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Immunological diseases are typically heterogeneous in clinical presentation, severity and response to therapy. Biomarkers of immune diseases often reflect this variability, especially compared to their regulated behaviour in health. This leads to a common difficulty that frustrates biomarker discovery and interpretation - namely, unequal dispersion of immune disease biomarker expression between patient classes necessarily limits a biomarker's informative range. To solve this problem, we introduce dataset restriction, a procedure that splits datasets into classifiable and unclassifiable samples. Applied to synthetic flow cytometry data, restriction identifies biomarkers that are otherwise disregarded. In advanced melanoma, restriction finds biomarkers of immune-related adverse event risk after immunotherapy and enables us to build multivariate models that accurately predict immunotherapy-related hepatitis. Hence, dataset restriction augments discovery of immune disease biomarkers, increases predictive certainty for classifiable samples and improves multivariate models incorporating biomarkers with a limited informative range. This principle can be directly extended to any classification task., (© 2024. The Author(s).)

Background: The increasing prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) incurs substantial morbidity, mortality and healthcare costs. Detection and clinical intervention at early stages of disease improves prognosis; however, we are currently limited by a lack of reliable diagnostic tests for population screening and monitoring responses to therapy. To address this unmet need, we investigated human invariant Natural Killer T cell (iNKT) activation by fat-loaded hepatocytes, leading to the discovery that circulating soluble CD46 (sCD46) levels accurately predict hepatic steatosis., Methods: sCD46 in plasma was measured using a newly developed immuno-competition assay in two independent cohorts: Prospective living liver donors (n = 156; male = 66, female = 90) and patients with liver tumours (n = 91; male = 58, female = 33). sCD46 levels were statistically evaluated as a predictor of hepatic steatosis., Findings: Interleukin-4-secreting (IL-4 + ) iNKT cells were over-represented amongst intrahepatic lymphocytes isolated from resected human liver samples. IL-4 + iNKT cells preferentially developed in cocultures with a fat-loaded, hepatocyte-like cell line, HepaRG. This was attributed to induction of matrix metalloproteases (MMP) in fat-loaded HepaRG cells and primary human liver organoids, which led to indiscriminate cleavage of immune receptors. Loss of cell-surface CD46 resulted in unrepressed differentiation of IL-4 + iNKT cells. sCD46 levels were elevated in patients with hepatic steatosis. Discriminatory cut-off values for plasma sCD46 were found that accurately classified patients according to histological steatosis grade., Interpretation: sCD46 is a reliable clinical marker of hepatic steatosis, which can be conveniently and non-invasively measured in serum and plasma samples, raising the possibility of using sCD46 levels as a diagnostic method for detecting or grading hepatic steatosis., Funding: F.B. was supported by the Else Kröner Foundation (Award 2016_kolleg.14). G.G. was supported by the Bristol Myers Squibb Foundation for Immuno-Oncology (Award FA-19-009). N.S. was supported by a Wellcome Trust Fellowship (211113/A/18/Z). J.A.H. received funding from the European Union's Horizon 2020 research and innovation programme (Award 860003). J.M.W. received funding from the Else Kröner Foundation (Award 2015_A10)., Competing Interests: Declaration of interests University Hospital Regensburg has filed a not yet published European patent application (Registration Nr. 23 183 382.3) for sCD46 as a clinical biomarker of hepatic steatosis. J.A.H. received in-kind support from Beckman Coulter. The authors have no other conflicts of interest to declare., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Soluble urokinase plasminogen activator receptors (suPARs) are a biomarker for inflammatory diseases. This study aims to investigate its diagnostic properties regarding periprosthetic joint infections (PJI). This retrospective cohort study included adult patients who underwent joint puncture for suspected PJI. The presence of PJI was determined according to the criteria of the European Bone and Joint Infection Society (EBJIS). Laboratory study analyses included the determination of white blood cells (WBC) in whole blood, C-reactive protein (CRP) in blood plasma, and suPAR in both blood plasma and synovial fluid. Appropriate diagnostic cut-off values were identified utilizing Youden's J, and their diagnostic performance was determined by calculating the positive (PPV) and negative predictive value (NPV) for each marker. Sixty-seven cases were included in the final analysis. Forty-three samples (64%) were identified as periprosthetic joint infection (PJI) and twenty-four specimen (36%) were PJI negative cases. The PPV and NPV were 0.80 and 0.70 for synovial suPAR, 0.86 and 0.55 for CRP, 0.84 and 0.31 for WBC and 1.00 and 0.31 for plasma suPAR. Synovial suPAR showed a solid diagnostic performance in this study and has the potential to be an alternative or complementary biomarker for PJI. Further investigations in larger patient collectives are indicated.

Brain metastasis still encompass very grim prognosis and therefore understanding the underlying mechanisms is an urgent need toward developing better therapeutic strategies. We uncover the intricate interactions between recruited innate immune cells and resident astrocytes in the brain metastatic niche that facilitate metastasis of melanoma and breast cancer. We show that granulocyte-derived lipocalin-2 (LCN2) induces inflammatory activation of astrocytes, leading to myeloid cell recruitment to the brain. LCN2 is central to inducing neuroinflammation as its genetic targeting or bone-marrow transplantation from LCN2 -/- mice was sufficient to attenuate neuroinflammation and inhibit brain metastasis. Moreover, high LCN2 levels in patient blood and brain metastases in multiple cancer types were strongly associated with disease progression and poor survival. Our findings uncover a previously unknown mechanism, establishing a central role for the reciprocal interactions between granulocytes and astrocytes in promoting brain metastasis and implicate LCN2 as a prognostic marker and potential therapeutic target., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Immune checkpoint inhibitors have revolutionized treatment of advanced melanoma, but commonly cause serious immune-mediated complications. The clinical ambition of reserving more aggressive therapies for patients least likely to experience immune-related adverse events (irAE) has driven an extensive search for predictive biomarkers. Here, we externally validate the performance of 59 previously reported markers of irAE risk in a new cohort of 110 patients receiving Nivolumab (anti-PD1) and Ipilimumab (anti-CTLA-4) therapy. Alone or combined, the discriminatory value of these routine clinical parameters and flow cytometry biomarkers was poor. Unsupervised clustering of flow cytometry data returned four T cell subsets with higher discriminatory capacity for colitis than previously reported populations, but they cannot be considered as reliable classifiers. Although mechanisms predisposing some patients to particular irAEs have been described, we are presently unable to capture adequate information from pre-therapy flow cytometry and clinical data to reliably predict risk of irAE in most cases., Competing Interests: MK is a Beckman Coulter Life Sciences associate. SH has received consulting fees and speaker’s honoraria from BMS and Merck Sharp & Dohme (MSD). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Glehr, Riquelme, Yang Zhou, Cordero, Schilling, Kapinsky, Schlitt, Geissler, Burkhardt, Schmidt, Haferkamp, Hutchinson and Kronenberg.)

Background: Steatotic livers are more prone to rejection, but are often transplanted owing to the shortage of available organs. Type II NKT (T2NKT) cells are liver-resident lymphocytes that react to lipids presented by CD1d. The role of T2NKT cells in rejection of fatty liver transplants is unclear, partly because of a lack of T2NKT cell markers and their very low frequency in blood. Here, we quantify human T2NKT cells in blood and liver tissue by flow cytometry and provide a strategy for their enrichment and expansion., Methods: Human T2NKT cells were identified as CD3 + CD56 + CD161 + TCR-γᵹ - TCRVα7.2 - and TCRVα24 - cells. T2NKT cells were enriched from blood by sequential positive selection using CD56 and CD3 microbeads. These were subsequently FACS-sorted to purity then expanded in vitro for 3 weeks using anti-CD3/CD28 beads and TGF-β1., Results: The frequency of human T2NKT cells in blood was very low (0.8 ± 0.4% of CD3 + T cells) but they were a more abundant population in liver (6.3 ± 0.9%). Enriched T2NKT cells expressed the transcription factor PLZF. A novel subset of FoxP3 + T2NKT cells was discovered in blood and liver tissue. T2NKT cells were expanded in culture by 15- to 28-fold over 3 weeks, during which time they maintained expression of all identifying markers, including PLZF and FoxP3., Conclusions: Our work defines new strategies for identifying and isolating T2NKT cells from human blood and liver tissue. We showed that this rare population can be expanded in vitro in order to obtain experimentally amenable cell numbers. Further, we identified a novel T2NKT cell subset that stably expresses FoxP3, which might play a role in regulating innate-like lymphocyte responses in steatotic liver transplants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yang Zhou, Werner, Glehr, Geissler, Hutchinson and Kronenberg.)

Treatment of advanced melanoma with combined immune checkpoint inhibitor (ICI) therapy is complicated in up to 50% of cases by immune-related adverse events (irAE) that commonly include hepatitis, colitis and skin reactions. We previously reported that pre-therapy expansion of cytomegalovirus (CMV)-reactive CD4 + effector memory T cells (T EM ) predicts ICI-related hepatitis in a subset of patients with Stage IV melanoma given αPD-1 and αCTLA-4. Here, we develop and validate a 10-color flow cytometry panel for reliably quantifying CD4 + T EM cells and other biomarkers of irAE risk in peripheral blood samples. Compared to previous methods, our new panel performs equally well in measuring CD4 + T EM cells (agreement = 98%) and is superior in resolving CD4 + CD197 + CD45RA - central memory T cells (T CM ) from CD4 + CD197 + CD45RA + naive T cells (T naive ). It also enables us to precisely quantify CD14 + monocytes (CV = 6.6%). Our new "monocyte and T cell" (MoT) assay predicts immune-related hepatitis with a positive predictive value (PPV) of 83% and negative predictive value (NPV) of 80%. Our essential improvements open the possibility of sharing our predictive methods with other clinical centers. Furthermore, condensing measurements of monocyte and memory T cell subsets into a single assay simplifies our workflows and facilitates computational analyses., Competing Interests: MK is a Beckman Coulter Life Sciences associate. SH has received consulting fees and speaker’s honoraria from BMS and Merck Sharp & Dohme (MSD). NA is employed by MVZ for Laboratory Medicine Raubling. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Schilling, Glehr, Kapinsky, Ahrens, Riquelme, Cordero, Bitterer, Schlitt, Geissler, Haferkamp, Hutchinson and Kronenberg.)

In order to differentiate prognostic subgroups of patients with aggressive B-cell lymphoma, we analyzed the expression of 800 miRNAs with the NanoString nCounter human miRNA assay on a cohort of 228 FFPE samples of patients enrolled in the RICOVER-60 and MegaCHOEP trials. We identified significant miRNA signatures for overall survival (OS) and progression-free survival (PFS) by LASSO-penalized linear Cox-regression. High expression levels of miR-130a-3p and miR-423-5p indicate a better prognosis, whereas high levels of miR-374b-5p, miR-590-5p, miR-186-5p, and miR-106b-5p increase patients' risk levels for OS. Regarding PFS high expression of miR-365a-5p in addition to the other two miRNAs improves the prognosis and high levels of miR374a-5p, miR-106b-5p, and miR-590-5p, connects with increased risk and poor prognosis. We identified miRNA signatures to subdivide patients into two different risk groups. These prognostic models may be used in risk stratification in future clinical trials and help making personalized therapy decisions.

Treatment of advanced melanoma with combined PD-1/CTLA-4 blockade commonly causes serious immune-mediated complications. Here, we identify a subset of patients predisposed to immune checkpoint blockade-related hepatitis who are distinguished by chronic expansion of effector memory CD4 + T cells (T EM cells). Pre-therapy CD4 + T EM cell expansion occurs primarily during autumn or winter in patients with metastatic disease and high cytomegalovirus (CMV)-specific serum antibody titres. These clinical features implicate metastasis-dependent, compartmentalised CMV reactivation as the cause of CD4 + T EM expansion. Pre-therapy CD4 + T EM expansion predicts hepatitis in CMV-seropositive patients, opening possibilities for avoidance or prevention. 3 of 4 patients with pre-treatment CD4 + T EM expansion who received αPD-1 monotherapy instead of αPD-1/αCTLA-4 therapy remained hepatitis-free. 4 of 4 patients with baseline CD4 + T EM expansion given prophylactic valganciclovir and αPD-1/αCTLA-4 therapy remained hepatitis-free. Our findings exemplify how pathogen exposure can shape clinical reactions after cancer therapy and how this insight leads to therapeutic innovations.

Diffuse large B-cell lymphoma (DLBCL) is a disease with heterogeneous outcome. Stromal signatures have been correlated to survival in DLBCL. Their use, however, is hampered by the lack of assays for formalin-fixed paraffin-embedded material (FFPE). We constructed a lymphoma-associated macrophage interaction signature (LAMIS) interrogating features of the microenvironment using a NanoString assay applicable to FFPE. The clinical impact of the signature could be validated in a cohort of 466 patients enrolled in prospective clinical trials of the German High-Grade Non-Hodgkin Lymphoma Study Group (DSHNHL). Patients with high expression of the signature (LAMIS high ) had shorter EFS, PFS, and OS. Multivariate analyses revealed independence from IPI factors in EFS (HR 1.7, 95% CI 1.2-2.4, p-value = 0.001), PFS (HR 1.8, 95% CI 1.2-2.5, p-value = 0.001) and OS (HR 1.8, 95% CI 1.3-2.7, p-value = 0.001). Multivariate analyses adjusted for the IPI factors showed the signature to be independent from COO, MYC rearrangements and double expresser status (DE). LAMIS high and simultaneous DE status characterized a patient subgroup with dismal prognosis and early relapse. Our data underline the importance of the microenvironment in prognosis. Combined analysis of stromal features, the IPI and DE may provide a new rationale for targeted therapy.