Rheumatoid arthritis (RA) is a chronic autoimmune disease marked by persistent synovial inflammation, yet the processes driving disease progression are not completely understood. Here, we examined the role of fibroblast-like synoviocytes (FLS) and neutrophils in RA pathophysiology, using primary FLS, neutrophils, and synovial fluid (SF) from RA and osteoarthritis (OA) patients, as well as healthy controls. Our findings demonstrate that FLS and neutrophils drive an immunothrombotic state in RA SF by expressing tissue factor (TF), an effect mediated by JAK1/2 signaling. Furthermore, we showed that RA SF stimulates IL-8 (CXCL8) expression in control FLS through PAR-1 signaling, and this response was attenuated by DNase I treatment and CIT-013, a monoclonal antibody targeting anti-citrullinated histones H2A and H4 in neutrophil extracellular traps (NETs), supporting the hypothesis that the effect is mediated by NETs. Notably, FLS derived from RA patients exhibit enhanced CXCL8 expression, and elevated IL-8 levels were detected in RA SF, both contributing to neutrophil recruitment, a process that could be mitigated through blockade with an anti-CXCL-8 neutralizing antibody. These results suggest an amplification loop in which TF expression, thrombin activity, and NET formation converge to activate FLS, sustain IL-8 mediated neutrophil migration, and perpetuate synovial inflammation, revealing how stromal and immune cells interact to propagate RA pathophysiology.

BackgroundSystemic inflammation plays a critical role in hepatocellular carcinoma (HCC) progression and postoperative outcomes. This study assessed the prognostic value of the neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) for overall (OS) and recurrence-free survival (RFS) in patients undergoing curative hepatectomy or orthotopic liver transplantation (OLT).MethodsIn this multicenter retrospective cohort of 74 HCC patients (86.5% male; median age 68.0 years), NLR and PLR were evaluated preoperatively and at 1-, 3-, 6-, and 12-months post-surgery. Associations with OS and RFS were evaluated using univariate, multivariable, and Cox proportional hazards models with time-varying covariates, while model discrimination was assessed using Harrell’s concordance index.ResultsElevated preoperative PLR predicted major postoperative complications (AUROC = 0.667, p = 0.030). The 3-month NLR demonstrated strong discriminative performance for OS (C-index 0.79). In time-varying Cox models, postoperative NLR emerged as a robust, time-independent predictor of OS (HR 1.35; p = 0.033), independent of the surgical procedure and Barcelona Clinic Liver Cancer stage. Multivariable analyses demonstrated that elevated NLR at 3-, 6-, and 12-months and PLR at 12 months independently predicted reduced OS. Hepatectomy was the dominant determinant of reduced RFS compared with OLT (p < 0.001), while an NLR threshold >2.5 identified a subgroup at markedly increased mortality risk, particularly following hepatectomy.ConclusionsPostoperative NLR is a powerful, time-independent prognostic biomarker for OS in surgically treated HCC, especially after liver resection, while PLR provides complementary prognostic information. These findings support the clinical integration of inflammation-based biomarkers into postoperative risk stratification and surveillance strategies.

Chronic inflammation perturbs hematopoietic homeostasis, promoting aberrant myelopoiesis and clonal expansion of mutated stem cells. Here, we develop a mathematical model that integrates both local (bone marrow-intrinsic) and global (systemic/peripheral) inflammation-driven feedback mechanisms to investigate their roles in hematopoietic regulation and disease progression. Our model captures the nonlinear interplay between self-renewal, progenitor proliferation, and inflammatory cues, enabling classification of healthy, myelodysplastic, and leukemic states based on stem cell population dynamics. We show that global inflammatory feedback enhances the resilience of hematopoiesis, while excessive feedback on progenitor cells under chronic inflammation drives instability and clonal dominance. Using sensitivity analysis and parameter space mapping, we identify critical feedback thresholds governing transitions between hematopoietic states and reveal how mutated clones exploit inflammation to outcompete wild-type cells. This systems-level framework offers mechanistic insights into the emergence of myeloid malignancies and provides a computational platform for exploring potential anti-inflammatory therapeutic strategies.

Abstract Introduction Complement and coagulation are tightly interconnected systems that contribute to immunothrombosis and can drive inflammatory or thrombotic diseases. Leveraging this relationship and crosstalk we developed a method to functionally evaluate complement-induced coagulation activity using thromboelastometry (thermoelastometry of complement-driven immunothrombosis; TCDI). Methods To study the complement-dependent activation of coagulation, platelet-poor plasma (PPP) from patients was mixed with healthy blood in the presence or absence of the compstatin-based C3 inhibitor Cp40. PPP from healthy controls (n=10), or from patients with antiphospholipid syndrome (APS; n=6), severe COVID-19 (n=13), rheumatoid arthritis (RA; n=7), or synovial fluid (SF) from RA patients, were analyzed for their capacity to induce complement activation in healthy blood. Whole blood coagulation was analyzed by thromboelastometry and complement-driven immunothrombosis was quantified as clotting time (CT) prolongation following Cp40 treatment, expressed as fractional difference percentage (FD%). In parallel, C3a generation was measured by ELISA to monitor the C3 inhibitory activity of Cp40. Results Plasma from patients with APS and COVID-19 induced significant CT prolongation following C3 inhibition by Cp40 and increased FD% values compared with controls, indicating active complement-driven immunothrombosis. Higher TCDI levels were associated with mortality in severe COVID-19. In RA, TCDI positivity was detected in synovial fluid (SF) rather than peripheral plasma. Moreover, TCDI-positive samples treated with Cp40 exhibited significant inhibition of C3a generation, which strongly correlated with FD% values (r=0.67, p=0.0005). Conclusion The TCDI assay may provide a rapid, real-time evaluation of immunothrombotic activity in inflammatory and thrombotic disorders, which could inform timely medical prevention.

Chimeric antigen receptor-T (CAR-T) cell immunotherapy constitutes a cornerstone in the management of patients with relapsed/refractory B-cell lineage lymphoid malignancies. Toxicities such as cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and hematotoxicity (ICAHT) have been recognized in the post-infusion period. The initial interplay between CAR-T cells and tumor cells, followed by cytokine release and the bystander activation of the innate immunity cells, result in endothelial cell injury. In the current review, the ongoing research regarding endothelial injury in CAR-T cell recipients is summarized. Various markers of endothelial injury have been investigated in CAR-T cell recipients, including markers of complement activation, such as soluble C5b-9, endothelial dysfunction (angiopoietin-2, VCAM1, ICAM-1), inflammation, and thrombosis (von Willebrand antigen, ADAMTS13, thrombomodulin). The expression level of these endothelial injury markers has been identified as impaired in CAR-T cell recipients, not only when compared with healthy controls but also among patients with severe CRS/ICANS and those with mild toxicities or without toxicities. Furthermore, the Endothelial Activation and Stress Index (EASIX) and modified versions of this score, calculated in the pre- and early post-infusion period, seem to predict development of severe toxicities, ICAHT, and, thus, poor overall survival in CAR-T cell patients. More data concerning the role of these endothelial injury markers and clinical outcomes in CAR-T cell settings are essential.

Hepatitis E virus (HEV) infection is a frequent cause of acute viral hepatitis. Immunocompromised patients, especially those under anti-CD20 regimens, are prone to chronic or treatment-resistant courses of hepatitis E. We report a case of chronic HEV infection in a 36-year-old man with a history of thrombotic thrombocytopenic purpura treated with rituximab 6 months ago, who presented with new-onset painless jaundice and malaise. Laboratory tests and imaging revealed signs of inflammation and hepatic dysfunction. Due to initial suspicion of autoimmune hepatitis, corticosteroid therapy was started. However, liver biopsy and positive HEV RNA value redefined the diagnosis. Serology tests revealed initially acute infection, which later progressed to chronic hepatitis E infection. Treatment with ribavirin, along with supportive care, achieved significant clinical and laboratory improvement, resolving jaundice, restoring normal transaminase and suppressing HEV RNA values. Further review of the literature highlights the impact of immunosuppression caused by anti-CD20 therapies on HEV infection, as well as the challenges in both treatment and achieving sustained virus clearance in such patients. Moreover, this report underlines the importance of HEV screening in patients with hepatitis who have undergone anti-CD20 therapies, shedding light on a situation that is not well described in the literature and should not be overlooked, even in developed countries.

Physical activity exerts systemic anti-inflammatory effects and reduces the risk for multiple non-communicable diseases, with 7.2% of all-cause deaths globally being attributed to physical inactivity. However, the cellular and molecular components of the exercise-induced anti-inflammatory effects remain only partly understood. Herein we show that moderate-intensity exercise promotes anti-inflammatory reprograming of macrophages orchestrated by the skeletal muscle cells secretome. Primary bone marrow-derived macrophages (BMDMs) exposed to the secretome of mechanically-loaded myotubes (exercise-conditioned medium, exCM) acquire an anti-inflammatory transcriptional profile and increased reliance on oxidative phosphorylation, as shown by Seahorse real-time cell metabolic analysis, compatible with an M2-like phenotypic switch. Using an unbiased proteomic analysis of the exCM we identify the chaperonin Hsp60 as a key mediator of the anti-inflammatory effects of exercise. Hsp60 expression increases in mechanically loaded myotubes in vitro, in the quadriceps muscle and serum of mice following an 8-week program of moderate-intensity aerobic exercise, as well as in human muscle after resistance training. Importantly, treatment of BMDMs with Hsp60 in vitro recapitulates the exCM-induced transcriptional reprograming, promoting an M2-like phenotype. Taken together, our data highlight Hsp60 as a novel component of the skeletal muscle cell-macrophage crosstalk, providing mechanistic insights into the anti-inflammatory effects of exercise. ### Competing Interest Statement The authors have declared no competing interest.

Α shared pathobiology between MDS and cardiovascular disease(CVD) is postulated, but many MDS patients do not develop CVD and only about 10% die from CVD, whereas there is lack of MDS-specific predictors of CVD risk and death. We conducted a prospective observational single-centre cohort study in lower risk(LR) MDS patients to identify patients at risk for CVD development and death. Patients underwent evaluation for CVD every 6 months by ultrasound and coronary artery calcium(CAC) scan. The carotid plaque score(CPS) was calculated as reported previously (Stein J, 2008). MESA, Framingham(FRS), PREVENT, HELLENIC and SMART risk scores and serum markers of CVD were also assessed. Progression free survival (PFS) was defined as the time from first evaluation to CVD event, MDS progression or death. Of 37 patients recruited in the study 18 had preexisting CVD(pCVD) and 19 had no prior history of CVD(naïve, nCVD). No differences between pCVD and nCVD patients were found for age, sex, WHO subtype according to 2022 classification, IPSSM and mutational profile, but pCVD patients were more often transfusion dependent (p=0.005) and had higher NT-proBNP levels (p=0.037) at baseline. In sharp contrast to general population (Okwuosa, TM et al. JACC 2012), both CAC and CPS scores were not associated with FRS, PREVENT and HELLENIC scores and were increased in low and intermediate risk patients, suggesting that the current CVD risk prediction tools are imprecise in MDS. 27 patients (pCVD:14, nCVD:13) had >1 evaluation. The mean annual CAC increase was 60±31% in pCVD and 54.5±20% in nCVD (p=0.5), markedly higher than expected in the general population (McCullough PA et al. Arch Int Med 2009). We ranked patients from slow (SP) to fast (FP) progressors for accelerated subclinicalatherosclerosis based on weighted annual absolute and relative differences in CAC and CPS, adjusted for the corresponding CVD risk scores. Of note, 7 nCVD patients were ranked as faster progressors from 7 pCVD ones. No associations of SP/FP rankings with age, sex, WHO subtype, IPSSM and mutational profile were found. By contrast, there was a strong correlation with transfusion status (p=0.007) and NT-proBNP levels (p=0.002) at baseline. With a median F/U of 38.8(95% CI 19.6-28) months 8 patients suffered CVD events and 10 died. No difference in PFS was found in SP vs FP and pCVD vs nCVD comparisons. As peripheral blood monocytes are directly involved in the development and progression of CVD we assessed their immunophenotypic profile by multiparametric flow cytometry. In line with their proatherogenic role we observed higher levels of intermediate (p=0.002) and CCR2+ intermediate (p=0.002) monocytes in pCVD vs nCVD patients, whereas SP/FP ranking also correlated with CCR2+ intermediate monocytes (p=0.013). Bulk RNA-seq was then performed on purified intermediate monocytes from 4 SP and 5 FP patients. We identified 176 upregulated and 182 downregulated genes in FP compared to SP patients. The 2 top downregulated genes wereDUSP1 and JUNB, both of which have a protective role against atherosclerosis by modulating monocyte behavior and inflammation. Another downregulated gene, shared with the atherosclerosis monocyte signature of the MESA cohort (Liu et al, Nat Commun 2017), was ALDH1A1, which protects against vascular calcification. GO enrichment revealed upregulation of genes associated with mitochondrial function and enrichment of pathways related to ribosomal small subunit assembly in FP patients, whereas genes associated with the integrated stress response, responses to hypoxia and calcium were downregulated. GSEA against MSignDB and KEGG2 showed a positive correlation of the molecular signature of FP patients to that of TNF signaling and lipid and atherosclerosis pathways. Collectively, the transcriptomic profile of intermediate monocytes from FP patients indicates a state of increased cellular stress triggered by chronic inflammation with a parallel loss of compensatory defense mechanisms. To our knowledge this is the first study in LR-MDS patients assessing longitudinally essential clinical and laboratory factors associated with CVD and providing objective measurements of subclinical atherosclerosis. Our results suggest that the established CVD risk models are dysfunctional in LR-MDS patients. Transfusion status, NT-proBNP levels and the molecular signature of intermediate monocytes may act as novel indicators of CVD risk in MDS patients.

CD8+ T cells are critical players in anti-tumor immunity against solid tumors, targeted by immunotherapies. Emerging evidence suggests that CD8+ T cells also play a crucial role in anti-tumor responses and determining treatment outcomes in hematologic malignancies like myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML). In this review, we focus on the implication of CD8+ T cells in the treatment response of patients with MDS and AML. First, we review reported studies of aberrant functionality and clonality of CD8+ T cells in MDS and AML, often driven by the immunosuppressive bone marrow microenvironment, which can hinder effective antitumor immunity. Additionally, we discuss the potential use of CD8+ T cell subpopulations, including memory and senescent-like subsets, as predictive biomarkers for treatment response to a variety of treatment regimens, such as hypomethylating agents, which is the standard of care for patients with higher-risk MDS, and chemotherapy which is the main treatment of patients with AML. Understanding the multifaceted role of CD8+ T cells and their interaction with malignant cells in MDS and AML will provide useful insights into their potential as prognostic/predictive biomarkers, but also uncover alternative approaches to novel treatment strategies that could reshape the therapeutic landscape, thus improving treatment efficacy, aiding in overcoming treatment resistance and improving patient survival in these challenging myeloid neoplasms.

Bone marrow CD8+ T cells from patients with MDS and CMML show decreased TCR diversity compared to AML. Treatment with the hypomethylating agent azacitidine in MDS and CMML alters TCR repertoire of CD8+ T cells without affecting TCR diversity.

Association between the frequency of CD57+CXCR3+CD8+ T cells and outcome in patients with HR-MDS and AML under treatment with AZA. A, Box plots displaying the percentage of the CD57+CXCR3+ cells within CD8+ T cells, assessed by flow cytometry in responders and nonresponders (HR-MDS, n = 12 responders and 9 nonresponders; AML, n = 9 responders and 11 nonresponders; CMML, n = 5 responders and 5 nonresponders). B, After stratification of patients with HR-MDS and AML to responders (n = 12) and nonresponders (n = 19), FlowSOM analysis was performed on BM CD8+ T cells, which generated six metaclusters that are projected onto the viSNE plots. Representative viSNE plots (one for each group) are shown. C, Box plots showing the proportion of all metaclusters, expressed as the frequency within CD8+ T cells. D, Heatmap depicting the expression levels of all T-related markers. E, Kaplan–Meier curves for OS in patients which received AZA treatment, with ≤29% (n = 51) and >29% (n = 26) CD57+CXCR3+ CD8+ T cells before treatment initiation. The survival curves were compared by the log-rank (Mantel–Cox) test, and the P value is shown. The median OS of the ≤29% group was 20.98 months, whereas the median OS of the >29% group was 12.05 months. F, Survival curves for each disease subgroup. Increased (%) CD57+CXCR3+ correlates significantly with worse survival in patients with HR-MDS and AML, whereas no association is observed in patients with CMML. G, Patients with HR-MDS and AML with ≤29% CD57+CXCR3+ exhibited higher response rates. No association between the frequency of CD57+CXCR3+CD8+ T cells and response to therapy was observed in patients with CMML. An unpaired Student t test was used in A. A Mann–Whitney U test was used in D. **, P < 0.01; ***, P < 0.001.

Luspatercept (LUSPA) a transforming growth factor-beta (TGF-β) superfamily inhibitor is a novel agent for treating the anemia of patients with lower-risk Myelodysplastic Neoplasms (LR-MDS). Although late erythroblasts (Ery) are considered as the target cells of LUSPA (Suragani RNVS et al, Nat Med 2014) limited data exist regarding its impact on the transcriptome of other cell types. We investigated the effect of LUSPA therapy on the molecular signature of FACS sorted bone marrow hematopoietic stem and progenitor cells (HSPCs), erythroblasts (Ery) and CD3+ T cells from five patients with LR-MDS, by utilizing single cell transcriptomic analysis (10x Genomics). All patients harbored SF3B1 mutations; two patients had mutations in TET2, one in DNMT3A and one in RUNX1. In pretreatment samples cells were partitioned into 12 cell clusters and annotated based on their distinct gene expression profile. Pathway enrichment analysis (EnrichR) of each cluster's molecular signature identified enhanced TGF-β signaling in HSPCs and myeloid-primed progenitor clusters (My P), rather than erythroblast (Ery) or megakaryocyte-erythroid progenitors (MEP), indicating that HSPCs could also be targeted by LUSPA. After treatment with LUSPA we observed a downregulation of the expression of genes associated with TNF signaling via NF-κB and interferon-γ (IFN-γ) signaling in all clusters except from the late erythroblast (Late Ery) cluster. Downregulation of genes associated with cholesterol homeostasis, a pathway associated with proliferation and myeloid bias of hematopoietic progenitors (Mitroulis et al, Cell, 2018), was also observed in the clusters of HSPC, multipotent progenitors (MPP) and myeloid-biased progenitors (My P). Of note, downregulation of genes associated with TGF-β signaling was observed in the clusters of HSCs, myeloid progenitor (My P), and megakaryocyte-erythroid progenitors (MEP), but not in erythroblasts (Ery). Increased cell proliferation signatures, such as the G2M cell cycle pathway and E2F targets, were also observed after Luspatercept in MEPs and myeloid progenitor clusters (My P). Members of the TGF-β superfamily are known to suppress anti-tumor immunity by actin on T cells (Pinjusic K et al, J Immunother Cancer 2022), therefore we also analyzed T cells. In pretreatment samples, cells were partitioned into 11 cell clusters, including 5 clusters of CD8+ T cells, 4 clusters of CD4+ T cells, a cluster of proliferating cells and a cluster of progenitor T cells. After treatment with LUSPA we observed an increase in the cytotoxic score in three cytotoxic T lymphocyte (CTL) cell clusters (IFN-CTL, KLRB1-CTL and NKG7-CTL). Specifically, there was a significant upregulation in the expression of GZMA, GZMH, NKG7, CX3CR1, GNLY in NKG7-CTL cluster, of GZMA, GZMK in KLRB1-CTL and GZMA and GZMH in IFN-CTL. Additionally, there was a downregulation in the exhaustion score of the NKG7-CTL cluster. Regarding cell exhaustion score, genes that were downregulated after treatment with LUSPA included TNFRSF1B, TNFRSF9, FAM3C, IFNG, LYST, and GBP2. IFN signaling has been previously associated with cytotoxic activity in bone marrow CD8+ T cells in MDS (Tasis et al, Cancer Res Commun, 2024)To this direction, we observed an increase in the IFN score of the NKG7-CTL cluster, which was associated with an upregulation in the expression of IFITM1, IFIT3, OASL, XAF1, LY6E, IRF9, MX1, and EIF2AK2. Also, in the Treg-like cluster pathway analysis showed that LUSPA treatment enhanced the IFN response signature associated with the upregulation in the expression of SAMD9L, EIF2AK2, OASL and downregulated the apoptosis signature associated with a decrease in the expression of DNAJA1, JUN, DDIT3, SAT1, BIRC3. In the other CD4+ T cell clusters, the upregulation of IFN response signature was also observed in IL7R-CD4+ and CCR7-CD4+ clusters. Taken together our preliminary findings point to a direct effect of LUSPA on HSPC subsets by modulating the expression of genes associated with inflammatory, cholesterol, TGF-β and cell proliferation pathways, but also on T cells by enhancing the cytotoxic signature of CTL, paired with the induction of an IFN related response in several T cell clusters. Our results are consistent with the reported trilineage response after LUSPA (Garcia-Manero G et al, Blood 2022) and suggest a pleiotropic effect of LUSPA potentially including a beneficial immunomodulation in patients with MDS.

Untargeted analysis of CD45+ immune cells in patients with MDS, AML, and CMML by CyTOF. A, Multidimensional scale plot depicting the relationship between BM samples of patients with LR-MDS (n = 12), HR-MDS (n = 15), AML (n = 16), and CMML (n = 5). B, Heatmap showing the expression of the markers used for the characterization of each cell cluster. C, UMAP displaying the major immune cell clusters. D, Box charts displaying the frequency of each cell cluster. E, Violin plots showing the expression level of CXCR3 in the CD8 T1, CD8 T2, and CD4 T2 clusters, respectively. Kruskal–Wallis followed by the “two-stage” Benjamini, Krieger, and Yekutieli multiple comparison test was used in D. One-way ANOVA followed by the “two-stage” Benjamini, Krieger, and Yekutieli multiple comparison test was used in E. *, P < 0.05; **, P < 0.01.

Sickle cell disease (SCD) results from mutations in the β-globin gene, producing abnormal hemoglobin S (HbS) and leading to complications causing significant morbidity and mortality.1 One of the hallmark consequences of SCD is the occurrence of vaso-occlusive crises (VOCs), which arise from the interplay of factors in the disease's pathophysiology, involving abnormal hemoglobin polymerization, inflammation, endothelial dysfunction, and activation of the immune system, culminating in the painful obstruction of blood vessels by sickled red blood cells, that tend to obstruct blood vessels, leading to reduced blood flow and oxygen supply. This vicious cycle of ischemia followed by reperfusion constitutes the ischemia-reperfusion model.2 The complement system, a complex defense mechanism, is implicated in various diseases through unregulated activation.3 However, diagnostic challenges hinder patient selection for complement inhibition.4 Preliminary data from our group using novel assays indicate complement activation even at a steady state in a limited patient population.5 Limited information exists on additional markers in the complement activation and endothelial dysfunction cycle in SCD. Neutrophil extracellular traps (NETs), indicative of thromboinflammation, are elevated in SCD patients, even during steady state.6 ADAMTS13 (A Disintegrin and Metalloproteinase with Thrombospondin motifs), studied for its role in SCD vasculopathy, shows conflicting results as a potential biomarker.7, 8 Genetic variants and autoantibodies leading to unregulated complement activation are implicated in the pathogenesis of various human diseases.9 Despite the lack of specific biomarkers or targeted treatments for crises, our hypothesis posits the presence of complement activation and thromboinflammation in SCD, particularly during complications, with distinct yet unexplored clinical or genetic features in these patients. Our study's methods regarding patient population, observation period, functional assays, and genetic, bioinformatic, and statistical analysis are demonstrated in supplementary materials. Our study included 81 adult SCD patients who are treated in different Hemoglobinopathies Units across Northern Greece. Their median age was 41 years, and 50 were female (61.7%). As expected in our population, the majority had the S/beta genotype (62), while 19 patients had the S/S genotype. Twenty-three presented SCD complications during the observation period (17 vaso-occlusive crises and six proteinuria/nephropathy) and were studied during this complication. Importantly, none of the patients that presented with renal damage, was on deferasirox, or other iron chelation therapy. The remaining 58 patients were studied at the end of the observation period. The comprehensive characteristics and parameters studied are summarized in Table 1. First, we measured soluble C5b-9 and modified Ham test at a steady state and during the observation period. At steady state, soluble C5b-9 was above normal limits in a significantly higher percentage of patients who then presented a SCD complication compared to those who did not (7/23, 30%, p = 0.028). Similarly, patients who presented a complication had a significantly higher rate of modified Ham test (5/23, 21%, p = 0.001, Figure 1A) at steady state. During the observation period, we detected a significant increase in soluble C5b-9 (p < 0.001). This increase was significantly higher in patients with complications (p = 0.046, Figure 1B). One patient who did not experience complications had a positive Ham test and sC5b-9, while 42 patients who did not present complications had only elevated C5b-9. Since VOC was the most common complication, significant results were replicated in patients with VOC (data not shown). ADAMTS13 was similar and within normal limits at steady state and follow-up (Figure 2B). One other parameter studied as a marker of thromboinflammation was NETs. We were able to measure NETS on 60 of our 81 patients. NETs at steady state were significantly increased in patients who then developed complications related to the disease. Additionally, there was a significant increase of NETs measured at follow-up compared to steady state (p < 0.001, Figure 2B). Patients with a positive modified Ham test had increased C5b9 (p < 0.001). No significant association was found between NETs and C5b-9 in our population. We started our genetic analysis from rare variants with a minor allele frequency (MAF) less than 1%, since rare variants have been commonly described in patients with complement-related disorders10. We detected 23 rare variants, as shown in detail in Supporting Information S1: Table 1. Almost all rare variants were documented in unique patients. Only rs35836460 in CFH, rs186530184 in CFHR1, rs183647515 in THBD, and rs202206149 in ADAMTS13 were found in two patients, with rs3176136 in THBD detected in three patients. Among 23 rare variants, only three were characterized as pathogenic by bioinformatic tools as described in the next paragraph. To further understand the clinical significance of the remaining detected variants, we used four bioinformatic tools. We found 8 missense variants characterized as deleterious by these tools, as presented in detail in Supporting Information S1: Table 2. Among them, three were also rare variants (rs117793540 in C3, rs1800579 in THBD, and rs143568784 in ADAMTS13), potentially confirming its pathogenic role. Each rare variant was found in one patient. Additionally, rs144082872 in CFI was also detected in one patient. The remaining deleterious variants were detected in 2–15 patients each. Patients were heterozygous, except for one homozygous for rs28647808 (ADAMTS13). Next, we searched for detected variants in the database of complement gene variants. As shown in Supporting Information S1: Table 3, 21 variants have been described in this complement database. In agreement with the bioinformatic tools, rs144082872 in CFI has also been characterized as pathogenic in this database. To better characterize the phenotype of SCD patients, we investigated associations of functional and genetic assays with clinical characteristics. All patients with pathogenic or deleterious variants had increased complement activation by functional assays (modified Ham test or soluble C5b9), except for two patients with CFB and two with ADAMTS13 variants. Interestingly, patients with a combination of pathogenic or deleterious variants had a significantly higher transfusion dependency rate (65% vs. 35%, p = 0.031). Finally, we sought to determine predictors of complications. Among studied baseline patient characteristics (Supporting Information S1: Table 4), there was no univariate association with complications. Therefore, we included steady-state soluble C5b-9 and NETs, as well as the presence of a pathogenic or deleterious variant in multivariate analysis. The only independent predictor was C5b-9 levels. In our study, we reveal activation of complement and thromboinflammation, even in the steady state of SCD patients who later develop complications. Notably, these markers experience a significant surge during the onset of complications. Germline genetic variants may contribute to the predisposition to complement activation, linked to clinical characteristics. Importantly, C5b-9 independently predicts complications in our multivariate analysis. The inflammatory response becomes a key driver in SCD's pathophysiology, fueled by recurrent ischemic reperfusion events. Besides direct tissue damage, chronic inflammation contributes to cumulative damage in SCD. Complement activation plays a crucial role in both inflammation and the prominent hemolysis seen in SCD. Given the disease's multifactorial nature, markers such as NETs and ADAMTS13 are explored as potential treatment targets. Assessing complement activation in clinical labs faces challenges due to limited access to robust assays.9 Our study addresses this by introducing a modified Ham test for early and robust detection of complement activation, alongside confirming soluble C5b-9 as a practical marker for complications' evaluation. The increase in Bb fragments supports alternative pathway activation. Previous research using less precise assays indicated alternative pathway activation in SCD.11 Exploring the genomic landscape of complement-related variants in SCD is a novel aspect of our study. Genetic analysis using multiple tools identifies pathogenic variants, highlighting an association between variants and clinical characteristics.12 Our study has approached genetic analysis with multiple tools, while previous studies in complement-related disorders have reported only rare variants.13 As our group has shown in thrombotic microangiopathies (TMA),14 additional variants might also harbor clinical significance. In an effort to better understand the clinical significance of rare variants, novel databases have been created, such as the Database of Complement Gene Variants.12 These databases are considerably helpful in linking the genotype with the phenotype in these patients. However, this approach does not take into account the majority of detected variants, whose functional and clinical significance remains to be studied. Variants such as rs1047286, rs2230199, rs800292 for C3, as well as rs1061147, rs1061170 for CFH, and the deleterious variant rs12614 for CFB have been implicated in the development of age-related macular degeneration (AMD).15, 16 Current SCD pharmacological treatments, including hydroxyurea and voxelotor, show positive effects, but the need for novel therapeutic approaches persists.17 Crizanlizumab, initially promising, was discontinued by the European Medicines Agency (EMA) due to efficacy issues.18 Eculizumab, a complement inhibitor, shows encouraging results, but challenges hinder wider study.19 Preclinical data suggest that MASP-2 or MASP-3 inhibition may prevent SCD complications.20 The CROSSWALK trial is investigating the benefits of a C5 inhibitor in VOC prevention.21 Our study has limitations, including varying patient treatments across centers and limited population size. Long-term observations in a larger cohort could provide additional insights. Data gaps on ADAMTS13Ag hinder a full understanding of its role in SCD. The predominantly S/β genotype in our population limits generalizability, and the impact of hydroxyurea on complement profiles is explored but inconclusive. The study's timing during the COVID-19 pandemic affected sample collection and patient visits. One other limitation is that 20 of our patients were on a chronic transfusion schedule. Moreover, our study's cohort is rather small, and further studies with bigger cohorts are needed to determine whether or not genetic variants play a role. In conclusion, complement activation and thromboinflammation are evident in SCD patients, especially during complications. Considering the safety and efficacy of complement inhibitors in other complementopathies and preliminary data from SCD, our study highlights useful tools to early detect patients that might benefit from complement inhibition. The authors would like to thank the immunobiologist Maria Spachidou for their technical assistance. Eleni Gavriilaki was supported by the ASH Global Research Award. Christos Varelas and Eleni Gavriilaki designed research. Christos Varelas, Efthymia Vlachaki, Philippos Klonizakis, Despoina Pantelidou, Michael Diamantidis, Nikolaos Sabanis, Ioanna Christodoulou, Despina Papadopoulou, Evdoxia Koravou, Ioanna Sakellari, Stamatia Theodoridou, and Ioannis Mitroulis performed research. Christos Varelas, Fani Minti, Apostolia Papalexandri, Tasoula Touloumenidou, and Eleni Gavriilaki analyzed data and wrote the paper. Efthymia Vlachaki, Sofia Vakalopoulou, Vasilis Perifanis, George Vassilopoulos, and Eleni Gavriilaki contributed to the research design and edited and approved the paper. Eleni Gavriilaki has consulted for Alexion, Omeros, and Sanofi Cooperation. The other authors declare no competing financial interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. The work was in part funded by the General Secretariat for Research and Technology Management and Implementation Authority for Research, Technological Development and Innovation Actions (MIA-RTDI) (grant T2EDK-02288, MDS-TARGET). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Identification of a CD8+ subpopulation (CD57+CXCR3+) which distinguishes patients with MDS from patients with AML and CMML. A, Representative viSNE plots, derived from the FlowSOM analysis of BM CD8+ T cells from patients with LR-MDS (n = 12), HR-MDS (n = 15), AML (n = 16), and CMML (n = 5). B, Bar plots displaying the proportion of the metaclusters between the groups, expressed as percentage within CD8+ T cells. C, Heatmap depicting the expression level of the T-related markers between the metaclusters. D, Violin plots showing the expression level of CXCR3 in metacluster 1. E, Representative flow cytometry plots for the identification of the CD57+CXCR3+CD8+ T cell subpopulation in a cohort of patients with LR-MDS (n = 7), HR-MDS (n = 27), AML (n = 20), and CMML (n = 10). F, Percentage of CD57+CXCR3+ cells within CD8+ T cells. Kruskal–Wallis was used in B and D. One-way ANOVA followed by the “two-stage” Benjamini, Krieger, and Yekutieli multiple comparison test was used in F. *, P < 0.05; ***, P < 0.001.

IntroductionImmune checkpoint blockade (ICB) immunotherapy has revolutionized cancer treatment, demonstrating exceptional clinical responses in a wide range of cancers. Despite the success, a significant proportion of patients still fail to respond, highlighting the existence of unappreciated mechanisms of immunotherapy resistance. Delineating such mechanisms is paramount to minimize immunotherapy failures and optimize the clinical benefit.MethodsIn this study, we treated tumour-bearing mice with PD-L1 blockage antibody (aPD-L1) immunotherapy, to investigate its effects on cancer-induced emergency myelopoiesis, focusing on bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs). We examined the impact of aPD-L1 treatment on HSPC quiescence, proliferation, transcriptomic profile, and functionality.ResultsHerein, we reveal that aPD-L1 in tumour-bearing mice targets the HSPCs in the BM, mediating their exit from quiescence and promoting their proliferation. Notably, disruption of the PDL1/PD1 axis induces transcriptomic reprogramming in HSPCs, observed in both individuals with Hodgkin lymphoma (HL) and tumour-bearing mice, shifting towards an inflammatory state. Furthermore, HSPCs from aPDL1-treated mice demonstrated resistance to cancer-induced emergency myelopoiesis, evidenced by a lower generation of MDSCs compared to control-treated mice.DiscussionOur findings shed light on unrecognized mechanisms of action of ICB immunotherapy in cancer, which involves targeting of BM-driven HSPCs and reprogramming of cancer-induced emergency myelopoiesis.