An immune-competent human gut microphysiological system enables inflammation-modulation by Faecalibacterium prausnitzii.

Crosstalk of microbes with human gut epithelia and immune cells is crucial for gut health. However, there is no existing system for a long-term co-culture of human innate immune cells with epithelium and oxygen-intolerant commensal microbes, hindering the understanding of microbe-immune interactions in a controlled manner. Here, we established a gut epithelium-microbe-immune (GuMI) microphysiological system to maintain the long-term continuous co-culture of Faecalibacterium prausnitzii/Faecalibacterium duncaniae with colonic epithelium, antigen-presenting cells (APCs, herein dendritic cells and macrophages), and CD4+ naive T cells circulating underneath the colonic epithelium. In GuMI-APC condition, multiplex cytokine assays suggested that APCs contribute to the elevated level of cytokines and chemokines secreted into both apical and basolateral compartments compared to GuMI condition that lacks APC. In GuMI-APC with F. prausnitzii (GuMI-APC-FP), F. prausnitzii increased the transcription of pro-inflammatory genes such as toll-like receptor 1 (TLR1) and interferon alpha 1 (IFNA1) in the colonic epithelium, without a significant effect on cytokine secretion, compared to the GuMI-APC without bacteria (GuMI-APC-NB). In contrast, in the presence of CD4+ naive T cells (GuMI-APCT-FP), TLR1, IFNA1, and IDO1 transcription levels decreased with a simultaneous increase in F. prausnitzii-induced secretion of pro-inflammatory cytokines (e.g., IL8) compared to GuMI-APC-FP that lacks T cells. These results highlight the contribution of individual innate immune cells in regulating the immune response triggered by the gut commensal F. prausnitzii. The integration of defined populations of immune cells in the gut microphysiological system demonstrated the usefulness of GuMI physiomimetic platform to study microbe-epithelial-immune interactions in healthy and disease conditions.

Potential relevance of a motor skill "proficiency barrier" on health-related fitness in youth.

This study investigated the potential impact of a motor skill proficiency barrier on measures of cardiorespiratory (CRF) and musculoskeletal (MSF) fitness in youth. A sample of 241 youth (114 girls) aged 10 - 18 years, completed the Motor Competence Assessment battery with composite scores indexed according to age- and gender-adjusted percentile scores. Motor competence (MC) levels were categorized as low (≤ 25%tile - proficiency barrier), moderate (≥ 26%tile to < 75%tile), and high (≥ 75%tile). CRF levels (Health Risk, Needs Improvement, and Healthy) were assessed using the Fitnessgram® 20 m PACER test. Low (≤ 20%tile), moderate (≥ 21%tile to ≤ 80%tile), and high (≥ 80%tile) MSF levels were assessed using grip strength normative data. Two 3 × 3 chi-square tests were conducted to determine the probability of MC level predicting CRF and MSF levels. Results demonstrated statistically significant models for performance on both the PACER (χ2[4, N = 241] = 22.65, p < .001) and grip strength (χ2[4, N = 241] = 23.95, p < .001). Strong evidence of a proficiency barrier impacting CRF was noted, as no low skilled youth met the "Healthy" fitness zone standards for PACER performance. Evidence supporting a barrier with grip strength was not as strong, as 20.8% of youth exhibiting low MC displayed high grip strength. However, all individuals with high levels of MC demonstrated at least moderate grip strength. Results emphasize the importance of developing MC during childhood as it may provide a protective effect against unhealthy CRF and MSF across youth.HighlightsThese data support the notion of Seefeldt's (1980) proficiency barrier as it relates to CRF, as no youth demonstrating low MC met the healthy fitness zone criteria for PACER performance. The development of MC may both directly and indirectly provide a protective effect against unhealthy CRF levels across childhood and adolescence.Evidence supporting a proficiency barrier with MSF as measured by grip strength was not as strong; however, all individuals with high levels of MC demonstrated at least moderate grip strength. Thus, the development of MC may be a protective factor to mitigate low levels of MSF via enhanced neuromuscular function.Promoting the development of MC in a variety of developmentally appropriate activities and settings (e.g. MC skills practice, structured and unstructured play, and performance contexts) is important to promote positive trajectories of CRF and MSF across childhood and adolescence.

An immune-competent human gut microphysiological system enables inflammation-modulation of Faecalibacterium prausnitzii.

Crosstalk of microbes with human gut epithelia and immune cells is crucial for gut health. However, there is no existing system for a long-term co-culture of human innate immune cells with epithelium and oxygen-intolerant commensal microbes, hindering the understanding of microbe-immune interactions in a controlled manner. Here, we establish a gut epithelium-microbe-immune microphysiological system to maintain the long-term continuous co-culture of Faecalibacterium prausnitzii/Faecalibacterium duncaniae with colonic epithelium, antigen-presenting cells (APCs, herein dendritic cells and macrophages), with CD4+ naïve T cells circulating underneath the colonic epithelium. Multiplex cytokine assays suggested that APCs contribute to the elevated level of cytokines and chemokines being secreted into both apical and basolateral compartments. In contrast, the absence of APCs does not allow reliable detection of these cytokines. In the presence of APCs, F. prausnitzii increased the transcription of pro-inflammatory genes such as toll-like receptor 1 (TLR1) and interferon alpha 1 (IFNA1) in the colonic epithelium, but no significant change on the secreted cytokines. In contrast, integration of CD4+ naïve T cells reverses this effect by decreasing the transcription of TLR1, IFNA1, and indoleamine 2,3-dioxygenase, and increasing the F. prausnitzii-induced secretion of pro-inflammatory cytokines such as IL-8, MCP-1/CCL2, and IL1A. These results highlight the contribution of individual innate immune cells in the regulation of the immune response triggered by the gut commensal F. prausnitzii. The successful integration of defined populations of immune cells in this gut microphysiological system demonstrated the usefulness of the GuMI physiomimetic platform to study microbe-epithelial-immune interactions in health and disease.

Influence of ethanol on cannabinoid pharmacokinetic parameters in chronic users.

Cannabis is not only the most widely used illicit drug worldwide but is also regularly consumed along with ethanol. In previous studies, it was assumed that cannabis users develop cross-tolerance to ethanol effects. The present study was designed to compare the effects of ethanol in comparison to and in combination with a cannabis joint and investigate changes in pharmacokinetics. In this study, 19 heavy cannabis users participated and received three alcohol dosing conditions that were calculated to achieve steady blood alcohol concentrations (BAC) of about 0, 0.5 and 0.7 g/l during a 5-h time window. Subjects smoked a Δ(9)-tetrahydrocannabinol (THC) cigarette (400 µg/kg) 3 h post-onset of alcohol dosing. Blood samples were taken between 0 and 4 h after smoking. During the first hour, samples were collected every 15 min and every 30 min thereafter. Mean steady-state BACs reached 0, 0.36 and 0.5 g/l. The apparent elimination half-life of THC was slightly prolonged (1.59 vs. 1.93 h, p < 0.05) and the concentration 1 h after smoking was slightly lower (24 vs. 17 ng/ml, p < 0.05) with the higher ethanol dose. The prolonged THC elimination might be explained by a small ethanol-mediated change in distribution to and from deep compartments. Concentrations and pharmacokinetics of 11-hydroxy-THC and 11-nor-9-carboxy-THC (THCA) were not significantly influenced by ethanol. However, THCA concentrations appeared lower in both ethanol conditions, which might also be attributable to changes in distribution. Though not significant in the present study, this might be relevant in the interpretation of cannabinoid concentrations in blood.

Coculture of primary human colon monolayer with human gut bacteria.

The presence of microbes in the colon impacts host physiology. Therefore, microbes are being evaluated as potential treatments for colorectal diseases. Humanized model systems that enable robust culture of primary human intestinal cells with bacteria facilitate evaluation of potential treatments. Here, we describe a protocol that can be used to coculture a primary human colon monolayer with aerotolerant bacteria. Primary human colon cells maintained as organoids are dispersed into single-cell suspensions and then seeded on collagen-coated Transwell inserts, where they attach and proliferate to form confluent monolayers within days of seeding. The confluent monolayers are differentiated for an additional 4 d and then cocultured with bacteria. As an example application, we describe how to coculture differentiated colon cells for 8 h with four strains of Bacteroides thetaiotaomicron, each engineered to detect different colonic microenvironments via genetically embedded logic circuits incorporating deoxycholic acid and anhydrotetracycline sensors. Characterization of this coculture system reveals that barrier function remains intact in the presence of engineered B. thetaiotaomicron. The bacteria stay close to the mucus layer and respond in a microenvironment-specific manner to the inducers (deoxycholic acid and anhydrotetracycline) of the genetic circuits. This protocol thus provides a useful mucosal barrier system to assess the effects of bacterial cells that respond to the colonic microenvironment, and may also be useful in other contexts to model human intestinal barrier properties and microbiota-host interactions.

Human physiomimetic model integrating microphysiological systems of the gut, liver, and brain for studies of neurodegenerative diseases.

Slow progress in the fight against neurodegenerative diseases (NDs) motivates an urgent need for highly controlled in vitro systems to investigate organ-organ- and organ-immune-specific interactions relevant for disease pathophysiology. Of particular interest is the gut/microbiome-liver-brain axis for parsing out how genetic and environmental factors contribute to NDs. We have developed a mesofluidic platform technology to study gut-liver-cerebral interactions in the context of Parkinson's disease (PD). It connects microphysiological systems (MPSs) of the primary human gut and liver with a human induced pluripotent stem cell-derived cerebral MPS in a systemically circulated common culture medium containing CD4+ regulatory T and T helper 17 cells. We demonstrate this approach using a patient-derived cerebral MPS carrying the PD-causing A53T mutation, gaining two important findings: (i) that systemic interaction enhances features of in vivo-like behavior of cerebral MPSs, and (ii) that microbiome-associated short-chain fatty acids increase expression of pathology-associated pathways in PD.

Primary human colonic mucosal barrier crosstalk with super oxygen-sensitive Faecalibacterium prausnitzii in continuous culture.

BACKGROUND: The gut microbiome plays an important role in human health and disease. Gnotobiotic animal and in vitro cell-based models provide some informative insights into mechanistic crosstalk. However, there is no existing system for a long-term co-culture of a human colonic mucosal barrier with super oxygen-sensitive commensal microbes, hindering the study of human-microbe interactions in a controlled manner. METHODS: Here, we investigated the effects of an abundant super oxygen-sensitive commensal anaerobe, Faecalibacterium prausnitzii, on a primary human mucosal barrier using a Gut-MIcrobiome (GuMI) physiome platform that we designed and fabricated. FINDINGS: Long-term continuous co-culture of F. prausnitzii for two days with colon epithelia, enabled by continuous flow of completely anoxic apical media and aerobic basal media, resulted in a strictly anaerobic apical environment fostering growth of and butyrate production by F. prausnitzii, while maintaining a stable colon epithelial barrier. We identified elevated differentiation and hypoxia-responsive genes and pathways in the platform compared with conventional aerobic static culture of the colon epithelia, attributable to a combination of anaerobic environment and continuous medium replenishment. Furthermore, we demonstrated anti-inflammatory effects of F. prausnitzii through HDAC and the TLR-NFKB axis. Finally, we identified that butyrate largely contributes to the anti-inflammatory effects by downregulating TLR3 and TLR4. CONCLUSIONS: Our results are consistent with some clinical observations regarding F. prausnitzii, thus motivating further studies employing this platform with more complex engineered colon tissues for understanding the interaction between the human colonic mucosal barrier and microbiota, pathogens, or engineered bacteria.

Publisher Correction: Palm Fruit Bioactives augment expression of Tyrosine Hydroxylase in the Nile Grass Rat basal ganglia and alter the colonic microbiome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Gut-Liver Physiomimetics Reveal Paradoxical Modulation of IBD-Related Inflammation by Short-Chain Fatty Acids.

Although the association between the microbiome and IBD and liver diseases is known, the cause and effect remain elusive. By connecting human microphysiological systems of the gut, liver, and circulating Treg and Th17 cells, we created a multi-organ model of ulcerative colitis (UC) ex vivo. The approach shows microbiome-derived short-chain fatty acids (SCFAs) to either improve or worsen UC severity, depending on the involvement of effector CD4 T cells. Using multiomics, we found SCFAs increased production of ketone bodies, glycolysis, and lipogenesis, while markedly reducing innate immune activation of the UC gut. However, during acute T cell-mediated inflammation, SCFAs exacerbated CD4+ T cell-effector function, partially through metabolic reprograming, leading to gut barrier disruption and hepatic injury. These paradoxical findings underscore the emerging utility of human physiomimetic technology in combination with systems immunology to study causality and the fundamental entanglement of immunity, metabolism, and tissue homeostasis.

Palm Fruit Bioactives augment expression of Tyrosine Hydroxylase in the Nile Grass Rat basal ganglia and alter the colonic microbiome.

Tyrosine hydroxylase (TH) catalyzes the hydroxylation of L-tyrosine to L-DOPA. This is the rate-limiting step in the biosynthesis of the catecholamines - dopamine (DA), norepinephrine (NE), and epinephrine (EP). Catecholamines (CA) play a key role as neurotransmitters and hormones. Aberrant levels of CA are associated with multiple medical conditions, including Parkinson's disease. Palm Fruit Bioactives (PFB) significantly increased the levels of tyrosine hydroxylase in the brain of the Nile Grass rat (NGR), a novel and potentially significant finding, unique to PFB among known botanical sources. Increases were most pronounced in the basal ganglia, including the caudate-putamen, striatum and substantia nigra. The NGR represents an animal model of diet-induced Type 2 Diabetes Mellitus (T2DM), exhibiting hyperglycemia, hyperinsulinemia, and insulin resistance associated with hyperphagia and accelerated postweaning weight gain induced by a high-carbohydrate diet (hiCHO). The PFB-induced increase of TH in the basal ganglia of the NGR was documented by immuno-histochemical staining (IHC). This increase in TH occurred equally in both diabetes-susceptible and diabetes-resistant NGR fed a hiCHO. PFB also stimulated growth of the colon microbiota evidenced by an increase in cecal weight and altered microbiome.  The metabolites of colon microbiota, e.g. short-chain fatty acids, may influence the brain and behavior significantly.

Specific remodeling of splenic architecture by cytomegalovirus.

Efficient immune defenses are facilitated by the organized microarchitecture of lymphoid organs, and this organization is regulated by the compartmentalized expression of lymphoid tissue chemokines. Mouse cytomegalovirus (MCMV) infection induces significant remodeling of splenic microarchitecture, including loss of marginal zone macrophage populations and dissolution of T and B cell compartmentalization. MCMV preferentially infected the splenic stroma, targeting endothelial cells (EC) as revealed using MCMV-expressing green fluorescent protein. MCMV infection caused a specific, but transient transcriptional suppression of secondary lymphoid chemokine (CCL21). The loss of CCL21 was associated with the failure of T lymphocytes to locate within the T cell zone, although trafficking to the spleen was unaltered. Expression of CCL21 in lymphotoxin (LT)-alpha-deficient mice is dramatically reduced, however MCMV infection further reduced CCL21 levels, suggesting that viral modulation of CCL21 was independent of LTalpha signaling. Activation of LTbeta-receptor signaling with an agonistic antibody partially restored CCL21 mRNA expression and redirected transferred T cells to the splenic T cell zone in MCMV-infected mice. These results indicate that virus-induced alterations in lymphoid tissues can occur through an LT-independent modulation of chemokine transcription, and targeting of the LT cytokine system can counteract lymphoid tissue remodeling by MCMV.

Palm Fruit Bioactives modulate human astrocyte activity in vitro altering the cytokine secretome reducing levels of TNFα, RANTES and IP-10.

Neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease, are becoming more prevalent and an increasing burden on society. Neurodegenerative diseases often arise in the milieu of neuro-inflammation of the brain. Reactive astrocytes are key regulators in the development of neuro-inflammation. This study describes the effects of Palm Fruit Bioactives (PFB) on the behavior of human astrocytes which have been activated by IL-1ß. When activated, the astrocytes proliferate, release numerous cytokines/chemokines including TNFα, RANTES (CCL5), IP-10 (CXCL10), generate reactive oxygen species (ROS), and express specific cell surface biomarkers such as the Intercellular Adhesion Molecule (ICAM), Vascular Cellular Adhesion Molecule (VCAM) and the Neuronal Cellular Adhesion Molecule (NCAM). Interleukin 1-beta (IL-1ß) causes activation of human astrocytes with marked upregulation of pro-inflammatory genes. We show significant inhibition of these pro-inflammatory processes when IL-1ß-activated astrocytes are exposed to PFB. PFB causes a dose-dependent and time-dependent reduction in specific cytokines: TNFα, RANTES, and IP-10. We also show that PFB significantly reduces ROS production by IL-1ß-activated astrocytes. Furthermore, PFB also reduces the expression of ICAM and VCAM, both in activated and naïve human astrocytes in vitro. Since reactive astrocytes play an essential role in the neuroinflammatory state preceding neurodegenerative diseases, this study suggests that PFB may have a potential role in their prevention and/or treatment.

Chicken toll-like receptor 3 recognizes its cognate ligand when ectopically expressed in human cells.

Recognition of pathogens by toll-like receptors (TLRs) causes activation of signaling cascades that trigger cytokine secretion and, ultimately, innate immunity. Genes encoding proteins with substantial homology to mammalian TLR1, TLR2, TLR3, TLR4, TLR5, and TLR7 are present in the chicken genome, whereas orthologs of TLR8, TLR9, and TLR10 seem to be defective or missing. Except for chicken TLR2 (ChTLR2), which was previously shown to recognize lipopeptides and lipopolysaccharides (LPS), the ligand specificity of ChTLRs had not been determined. We found that polyI:C, LPS, R848, S-28463, and ODN2006, which are specifically recognized by TLR3, TLR4, TLR7/8, and TLR9 in mammals, induced substantial amounts of type I interferon (IFN) and interleukin-6 (IL-6) in freshly prepared chicken splenocytes. To determine the ligand specificity of ChTLR3 and ChTLR7, we used a standard reporter assay frequently employed for analysis of mammalian TLRs. Neither S-28463 nor any other TLR ligand induced reporter activity in human 293 cells expressing ChTLR7. However, human 293 cells expressing ChTLR3 strongly and specifically responded to polyI:C, demonstrating that this chicken receptor represents a true ortholog of mammalian TLR3.

Influence of ethanol on the pharmacokinetic properties of Δ9-tetrahydrocannabinol in oral fluid.

Oral fluid (OF) tests aid in identifying drivers under the influence of drugs. In this study, 17 heavy cannabis users consumed alcohol to achieve steady blood alcohol concentrations of 0 to 0.7 g/L and smoked cannabis 3 h afterward. OF samples were obtained before and up to 4 h after smoking and on-site tests were performed (Dräger DrugTest 5000 and Securetec DrugWipe 5+). Maximum concentrations of tetrahydrocannabinol (THC) immediately after smoking (up to 44,412 ng/g) were below 4,300 (median 377) ng/g 1 h after smoking and less than 312 (median 88) ng/g 3 h later with 5 of 49 samples negative, suggesting that recent cannabis use might occasionally not be detectable. An influence of alcohol was not observed. Drinking 300 mL variably influenced THC concentrations (median only -29.6%), which suggests that drinking does not markedly affect on-site test performance. Many (92%) Dräger tests performed 4 h after smoking were still positive, indicating sufficient sensitivity for recent cannabis use. Differences in the results of a roadside study with DrugTest 5000 (sensitivity 84.8%, specificity 96.0%, accuracy 84.3%) could be explained by a higher number of true negatives, differences between OF and serum and differences between occasional and chronic users.

The function of hypoxia-inducible factor (HIF) is independent of the endoplasmic reticulum protein OS-9.

The protein "amplified in osteosarcoma-9" (OS-9) has been shown previously to interact with the prolyl hydroxylases PHD2 and PHD3. These enzymes initiate oxygen-dependent degradation of the α-subunit of hypoxia-inducible factor (HIF), a transcription factor that adapts cells to insufficient oxygen supply (hypoxia). A new model has been proposed where OS-9 triggers PHD dependent degradation of HIF-α. It was the aim of our study to define the molecular mode of action of OS-9 in the regulation of PHD and HIF activity. Although initial co-immunoprecipitation experiments confirmed physical interaction between OS-9 and PHD2, neither overexpression nor lentiviral inhibition of OS-9 expression affected HIF regulation. Subcellular localization experiments revealed a distinct reticular staining pattern for OS-9 while PHD2 was mainly localized in the cytoplasm. Further cell fractionation experiments and glycosylation tests indicated that OS-9 is a luminal ER protein. In vivo protein interaction analysis by fluorescence resonance energy transfer (FRET) showed no significant physical interaction of overexpressed PHD2-CFP and OS-9-YFP. We conclude that OS-9 plays no direct functional role in HIF degradation since physical interaction of OS-9 with oxygen sensing HIF prolyl hydroxylases cannot occur in vivo due to their different subcellular localization.

Lymphotoxin-mediated crosstalk between B cells and splenic stroma promotes the initial type I interferon response to cytomegalovirus.

Toll-like receptor (TLR)-dependent pathways control the production of IFNalphabeta, a key cytokine in innate immune control of viruses including mouse cytomegalovirus (MCMV). The lymphotoxin (LT) alphabeta-LTbeta receptor signaling pathway is also critical for defense against MCMV and thought to aid in the IFNbeta response. We find that upon MCMV infection, mice deficient for lymphotoxin (LT)alphabeta signaling cannot mount the initial part of a biphasic IFNalphabeta response, but show normal levels of IFNalphabeta during the sustained phase of infection. Significantly, the LTalphabeta-dependent, IFNalphabeta response is independent of TLR signaling. B, but not T, cells expressing LTbeta are essential for promoting the initial IFNalphabeta response. LTbetaR expression is required strictly in splenic stromal cells for initial IFNalphabeta production to MCMV and is dependent upon the NF-kappaB-inducing kinase (NIK). These results reveal a TLR-independent innate host defense strategy directed by B cells in communication with stromal cells via the LTalphabeta cytokine system.

The inhibitory HVEM-BTLA pathway counter regulates lymphotoxin receptor signaling to achieve homeostasis of dendritic cells.

Proliferation of dendritic cells (DC) in the spleen is regulated by positive growth signals through the lymphotoxin (LT)-beta receptor; however, the countering inhibitory signals that achieve homeostatic control are unresolved. Mice deficient in LTalpha, LTbeta, LTbetaR, and the NFkappaB inducing kinase show a specific loss of CD8- DC subsets. In contrast, the CD8alpha- DC subsets were overpopulated in mice deficient in the herpesvirus entry mediator (HVEM) or B and T lymphocyte attenuator (BTLA). HVEM- and BTLA-deficient DC subsets displayed a specific growth advantage in repopulating the spleen in competitive replacement bone marrow chimeric mice. Expression of HVEM and BTLA were required in DC and in the surrounding microenvironment, although DC expression of LTbetaR was necessary to maintain homeostasis. Moreover, enforced activation of the LTbetaR with an agonist Ab drove expansion of CD8alpha- DC subsets, overriding regulation by the HVEM-BTLA pathway. These results indicate the HVEM-BTLA pathway provides an inhibitory checkpoint for DC homeostasis in lymphoid tissue. Together, the LTbetaR and HVEM-BTLA pathways form an integrated signaling network regulating DC homeostasis.

Unique and conserved functions of B cell-activating factor of the TNF family (BAFF) in the chicken.

The chicken represents the best-characterized animal model for B cell development in the so-called gut-associated lymphoid tissue (GALT) and the molecular processes leading to B cell receptor diversification in this species are well investigated. However, the mechanisms regulating B cell development and homeostasis in GALT species are largely unknown. Here we investigate the role played by the avian homologue of B cell-activating factor of the tumor necrosis factor family (BAFF). Flow cytometric analysis showed that the receptor for chicken B cell-activating factor of the tumor necrosis factor family (chBAFF) is expressed by mature and immature B cells. Unlike murine and human BAFF, chBAFF is primarily produced by B cells both in peripheral lymphoid organs and in the bursa of Fabricius, the chicken's unique primary lymphoid organ. In vitro and in vivo studies revealed that chBAFF is required for mature B cell survival. In addition, in vivo neutralization with a decoy receptor led to a reduction of the size and number of B cell follicles in the bursa, demonstrating that, in contrast to humans and mice, in chickens BAFF is also required for the development of immature B cells. Collectively, we show that chBAFF has phylogenetically conserved functions in mature B cell homeostasis but displays unique and thus far unknown properties in the regulation of B cell development in birds.

OX40 costimulation promotes persistence of cytomegalovirus-specific CD8 T Cells: A CD4-dependent mechanism.

The mechanisms that regulate CMV-specific T cell responses in vivo are poorly understood. During murine CMV infection of B6 mice, primary responses in the spleen are dominated by CD8 T cells reactive with antigenic epitopes in M45, M57, and m139 murine CMV gene products. However, during the later persistent phase of infection, CD8 T cell responses to epitopes in m139 and M38 viral gene products predominate. The basis for this shift in CD8 T populations is unknown. In this study, we demonstrate that OX40, a TNFR superfamily member, specifically regulates the accumulation of CD8 T cells reactive with the persistent-phase epitopes. Defective CD8 T cell responses in OX40(-/-) mice were replicated in MHC class II(-/-) mice implying that CD4 T cells in part controlled the differentiation of the CD8 T cell clones responsive to these epitopes during persistent infection. Furthermore, treatment of infected mice with an agonist OX40 Ab induced expansion of protective primary virus-specific CD8 T cells independent of CD4 T cell help, but CD4 T cells were crucial for anti-OX40 to promote CD8 T cells reactive to the persistent dominant epitopes. Collectively, these results indicate manipulation of OX40 may be useful in improving cellular immunotherapy regimes for treatment of persistent virus infections.