Background Solar urticaria is a rare photodermatosis characterised by rapid onset sunlight-induced urticaria, but its pathophysiology is not well understood. Objective To define the cutaneous cellular and molecular events in the evolution of solar urticaria following its initiation by solar simulated ultraviolet radiation (SSR), and compare with healthy controls (HC). Methods Cutaneous biopsies were taken from unexposed skin and skin exposed to a single low (physiological) dose of SSR, at 30 minutes, 3 and 24 hours post-exposure, in n=6 solar urticaria patients and n=6 HC. Biopsies were assessed by immunohistochemistry and bulk RNA-sequencing analysis. Results In solar urticaria, there was enrichment of several innate immune pathways, with striking early involvement of neutrophils, which was not observed in HC. Multiple pro-inflammatory cytokine and chemokine genes were upregulated (including IL20, IL6, CXCL8) or identified as upstream regulators (including TNF, IL1β, IFNγ). IgE and FcεR1 were identified as upstream regulators, and phosphorylated STAT3 expression in mast cells was increased in solar urticaria at 30 minutes and 3 hours post-SSR exposure, suggesting a mechanism of mast cell activation. Clinical resolution of solar urticaria by 24 hours mirrored resolution of inflammatory gene signature profiles. Comparison with available datasets of chronic spontaneous urticaria showed transcriptomic similarities relating to immune activation but several transcripts were identified solely in solar urticaria including CXCL8 and CSF2/3. Conclusions Solar urticaria is characterised by rapid STAT3 activation in mast cells and involvement of multiple chemotactic and innate inflammatory pathways, with FcεR1 engagement indicated as an early event.

Abstract Photoaggravated atopic dermatitis (PAD) is a subtype of atopic dermatitis (AD) that is exacerbated or provoked by exposure to ultraviolet radiation. However, its early pathomechanisms are poorly understood. Our aims were (i) to investigate cutaneous clinical, barrier, cellular and molecular changes in the evolution of PAD following low-dose solar simulated ultraviolet radiation (SSR) exposure, and (ii) to compare the findings in healthy controls exposed to the same SSR challenge. Three consecutive daily low-dose (10 J cm−2) SSR exposures were delivered to upper buttock skin of six patients with PAD and six healthy controls. Biopsies were taken from unexposed skin and from SSR-exposed skin at 30 min, 3 h and 24 h after the first exposure, and at 24 h after the third exposure (4 days from baseline). Biopsies were analysed by RNA sequencing using immunohistochemistry and Ingenuity Pathway Analysis. Baseline (unexposed) comparison of PAD vs. healthy control skin revealed 838 differentially expressed genes including significant upregulation of molecules associated with AD, such as inflammatory cytokines and chemokines (CCL1, CCL13, CCL18; all P < 0.01) and epidermal differentiation and proliferation markers (LCE3A, SPRR2B, S100A7; all P < 0.01), along with functional enrichment of immune cell trafficking, T helper (Th)2 pathway response, and cell signalling. Following SSR, clinically and histologically eczematous responses were provoked in patients with PAD, with histological abnormalities becoming progressively more abnormal from baseline to 4 days. Erythema scores were higher in PAD skin than in healthy controls at 24 h and 4 days (24-h erythema score 11.3 vs. 6.8, P < 0.01). In PAD 3 h post-SSR, transcriptomic analysis indicated activation or recruitment of lymphocytes, neutrophils and antigen-presenting cells (all P < 0.001 vs. unexposed skin). Upregulated cytokine genes included IL22, IL20 and IL6 (all P < 0.01 vs. unexposed skin). Interleukin (IL)-1α, tumour necrosis factor and several Toll-like receptors were identified as activated upstream regulators. These findings were not seen in healthy controls, and indicated that innate, Th1 and Th22 activation was provoked by SSR at an early stage. By 24 h post-SSR, additional upregulation of IL13 and CCL17 was consistent with augmented Th2 responses alongside more histologically eczematous features, including lymphocytic infiltration, acanthosis and spongiosis. The evolving dermal inflammatory infiltrate included dual positive CD4+ GATA3+, as well as CD69+ and CD11c+ cells, suggesting involvement of resident memory cells, myeloid dendritic cells and Th2 lymphocytes. In conclusion, PAD was characterized by a predominant Th2 signature, with the additional early involvement of pattern recognition responses, and innate and adaptive immune cells. These findings may inform treatment strategies for flares of both PAD and AD.

Background: Solar urticaria is a rare photodermatosis characterized by rapid-onset sunlight-induced urticaria, but its pathophysiology is not well understood. Objective: We sought to define cutaneous cellular and molecular events in the evolution of solar urticaria following its initiation by solar-simulated UV radiation (SSR) and compare with healthy controls (HC). Methods: Cutaneous biopsy specimens were taken from unexposed skin and skin exposed to a single low (physiologic) dose of SSR at 30 minutes, 3 hours, and 24 hours after exposure in 6 patients with solar urticaria and 6 HC. Biopsy specimens were assessed by immunohistochemistry and bulk RNAsequencing analysis. Results: In solar urticaria specimens, there was enrichment of several innate immune pathways, with striking early involvement of neutrophils, which was not observed in HC. Multiple proinflammatory cytokine and chemokine genes were upregulated (including IL20 , IL6 , and CXCL8 ) or identified as upstream regulators (including TNF, IL-1 b , and IFN- g ). IgE and Fc epsilon RI were identified as upstream regulators, and phosphorylated signal transducer and activator of transcription 3 expression in mast cells was increased in solar urticaria at 30 minutes and 3 hours after SSR exposure, suggesting a mechanism of mast cell activation. Clinical resolution of solar urticaria by 24 hours mirrored resolution of inflammatory gene signature profiles. Comparison with available datasets of chronic spontaneous urticaria showed transcriptomic similarities relating to immune activation, but several transcripts were identified solely in solar urticaria, including CXCL8 and CSF2/3 . Conclusions: Solar urticaria is characterized by rapid signal transducer and activator of transcription 3 activation in mast cells and involvement of multiple chemotactic and innate inflammatory pathways, with Fc epsilon RI engagement indicated as an early event. (J Allergy Clin Immunol 2024;153:1369-80.)

Regulation of cutaneous immunity is severely compromised in inflammatory skin disease. To investigate the molecular crosstalk underpinning tolerance versus inflammation in atopic dermatitis, we utilise a human in vivo allergen challenge study, exposing atopic dermatitis patients to house dust mite. Here we analyse transcriptional programmes at the population and single cell levels in parallel with immunophenotyping of cutaneous immunocytes revealed a distinct dichotomy in atopic dermatitis patient responsiveness to house dust mite challenge. Our study shows that reactivity to house dust mite was associated with high basal levels of TNF-expressing cutaneous Th17 T cells, and documents the presence of hub structures where Langerhans cells and T cells co-localised. Mechanistically, we identify expression of metallothioneins and transcriptional programmes encoding antioxidant defences across all skin cell types, that appear to protect against allergen-induced inflammation. Furthermore, single nucleotide polymorphisms in the MTIX gene are associated with patients who did not react to house dust mite, opening up possibilities for therapeutic interventions modulating metallothionein expression in atopic dermatitis.

Introduction: Neuroendocrine tumours (NETs) are rare tumours with an increasing incidence. While low- and intermediate-grade pancreatic NET (PanNET) and small intestinal NET (siNET) are slow growing, they have a relatively high rate of metastasizing to the liver, leading to substantially worse outcomes. In many solid tumours, the outcome is determined by the quality of the antitumour immune response. However, the quality and significance of antitumour responses in NETs are incompletely understood. This study provides clinico-pathological analyses of the tumour immune microenvironment in PanNET and siNETs. Methods: Formalin-fixed paraffin-embedded tissue from consecutive resected PanNETs (61) and siNETs (131) was used to construct tissue microarrays (TMAs); 1-mm cores were taken from the tumour centre, stroma, tumour edge, and adjacent healthy tissue. TMAs were stained with antibodies against CD8, CD4, CD68, FoxP3, CD20, and NCR1. T-cell counts were compared with counts from lung cancers. Results: For PanNET, median counts were CD8+ 35.4 cells/mm2, CD4+ 7.6 cells/mm2, and CD68+ macrophages 117.7 cells/mm2. For siNET, there were CD8+ 39.2 cells/mm2, CD4+ 24.1 cells/mm2, and CD68+ 139.2 cells/mm2. The CD8+ cell density in the tumour and liver metastases were significantly lower than in the adjacent normal tissues, without evidence of a cell-rich area at the tumour edge that might have suggested immune exclusion. T-cell counts in lung cancer were significantly higher than those in PanNET and siNETs: CD8+ 541 cells/mm2 and CD4+ 861 cells/mm2 (p ≤ 0.0001). Conclusion: PanNETs and siNETs are immune cold with no evidence of T cell exclusion; the low density of immune infiltrates indicates poor antitumour immune responses.

NK cells are the predominant innate lymphocyte subset that contribute to anti-tumor immunity. The molecular profile of human tumor-infiltrating NK cells, and particularly, their characteristics in relation to CD8 + cytotoxic T lymphocyte (CTL) density within tumors is poorly understood. Here, we performed single-cell and bulk transcriptomic analysis of NK cells within tumor and adjacent normal lung tissue from patients with treatment-naïve lung cancer. We found divergent NK cell subsets with distinct functional features that were associated with the magnitude of CD8 + CTL response in tumors. CD16 pos NK cells present in tumors with high density of CD8 + CTLs were enriched for transcripts linked to cytotoxicity, pro-inflammatory chemokines and effector function. CD16 neg NK cells predominantly infiltrated tumors relative to lung tissue and harbored sub-populations with unique gene expression programs. Notably, tumors with high density of CD8 + CTLs were enriched for a CD16 neg NK cell subset with features of tissue residency and NK cell memory as well as a CD16 neg ILC3-like subset that displayed transcriptional features linked to activation, NFκB signaling, dendritic cell recruitment and lymphoid-tissue inducing properties. These data reveal that a potentially synergistic NK cell innate immune program may co-operate with CD8 + CTLs to mediate tumor clearance in tumors with a high tumor-infiltrating lymphocyte (TIL) density.

Background Determination of culprit drug in drug reaction with eosinophilia and systemic symptoms (DRESS) is crucial. Skin tests have been utilised, although it remains unclear how sensitive these are. We set out to determine the value of skin tests in the assessment of drug causality in DRESS. Methods A systematic literature search was conducted for publications from 1996 onwards of skin tests (skin prick test = SPT, patch test = PT, intradermal test = IDT) performed in clearly defined DRESS cases. Outcomes of testing, drug culpability assessments and challenge tests data were extracted. Results 17 articles met inclusion criteria. In 290 DRESS patients, patch testing was most frequent [PT = 97.2% (n=282), IDT = 12.4% (n=36), SPT = 3.1% (n=9)]. Positive results were noted in 58.4% (n=160/282) of PT, 66.5% IDT and 25% SPT. When confidence of drug causality was high (n=73 of 194), testing did not correlate well with clinical suspicion: PT 37.6%; IDT 36.5% (n=19 of 52). Direct comparison of skin testing with provocation testing (n=12) showed 83.3% correlation. Positive IDTs were reported in 8 negative PT cases. Conclusion Skin tests, particularly PT and IDT, have been reported as tools for diagnosis of causal drugs in DRESS. Heterogeneity in methodology, results analysis and reporting of cohorts, makes meta-analysis to determine sensitivity and specificity of published literature impossible and highlights weaknesses in the field. We propose that international collaboration is essential to harmonise the methodology and reporting measures from hypersensitivity testing studies in larger cohorts.

Abstract One of the most severe forms of cutaneous adverse drug reactions is “drug reaction with eosinophilia and systemic symptoms” (DRESS), hence subsequent avoidance of the causal drug is imperative. However, attribution of drug culpability in DRESS is challenging and standard skin allergy tests are not recommended due to patient safety reasons. Whilst incidence of DRESS is relatively low, between 1:1000 and 1:10 000 drug exposures, antibiotics are a commoner cause of DRESS and absence of confirmatory diagnostic test can result in unnecessary avoidance of efficacious treatment. We therefore sought to identify potential biomarkers for development of a diagnostic test in antibiotic-associated DRESS. Peripheral blood mononuclear cells from a “discovery” cohort (n = 5) challenged to causative antibiotic or control were analyzed for transcriptomic profile. A panel of genes was then tested in a validation cohort (n = 6) and compared with tolerant controls and other inflammatory conditions which can clinically mimic DRESS. A scoring system to identify presence of drug hypersensitivity was developed based on gene expression alterations of this panel. The DRESS transcriptomic panel identified antibiotic-DRESS cases in a validation cohort but was not altered in other inflammatory conditions. Machine learning or differential expression selection of a biomarker panel consisting of 6 genes (STAC, GPR183, CD40, CISH, CD4, and CCL8) showed high sensitivity and specificity (100% and 85.7%–100%, respectively) for identification of the culprit drug in these cohorts of antibiotic-associated DRESS. Further work is required to determine whether the same panel can be repeated for larger cohorts, different medications, and other T-cell-mediated drug hypersensitivity reactions.

Background: One of the most severe forms of T cell mediated cutaneous adverse drug reactions is drug reaction with eosinophilia and systemic symptoms (DRESS), hence subsequent avoidance of the causal drug is imperative. However, attribution of drug culpability in DRESS is challenging and standard skin allergy tests are not recommended due to for patient safety reasons. We sought to identify potential biomarkers for development of a diagnostic test. Methods: Peripheral blood mononuclear cells (PBMCs) from a discovery cohort (n=5) challenged to drug or control were analysed for transcriptomic profile. A signature panel of genes was then tested in a validation cohort (n=6), and compared to tolerant controls and other inflammatory conditions which can clinically mimic DRESS. A scoring system to identify presence of drug hypersensitivity was developed based on gene expression alterations of this panel. Results: Whole transcriptome analysis identified 4 major gene clusters including those regulating T cell activation via NFAT and cytokine receptor activity. 22 differentially expressed gene transcripts were identified as a DRESS signature including Type 1 interferon pathways and Th2 activation. The DRESS transcriptomic panel identified antibiotic-DRESS cases in a validation cohort but was not altered in other inflammatory conditions. Machine learning or differential expression selection of a biomarker panel showed high sensitivity and specificity (100% and 85.7-100% respectively) for identification of the culprit drug in DRESS. Conclusion: Transcriptomic analysis of DRESS revealed important insights into the key activated pathways and identified a transcriptional signature which shows potential as a test with high sensitivity for drug culpability attribution.

Einleitung: Das humane Papillomavirus 16 (HPV16) kann oropharyngeale Plattenepithelkarzinome (OPSCC) verursachen. Um die virale Genexpression in primärem Krebsgewebe zu bewerten, analysierten wir die Korrelation zwischen viraler genomischer und transkriptomischer Genexpression.

Introduction: Human Papillomavirus 16 (HPV16) can cause oropharyngeal squamous cell carcinoma (OPSCC). To assess the viral gene expression in primary cancer tissue we analysed the correlation of viral genomic and transcriptomic gene expression.

Accurate regulation of cutaneous immunity is fundamental for human health and quality of life but is severely compromised in inflammatory skin disease. To investigate the molecular crosstalk underpinning tolerance vs inflammation in human skin, we set up a human in vivo allergen challenge study, exposing patients with atopic dermatitis (AD) to house dust mite (HDM). Analyses of transcriptional programmes at the population and single cell levels in parallel with immunophenotyping of resident and infiltrating immune cells indicated that inflammatory responses to HDM were associated with immune activation in Langerhans cells (LCs) and cutaneous T cells. High basal level of TNF production by cutaneous Th17 T cells predisposed to an inflammatory reaction and resulted in formation of hub structures where LCs and T cells interacted, leading to loss of functional programming in LCs. Additionally, single nucleotide polymorphisms in MT1X gene associated with enhanced expression of metallothioneins and transcriptional programmes encoding antioxidant defences across skin cell types in non-reactive patients, were protective against T cell mediated inflammation. Our results provide a unique insight into the dynamics of immune regulation in the human skin and define regulatory circuits that can be harnessed to improve skin health and treat disease.

Immune-checkpoint blockade (ICB) has shown remarkable clinical success in boosting antitumor immunity. However, the breadth of its cellular targets and specific mode of action remain elusive. We find that tumor-infiltrating follicular regulatory T (TFR) cells are prevalent in tumor tissues of several cancer types. They are primarily located within tertiary lymphoid structures and exhibit superior suppressive capacity and in vivo persistence as compared with regulatory T cells, with which they share a clonal and developmental relationship. In syngeneic tumor models, anti-PD-1 treatment increases the number of tumor-infiltrating TFR cells. Both TFR cell deficiency and the depletion of TFR cells with anti-CTLA-4 before anti-PD-1 treatment improve tumor control in mice. Notably, in a cohort of 271 patients with melanoma, treatment with anti-CTLA-4 followed by anti-PD-1 at progression was associated with better a survival outcome than monotherapy with anti-PD-1 or anti-CTLA-4, anti-PD-1 followed by anti-CTLA-4 at progression or concomitant combination therapy. Vijayanand and colleagues show highly suppressive CD4+CTLA-4+PD-1+ follicular regulatory T (TFR) cells reside within tumor microenvironments. Depleting TFR cells or blocking their activity with CTLA-4-depleting antibodies before anti-PD-1 checkpoint blockade therapy improved the efficacy of anti-PD-1 treatment in mouse tumor models and was also associated with better survival outcomes in a large cohort of patients with melanoma.

Background: beta-lactam antibiotics are associated with a variety of immune-mediated or hypersensitivity reactions, including immediate (type I) reactions mediated by antigen-specific IgE. Objective: We sought to identify genetic predisposing factors for immediate reactions to beta-lactam antibiotics. Methods: Patients with a clinical history of immediate hypersensitivity reactions to either penicillins or cephalosporins, which were immunologically confirmed, were recruited from allergy clinics. A genome-wide association study was conducted on 662 patients (the discovery cohort) with a diagnosis of immediate hypersensitivity and the main finding was replicated in a cohort of 98 Spanish cases, recruited using the same diagnostic criteria as the discovery cohort. Results: Genome-wide association study identified rs71542416 within the Class II HLA region as the top hit (P = 2 x 10(-14)); this was in linkage disequilibrium with HLA-DRB1*10:01 (odds ratio, 2.93; P = 5.4 x 10(-7)) and HLA-DQA1*01:05 (odds ratio, 2.93, P = 5.4 x 10(-7)). Haplotype analysis identified that HLADRB1*10:01 was a risk factor even without the HLADQA1*01:05 allele. The association with HLA-DRB1*10:01 was replicated in another cohort, with the meta-analysis of the discovery and replication cohorts showing that HLA-DRB1*10:01 increased the risk of immediate hypersensitivity at a genome-wide level (odds ratio, 2.96; P = 4.1 x 10(-9)). No association with HLA-DRB1*10:01 was identified in 268 patients with delayed hypersensitivity reactions to beta-lactams. Conclusions: HLA-DRB1*10:01 predisposed to immediate hypersensitivity reactions to penicillins. Further work to identify other predisposing HLA and non-HLA loci is required.

IntroductionHuman papillomavirus 16 (HPV16) is the main cause of oropharyngeal squamous cell carcinoma (OPSCC). To date, the links between HPV16 gene expression and adaptive immune responses have not been investigated. We evaluated the correlation of HPV16 DNA, RNA transcripts and features of adaptive immune response by evaluating antibody isotypes against E2, E7 antigens and density of tumor-infiltrating lymphocytes (TIL).Material and MethodsFFPE-tissue from 27/77 p16-positive OPSCC patients was available. DNA and RNA were extracted and quantified using qPCR for all HPV16 genes. The TIL status was assessed. Immune responses against E2 and E7 were quantified by ELISA (IgG, IgA, and IgM; 77 serum samples pre-treatment, 36 matched post-treatment).ResultsAmounts of HPV16 genes were highly correlated at DNA and RNA levels. RNA co-expression of all genes was detected in 37% (7/19). E7 qPCR results were correlated with higher anti-E7 antibody (IgG, IgA) level in the blood. Patients with high anti-E2 IgG antibody (>median) had better overall survival (p=0.0311); anti-E2 and anti-E7 IgA levels had no detectable effect. During the first 6 months after treatment, IgA but not IgG increased significantly, and >6 months both antibody classes declined over time. Patients with immune cell-rich tumors had higher levels of circulating antibodies against HPV antigens.ConclusionWe describe an HPV16 qPCR assay to quantify genomic and transcriptomic expression and correlate this with serum antibody levels against HPV16 oncoproteins. Understanding DNA/RNA expression, relationship to the antibody response in patients regarding treatment and outcome offers an attractive tool to improve patient care.

Immune checkpoint blockade (ICB) has shown remarkable clinical success in boosting anti-tumor immunity. However, the breadth of its cellular targets and specific mode of action remain elusive. We find that T follicular regulatory (TFR) cells are present in high numbers in multiple tumors, inhibit anti-tumor immunity and are responsive to ICB. TCR-seq data, trajectory analyses and adoptive transfer studies indicate intratumoral TREG to TFR cell conversion. When compared to TREG cells, TFR cells exhibited enhanced suppressive capacity. In syngeneic tumor models, anti-PD-1 treatment increased the number of tumor-infiltrating TFR cells. Conditional knockout of TFR cells or depletion of TFR cells with anti-CTLA-4 antibody prior to anti-PD-1 treatment, improved tumor control in mice. Notably, in a cohort of melanoma patients, treatment with anti-CTLA-4 followed by anti-PD-1 at progression was associated with better long-term survival outcomes than anti-PD-1 or anti-CTLA-4 monotherapy, anti-PD-1 followed by anti CTLA-4 at progression or concomitant combination therapy.

Background The role of tumor-associated macrophages (TAMs) in determining the outcome between the antitumor effects of the adaptive immune system and the tumor’s anti-immunity stratagems, is controversial. Macrophages modulate their activities and phenotypes by integration of signals in the tumor microenvironment. Depending on how macrophages are activated, they may adopt so-called M1-like, antitumor or M2-like, protumor profiles. In many solid tumors, a dominance of M2-like macrophages is associated with poor outcomes but in some tumor types, strong M1-like profiles are linked to better outcomes. We aimed to investigate the interrelationship of these TAM populations to establish how they modulate the efficacy of the adaptive immune system in early lung cancer. Methods Macrophages from matched lung (non-tumor-associated macrophages (NTAMs)) and tumor samples (TAMs) from resected lung cancers were assessed by bulk and single-cell transcriptomic analysis. Protein expression of genes characteristic of M1-like (chemokine (C-X-C motif) ligand 9) or M2-like (matrix metallopeptidase 12) functions was confirmed by confocal microscopy. Immunohistochemistry related the distribution of TAM transcriptomic signatures to density of CD8 + tissue-resident memory T cells (T RM ) in tumors and survival data from an independent cohort of 393 patients with lung cancer. Results TAMs have significantly different transcriptomic profiles from NTAMs with >1000 differentially expressed genes. TAMs displayed a strong M2-like signature with no significant variation between patients. However, single-cell RNA-sequencing supported by immuno-stained cells revealed that additionally, in 25% of patients the M2-like TAMs also co-expressed a strong/hot M1-like signature (M1 hot ). Importantly, there was a strong association between the density of M1 hot TAMs and T RM cells in tumors that was in turn linked to better survival. Our data suggest a mechanism by which M1 hot TAMs may recruit T RM cells via CXCL9 expression and sustain them by making available more of the essential fatty acids on which T RM depend. Conclusions We showed that in early lung cancer, expression of M1-like and M2-like gene signatures are not mutually exclusive since the same TAMs can simultaneously display both gene-expression profiles. The presence of M1 hot TAMs was associated with a strong T RM tumor-infiltrate and better outcomes. Thus, therapeutic approaches to re-program TAMs to an M1 hot phenotype are likely to augment the adaptive antitumor responses.

Eczema is a characteristic pattern of skin inflammation that has many subtypes, with some induced by external factors such as irritants or skin sensitizers. Atopic eczema is due partly to a genetic susceptibility, which programmes altered immune responses and skin physiology, together with reactions to exogenous allergens and microbes, but several eczema patterns do not appear to have external causes. Management requires identification and avoidance of provoking factors. The inflammation is treated with topical steroids of different potencies, supplemented with moisturizers. Newer therapies include topical calcineurin antagonists, with a range of systemic therapies being used to control the most severe types of disease.