Contrasting functions of ATP hydrolysis by MDA5 and LGP2 in viral RNA sensing.

Cytosolic long dsRNA, among the most potent proinflammatory signals, is recognized by melanoma differentiation-associated protein 5 (MDA5). MDA5 binds dsRNA cooperatively forming helical filaments. ATP hydrolysis by MDA5 fulfills a proofreading function by promoting dissociation of shorter endogenous dsRNs from MDA5 while allowing longer viral dsRNAs to remain bound leading to activation of interferon-ß responses. Here, we show that adjacent MDA5 subunits in MDA5-dsRNA filaments hydrolyze ATP cooperatively, inducing cooperative filament disassembly. Consecutive rounds of ATP hydrolysis amplify the filament footprint, displacing tightly bound proteins from dsRNA. Our electron microscopy and biochemical assays show that LGP2 binds to dsRNA at internal binding sites through noncooperative ATP hydrolysis. Unlike MDA5, LGP2 has low nucleic acid selectivity and can hydrolyze GTP and CTP as well as ATP. Binding of LGP2 to dsRNA promotes nucleation of MDA5 filament assembly resulting in shorter filaments. Molecular modeling identifies an internally bound MDA5-LGP2-RNA complex, with the LGP2 C-terminal tail forming the key contacts with MDA5. These contacts are specifically required for NTP-dependent internal RNA binding. We conclude that NTPase-dependent binding of LGP2 to internal dsRNA sites complements NTPase-independent binding to dsRNA ends, via distinct binding modes, to increase the number and signaling output of MDA5-dsRNA complexes.

DNA-PKcs is required for cGAS/STING-dependent viral DNA sensing in human cells.

To mount an efficient interferon response to virus infection, intracellular pattern recognition receptors (PRRs) sense viral nucleic acids and activate anti-viral gene transcription. The mechanisms by which intracellular DNA and DNA viruses are sensed are relevant not only to anti-viral innate immunity, but also to autoinflammation and anti-tumour immunity through the initiation of sterile inflammation by self-DNA recognition. The PRRs that directly sense and respond to viral or damaged self-DNA function by signaling to activate interferon regulatory factor (IRF)-dependent type one interferon (IFN-I) transcription. We and others have previously defined DNA-dependent protein kinase (DNA-PK) as an essential component of the DNA-dependent anti-viral innate immune system. Here, we show that DNA-PK is essential for cyclic GMP-AMP synthase (cGAS)- and stimulator of interferon genes (STING)-dependent IFN-I responses in human cells during stimulation with exogenous DNA and infection with DNA viruses.

Mechanisms of type I interferon production by chicken TLR21.

The innate immune response relies on the ability of host cells to rapidly detect and respond to microbial nucleic acids. Toll-like receptors (TLRs), a class of pattern recognition receptors (PRRs), play a fundamental role in distinguishing self from non-self at the molecular level. In this study, we focused on TLR21, an avian TLR that recognizes DNA motifs commonly found in bacterial genomic DNA, specifically unmethylated CpG motifs. TLR21 is believed to act as a functional homologue to mammalian TLR9. By analysing TLR21 signalling in chickens, we sought to elucidate avian TLR21 activation outputs in parallel to that of other nucleic acid species. Our analyses revealed that chicken TLR21 (chTLR21) triggers the activation of NF-κB and induces a potent type-I interferon response in chicken macrophages, similar to the signalling cascades observed in mammalian TLR9 activation. Notably, the transcription of interferon beta (IFNB) by chTLR21 was found to be dependent on both NF-κB and IRF7 signalling, but independent of the TBK1 kinase, a distinctive feature of mammalian TLR9 signalling. These findings highlight the conservation of critical signalling components and downstream responses between avian TLR21 and mammalian TLR9, despite their divergent evolutionary origins. These insights into the evolutionarily conserved mechanisms of nucleic acid sensing contribute to the broader understanding of host-pathogen interactions across species.

LUBAC is required for RIG-I sensing of RNA viruses.

The ability of cells to mount an interferon response to virus infections depends on intracellular nucleic acid sensing pattern recognition receptors (PRRs). RIG-I is an intracellular PRR that binds short double-stranded viral RNAs to trigger MAVS-dependent signalling. The RIG-I/MAVS signalling complex requires the coordinated activity of multiple kinases and E3 ubiquitin ligases to activate the transcription factors that drive type I and type III interferon production from infected cells. The linear ubiquitin chain assembly complex (LUBAC) regulates the activity of multiple receptor signalling pathways in both ligase-dependent and -independent ways. Here, we show that the three proteins that constitute LUBAC have separate functions in regulating RIG-I signalling. Both HOIP, the E3 ligase capable of generating M1-ubiquitin chains, and LUBAC accessory protein HOIL-1 are required for viral RNA sensing by RIG-I. The third LUBAC component, SHARPIN, is not required for RIG-I signalling. These data cement the role of LUBAC as a positive regulator of RIG-I signalling and as an important component of antiviral innate immune responses.

Trunk-to-leg-volume ratio is not associated with bone density or fracture risk in middle-aged adults: results from the National Health and Nutrition Examination Survey.

Unlike a high body mass index or waist circumference, a high trunk-to-leg-volume ratio does not associate with a lower risk of osteopenia/osteoporosis at the femoral neck. While elevated TLVR showed a suggestive association with a history of wrist fracture in women, additional research is needed to confirm this suggestion. PURPOSE: Body mass index (BMI) and waist circumference (WC) are commonly used to predict bone health, which is typically assessed via bone mineral density (BMD). Trunk-to-leg-volume ratio (TLVR), a relatively novel measure, predicts cardiometabolic outcomes, but its relationships with BMD and fracture remain unstudied. This study evaluated these anthropometric measures' associations with BMD and fracture in Americans aged 40-60 years. METHODS: Analyses of middle-aged adults from the 2013-2014 to 2017-2018 National Health and Nutrition Examination Survey were conducted. Whole-body, dual-energy X-ray absorptiometry was used to quantify TLVR as well as BMD at the lumbar spine, while a femur-specific scan was used to quantify femoral neck BMD. Fracture history was self-reported. Linear and logistic regression models were constructed with age, diabetes, smoking, race/ethnicity, education, and physical activity as confounding variables. RESULTS: TLVR was generally not associated with BMD, while WC and BMI showed positive associations with femoral neck BMD. Odds of osteopenia/osteoporosis at the femoral neck were ~ 65-80% lower among participants in the highest tertile of BMI and WC versus the lowest (p < 0.001). There were no statistically significant associations between anthropometric predictors and fracture. Women in tertiles 2 or 3 of TLVR (p = 0.097 and 0.079, respectively) did have 2.66 times the odds of wrist fracture than women in tertile 1, but this was not significant. CONCLUSION: As shown in previous research, BMI and WC show positive associations with femoral BMD. In contrast, the more novel anthropometric marker TLVR shows no association with femoral BMD, and no clear association with fracture.

PKR-mediated stress response enhances dengue and Zika virus replication.

IMPORTANCE: One of the fundamental features that make viruses intracellular parasites is the necessity to use cellular translational machinery. Hence, this is a crucial checkpoint for controlling infections. Here, we show that dengue and Zika viruses, responsible for nearly 400 million infections every year worldwide, explore such control for optimal replication. Using immunocompetent cells, we demonstrate that arrest of protein translations happens after sensing of dsRNA and that the information required to avoid this blocking is contained in viral 5'-UTR. Our work, therefore, suggests that the non-canonical translation described for these viruses is engaged when the intracellular stress response is activated.

Transcriptional reprogramming of natural killer cells by vaccinia virus shows both distinct and conserved features with mCMV.

Natural killer (NK) cells have an established role in controlling poxvirus infection and there is a growing interest to exploit their capabilities in the context of poxvirus-based oncolytic therapy and vaccination. How NK cells respond to poxvirus-infected cells to become activated is not well established. To address this knowledge gap, we studied the NK cell response to vaccinia virus (VACV) in vivo, using a systemic infection murine model. We found broad alterations in NK cells transcriptional activity in VACV-infected mice, consistent with both direct target cell recognition and cytokine exposure. There were also alterations in the expression levels of specific NK surface receptors (NKRs), including the Ly49 family and SLAM receptors, as well as upregulation of memory-associated NK markers. Despite the latter observation, adoptive transfer of VACV-expercienced NK populations did not confer protection from infection. Comparison with the NK cell response to murine cytomegalovirus (MCMV) infection highlighted common features, but also distinct NK transcriptional programmes initiated by VACV. Finally, there was a clear overlap between the NK transcriptional response in humans vaccinated with an attenuated VACV, modified vaccinia Ankara (MVA), demonstrating conservation between the NK response in these different host species. Overall, this study provides new data about NK cell activation, function, and homeostasis during VACV infection, and may have implication for the design of VACV-based therapeutics.

Anxiety and visceral sensitivity relate to gastrointestinal symptoms in runners but not pre- or during-event nutrition intake.

BACKGROUND: Previous research has shown anxiety to relate to gastrointestinal (GI) symptoms in endurance athletes, but it remains unclear whether competition-related fueling is impacted by anxiety. This study examined whether general anxiety, pre-race anxiety, and visceral sensitivity were associated with nutritional intake before and during endurance running races. METHODS: A total of 149 (86 female, 63 male) runners participated in this cross-sectional survey study. Assessments, which were carried out at a median of eight hours after races finished, included the State-Trait Inventory for Cognitive and Somatic Anxiety (STICSA)-Trait, Visceral Sensitivity Index (VSI), perceived pre-race anxiety (0-10), during-race GI symptoms (total, upper, and lower), and intakes of energy, macronutrients, fluid, and caffeine for the pre-race period (4 h before) and during races. Spearman's correlations were used to examine associations between variables. A two-sided P value ≤0.01 was used as the threshold for significance. RESULTS: Median race durations were 139.5 and 126.9 min for women and men, respectively. VSI scores were positively correlated with total during-race GI symptoms, while STICSA-Trait scores were positively correlated with total and upper during-race GI symptoms (ρ=0.22-0.25; P<0.01). No significant correlations were observed between measures of anxiety/visceral sensitivity and nutrient intakes before or during races. CONCLUSIONS: In a sample of primarily recreational runners, greater anxiety and visceral sensitivity did not translate to lower intakes of energy, macronutrients, fluid, etc. around competition. Further work is needed in other settings, especially with high-level athletes, to understand the impact of competition anxiety on fueling choices.

Ordered Eating and Its Effects on Various Postprandial Health Markers: A Systematic Review.

OBJECTIVE: Aberrations in glucose, insulin, and other postprandial (PP) markers are common in obesity and cardiometabolic disorders. One potentially simple lifestyle/dietary modification to manage these issues is to change the order in which foods are consumed within meals. Carbohydrate exerts the largest effect on PP glucose, and there is some evidence that ingesting dietary fat or protein before carbohydrate delays gastric emptying of carbohydrate and reduces PP glucose. Additionally, certain dietary proteins may augment insulin release if ingested with carbohydrate, thereby improving blood glucose clearance. This review aimed to systematically evaluate evidence from acute experiments that modified the order in which foods were consumed in isocaloric meals. METHODS: Outcomes of interest were PP glucose and insulin (including area under the curve for both), C-peptide, gut hormones, and perceptual responses. Three databases were searched (PubMed, Cochrane CENTRAL, Web of Science) in February 2022. Additionally, reference lists of identified reports were searched, and an author of several studies was consulted to verify that relevant literature was included. The review included acute interventions that administered isocaloric meals of the same foods but with foods eaten in different orders. Studies were not excluded based on participant characteristics. RESULTS: Eleven reports were identified. All reports that assessed glucose and insulin showed a tendency toward lower levels, at least over parts of the PP period, by consuming carbohydrates last. GLP-1 tended to be higher in carbohydrate-last conditions, though this was only measured in a few studies. Perceptual responses (hunger, fullness, etc.) were not consistently different between conditions in two studies, but the certainty of evidence was very low. CONCLUSIONS: Findings indicate that, at least acutely, there may be benefits to eating carbohydrate after vegetable and/or protein-rich foods. The most consistent effect (judged as moderate certainty) is that carbohydrate-last meal orders tend to lower blood glucose and insulin excursions.

Intramuscular Tranexamic Acid Administration on the Battlefield.

Background: Tranexamic acid (TXA) is routinely administered intravenously (IV) and intraosseously (IO) in response to exsanguination. Case: This report describes a patient who sustained multiple high-powered rifle gunshot wounds that received battlefield-environment intramuscular (IM) administration of TXA due to inability to obtain IV / IO access. This case represents the unlikely positive outcome in the setting of multiple remarkable obstacles, which may have been ameliorated by novel administration of TXA. Conclusion: Cases of IM TXA administration as a primary intervention are not well represented in the current body of medical literature. This case report highlights a clinical scenario where IM TXA was utilized as part of first-line treatment that led to a positive clinical outcome. Although IM TXA is not yet endorsed by current trauma guidelines, this case suggests that IM route administration of TXA should be further investigated. If indeed IM administration of TXA proves just as efficacious as alternative routes, this would hold considerable advantageous implications for austere situations were sterility and IV / IO placement are impractical. This would also represent another avenue by which to decrease the time-to-TXA for patients, allowing sooner correction of hemorrhage and trauma-associated coagulopathy.

DNA-PKcs restricts Zika virus spreading and is required for effective antiviral response.

Zika virus (ZIKV) is a single-strand RNA mosquito-borne flavivirus with significant public health impact. ZIKV infection induces double-strand DNA breaks (DSBs) in human neural progenitor cells that may contribute to severe neuronal manifestations in newborns. The DNA-PK complex plays a critical role in repairing DSBs and in the innate immune response to infection. It is unknown, however, whether DNA-PK regulates ZIKV infection. Here we investigated the role of DNA-PKcs, the catalytic subunit of DNA-PK, during ZIKV infection. We demonstrate that DNA-PKcs restricts the spread of ZIKV infection in human epithelial cells. Increased ZIKV replication and spread in DNA-PKcs deficient cells is related to a notable decrease in transcription of type I and III interferons as well as IFIT1, IFIT2, and IL6. This was shown to be independent of IRF1, IRF3, or p65, canonical transcription factors necessary for activation of both type I and III interferon promoters. The mechanism of DNA-PKcs to restrict ZIKV infection is independent of DSB. Thus, these data suggest a non-canonical role for DNA-PK during Zika virus infection, acting downstream of IFNs transcription factors for an efficient antiviral immune response.

Selective modulation of cell surface proteins during vaccinia infection: A resource for identifying viral immune evasion strategies.

The interaction between immune cells and virus-infected targets involves multiple plasma membrane (PM) proteins. A systematic study of PM protein modulation by vaccinia virus (VACV), the paradigm of host regulation, has the potential to reveal not only novel viral immune evasion mechanisms, but also novel factors critical in host immunity. Here, >1000 PM proteins were quantified throughout VACV infection, revealing selective downregulation of known T and NK cell ligands including HLA-C, downregulation of cytokine receptors including IFNAR2, IL-6ST and IL-10RB, and rapid inhibition of expression of certain protocadherins and ephrins, candidate activating immune ligands. Downregulation of most PM proteins occurred via a proteasome-independent mechanism. Upregulated proteins included a decoy receptor for TRAIL. Twenty VACV-encoded PM proteins were identified, of which five were not recognised previously as such. Collectively, this dataset constitutes a valuable resource for future studies on antiviral immunity, host-pathogen interaction, poxvirus biology, vector-based vaccine design and oncolytic therapy.

Smallpox vaccination induces a substantial increase in commensal skin bacteria that promote pathology and influence the host response.

Interactions between pathogens, host microbiota and the immune system influence many physiological and pathological processes. In the 20th century, widespread dermal vaccination with vaccinia virus (VACV) led to the eradication of smallpox but how VACV interacts with the microbiota and whether this influences the efficacy of vaccination are largely unknown. Here we report that intradermal vaccination with VACV induces a large increase in the number of commensal bacteria in infected tissue, which enhance recruitment of inflammatory cells, promote tissue damage and influence the host response. Treatment of vaccinated specific-pathogen-free (SPF) mice with antibiotic, or infection of genetically-matched germ-free (GF) animals caused smaller lesions without alteration in virus titre. Tissue damage correlated with enhanced neutrophil and T cell infiltration and levels of pro-inflammatory tissue cytokines and chemokines. One month after vaccination, GF and both groups of SPF mice had equal numbers of VACV-specific CD8+ T cells and were protected from disease induced by VACV challenge, despite lower levels of VACV-neutralising antibodies observed in GF animals. Thus, skin microbiota may provide an adjuvant-like stimulus during vaccination with VACV and influence the host response to vaccination.

Self-compassion in individuals with substance use disorder: the association with personal growth and well-being.

Individuals with Substance Use Disorder (SUD) who do not have empathy toward oneself, or self-compassion, may limit their opportunities for personal growth and overall well-being. Due to scarce empirical studies examining interactions between self-compassion, personal growth and well-being in persons with SUD, the goal of this research was to examine associations among these concepts. A survey was administered to patients in treatment for SUD using validated scales (Sussex-Oxford Compassion for the Self Scale (SSOCS-S), Personal Growth Initiative (PGI) Scale-II, and World Health Organization (WHO)-5 Well-Being Index) and 153 responses were collected. Over two-thirds (69.9%) of the sample were seeking treatment for addiction to prescription opioids; other substances used included heroin (37.3%), methamphetamine (30.7%), benzodiazepines (17%), fentanyl (15.7%), cocaine (9.8%), alcohol (9.8%), and other substances (3.9%). Gender identity, age, and length of treatment did not correlate with personal growth or well-being. Total self-compassion was significantly associated with personal growth initiative (r = .568, p < 0.001) and each of PGI subscales. Additionally, total self-compassion was significantly associated with well-being (r = .567, p < 0.001). When interacting with persons with SUD, we should instill the practice of self-compassion which may help accomplish personal growth and enhance well-being. These individuals need support and humanity from not only providers and loved ones, but also themselves.

Suppression of innate immunity by the vaccinia virus protein N1 promotes skin microbiota expansion and increased immune infiltration following vaccination.

Vaccinia virus (VACV) protein N1 is an intracellular immunomodulator that contributes to virus virulence via inhibition of NF-κB. Intradermal infection with a VACV lacking gene N1L (vΔN1) results in smaller skin lesions than infection with wild-type virus (WT VACV), but the impact of N1 deletion on the local microbiota as well as the innate and cellular immune responses in infected ear tissue is mostly uncharacterized. Here, we analysed the bacterial burden and host immune response at the site of infection and report that the presence of protein N1 correlated with enhanced expansion of skin microbiota, even before lesion development. Furthermore, early after infection (days 1-3), prior to lesion development, the levels of inflammatory mediators were higher in vΔN1-infected tissue compared to WT VACV infection. In contrast, infiltration of ear tissue with myeloid and lymphoid cells was greater after WT VACV infection and there was significantly greater secondary bacterial infection that correlated with greater lesion size. We conclude that a more robust innate immune response to vΔN1 infection leads to better control of virus replication, less bacterial growth and hence an overall reduction of tissue damage and lesion size. This analysis shows the potent impact of a single viral immunomodulator on the host immune response and the pathophysiology of VACV infection in the skin.

Red blood cell ATP release correlates with red blood cell hemolysis.

The precise matching of blood flow to skeletal muscle during exercise remains an important area of investigation. Release of adenosine triphosphate (ATP) from red blood cells (RBCs) is postulated as a mediator of peripheral vascular tone in response to shear stress, hypoxia, and mechanical deformation. We tested the following hypotheses: 1) RBCs of different densities contain different quantities of ATP; 2) hypoxia is a stimulus for ATP release from RBCs; and 3) hypoxic ATP release from RBCs is related to RBC lysis. Human blood was drawn from male and female volunteers (n = 11); the RBCs were isolated and washed. A Percoll gradient was used to separate RBCs based on cellular density. Density groups were then resuspended to 4% hematocrit and exposed to normoxia or hypoxia in a tonometer. Equilibrated samples were drawn and centrifuged; paired analyses of ATP (luminescence via a luciferase-catalyzed reaction) and hemolysis (Harboe spectrophotometric absorbance assay) were measured in the supernatant. ATP release was not different among low-density cells versus middle-density versus high-density cells. Similarly, hemoglobin (Hb) release was not different among the red blood cell subsets. No difference was found for either ATP release or Hb release following matched exposure to normoxic or hypoxic gas. The concentrations of ATP and Hb for all subsets combined were linearly correlated (r = 0.59, P ≤ 0.001). With simultaneous probing for Hb and ATP in the supernatant of each sample, we conclude that ATP release from RBCs can be explained by hemolysis and that hypoxia per se does not stimulate either ATP release or Hb release from RBCs.

Exploiting Connections for Viral Replication.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the COVID-19 (coronavirus disease 2019) pandemic, is a positive strand RNA (+RNA) virus. Like other +RNA viruses, SARS-CoV-2 is dependent on host cell metabolic machinery to survive and replicate, remodeling cellular membranes to generate sites of viral replication. Viral RNA-containing double-membrane vesicles (DMVs) are a striking feature of +RNA viral replication and are abundant in SARS-CoV-2-infected cells. Their generation involves rewiring of host lipid metabolism, including lipid biosynthetic pathways. Viruses can also redirect lipids from host cell organelles; lipid exchange at membrane contact sites, where the membranes of adjacent organelles are in close apposition, has been implicated in the replication of several +RNA viruses. Here we review current understanding of DMV biogenesis. With a focus on the exploitation of contact site machinery by +RNA viruses to generate replication organelles, we discuss evidence that similar mechanisms support SARS-CoV-2 replication, protecting its RNA from the host cell immune response.

An intron variant of the GLI family zinc finger 3 (GLI3) gene differentiates resistance training-induced muscle fiber hypertrophy in younger men.

We examined the association between genotype and resistance training-induced changes (12 wk) in dual x-ray energy absorptiometry (DXA)-derived lean soft tissue mass (LSTM) as well as muscle fiber cross-sectional area (fCSA; vastus lateralis; n = 109; age = 22 ± 2 y, BMI = 24.7 ± 3.1 kg/m2 ). Over 315 000 genetic polymorphisms were interrogated from muscle using DNA microarrays. First, a targeted investigation was performed where single nucleotide polymorphisms (SNP) identified from a systematic literature review were related to changes in LSTM and fCSA. Next, genome-wide association (GWA) studies were performed to reveal associations between novel SNP targets with pre- to post-training change scores in mean fCSA and LSTM. Our targeted investigation revealed no genotype-by-time interactions for 12 common polymorphisms regarding the change in mean fCSA or change in LSTM. Our first GWA study indicated no SNP were associated with the change in LSTM. However, the second GWA study indicated two SNP exceeded the significance level with the change in mean fCSA (P = 6.9 × 10-7 for rs4675569, 1.7 × 10-6 for rs10263647). While the former target is not annotated (chr2:205936846 (GRCh38.p12)), the latter target (chr7:41971865 (GRCh38.p12)) is an intron variant of the GLI Family Zinc Finger 3 (GLI3) gene. Follow-up analyses indicated fCSA increases were greater in the T/C and C/C GLI3 genotypes than the T/T GLI3 genotype (P < .05). Data from the Auburn cohort also revealed participants with the T/C and C/C genotypes exhibited increases in satellite cell number with training (P < .05), whereas T/T participants did not. Additionally, those with the T/C and C/C genotypes achieved myonuclear addition in response to training (P < .05), whereas the T/T participants did not. In summary, this is the first GWA study to examine how polymorphisms associate with the change in hypertrophy measures following resistance training. Future studies are needed to determine if the GLI3 variant differentiates hypertrophic responses to resistance training given the potential link between this gene and satellite cell physiology.

Vaccinia Virus Infection Inhibits Skin Dendritic Cell Migration to the Draining Lymph Node.

There is a paucity of information on dendritic cell (DC) responses to vaccinia virus (VACV), including the traffic of DCs to the draining lymph node (dLN). In this study, using a mouse model of infection, we studied skin DC migration in response to VACV and compared it with the tuberculosis vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG), another live attenuated vaccine administered via the skin. In stark contrast to BCG, skin DCs did not relocate to the dLN in response to VACV. Infection with UV-inactivated VACV or modified VACV Ankara promoted DC movement to the dLN, indicating that interference with skin DC migration requires replication-competent VACV. This suppressive effect of VACV was capable of mitigating responses to a secondary challenge with BCG in the skin, ablating DC migration, reducing BCG transport, and delaying CD4+ T cell priming in the dLN. Expression of inflammatory mediators associated with BCG-triggered DC migration were absent from virus-injected skin, suggesting that other pathways invoke DC movement in response to replication-deficient VACV. Despite adamant suppression of DC migration, VACV was still detected early in the dLN and primed Ag-specific CD4+ T cells. In summary, VACV blocks skin DC mobilization from the site of infection while retaining the ability to access the dLN to prime CD4+ T cells.