Soluble immune checkpoints (sICs) have emerged as potential biomarkers in various cancers. However, their role in extensive-stage small cell lung cancer (ES-SCLC), an aggressive tumor type with limited therapeutic options and poor prognosis, remains poorly characterized. We analyzed 14 circulating sICs, leukocyte-platelet (PLT) complexes, and PD-L1 surface expression in ES-SCLC patients treated with chemoimmunotherapy (n = 41) prior to treatment and healthy donors (HD, n = 10). We assessed their associations with clinical and demographic factors and performed survival analyses using Kaplan-Meier and log-rank tests. Levels of soluble (s) PD-L1, PD-L2, BTLA, HVEM, TIM-3, and CD27 were higher in plasma from ES-SCLC patients compared to those from HD (p < 0.05). Patients with liver metastases exhibited higher sIC levels, and a multivariate model demonstrated discriminative power. Network analyses revealed distinct patterns of immune dysregulation between ES-SCLC and HD. We observed correlations among sICs and leukocyte-PLT complexes and leukocyte PD-L1 expression, indicating links between systemic immune status and tumor-immune interactions. Furthermore, increased concentrations of sTIM-3, sCD27, sHVEM and sPD-L1 were associated with a poor prognosis. Overall, sICs reflect relevant clinical, prognostic, and immunological features in ES-SCLC. Their plasma measurement could aid in non-invasive patient stratification regarding liver metastases and survival risk.

Despite having endured numerous pandemics throughout history and contemporary technological advancements, the world was still unprepared for the emergence of the SARS-CoV-2 pathogen. Prior to the development and distribution of effective vaccines, health officials relied heavily on diagnostics to contain the spread of the virus. Reverse transcription-polymerase chain reaction (RT-PCR) is employed for diagnosing SARS-CoV-2 due to its exceptional sensitivity and specificity. However, clinical sample preparation techniques for SARS-CoV-2 may have limited detection sensitivity due to the large volumes of viral transport medium in which nasopharyngeal swabs are stored, and where only a fraction of the sample was used for analysis. Consequently, we have developed a sample processing method that improves the sensitivity and specificity of SARS-CoV-2 diagnostics by capturing viral particles using aptamers specific to the S-protein functionalized on magnetic particles ("apta-beads") and thereby concentrating the analyte. The eluted viral particles are then directly amplified on the Kimera P-IV point-of-care plasmonic qPCR platform. With our protocols, we have been able to capture various forms of SARS-CoV-2 analytes, including recombinant spike protein from both Wuhan and Omicron strains, spike-expressing pseudoviral particles, and SARS-CoV-2 viruses. Furthermore, the capture protocol can be performed in as little as 2 minutes, with the added effect of removing unwanted inhibitory components to PCR reactions. In conclusion, our "apta-bead" sample purification, combined with the rapid plasmonic PCR analysis, can play a critical role in limiting sample-to-result turnover time, and thereby mitigate the spread of the infectious disease.

Abstract Introduction Activating NK cell receptors are critical for controlling acute murine cytomegalovirus (MCMV) infection, most clearly demonstrated in C57BL/6 mice, where resistance is mediated by the Ly49H receptor through recognition of the viral ligand m157 expressed on infected cells. NK cell—dependent resistance to MCMV is also observed in Ly49H-deficient strains, such as MA/My, where Ly49P and Ly49R can recognize MCMV-infected cells in a class I MHC—dependent manner in vitro. However, the role of these receptors in vivo has not been explored. In addition, these activating receptors may engage self—MHC class I ligands in vivo, yet the physiological relevance of this interaction remains poorly defined. Methods To selectively assess Ly49P function in vivo, we generated Ly49- and MHC class I—deficient mice expressing a single Ly49P receptor on NK cells by knocking in Ly49P cDNA into the Ncr1 locus. Founder mice were then subsequently crossed onto distinct MHC class I backgrounds. Results NK cells from Ly49P-KkDk or Ly49P-KdDd mice exhibited reduced surface expression of Ly49P compared with Ly49P-KbDb mice, suggesting MHC class I—dependent masking of Ly49P. Consistently, Ly49P-KkDk NK cells showed diminished degranulation and IFN-γ production compared with controls following anti-Ly49P or NK1.1 stimulation, indicating a generalized hyporesponsive state to ITAM-associated signaling. This anergic state appeared to be regulated at the level of proximal signaling, as NK cell responsiveness was restored by PMA/ionomycin stimulation, which bypasses early signaling events. Notably, disruption of Ly49P—MHC class I cis interactions by citric acid treatment restored responsiveness of hyporesponsive NK cells to both Ly49P and NK1.1 crosslinking. Conclusion Thus, sustained stimulation of a self-specific activating receptor in vivo induces a hyporesponsive state to stimulation through both cognate and unrelated activating receptors. Funding Source NIH Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Alcohol-associated hepatitis (AH) is the most severe clinical manifestation of alcohol-associated liver disease. Corticosteroids are the only disease-specific therapy shown to improve short-term survival. Currently, no non-invasive markers are available to predict patient response to corticosteroids or long-term survival in AH. This study investigates whether surface antigens on plasma extracellular vesicles (EVs), key mediators of intercellular communication, can reflect the underlying immune dysregulation in AH and serve as prognostic markers. Patients with AH were prospectively enrolled between 2020 and 2024. Blood samples were collected before corticosteroid initiation during the first 24 h of hospitalization. EVs were characterized using nanoparticle tracking analysis, cryo-electron microscopy, and flow cytometry. Interleukin-6 (IL-6), soluble (s)CD62p, Circulating Vascular Cell Adhesion Molecule-1 (sVCAM), tumor necrosis factor receptor superfamily member 1 (TNRFS1a), and Intercellular Adhesion Molecule 1 (ICAM-1) were quantified by ELISA. Key outcome variables included response to corticosteroids and mortality. A total of 46 patients with AH and 28 healthy donors (HD) were included. EV concentration was significantly higher in AH patients than in HD (9.3 × 1011 [IQR 4-24] versus 2.4 × 1011 [IQR 2-4], p = 0.03). Specific EV antigens were associated with key clinical outcomes: CD20 and CD2 levels differed between patients with or without infections (bacterial, viral, and fungal) developed during hospitalization; CD40 and CD146 were elevated in patients who developed acute kidney injury. EVs enriched in monocyte (CD14) and T-reg (CD25) markers were associated with plasma IL-6 levels, while endothelial markers CD105 and CD146 correlated with sVCAM and sCD62p. EVs enriched in platelet (CD49e) and endothelial (CD31) markers were associated with corticosteroid response, whereas EVs enriched with endothelial (CD105 and CD146) and B lymphocyte (CD19) markers were associated with mortality. Overall, EVs enriched in endothelial and monocyte markers may represent a candidate non-invasive tool for predicting corticosteroid response and mortality in AH, aiding risk stratification and early identification of non-responders for timely transplant evaluation.

Infections caused by multidrug-resistant organisms (MDRO) are linked to poor outcomes, particularly in patients with cirrhosis. The underlying mechanisms are not fully understood and may involve a different immune response against MDRO. This study aimed to compare the in vitro immune response between multidrug-resistant (MDR) Escherichia coli and antibiotic-susceptible E. coli strains. Surface protein extract and DNA extract were obtained from MDR E. coli (n = 6) and antibiotic-susceptible E. coli (n = 6) strains isolated from infected patients with cirrhosis. The extracts were used to stimulate in vitro peripheral blood mononuclear cells from healthy donors. After 48 h, cytokine levels (IFN-γ, IL-1β, IL-10, IL-12p70, MCP-1, IL-8, IL-6, MIP-1α, and MIP-1β) were measured. We observed no significant differences in cytokine production between MDR and susceptible strains. However, we identified notable interindividual variability in cytokine production for most of the cytokines studied. Only IFN-γ and IL-6 in surface extract and MCP-1 in DNA extract showed similar levels across all donors. We conclude that the cytokine profiles induced by MDR E. coli in vitro were similar to those in susceptible strains. These findings suggest that the poor prognosis associated with MDR E. coli infections is not due to a differential immune response but rather to other factors.

OBJECTIVES:Systemic sclerosis (SSc) is a complex autoimmune disease characterized by microvascular damage, immune dysregulation, and tissue fibrosis. While lymphocyte-platelet (PLT) complexes have been implicated in autoimmune diseases, their role in SSc is not well understood. METHODS:In a study of 21 predominantly female SSc patients, 66.7 % had limited SSc (lcSSc), with anti-centromere antibodies (ACA) being the most common autoantibody pattern. We applied flow cytometry to analyze B cells with bound PLTs, enzyme-linked immunosorbent assay (ELISA) to determine plasma levels of activated PLT soluble factors, and co-culture assays to evaluate B cell cytokine secretion and plasma cell differentiation. RESULTS:SSc patients had a higher percentage of B cells, but not T cells, with bound PLTs compared to healthy donors (HD). Despite similar PLT counts, SSc patients showed higher plasmatic levels of P-selectin (CD62P), soluble CD40 ligand (sCD40L), platelet-derived growth factor (PDGF), and transforming growth factor-β (TGF-β). Plasma IL-10 levels were also higher in SSc patients, with increased intracellular IL-10 in B cells with bound PLTs. We observed an increased IL-10 production and plasma cell differentiation when B cells were co-cultured with PLTs, especially from SSc patients. B cells with bound PLTs were associated with calcinosis, digital ulcers, and ACA status, with no effect from previous corticosteroid or aspirin therapy. Logistic regression identified B cells with bound PLTs as a predictor for distinguishing lcSSc patients. CONCLUSIONS:B cells with bound PLTs play a significant role in SSc by modulating B cell function and contributing to disease pathogenesis. Their association with clinical parameters suggests their potential as biomarkers for disease severity and subtype classification in SSc.

Rheumatoid arthritis affects ~0.5-1% of the adult population and results in joint inflammation, chronic pain, and many systemic comorbidities. Immune and inflammatory tissue damage is the main pathogenic mechanism in rheumatoid arthritis, and involves the hyperactivation of both innate and adaptive immune systems. Trained immunity has become well established as an important feature of the innate immune system that allows the host to mount functionally altered immune responses based on their previous history of immune exposures, independently of the classical adaptive immunological memory. However, the role of trained immunity in systemic autoimmune and inflammatory disorders remains poorly understood. In the current work, we demonstrate that emergency myelopoiesis is induced in chronic rheumatoid arthritis in murine models and acts not only to enhance innate immune cell numbers but also to produce functionally altered innate immune cells. Such effects are cell-intrinsic to hematopoietic stem and progenitor cells (HSPCs) and persist independently of the inflammatory disease milieu. Importantly, these trained immunity mechanisms impact not only macrophages but also dendritic cells, which are the major antigen presenting cells that bridge the innate and adaptive immune responses. "Trained" dendritic cells show changes in global gene expression profiles, and significantly altered responses to recall immune stimulation and capacity for T cell activation. This study therefore represents the first demonstration of "dendritic cell trained immunity" in rheumatoid arthritis. ### Competing Interest Statement The authors have declared no competing interest.

Background Thrombocytopenia is frequently associated with increased mortality in sepsis; however, the underlying immunological mechanisms remain poorly understood. Objectives This study characterizes molecular and phenotypic changes associated with thrombocytopenia in sepsis, focusing on platelets (PLTs), neutrophils, and their interactions, and evaluates their contributions to poor outcomes. Methods Blood samples were collected from 19 healthy donors, 27 nonthrombocytopenic (N-TP) and 22 thrombocytopenic (TP) patients with community-acquired sepsis at baseline (diagnosis), and at 24, 48, and 72 hours after admission. Plasma levels of PLT-derived mediators and neutrophil surrogate markers were quantified by ELISA. PLT and neutrophil phenotypes and neutrophil degranulation were assessed by flow cytometry, while extracellular DNA release, as a surrogate approximation of NETosis, was measured by SYTOX Green fluorescence and validated by myeloperoxidase (MPO)-DNA ELISA. Results TP patients showed higher percentages of PD-L1+ PLTs and increased CXCR4 expression on PLTs compared with N-TP patients. Their plasma also contained elevated soluble P-selectin and vascular endothelial growth factor levels. Compared with healthy donors, TP patients exhibited fewer neutrophil-PLT complexes; however, a greater proportion of these complexes were PD-L1+ and expressed higher CXCR4 levels than those in N-TP patients. Despite reduced extracellular DNA release upon stimulation, TP patients had higher plasma MPO-DNA complexes and MPO, neutrophil elastase, and S100A8/A9 levels. Clinically, TP patients had worse outcomes, with lower soluble CD40L levels and impaired in vitro extracellular DNA release correlating with disease severity (Sequential Organ Failure Assessment score ≥8). Conclusions Our findings suggest a distinct immune signature in TP septic patients, defined by enhanced PLT activation and neutrophil dysfunction, potentially contributing to poor prognosis.

Introduction:Achieving the primary treat-to-target (T2T) goal in rheumatoid arthritis (RA) remains challenging for many patients, reflecting limitations in the effectiveness of existing treatments. Our study examines factors influencing Janus kinase (JAK) inhibitor effectiveness by analyzing interindividual variability in demographic and clinical characteristics of real-world RA patients. Materials and methods:This observational retrospective study involves RA patients receiving tofacitinib, baricitinib, upadacitinib, or filgotinib between September 2017 and January 2025. Predictive factors of achieving the T2T goal at 6 months were identified through logistic regression analyses. Disparities in the treatment effectiveness retention based on predictive factors were assessed using the Kaplan-Meier estimate and compared with the log-rank test. The Cox model was applied to analyze whether the predictive factors identified could influence the retention of JAK inhibitor treatment effectiveness. Results:One hundred fifty patients were included: 81 (54%) achievers and 69 (46%) non-achievers of remission or, at least, low disease activity at 6 months of treatment. High disease activity at baseline, with respect to moderate activity, was identified as an unfavorable factor for achieving the T2T goal (Odds ratio adjusted: 0.96; 95% confidence interval: 0.92-0.99; p = 0.028). In treatment effectiveness retention rates, no differences were observed between patients with high versus moderate disease activity (p = 0.103). RA disease activity at baseline was not found to impact the survival of JAK inhibitor effectiveness (p = 0.106). Conclusion:In RA, high disease activity at the initiation of treatment with tofacitinib, baricitinib, upadacitinib, or filgotinib does not preclude an effective treatment response but is associated with an increased risk of therapeutic failure. Factors not related to the achievement of the T2T goal at 6 months of JAK inhibitor treatment include: age, female sex, body mass index, RA disease duration, seropositivity for rheumatoid factor, seropositivity for anti-cyclic citrullinated peptides, JAK inhibitor selectivity, type and number of prior biologic treatments, concomitant use and number of prior conventional synthetic disease-modifying antirheumatic drugs, and number of prior JAK inhibitors. These conclusions are derived from a retrospective real-world study and should be confirmed in prospective studies.

Psoriasis is a chronic inflammatory skin disease driven by genetic, environmental, and immune factors. While biologics like adalimumab (anti-TNFα) and risankizumab (anti-IL-23) have improved outcomes, patient response variability remains unclear. This study examined immune-related transcriptomic differences between lesional (L) and non-lesional (NL) psoriatic skin, focusing on immune-related hub genes, their plasma levels, and their correlations with severity and treatment response. Patients with moderate-to-severe psoriasis were enrolled before treatment with anti-TNFα (n = 16) or anti-IL-23 (n = 18). Plasma and paired L and NL skin biopsies were collected for RNA sequencing. Gene ontology enrichment analysis found four immune-related terms enriched in L skin: T-helper 17, granulocyte and lymphocyte chemotaxis, and antimicrobial humoral response. A protein–protein interaction network identified ten immune-related hub genes upregulated in L skin that correlated with clinical severity. Patients with prior treatments expressed distinctive gene profiles. Plasma levels of CCL20 strongly correlated with disease severity. Decision tree models identified CCL20 expression in skin and plasma levels of IL-6 and CXCL8 as candidate predictors for anti-TNFα response. Similarly, skin expression of CXCL8, IL-6, and CXCL10, alongside plasma levels of CCL20, IL-6, and CXCL8, may predict anti-IL-23 response. Ten immune-related hubs may serve as possible biomarkers for disease severity and therapeutic response in psoriasis.

Antecedentes y objetivo La seguridad de los inhibidores de quinasa Janus (JAK) ha ganado atención debido a reacciones adversas observadas poscomercialización. El estudio se centra en el análisis de reacciones adversas relacionadas con tofacitinib, baricitinib, upadacitinib y filgotinib en pacientes con artritis reumatoide, incluyendo la identificación de factores predictivos relacionados con su aparición. Pacientes y métodos Estudio retrospectivo observacional. Se incluyeron pacientes adultos en seguimiento por artritis reumatoide en un hospital universitario, en tratamiento con inhibidor de JAK entre septiembre 2017 y enero 2024. Se calculó la incidencia acumulada de cada reacción adversa mediante la escala de Naranjo. Se identificaron los factores predictivos para la aparición de reacciones adversas mediante análisis de regresión logística. Resultados Se incluyeron 223 pacientes, el 28,7% presentó reacción adversa a inhibidor de JAK. Las reacciones adversas con mayor incidencia acumulada fueron infecciones y trastornos gastrointestinales. Las infecciones comprendieron: tracto respiratorio superior (4,5%), celulitis (3,1%), tracto urinario (2,7%), herpes zóster (1,8%). Los trastornos gastrointestinales englobaron: dolor abdominal (4,0%), diarrea (3,6%), náuseas y vómitos (3,6%), perforación gastrointestinal (1,3%) y diverticulitis (0,9%). Con un 0,5% se clasificaron: cefalea, parestesias, erupción cutánea, neutropenia severa, insomnio, disnea y crisis hipertensiva. Como factores de riesgo, se identificaron: el tratamiento con inhibidor de JAK no selectivo (OR ajustado: 4,03; IC 95%: 1,15-14,10; p=0,029) y una mayor edad (OR ajustado: 1,03; IC 95%: 1,00-1,05; p=0,036). Conclusiones Infecciones y trastornos gastrointestinales representaron las reacciones adversas a inhibidor de JAK con mayor incidencia acumulada, siendo los factores de riesgo para su aparición el tratamiento con inhibidor de JAK no selectivo y una mayor edad del paciente.

PD-(L)1 inhibitors are part of the treatment strategy for non-small cell lung cancer (NSCLC) although its efficacy is limited to certain patients. Our study aimed to identify patients who might benefit from anti-PD-(L)1 inhibitors by analyzing the PD-L1 expression on circulating leukocytes and its evolution during treatment. One hundred thirteen NSCLC patients, according to their radiological response after 10–12 weeks of treatment, were classified into responders, stable, and progressive disease. Percentages of circulating PD-L1+ leukocytes, PD-L1+ platelets (PLTs), and leukocyte-PLT complexes were assessed using flow cytometry, and plasma concentrations of soluble immunomodulatory factors were quantified by ELISA. Responders exhibited significantly higher pre-treatment percentages of PD-L1+ neutrophils, PD-L1+ CD14+ cells, and PD-L1+ PLTs than progressors. The percentages of these populations decreased in responders post-treatment, contrasting with stables and progressors. PLTs notably contributed to PD-L1 expression in CD14+ cells and neutrophils. Plasma cytokine analysis revealed baseline differences only in IL-17 concentration among groups, whereas network analyses highlighted distinct association patterns between plasma molecules and PD-L1+ leukocytes after 10–12 weeks of treatment. Our findings suggest that pre-treatment assessment of circulating PD-L1+ neutrophils, PD-L1+ CD14+ cells, and PD-L1+ PLTs may be helpful in identifying NSCLC patients who are potential candidates for anti-PD-(L)1 therapy.