Insomnia symptoms and neurofunctional correlates among adults receiving buprenorphine for opioid use disorder.

OBJECTIVES: Insomnia symptoms are negatively related to opioid use disorder (OUD) treatment outcomes, possibly reflecting the influence of sleep on neurofunctional domains implicated in addiction. Moreover, the intersection between OUD recovery and sleep represents an area well-suited for the development of novel, personalized treatment strategies. This study assessed the prevalence of clinically significant insomnia symptoms and characterized its neurofunctional correlates among a clinical sample of adults with OUD receiving buprenorphine. METHODS: Adults (N = 129) receiving buprenorphine for OUD from an outpatient clinic participated in a cross-sectional survey. Participants completed an abbreviated version of NIDA's Phenotyping Assessment Battery, which assessed 6 neurofunctional domains: sleep, negative emotionality, metacognition, interoception, cognition, and reward. Bivariate descriptive statistics compared those with evidence of clinically significant insomnia symptoms (Insomnia Severity Index [ISI] score of ≥11) to those with minimal evidence of clinically significant insomnia symptoms (ISI score of ≤10) across each of the neurofunctional domains. RESULTS: Roughly 60% of participants reported clinically significant insomnia symptoms (ISI score of ≥11). Experiencing clinically significant insomnia symptoms was associated with reporting greater levels of depression, anxiety, post-traumatic stress, stress intolerance, unhelpful metacognition, and interoceptive awareness (ps<0.05). Participants with evidence of clinically significant insomnia were more likely to report that poor sleep was interfering with their OUD treatment and that improved sleep would assist with their treatment (ps<0.05). CONCLUSIONS: Insomnia was prevalent among adults receiving buprenorphine for OUD. Insomnia was associated with neurofunctional performance, which may impact OUD treatment trajectories. Our findings indicate potential targets in the development of personalized treatment plans for patients with co-morbid insomnia and OUD. To inform the development of novel treatment strategies, more research is needed to understand the potential mechanistic links between sleep disturbances and substance use.

Impact of Smoking Cessation Motivations and Barriers on Quit Intentions Following a Hypothetical Flavor Ban Among African American/Black Individuals Who Use Menthol Cigarettes: A Mixed-Methods Study.

INTRODUCTION: The Food and Drug Administration (FDA) has proposed banning cigarettes and cigars with characterizing flavors-products used disproportionately by African American/black (AA/B) individuals. Little is known about how AA/B individuals who smoke menthol cigarettes will respond to flavor bans or how to amplify the intended benefits. This study explored predictors of quit intentions following a hypothetical flavor ban and further probed anticipated ban-related responses. AIMS AND METHODS: We recruited 213 AA/B individuals who use menthol cigarettes from Richmond, VA (September 2021-August 2022) for a mixed-methods study. Participants rated seven motivations for quitting and six barriers to quitting (Not a motivation or challenge[1]-Major motivation or challenge[4]), then reported how likely they were to quit smoking if characterizing flavors were banned in cigarettes and cigars. A subsample of 31 participants completed semi-structured interviews to further explore reactions to flavor restriction policies. RESULTS: Multivariable linear regressions suggested that participants who were more motivated to quit smoking because of "information about health hazards" and the "cost of cigarettes" reported higher quit intentions following a hypothetical menthol ban (p < .05). Additionally, those with cessation-related weight concerns reported lower post-ban quit intentions (p < .05). Interview themes highlighted smoking for stress reduction, harm/addiction perceptions of flavored tobacco products, trusted sources of tobacco-related information (including testimonials from people who formerly smoked), potential ban responses, and varying experiences with cessation strategies. CONCLUSIONS: Culturally specific cessation strategies that emphasize the health-related benefits of quitting, particularly those featuring the experiences of people who formerly smoked, may help AA/B individuals who smoke menthol cigarettes quit following a menthol ban. IMPLICATIONS: For the FDA's proposed bans on characterizing flavors in cigarettes and cigars to advance racial health equity, they must maximize cessation among African American/black (AA/B) individuals who use menthol cigarettes. This work suggests information on the health hazards and costs of smoking, as well as concerns over gaining weight, were predictors of quit intentions in a hypothetical flavor ban. Qualitative data suggest messaging highlighting the experiences of individuals who successfully quit may constitute an effective communication strategy. These insights can be used in the development of culturally specific cessation strategies for AA/B individuals who smoke menthol cigarettes.

Expanding on cross-price elasticity: Understanding tobacco product demand and substitution from the cross-price purchase task.

We examine whether cigarettes serve as substitutes for electronic nicotine delivery systems (ENDS) among ENDS users and demonstrate methodological extensions of data from a cross-price purchase task to inform policies and interventions. During a clinical laboratory study, n = 19 exclusive ENDS users and n = 17 dual cigarette/ENDS users completed a cross-price purchase task with cigarettes available at a fixed price while prices of own-brand ENDS increased. We estimated cross-price elasticity using linear models to examine substitutability. We defined five additional outcomes: nonzero cross-price intensity (purchasing cigarettes if ENDS were free), constant null demand (not purchasing cigarettes at any ENDS price), cross-product crossover point (first price where participants purchased more cigarettes than ENDS), dual-demand score (percentage of prices where both products were purchased), and dual-use break point (minimum relative price to force complete substitution). The cross-price elasticity results indicated that cigarettes could serve as substitutes for ENDS among ENDS users on average, but this average effect masked substantial heterogeneity in profiles of demand (here, a measure of the drug's reinforcement potential). Policies and regulations that increase ENDS prices appear unlikely to steer most exclusive ENDS users toward cigarette use, as most would not purchase cigarettes at any ENDS price, but they could prompt some dual users to substitute cigarettes completely while others remain dual users. This heterogeneity in consumer responses suggests additional indices of cross-product demand are useful to characterize the anticipated and unanticipated effects of tobacco price policies more fully.

Vaccine adjuvant-elicited CD8+ T cell immunity is co-dependent on T-bet and FOXO1.

T-bet and FOXO1 are transcription factors canonically associated with effector and memory T cell fates, respectively. During an infectious response, these factors direct the development of CD8+ T cell fates, where T-bet deficiency leads to ablation of only short-lived effector cells, while FOXO1 deficiency results in selective loss of memory. In contrast, following adjuvanted subunit vaccination in mice, both effector- and memory-fated T cells are compromised in the absence of either T-bet or FOXO1. Thus, unlike responses to challenge with Listeria monocytogenes, productive CD8+ T cell responses to adjuvanted vaccination require coordinated regulation of FOXO1 and T-bet transcriptional programs. Single-cell RNA sequencing analysis confirms simultaneous T-bet, FOXO1, and TCF1 transcriptional activity in vaccine-elicited, but not infection-elicited, T cells undergoing clonal expansion. Collectively, our data show that subunit vaccine adjuvants elicit T cell responses dependent on transcription factors associated with effector and memory cell fates.

Racial/ethnic differences in the association between menthol cigarette use and mental illness among adults who smoke in the United States.

Data from the National Survey on Drug Use and Health (2012-2018) were used to characterize the association between menthol cigarette use and indicators of Any (AMI) and Serious (SMI) Mental Illness among adults who smoke in the United States. In general, people who smoke menthol cigarettes were more likely to have AMI (aOR = 1.123 [1.063-1.194]) than people who smoke non-menthol cigarettes, but not SMI (aOR = 1.065 [0.966-1.175]). However, among non-Hispanic African American/Black people who smoke, those that used menthol cigarettes had lower adjusted odds of both AMI (aOR = 0.740 [0.572-0.958]) and SMI (aOR = 0.592 [0.390-0.899]) than their counterparts who used non-menthol cigarettes. Results suggest there may be race/ethnicity-specific drivers of the association between menthol cigarette use and mental illness.

Responses of African American Individuals Who Use Menthol Cigarettes to Potential Flavored Tobacco Bans.

INTRODUCTION: The U.S. Food and Drug Administration has proposed new product standards regarding the availability of menthol cigarettes and flavored cigars in the U.S. However, it is unclear whether limiting characterizing flavors in cigarettes and cigars as proposed, or across all tobacco products, produces differential effects on the tobacco use behaviors of African American/Black individuals who use menthol cigarettes. This study assessed whether limiting characterizing flavors in combusted products only or across all tobacco products produces differential impacts on the tobacco use behaviors of African American/Black individuals who use menthol cigarettes. METHODS: Adult African American/Black individuals who use menthol cigarettes in the U.S. were recruited through Qualtrics (n=373) and in Richmond, VA (n=206) for an online experiment from September 2021 to August 2022. Participants reported how their tobacco use behaviors would change under 3 scenarios: maintenance of the status quo, limited flavor ban (ban characterizing flavors in cigarettes and cigars), and comprehensive flavor ban (ban characterizing flavors in all tobacco products). Seemingly unrelated regressions compared differences in expected responses to policy scenarios (p<0.05). RESULTS: Both flavor ban scenarios resulted in higher quitting intentions for cigarettes and all tobacco products than the status quo (p<0.05). The comprehensive ban resulted in greater intentions to quit all tobacco products and lower intentions to switch to certain alternative products (e.g., E-cigarettes, smokeless tobacco, heated tobacco products) than the limited ban (p<0.05). CONCLUSIONS: African American/Black individuals who use menthol cigarettes appear more likely to quit smoking if characterizing flavors in combusted products (e.g., menthol cigarettes) are banned, regardless of if characterizing flavors are available in noncombusted alternative tobacco products.

The electronic nicotine delivery system (ENDS) purchase task: Are results sensitive to price framing?

Extension of the cigarette purchase task (CPT) to electronic nicotine delivery systems (ENDS) is complicated by the heterogeneous nature of this product class, as ambiguity exists regarding the appropriate price-frame (i.e., unit of the product being purchased). We explored correlations between ENDS purchase task (E-CPTs) outcomes featuring two common price-frames: 10 puffs and 1 mL of liquid. Adult exclusive ENDS users (N = 19) and dual users of ENDS and cigarettes (N = 16) completed two own-brand E-CPTs. One E-CPT used "10 puffs" as its price-frame; the other used "1 mL of liquid." Five outcomes were generated for each E-CPT: breakpoint, intensity, Omax, Pmax, and α. Exploratory Factor Analyses (EFA) considered how these outcomes captured latent structures of demand for ENDS. Spearman correlations in E-CPT outcomes assessed within-person variation between price-frames. Analyses also considered whether correlations differed by user group. E-CPT outcomes were highly correlated across price-frames (ρs > 0.57, ps < .001), and EFA revealed little difference in how outcomes from the tasks loaded onto two latent structures of demand ("Persistence" and "Amplitude") reported in the previous literature. The magnitude of correlations for E-CPT outcomes tended to be higher for exclusive ENDS users than for dual users. Participant responses to purchase task outcomes were similar across two E-CPT price-frames. Using "10 puffs" as a price-frame may be a generalizable approach among heterogenous groups of ENDS users, but researchers should consider their target population and structure the E-CPT to reflect participants' knowledge and purchasing behaviors. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Limited accrual of myxospores of Kudoa inornata (Cnidaria: Myxosporea) in their wild fish hosts, Cynoscion nebulosus (Teleostei: Sciaenidae).

Kudoa inornata is a myxosporean that infects the seatrout Cynoscion nebulosus. Increased prevalence of infection as fish age and absence of inflammation against plasmodia led to the hypothesis that seatrout retain and accumulate myxospores throughout their lives. However, opportunistic observations that wild-caught seatrout cleared infection when maintained in aquaculture conditions and evidence of encapsulated infected necrotic myofibers suggested that fish develop an immunity against this parasite, or that myxospores have a limited life span. To evaluate myxospore clearance and to test putative resistance to re-infection, we examined 44 wild-caught seatrout broodstock maintained in parasite-free water for 2-6 yr. Twenty-five fish served as negative controls (time zero of experiment), and 19 were exposed to water-borne K. inornata infective stages for 18 wk. Over 73% of the exposed fish became infected, compared to ~12% of control fish, indicating that fish were susceptible to re-infection by K. inornata. Whether plasmodia degenerate because K. inornata myxospores have a limited life span or seatrout develop an adaptive immunity against these life stages remains unknown. To test for accumulation of myxospores over time, we compared myxospore densities and intensities between sexes and across ages and sizes of wild seatrout. There was no significant difference in myxospore densities with size, age, or sex. However, intensities increased significantly with increasing fish age and size, indicating accrual of myxospores over time. These results combined with evidence of infection clearance suggest that K. inornata myxospores do not persist but nevertheless accrue in wild seatrout due to continuous contact with infective stages.

The interaction of nicotine concentration and device power on electronic nicotine delivery system (ENDS) abuse liability among exclusive ENDS users and dual users of ENDS and combustible cigarettes.

Open-system electronic nicotine delivery systems (ENDS) permit modifications to device characteristics such as power, potentially increasing nicotine and toxicant delivery. Limiting liquid nicotine concentration may carry unintended consequences by prompting users to increase device power to increase nicotine delivery. This study examined the abuse liability of ENDS across nicotine concentration and power settings. In a clinical laboratory study, n = 19 exclusive ENDS users and n = 13 dual ENDS/cigarette users, aged 21-55 completed four Latin-square ordered conditions that varied by liquid nicotine concentration (10 mg/ml [low], 30 mg/ml [high]) and device power (15 watts [low], 30 watts [high]), that were followed by a fifth own brand (OB) condition. A progressive ratio task (PRT) using bar presses to earn ENDS puffs was used to assess abuse liability and compare between conditions using mixed effects linear regressions. The low nicotine/high watt condition was associated with a significantly higher number of bar presses and puffs earned relative to the OB ENDS, high nicotine/high watt, and high nicotine/low watt conditions (p < .05). Findings appeared to be driven largely by exclusive ENDS users; most comparisons were not significant among dual users. Participants worked significantly harder for puffs of low nicotine/high watt ENDS, highlighting previous findings that suggest limiting liquid nicotine concentration without addressing power settings may be insufficient to reduce the abuse liability of ENDS. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Perceptions of Tobacco Product-Specific COVID-19 Risk and Changes in Tobacco Use Behaviors Among Smokers, E-Cigarette Users, and Dual Users.

INTRODUCTION: Coronavirus Disease 2019 (COVID-19) is a public health crisis, but its effects on tobacco users remain ill-defined. This report aimed to assess the relationship between tobacco product-specific risk perceptions for COVID-19 and changes in tobacco use since the start of the pandemic. METHODS: A sample (n = 776) of past-30 day exclusive smokers (n = 238), exclusive e-cigarette users (n = 143), and dual users (n = 395) residing in the US and aged 18 or older were collected using Mechanical Turk from April 27 to June 8, 2020. Adjusted associations between tobacco product-specific COVID-19 risk perceptions (ie risk that smokers/vapers are at for COVID-19 relative to non-smokers/non-vapers) and changes in tobacco use since the pandemic began were assessed using partial proportional odds models. RESULTS: A majority of those who used cigarettes (63.7%) and e-cigarettes (56.1%) felt that the risk of COVID-19 was greater for users of their tobacco product than for non-users. Twenty-four percent of smokers had increased their cigarette use since the start of the pandemic and 28.0% had decreased. Similarly, 27.3% of e-cigarette users had increased their e-cigarette use since the start of the pandemic and 23.8% had decreased. Higher risk perceptions for COVID-19 were associated with reductions in tobacco use since the pandemic began for exclusive e-cigarette users and dual users. CONCLUSIONS: These findings provide the support that tobacco product-specific COVID-19 risk perceptions may be an important correlate of changes in tobacco use during the pandemic. Targeted information to inform tobacco users regarding their risks for COVID-19 is needed during this public health crisis. IMPLICATIONS: Few published studies have investigated the relationship between tobacco product-specific risk perceptions for COVID-19 and changes in tobacco product use since the pandemic began. This study enhances the current literature by providing evidence that higher tobacco product-specific risk perceptions for COVID-19 are associated with reductions in tobacco use since the pandemic began for exclusive e-cigarette users and dual users of cigarettes and e-cigarettes. Additionally, daily tobacco users may be more likely to have increased their tobacco use than non-daily users. These findings emphasize the importance of disseminating targeted health information to tobacco users regarding COVID-19 risks.

Early Identification of Argatroban Resistance and the Consideration of Factor VIII.

BACKGROUND: Argatroban, a synthetic, parenteral, nonheparin anticoagulant, is a direct thrombin inhibitor indicated for the prophylaxis or treatment of venous thromboembolism (VTE) in patients with heparin-induced thrombocytopenia with thrombosis (HITT) and for use during percutaneous coronary intervention (PCI) in patients who have or are at risk for developing HITT. Although heparin resistance occurs in approximately 0.5% to 5% of heparin-treated patients and is well documented in the literature, argatroban resistance is limited to a single case report. The objective of this case is to describe a case in which argatroban resistance was suspected in a patient with critical limb ischemia. METHODS: This is a case report of a single patient. RESULTS: A 68-year-old female admitted for critical limb ischemia requiring vascular intervention was treated for presumed HITT with argatroban. A therapeutic activated partial thromboplastin time (aPTT) was not attained (31 seconds) despite multiple uptitrations of the dose to 2.8 µg/kg/min (adjusted based on the institutional protocol and with consideration of organ dysfunction). A coagulopathy workup revealed a high level of factor VIII (265%). CONCLUSION: This case supports early assessment of factor VIII levels and the consideration of argatroban resistance and in patients who have a subtherapeutic aPTT, despite multiple increases in dose with an elevated factor VIII level. Early identification should prompt the use of an alternative anticoagulant to ensure efficacy.

The CXCR4-Dependent LASP1-Ago2 Interaction in Triple-Negative Breast Cancer.

The CXCR4-LASP1 axis is an emerging target in the field of breast cancer metastasis. C-X-C chemokine receptor type 4 (CXCR4) mediates directed cell migration when activated by its cognate ligand CXCL12. LIM and SH3 Protein 1 (LASP1) is a critical node in the CXCR4 signaling pathway, as its deficiency blocks CXCR4-dependent Matrigel invasion. The mechanism by which LASP1 facilitates this invasive ability of tumor cells when CXCR4 is activated is unknown. Our previous proteomics work had revealed several components of the RNA interference (RNAi) machinery as being potential LASP1 interacting proteins. Here we report that argonaute 2 (Ago2), a protein with central involvement in RNAi, associates with LASP1 in triple-negative breast cancer (TNBC) cells. We demonstrate that LASP1 co-immunoprecipitates with Ago2 endogenously in a CXCL12-dependent manner, with further confirmation of this interaction by proximity ligation assay. Furthermore, this association is specific to CXCR4 as it can be abrogated by the CXCR4 antagonist, AMD3465. By GST-pulldown approach, we identify that LASP1 directly binds to Ago2 through its LIM and SH3 domains, and that this binding is dictated by the S146 and Y171 phosphorylation sites of LASP1. Additionally, the phosphorylation status of LASP1 affected tumor suppressor microRNA (miRNA) Let-7a-guided Ago2 activity. Levels of several endogenous targets of Let-7a were found to be altered including C-C chemokine receptor type 7 (CCR7), which is another critical chemokine receptor involved in metastasis to lymph nodes. Our results suggest a novel role for the LASP1-Ago2 module in shaping the RNAi landscape, functionally impacting the invasive ability of cancer cells.

Transformation Foci in IDH1-mutated Gliomas Show STAT3 Phosphorylation and Downregulate the Metabolic Enzyme ETNPPL, a Negative Regulator of Glioma Growth.

IDH1-mutated gliomas are slow-growing brain tumours which progress into high-grade gliomas. The early molecular events causing this progression are ill-defined. Previous studies revealed that 20% of these tumours already have transformation foci. These foci offer opportunities to better understand malignant progression. We used immunohistochemistry and high throughput RNA profiling to characterize foci cells. These have higher pSTAT3 staining revealing activation of JAK/STAT signaling. They downregulate RNAs involved in Wnt signaling (DAAM2, SFRP2), EGFR signaling (MLC1), cytoskeleton and cell-cell communication (EZR, GJA1). In addition, foci cells show reduced levels of RNA coding for Ethanolamine-Phosphate Phospho-Lyase (ETNPPL/AGXT2L1), a lipid metabolism enzyme. ETNPPL is involved in the catabolism of phosphoethanolamine implicated in membrane synthesis. We detected ETNPPL protein in glioma cells as well as in astrocytes in the human brain. Its nuclear localization suggests additional roles for this enzyme. ETNPPL expression is inversely correlated to glioma grade and we found no ETNPPL protein in glioblastomas. Overexpression of ETNPPL reduces the growth of glioma stem cells indicating that this enzyme opposes gliomagenesis. Collectively, these results suggest that a combined alteration in membrane lipid metabolism and STAT3 pathway promotes IDH1-mutated glioma malignant progression.

cDC1 IL-27p28 Production Predicts Vaccine-Elicited CD8+ T Cell Memory and Protective Immunity.

Although adjuvants and formulations are often either empirically derived, or at best judged by their ability to elicit broad inflammation, it would be ideal if specific innate correlates of adaptive immunity could be identified to set a universally applicable benchmark for adjuvant evaluation. Using an IL-27 reporter transgenic mouse model, we show in this study that conventional type 1 dendritic cell IL-27 production in the draining lymph node 12 h after s.c. vaccination directly correlates with downstream CD8+ T cell memory and protective immunity against infectious challenge. This correlation is robust, reproducible, predictive, entirely unique to vaccine biology, and is the only innate correlate of CD8+ T cell immune memory yet to be identified. Our results provide new insights into the basic biology of adjuvant-elicited cellular immunity and have clear implications for the screening and evaluation of novel adjuvants.

Targeting of the Eukaryotic Translation Initiation Factor 4A Against Breast Cancer Stemness.

Breast cancer stem cells (BCSCs) are intrinsically chemoresistant and capable of self-renewal. Following chemotherapy, patients can develop minimal residual disease due to BCSCs which can repopulate into a relapsed tumor. Therefore, it is imperative to co-target BCSCs along with the bulk tumor cells to achieve therapeutic success and prevent recurrence. So, it is vital to identify actionable molecular targets against both BCSCs and bulk tumor cells. Previous findings from our lab and others have demonstrated that inhibition of the emerging drug target eIF4A with Rocaglamide A (RocA) was efficacious against triple-negative breast cancer cells (TNBC). RocA specifically targets the pool of eIF4A bound to the oncogenic mRNAs that requires its helicase activity for their translation. This property enables specific targeting of tumor cells. The efficacy of RocA against BCSCs is unknown. In this study, we postulated that eIF4A could be a vulnerable node in BCSCs. In order to test this, we generated a paclitaxel-resistant TNBC cell line which demonstrated an elevated level of eIF4A along with increased levels of cancer stemness markers (ALDH activity and CD44), pluripotency transcription factors (SOX2, OCT4, and NANOG) and drug transporters (ABCB1, ABCG2, and ABCC1). Furthermore, genetic ablation of eIF4A resulted in reduced expression of ALDH1A1, pluripotency transcription factors and drug transporters. This pointed out that eIF4A is likely associated with selected set of proteins that are critical to BCSCs, and hence targeting eIF4A may eliminate BCSCs. Therefore, we isolated BCSCs from two TNBC cell lines: MDA-Bone-Un and SUM-159PT. Following RocA treatment, the self-renewal ability of the BCSCs was significantly reduced as determined by the efficiency of the formation of primary and secondary mammospheres. This was accompanied by a reduction in the levels of NANOG, OCT4, and drug transporters. Exposure to RocA also induced cell death of the BCSCs as evaluated by DRAQ7 and cell viability assays. RocA treatment induced apoptosis with increased levels of cleaved caspase-3. Overall, we identified that RocA is effective in targeting BCSCs, and eIF4A is an actionable molecular target in both BCSCs and bulk tumor cells. Therefore, anti-eIF4A inhibitors could potentially be combined synergistically with existing chemo-, radio- and/or immunotherapies.

Novel and Alternative Targets Against Breast Cancer Stemness to Combat Chemoresistance.

Breast cancer stem cells (BCSCs) play a vital role in tumor progression and metastasis. They are heterogeneous and inherently radio- and chemoresistant. They have the ability to self-renew and differentiate into non-BCSCs. These determinants of BCSCs including the plasticity between the mesenchymal and epithelial phenotypes often leads to minimal residual disease (MRD), tumor relapse, and therapy failure. By studying the resistance mechanisms in BCSCs, a combinatorial therapy can be formulated to co-target BCSCs and bulk tumor cells. This review addresses breast cancer stemness and molecular underpinnings of how the cancer stemness can lead to pharmacological resistance. This might occur through rewiring of signaling pathways and modulated expression of various targets that support survival and self-renewal, clonogenicity, and multi-lineage differentiation into heterogeneous bulk tumor cells following chemotherapy. We explore emerging novel and alternative molecular targets against BC stemness and chemoresistance involving survival, drug efflux, metabolism, proliferation, cell migration, invasion, and metastasis. Strategic targeting of such vulnerabilities in BCSCs may overcome the chemoresistance and increase the longevity of the metastatic breast cancer patients.

Central memory CD8+ T cells become CD69+ tissue-residents during viral skin infection independent of CD62L-mediated lymph node surveillance.

Memory CD8+ T cells in the circulation rapidly infiltrate non-lymphoid tissues following infection and provide protective immunity in an antigen-specific manner. However, the subsequent fate of memory CD8+ T cells after entering non-lymphoid tissues such as the skin during a secondary infection is largely unknown. Furthermore, because expression of CD62L is often used to identify the central memory (TCM) CD8+ T cell subset, uncoupling the physical requirement for CD62L-mediated lymph node homing versus other functional attributes of TCM CD8+ T cells remains unresolved. Here, we show that in contrast to naïve CD8+ T cells, memory CD8+ T cells traffic into the skin independent of CD62L-mediated lymph node re-activation and provide robust protective immunity against Vaccinia virus (VacV) infection. TCM, but not effector memory (TEM), CD8+ T cells differentiated into functional CD69+/CD103- tissue residents following viral clearance, which was also dependent on local recognition of antigen in the skin microenvironment. Finally, we found that memory CD8+ T cells expressed granzyme B after trafficking into the skin and utilized cytolysis to provide protective immunity against VacV infection. Collectively, these findings demonstrate that TCM CD8+ T cells become cytolytic following rapid infiltration of the skin to protect against viral infection and subsequently differentiate into functional CD69+ tissue-residents.

Clonal expansion of vaccine-elicited T cells is independent of aerobic glycolysis.

In contrast to responses against infectious challenge, T cell responses induced via adjuvanted subunit vaccination are dependent on interleukin-27 (IL-27). We show that subunit vaccine-elicited cellular responses are also dependent on IL-15, again in contrast to the infectious response. Early expression of interferon regulatory factor 4 (IRF4) was compromised in either IL-27- or IL-15-deficient environments after vaccination but not infection. Because IRF4 facilitates metabolic support of proliferating cells via aerobic glycolysis, we expected this form of metabolic activity to be reduced in the absence of IL-27 or IL-15 signaling after vaccination. Instead, metabolic flux analysis indicated that vaccine-elicited T cells used only mitochondrial function to support their clonal expansion. Loss of IL-27 or IL-15 signaling during vaccination resulted in a reduction in mitochondrial function, with no corresponding increase in aerobic glycolysis. Consistent with these observations, the T cell response to vaccination was unaffected by in vivo treatment with the glycolytic inhibitor 2-deoxyglucose, whereas the response to viral challenge was markedly lowered. Collectively, our data identify IL-27 and IL-15 as critical to vaccine-elicited T cell responses because of their capacity to fuel clonal expansion through a mitochondrial metabolic program previously thought only capable of supporting quiescent naïve and memory T cells.

IL-27p28 Production by XCR1+ Dendritic Cells and Monocytes Effectively Predicts Adjuvant-Elicited CD8+ T Cell Responses.

It is well accepted that the innate response is a necessary prerequisite to the formation of the adaptive response. This is true for T cell responses against infections or adjuvanted subunit vaccination. However, specific innate parameters with predictive value for the magnitude of an adjuvant-elicited T cell response have yet to be identified. We previously reported how T cell responses induced by subunit vaccination were dependent on the cytokine IL-27. These findings were unexpected, given that T cell responses to an infection typically increase in the absence of IL-27. Using a novel IL-27p28-eGFP reporter mouse, we now show that the degree to which an adjuvant induces IL-27p28 production from dendritic cells and monocytes directly predicts the magnitude of the T cell response elicited. To our knowledge, these data are the first to identify a concrete innate correlate of vaccine-elicited cellular immunity, and they have significant practical and mechanistic implications for subunit vaccine biology.