._oral_taxon_133, was significantly lower in the highest level of total alcohol consumption compared to nondrinkers after adjustment for multiple comparisons. CONCLUSIONS: Alcohol consumption was associated with the diversity and composition of the subgingival microbiome.

SYK ubiquitination by CBL E3 ligases restrains cross-presentation of dead cell-associated antigens by type 1 dendritic cells.

Cross-presentation of dead cell-associated antigens by conventional dendritic cells type 1 (cDC1s) is critical for CD8+ T cells response against many tumors and viral infections. It is facilitated by DNGR-1 (CLEC9A), an SYK-coupled cDC1 receptor that detects dead cell debris. Here, we report that DNGR-1 engagement leads to rapid activation of CBL and CBL-B E3 ligases to cause K63-linked ubiquitination of SYK and terminate signaling. Genetic deletion of CBL E3 ligases or charge-conserved mutation of target lysines within SYK abolishes SYK ubiquitination and results in enhanced DNGR-1-dependent antigen cross-presentation. We also find that cDC1 deficient in CBL E3 ligases are more efficient at cross-priming CD8+ T cells to dead cell-associated antigens and promoting host resistance to tumors. Our findings reveal a role for CBL-dependent ubiquitination in limiting cross-presentation of dead cell-associated antigens and highlight an axis of negative regulation of cDC1 activity that could be exploited to increase anti-tumor immunity.

Lola-I is a promoter pioneer factor that establishes de novo Pol II pausing during development.

While the accessibility of enhancers is dynamically regulated during development, promoters tend to be constitutively accessible and poised for activation by paused Pol II. By studying Lola-I, a Drosophila zinc finger transcription factor, we show here that the promoter state can also be subject to developmental regulation independently of gene activation. Lola-I is ubiquitously expressed at the end of embryogenesis and causes its target promoters to become accessible and acquire paused Pol II throughout the embryo. This promoter transition is required but not sufficient for tissue-specific target gene activation. Lola-I mediates this function by depleting promoter nucleosomes, similar to the action of pioneer factors at enhancers. These results uncover a level of regulation for promoters that is normally found at enhancers and reveal a mechanism for the de novo establishment of paused Pol II at promoters.

An integrated genomic approach identifies follistatin as a target of the p63-epidermal growth factor receptor oncogenic network in head and neck squamous cell carcinoma.

Although numerous putative oncogenes have been associated with the etiology of head and neck squamous cell carcinoma (HNSCC), the mechanisms by which these oncogenes and their downstream targets mediate tumor progression have not been fully elucidated. We performed an integrative analysis to identify a crucial set of targets of the oncogenic transcription factor p63 that are common across multiple transcriptomic datasets obtained from HNSCC patients, and representative cell line models. Notably, our analysis revealed FST which encodes follistatin, a secreted glycoprotein that inhibits the transforming growth factor TGFß/activin signaling pathways, to be a direct transcriptional target of p63. In addition, we found that FST expression is also driven by epidermal growth factor receptor EGFR signaling, thus mediating a functional link between the TGF-ß and EGFR pathways. We show through loss- and gain-of-function studies that FST predominantly imparts a tumor-growth and migratory phenotype in HNSCC cells. Furthermore, analysis of single-cell RNA sequencing data from HNSCC patients unveiled cancer cells as the dominant source of FST within the tumor microenvironment and exposed a correlation between the expression of FST and its regulators with immune infiltrates. We propose FST as a prognostic biomarker for patient survival and a compelling candidate mediating the broad effects of p63 on the tumor and its associated microenvironment.

The dietary sweetener sucralose is a negative modulator of T cell-mediated responses.

Artificial sweeteners are used as calorie-free sugar substitutes in many food products and their consumption has increased substantially over the past years1. Although generally regarded as safe, some concerns have been raised about the long-term safety of the consumption of certain sweeteners2-5. In this study, we show that the intake of high doses of sucralose in mice results in immunomodulatory effects by limiting T cell proliferation and T cell differentiation. Mechanistically, sucralose affects the membrane order of T cells, accompanied by a reduced efficiency of T cell receptor signalling and intracellular calcium mobilization. Mice given sucralose show decreased CD8+ T cell antigen-specific responses in subcutaneous cancer models and bacterial infection models, and reduced T cell function in models of T cell-mediated autoimmunity. Overall, these findings suggest that a high intake of sucralose can dampen T cell-mediated responses, an effect that could be used in therapy to mitigate T cell-dependent autoimmune disorders.

The impact of nucleosome structure on CRISPR/Cas9 fidelity.

The clustered regularly interspaced short palindromic repeats (CRISPR) Cas system is a powerful tool that has the potential to become a therapeutic gene editor in the near future. Cas9 is the best studied CRISPR system and has been shown to have problems that restrict its use in therapeutic applications. Chromatin structure is a known impactor of Cas9 targeting and there is a gap in knowledge on Cas9's efficacy when targeting such locations. To quantify at a single base pair resolution how chromatin inhibits on-target gene editing relative to off-target editing of exposed mismatching targets, we developed the gene editor mismatch nucleosome inhibition assay (GEMiNI-seq). GEMiNI-seq utilizes a library of nucleosome sequences to examine all target locations throughout nucleosomes in a single assay. The results from GEMiNI-seq revealed that the location of the protospacer-adjacent motif (PAM) sequence on the nucleosome edge drives the ability for Cas9 to access its target sequence. In addition, Cas9 had a higher affinity for exposed mismatched targets than on-target sequences within a nucleosome. Overall, our results show how chromatin structure impacts the fidelity of Cas9 to potential targets and highlight how targeting sequences with exposed PAMs could limit off-target gene editing, with such considerations improving Cas9 efficacy and resolving current limitations.

Effect of Antigen Retrieval on Genomic DNA From Immunodissected Samples.

Immunohistochemical (IHC) staining is an established technique for visualizing proteins in tissue sections for research studies and clinical applications. IHC is increasingly used as a targeting strategy for procurement of labeled cells via tissue microdissection, including immunodissection, computer-aided laser dissection (CALD), expression microdissection (xMD), and other techniques. The initial antigen retrieval (AR) process increases epitope availability and improves staining characteristics; however, the procedure can damage DNA. To better understand the effects of AR on DNA quality and quantity in immunodissected samples, both clinical specimens (KRAS gene mutation positive cases) and model system samples (lung cancer patient-derived xenograft tissue) were subjected to commonly employed AR methods (heat induced epitope retrieval [HIER], protease digestion) and the effects on DNA were assessed by Qubit, fragment analysis, quantitative PCR, digital droplet PCR (ddPCR), library preparation, and targeted sequencing. The data showed that HIER resulted in optimal IHC staining characteristics, but induced significant damage to DNA, producing extensive fragmentation and decreased overall yields. However, neither of the AR methods combined with IHC prevented ddPCR amplification of small amplicons and gene mutations were successfully identified from immunodissected clinical samples. The results indicate for the first time that DNA recovered from immunostained slides after standard AR and IHC processing can be successfully employed for genomic mutation analysis via ddPCR and next-generation sequencing (NGS) short-read methods.

Multimodal Dimension Reduction and Subtype Classification of Head and Neck Squamous Cell Tumors.

Traditional analysis of genomic data from bulk sequencing experiments seek to group and compare sample cohorts into biologically meaningful groups. To accomplish this task, large scale databases of patient-derived samples, like that of TCGA, have been established, giving the ability to interrogate multiple data modalities per tumor. We have developed a computational strategy employing multimodal integration paired with spectral clustering and modern dimension reduction techniques such as PHATE to provide a more robust method for cancer sub-type classification. Using this integrated approach, we have examined 514 Head and Neck Squamous Carcinoma (HNSC) tumor samples from TCGA across gene-expression, DNA-methylation, and microbiome data modalities. We show that these approaches, primarily developed for single-cell sequencing can be efficiently applied to bulk tumor sequencing data. Our multimodal analysis captures the dynamic heterogeneity, identifies new and refines subtypes of HNSC, and orders tumor samples along well-defined cellular trajectories. Collectively, these results showcase the inherent molecular complexity of tumors and offer insights into carcinogenesis and importance of targeted therapy. Computational techniques as highlighted in our study provide an organic and powerful approach to identify granular patterns in large and noisy datasets that may otherwise be overlooked.

Relationship between the subgingival microbiome and menopausal hormone therapy use: The Buffalo OsteoPerio study.

BACKGROUND: This study investigated the association between menopausal hormone therapy (HT) use and the subgingival microbiome, for which published information is limited. METHODS: This cross-sectional study included 1270 postmenopausal women, aged 53-81 years, who completed clinical examinations. Detailed information on HT use (type, delivery mode, duration) was obtained from questionnaires. HT use was categorized into three groups (never, former, current). 16S rRNA sequencing was performed on subgingival plaque samples obtained during dental examinations. Operational taxonomic units were centered log2-ratio (CLR) transformed to account for the compositional data structure. Analysis of variance was used to compare mean microbial relative abundances across HT categories with Benjamini-Hochberg correction. RESULTS: Significantly higher alpha diversity (Shannon Index) and beta diversity (Aitchison distance) was observed in never compared with current HT users (p < 0.05, each). Of the total 245 microbial taxa identified, 18 taxa differed significantly among the three HT groups, 11 of which were higher in current users and seven of which were lower in current users as compared with never users (p < 0.05, each). Differences in relative abundance between never and current HT users were materially unchanged after adjustment for age, body mass index, and oral hygiene. CONCLUSIONS: Relative abundance of several subgingival bacteria differed significantly between never and current HT users in a cohort of postmenopausal women. Additional studies are needed to determine the extent that these relationships might account for the previously reported inverse association between HT use and periodontal disease in older women.

Oral Microbiome Is Associated With Incident Hypertension Among Postmenopausal Women.

Background Oral microbiota are thought to influence blood pressure (BP) regulation. However, epidemiological data supporting this hypothesis are limited. We examined associations between oral microbiota, BP, and incident hypertension in postmenopausal women. Methods and Results Baseline (1997-2001) examinations were completed on 1215 women (mean age, 63 years) during which subgingival plaque was collected, BP was measured, and medical and lifestyle histories and medication inventory were obtained. Microbiome composition of subgingival plaque was measured using 16S ribosomal RNA gene amplicon sequencing. Baseline measured BP was defined as normotensive (systolic <120 mm Hg and diastolic <80 mm Hg, no BP medication use; n=429); elevated (systolic ≥120 mm Hg or diastolic ≥80 mm Hg, no medication use; n=306); or prevalent treated hypertension (history of physician diagnosis treated with medications; n=480). Incident hypertension (375 cases among 735 without baseline treated hypertension) was defined as newly physician-diagnosed hypertension treated with medication reported on annual health surveys (mean follow-up, 10.4 years). Cross-sectional analysis identified 47 bacterial species (of 245 total) that differed significantly according to baseline BP status (P<0.05). Prospective analysis identified 15 baseline bacterial species significantly (P<0.05) associated with incident hypertension: 10 positively (age-adjusted hazard ratios [HRs], 1.10-1.16 per SD in bacterial abundance) and 5 inversely (HRs, 0.82-0.91) associated. Associations were materially unchanged after further adjustment for demographic, clinical, and lifestyle factors; were similar when analysis was restricted to the normotensive group; and were of consistent magnitudes between strata of baseline age, smoking, body mass index, and BP categories. Conclusions Specific oral bacteria are associated with baseline BP status and risk of hypertension development among postmenopausal women. Research to confirm these observations and elucidate mechanisms is needed.

Dietary carbohydrate intake is associated with the subgingival plaque oral microbiome abundance and diversity in a cohort of postmenopausal women.

Limited research exists on carbohydrate intake and oral microbiome diversity and composition assessed with next-generation sequencing. We aimed to better understand the association between habitual carbohydrate intake and the oral microbiome, as the oral microbiome has been associated with caries, periodontal disease, and systemic diseases. We investigated if total carbohydrates, starch, monosaccharides, disaccharides, fiber, or glycemic load (GL) were associated with the diversity and composition of oral bacteria in subgingival plaque samples of 1204 post-menopausal women. Carbohydrate intake and GL were assessed from a food frequency questionnaire, and adjusted for energy intake. The V3-V4 region of the 16S rRNA gene from subgingival plaque samples were sequenced to identify the relative abundance of microbiome compositional data expressed as operational taxonomic units (OTUs). The abundance of OTUs were centered log(2)-ratio transformed to account for the compositional data structure. Associations between carbohydrate/GL intake and microbiome alpha-diversity measures were examined using linear regression. PERMANOVA analyses were conducted to examine microbiome beta-diversity measures across quartiles of carbohydrate/GL intake. Associations between intake of carbohydrates and GL and the abundance of the 245 identified OTUs were examined by using linear regression. Total carbohydrates, GL, starch, lactose, and sucrose intake were inversely associated with alpha-diversity measures. Beta-diversity across quartiles of total carbohydrates, fiber, GL, sucrose, and galactose, were all statistically significant (p for PERMANOVA p < 0.05). Positive associations were observed between total carbohydrates, GL, sucrose and Streptococcus mutans; GL and both Sphingomonas HOT 006 and Scardovia wiggsiae; and sucrose and Streptococcus lactarius. A negative association was observed between lactose and Aggregatibacter segnis, and between sucrose and both TM7_[G-1] HOT 346 and Leptotrichia HOT 223. Intake of total carbohydrate, GL, and sucrose were inversely associated with subgingival bacteria alpha-diversity, the microbial beta-diversity varied by their intake, and they were associated with the relative abundance of specific OTUs. Higher intake of sucrose, or high GL foods, may influence poor oral health outcomes (and perhaps systemic health outcomes) in older women via their influence on the oral microbiome.

Microdissection Methods Utilizing Single-Cell Subtype Analysis and the Impact on Precision Medicine.

Improving the utilization of tumor tissue from diagnostic biopsies is an unmet medical need. This is especially relevant today in the rapidly evolving precision oncology field where tumor genotyping is often essential for the indication of many advanced and targeted therapies. National Comprehensive Cancer Network (NCCN) guidelines now mandate molecular testing for clinically actionable targets in certain malignancies. Utilizing advanced stage lung cancer as an example, an improved genotyping approach for solid tumors is possible. The strategy involves optimization of the microdissection process and analysis of a large number of identical target cells from formalin-fixed paraffin-embedded (FFPE) specimens sharing similar characteristics, in other words, single-cell subtype analysis. The shared characteristics can include immunostaining status, cell phenotype, and/or spatial location within a histological section. Synergy between microdissection and droplet digital PCR (ddPCR) enhances the molecular analysis. We demonstrate here a methodology that illustrates genotyping of a solid tumor from a small tissue biopsy sample in a time- and cost-efficient manner, using immunostain targeting as an example.

Remediation of Mucorales-contaminated Healthcare Linens at a Laundry Facility Following an Investigation of a Case Cluster of Hospital-acquired Mucormycosis.

BACKGROUND: In an investigation of hospital-acquired mucormycosis cases among transplant recipients, healthcare linens (HCLs) delivered to our center were found to be contaminated with Mucorales. We describe an investigation and remediation of Mucorales contamination at the laundry supplying our center. METHODS: We performed monthly RODAC cultures of HCLs upon hospital arrival, and conducted site inspections and surveillance cultures at the laundry facility. Remediation was designed and implemented by infection prevention and facility leadership teams. RESULTS: Prior to remediation, 20% of HCLs were culture-positive for Mucorales upon hospital arrival. Laundry facility layout and processes were consistent with industry standards. Significant step-ups in Mucorales and mold culture-positivity of HCLs were detected at the post-dryer step (0% to 12% [P = .04] and 5% to 29% [P = .01], respectively). Further increases to 17% and 40% culture-positivity, respectively, were noted during pre-transport holding. Site inspection revealed heavy Mucorales-positive lint accumulation in rooftop air intake and exhaust vents that cooled driers; intake and exhaust vents that were facing each other; rooftop and plant-wide lint accumulation, including in the pre-transport clean room; uncovered carts with freshly-laundered HCLs. Following environmental remediation, quality assurance measures and education directed toward these sources, Mucorales culture-positivity of newly-delivered HCLs was reduced to 0.3% (P = .0001); area of lint-contaminated rooftop decreased from 918 m2 to 0 m2 on satellite images. CONCLUSIONS: Targeted laundry facility interventions guided by site inspections and step-wise culturing significantly reduced Mucorales-contaminated HCLs delivered to our hospital. Collaboration between infection prevention and laundry facility teams was crucial to successful remediation.

Recruitment of dendritic cell progenitors to foci of influenza A virus infection sustains immunity.

Protection from infection with respiratory viruses such as influenza A virus (IAV) requires T cell­mediated immune responses initiated by conventional dendritic cells (cDCs) that reside in the respiratory tract. Here, we show that effective induction of T cell responses against IAV in mice requires reinforcement of the resident lung cDC network by cDC progenitors. We found that CCR2-binding chemokines produced during IAV infection recruit pre-cDCs from blood and direct them to foci of infection, increasing the number of progeny cDCs next to sites of viral replication. Ablation of CCR2 in the cDC lineage prevented this increase and resulted in a deficit in IAV-specific T cell responses and diminished resistance to reinfection. These data suggest that the homeostatic network of cDCs in tissues is insufficient for immunity and reveal a chemokine-driven mechanism of expansion of lung cDC numbers that amplifies T cell responses against respiratory viruses.

An isoform of Dicer protects mammalian stem cells against multiple RNA viruses.

In mammals, early resistance to viruses relies on interferons, which protect differentiated cells but not stem cells from viral replication. Many other organisms rely instead on RNA interference (RNAi) mediated by a specialized Dicer protein that cleaves viral double-stranded RNA. Whether RNAi also contributes to mammalian antiviral immunity remains controversial. We identified an isoform of Dicer, named antiviral Dicer (aviD), that protects tissue stem cells from RNA viruses-including Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-by dicing viral double-stranded RNA to orchestrate antiviral RNAi. Our work sheds light on the molecular regulation of antiviral RNAi in mammalian innate immunity, in which different cell-intrinsic antiviral pathways can be tailored to the differentiation status of cells.

Fever supports CD8+ effector T cell responses by promoting mitochondrial translation.

Fever can provide a survival advantage during infection. Metabolic processes are sensitive to environmental conditions, but the effect of fever on T cell metabolism is not well characterized. We show that in activated CD8+ T cells, exposure to febrile temperature (39 °C) augmented metabolic activity and T cell effector functions, despite having a limited effect on proliferation or activation marker expression. Transcriptional profiling revealed an up-regulation of mitochondrial pathways, which was consistent with increased mass and metabolism observed in T cells exposed to 39 °C. Through in vitro and in vivo models, we determined that mitochondrial translation is integral to the enhanced metabolic activity and function of CD8+ T cells exposed to febrile temperature. Transiently exposing donor lymphocytes to 39 °C prior to infusion in a myeloid leukemia mouse model conferred enhanced therapeutic efficacy, raising the possibility that exposure of T cells to febrile temperatures could have clinical potential.