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Tumour dendritic cells (DCs) internalise antigen and upregulate CCR7, which directs their migration to tumour-draining lymph nodes (dLN). CCR7 expression is coupled to an activation programme enriched in regulatory molecule expression, including PD-L1. However, the spatio-temporal dynamics of CCR7 + DCs in anti-tumour immune responses remain unclear. Here, we use photoconvertible mice to precisely track DC migration. We report that CCR7 + DCs are the dominant DC population that migrate to the dLN, but a subset remains tumour-resident despite CCR7 expression. These tumour-retained CCR7 + DCs are phenotypically and transcriptionally distinct from their dLN counterparts and heterogeneous. Moreover, they progressively downregulate the expression of antigen presentation and pro-inflammatory transcripts with more prolonged tumour dwell-time. Tumour-residing CCR7 + DCs co-localise with PD-1 + CD8 + T cells in human and murine solid tumours, and following anti-PD-L1 treatment, upregulate stimulatory molecules including OX40L, thereby augmenting anti-tumour cytolytic activity. Altogether, these data uncover previously unappreciated heterogeneity in CCR7 + DCs that may underpin a variable capacity to support intratumoural cytotoxic T cells. Recognition of tumour antigen induces dendritic cell activation and migration to the lymph node. Here, the authors use photoconvertible mice to demonstrate that some activated dendritic cells are retained in tumours and gradually lose function, but their ability to support local anti-tumour responses can be augmented by anti-PD-L1 blockade.

Genetic variations underlying susceptibility to complex autoimmune and allergic diseases are concentrated within noncoding regulatory elements termed enhancers 1 . The functions of a large majority of disease-associated enhancers are unknown, in part owing to their distance from the genes they regulate, a lack of understanding of the cell types in which they operate, and our inability to recapitulate the biology of immune diseases in vitro. Here, using shared synteny to guide loss-of-function analysis of homologues of human enhancers in mice, we show that the prominent autoimmune and allergic disease risk locus at chromosome 11q13.5 2 – 7 contains a distal enhancer that is functional in CD4 + regulatory T (T reg ) cells and required for T reg -mediated suppression of colitis. The enhancer recruits the transcription factors STAT5 and NF-κB to mediate signal-driven expression of Lrrc32 , which encodes the protein glycoprotein A repetitions predominant (GARP). Whereas disruption of the Lrrc32 gene results in early lethality, mice lacking the enhancer are viable but lack GARP expression in Foxp3 + T reg cells, which are unable to control colitis in a cell-transfer model of the disease. In human T reg cells, the enhancer forms conformational interactions with the promoter of LRRC32 and enhancer risk variants are associated with reduced histone acetylation and GARP expression. Finally, functional fine-mapping of 11q13.5 using CRISPR-activation (CRISPRa) identifies a CRISPRa-responsive element in the vicinity of risk variant rs11236797 capable of driving GARP expression. These findings provide a mechanistic basis for association of the 11q13.5 risk locus with immune-mediated diseases and identify GARP as a potential target in their therapy. Shared synteny guides loss-of-function analysis of human enhancer homologues in mice, identifying a distal enhancer at the autoimmune and allergic disease risk locus at chromosome 11q13.5 whose function in regulatory T cells provides a mechanistic basis for its role in disease.

Whereas effector CD4 + and CD8 + T cells promote immune activation and can drive clearance of infections and cancer, CD4 + regulatory T (T reg ) cells suppress their function, contributing to both immune homeostasis and cancer immunosuppression. The transcription factor BACH2 functions as a pervasive regulator of T cell differentiation, promoting development of CD4 + T reg cells and suppressing the effector functions of multiple effector T cell (T eff ) lineages. Here, we report the development of a stable cell-based bioluminescence assay of the transcription factor activity of BACH2. Tetracycline-inducible BACH2 expression resulted in suppression of phorbol 12-myristate 13-acetate (PMA)/ionomycin-driven activation of a luciferase reporter containing BACH2/AP-1 target sequences from the mouse Ifng  + 18k enhancer. BACH2 expression repressed the luciferase signal in a dose-dependent manner but this activity was abolished at high levels of AP-1 signalling, suggesting contextual regulation of AP-1 driven gene expression by BACH2. Finally, using the reporter assay developed, we find that the histone deacetylase 3 (HDAC3)-selective inhibitor, RGFP966, inhibits BACH2-mediated repression of signal-driven luciferase expression. In addition to enabling mechanistic studies, this cell-based reporter may enable identification of small molecule agonists or antagonists of BACH2 function for drug development.

Genetic variations underlying susceptibility to complex autoimmune and allergic diseases are concentrated within noncoding regulatory elements termed enhancers . The functions of a large majority of disease-associated enhancers are unknown, in part owing to their distance from the genes they regulate, a lack of understanding of the cell types in which they operate, and our inability to recapitulate the biology of immune diseases in vitro. Here, using shared synteny to guide loss-of-function analysis of homologues of human enhancers in mice, we show that the prominent autoimmune and allergic disease risk locus at chromosome 11q13.5 contains a distal enhancer that is functional in CD4 regulatory T (T ) cells and required for T -mediated suppression of colitis. The enhancer recruits the transcription factors STAT5 and NF-κB to mediate signal-driven expression of Lrrc32, which encodes the protein glycoprotein A repetitions predominant (GARP). Whereas disruption of the Lrrc32 gene results in early lethality, mice lacking the enhancer are viable but lack GARP expression in Foxp3 T cells, which are unable to control colitis in a cell-transfer model of the disease. In human T cells, the enhancer forms conformational interactions with the promoter of LRRC32 and enhancer risk variants are associated with reduced histone acetylation and GARP expression. Finally, functional fine-mapping of 11q13.5 using CRISPR-activation (CRISPRa) identifies a CRISPRa-responsive element in the vicinity of risk variant rs11236797 capable of driving GARP expression. These findings provide a mechanistic basis for association of the 11q13.5 risk locus with immune-mediated diseases and identify GARP as a potential target in their therapy.

Immune checkpoint inhibitors (ICI), such as anti-PD-1, have revolutionized cancer treatment, but they are only effective for a minority of patients. The gut microbiome plays a crucial role in modulating immunotherapy treatment responses, and previous studies correlated lipopolysaccharide (LPS)-producing gut microbes with poorer prognosis. However, LPS from diverse bacterial species have activities ranging from immunostimulatory to inhibitory. By functionally analyzing fecal metagenomes from 112 melanoma patients prior to anti-PD-1 therapy, we found that a subset of LPS-producing bacteria encoding immunostimulatory hexa-acylated LPS was enriched in the microbiomes of clinical responders. We confirmed robust activation of the NF-kB pathway by hexa-acylated LPS in vitro, and this activation was significantly inhibited by penta-acylated LPS in a dose-dependent manner. Importantly, oral administration of hexa-acylated LPS augmented anti-PD-1-mediated anti-tumor immunity in an in vivo mouse model of cancer immunotherapy. Microbiome hexa-acylated LPS may therefore represent an accessible predictor and potential enhancer of clinical anti- PD-1 immunotherapy responses. Functional rather than taxonomic profiling of patient gut microbiomes reveals hexa-acylated LPS as a novel biomarker of responsiveness and a targetable pathway for enhancing responses to anti-PD-1, informing future studies and current patient treatment.

Tumour dendritic cells (DCs) internalise antigen and upregulate CCR7, which directs their migration to tumour-draining lymph nodes (dLN). CCR7 expression is coupled to a maturation programme enriched in regulatory molecule expression, including PD-L1, termed mRegDC. However, the spatio- temporal dynamics and role of mRegDCs in anti-tumour immune responses remain unclear. Using photoconvertible mice to precisely track DC migration, we found that mRegDCs were the dominant DC population arriving in the dLN, but a subset remained tumour-resident despite CCR7 expression. These tumour-retained mRegDCs were phenotypically and transcriptionally distinct from their dLN counterparts and were heterogeneous. Specifically, they demonstrated a progressive reduction in the expression of antigen presentation and pro-inflammatory transcripts with more prolonged tumour dwell-time. Tumour mRegDCs spatially co-localised with PD-1+CD8+ T cells in human and murine solid tumours. Following anti-PD-L1 treatment, tumour-residing mRegDCs adopted a state enriched in lymphocyte stimulatory molecules, including OX40L, which was capable of augmenting anti- tumour cytolytic activity. Altogether, these data uncover previously unappreciated heterogeneity in mRegDCs that may underpin a variable capacity to support intratumoural cytotoxic T cells, and provide insights into their role in cancer immunotherapy.

Introduction: Methamphetamine use is on the rise with increasing emergency department (ED) visits, behavioral health crises, and deaths associated with use and overdose. Emergency clinicians describe methamphetamine use as a significant problem with high resource utilization and violence against staff, but little is known about the patient’s perspective. In this study our objective was to identify the motivations for initiation and continued methamphetamine use among people who use methamphetamine and their experiences in the ED to guide future ED-based approaches. Methods: This was a qualitative study of adults residing in the state of Washington in 2020, who used methamphetamine in the prior 30 days, met criteria for moderate- to high-risk use, reported recently receiving care in the ED, and had phone access. Twenty individuals were recruited to complete a brief survey and semistructured interview, which was recorded and transcribed prior to being coded. Modified grounded theory guided the analysis, and the interview guide and codebook were iteratively refined. Three investigators coded the interviews until consensus was reached. Data was collected until thematic saturation. Results: Participants described a shifting line that separates the positive attributes from the negative consequences of using methamphetamine. Many initially used methamphetamine to enhance social interactions, combat boredom, and escape difficult circumstances by numbing the senses. However, continued use regularly led to isolation, ED visits for the medical and psychological sequelae of methamphetamine use, and engagement in increasingly risky behaviors. Because of their overwhelmingly frustrating experiences in the past, interviewees anticipated difficult interactions with healthcare clinicians, leading to combativeness in the ED, avoidance of the ED at all costs, and downstream medical complications. Participants desired a non-judgmental conversation and linkage to outpatient social resources and addiction treatment. Conclusion: Methamphetamine use can lead patients to seek care in the ED, where they often feel stigmatized and are provided little assistance. Emergency clinicians should acknowledge addiction as a chronic condition, address acute medical and psychiatric symptoms adequately, and provide positive connections to addiction and medical resources. Future work should incorporate the perspectives of people who use methamphetamine into ED-based programs and interventions.

BackgroundRoutine population-based screening for depression is an essential part of evolving health care models integrating care for mental health in primary care. Depression instruments often include questions about suicidal thoughts, but how patients experience these questions in primary care is not known and may have implications for accurate identification of patients at risk.ObjectivesTo explore the patient experience of routine population-based depression screening/assessment followed, for some, by suicide risk assessment and discussions with providers.DesignQualitative, interview-based study.ParticipantsThirty-seven patients from Kaiser Permanente Washington who had recently screened positive for depression on the 2-item Patient Health Questionnaire [PHQ] and completed the full PHQ-9.ApproachCriterion sampling identified patients who had recently completed the PHQ-9 ninth question which asks about the frequency of thoughts about self-harm. Patients completed semi-structured interviews by phone, which were recorded and transcribed. Directive and conventional content analyses were used to apply knowledge from prior research and elucidate new information from interviews; thematic analysis was used to organize key content overall and across groups based on endorsement of suicide ideation.Key ResultsFour main organizing themes emerged from analyses: (1) Participants believed being asked about suicidality was contextually appropriate and valuable, (2) some participants described a mismatch between their lived experience and the PHQ-9 ninth question, (3) suicidality disclosures involved weighing hope for help against fears of negative consequences, and (4) provider relationships and acts of listening and caring facilitated discussions about suicidality.ConclusionsAll participants believed being asked questions about suicidal thoughts was appropriate, though some who disclosed suicidal thoughts described experiencing stigma and sometimes distanced themselves from suicidality. Direct communication with trusted providers, who listened and expressed empathy, bolstered comfort with disclosure. Future research should consider strategies for reducing stigma and encouraging fearless disclosure among primary care patients experiencing suicidality.

Purpose Peptide antigens have been administered by different approaches as cancer vaccine therapy, including direct injection or pulsed onto dendritic cells; however, the optimal delivery method is still debatable. In this study, we describe the immune response elicited by two vaccine approaches using the wild-type (wt) p53 vaccine. Experimental design Twenty-one HLA-A2.1 patients with stage III, IV, or recurrent ovarian cancer overexpressing the p53 protein with no evidence of disease were treated in two cohorts. Arm A received SC wt p53:264-272 peptide admixed with Montanide and GM-CSF. Arm B received wt p53:264-272 peptide-pulsed dendritic cells IV. Interleukin-2 (IL-2) was administered to both cohorts in alternative cycles. Results Nine of 13 patients (69%) in arm A and 5 of 6 patients (83%) in arm B developed an immunologic response as determined by ELISPOT and tetramer assays. The vaccine caused no serious systemic side effects. IL-2 administration resulted in grade 3 and 4 toxicities in both arms and directly induced the expansion of T regulatory cells. The median overall survival was 40.8 and 29.6 months for arm A and B, respectively; the median progression-free survival was 4.2 and. 8.7 months, respectively. Conclusion We found that using either vaccination approach generates comparable specific immune responses against the p53 peptide with minimal toxicity. Accordingly, our findings suggest that the use of less demanding SC approach may be as effective. Furthermore, the use of low-dose SC IL-2 as an adjuvant might have interfered with the immune response. Therefore, it may not be needed in future trials.

Despite demonstrated improvement in patient outcomes with use of the Pediatric Traumatic Brain Injury (TBI) Guidelines (Guidelines), there are differential rates of adherence. Provider perspectives on barriers and facilitators to adherence have not been elucidated. This study aimed to identify and explore in depth the provider perspective on factors associated with adherence to the Guidelines using 19 focus groups with nurses and physicians who provided acute management for pediatric patients with TBI at five university-affiliated Level 1 trauma centers. Data were examined using deductive and inductive content analysis. Results indicated that three inter-related domains were associated with clinical adherence: 1) perceived guideline credibility and applicability to individual patients, 2) implementation, dissemination, and enforcement strategies, and 3) provider culture, communication styles, and attitudes towards protocols. Specifically, Guideline usefulness was determined by the perceived relevance to the individual patient given age, injury etiology, and severity and the strength of the evidence. Institutional methods to formally endorse, codify, and implement the Guidelines into the local culture were important. Providers wanted local protocols developed using interdisciplinary consensus. Finally, a culture of collaboration, including consistent, respectful communication and interdisciplinary cooperation, facilitated adherence. Provider training and experience, as well as attitudes towards other standardized care protocols, mirror the use and attitudes towards the Guidelines. Adherence was determined by the interaction of each of these guideline, institutional, and provider factors acting in concert. Incorporating provider perspectives on barriers and facilitators to adherence into hospital and team protocols is an important step toward improving adherence and ultimately patient outcomes.