Although simvastatin plays a crucial role in lipid management, tumor therapy and acute liver injury, its potential effects in autoimmune hepatitis (AIH) has received limited investigative attention. Our study demonstrated that in the ConA-induced AIH model, HMG-CoA reductase (HMGCR), the pharmacological target of simvastatin (SIM), was significantly upregulated in T cells, particularly in CD4+ T cells. Furthermore, our results showed that simvastatin treatment in ConA-induced AIH model reduced the level of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and alleviated liver injury. Flow cytometric analysis revealed that simvastatin treatment promoted CD4+ T cell apoptosis while significantly reducing the secretion of crucial inflammatory cytokines in vivo and vitro, including IL-17A, IL-6, IFN-γ, and TNF-α. To explore the underlying mechanisms, we performed transcriptome sequencing on the CD4+ T cells from mice treated with or without simvastatin. RNA-sequencing analysis revealed the involvement of the calcium signaling pathway and transcription factor NFATC3 in the regulation of CD4+ T cells. qPCR and flow cytometry analyses further confirmed that simvastatin exerted its therapeutic effects by suppressing the calcium signaling pathway and downregulating the expression of nuclear factor of activated T cells 3 (NFATC3). Collectively, our study demonstrates that simvastatin alleviates CD4+ T cell inflammatory responses in AIH through calcium-dependent signaling pathway.

Intestinal acute graft-versus-host disease (aGVHD) is a common life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although tissue-resident memory T (TRM) cells are thought to play a pathophysiological role in animal models of aGVHD, little is known about the role of distinct subsets of TRM cells in human intestinal aGVHD. Herein, we combined multiplex immunohistochemical staining with single-cell RNA sequencing to elucidate the differentiation trajectory, lineage commitment, clonal expansion, and functional properties of distinct CD8 + TRM cell subsets in human intestinal aGVHD. We identified a predominant GZMK + CD8 + TRM subset, characterized by the GZMK and CD49A markers. Intestinal aGVHD was associated with infiltration of GZMK + CD8 + T cells with TRM features, which showed enhanced clonal expansion, IFN signaling pathway–associated proinflammatory pathway expression, and lineage bifurcation differentiation properties. High GZMK + CD8 + TRM subset infiltration was associated with greater human intestinal aGVHD severity and poor prognosis. Together, our studies highlight the importance of the GZMK + CD8 + TRM subset in human intestinal aGVHD, and interest for designing GZMK + CD8 + TRM cell–targeted therapies.

Acute graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, although the diverse roles of neutrophils in inflammation and cancer have been recognized, the specific contributions of distinct neutrophil subsets to acute GVHD remain unclear. Herein, we performed a prospective clinical study to investigate the role of distinct neutrophil subsets in G-CSF-mobilized peripheral blood (PB) graft on acute GVHD and to establish statistical prediction models. Our study demonstrated that the early committed neutrophil progenitor (proNeu1) was a risk factor, whereas immature neutrophil (immatureNeu) was a protective factor for grade Ⅱ-Ⅳ acute GVHD. The prediction model based on donor age, proNeu1 and immatureNeu showed significant accuracy for Ⅱ-Ⅳ acute GVHD diagnosis, with an area under the curve (AUC) of 0.782 (95% CI, 0.67-0.88) for the training set and an AUC of 0.771 (95% CI, 0.63-0.91) for the validation set. In addition, Decision Curve Analysis (DCA) indicated that the prediction model possessed high clinical utility. High risk group based on the prediction model is correlated with higher 100-day cumulative incidence of acute GVHD and 1-year cumulative incidence of chronic GVHD. The prediction model based on proNeu1 and immatureNeu subsets provides a risk assessment tool for clinicians to better predict and manage the course of acute GVHD. Moreover, we demonstrated that donor-derived CD10-immature neutrophils in the G-CSF-mobilized PB grafts as an immunoregulatory neutrophil were associated with attenuated acute GVHD, potentially through increased Reactive Oxygen Species (ROS) levels.

Immune dysregulation is a key driver of metabolic dysfunction-associated steatotic liver disease (MASLD). Double-negative regulatory T (DNT) cells, which are essential for hepatic immune homeostasis, are functionally impaired in MASLD. CD39, an ectoenzyme that converts ATP to adenosine, sustains DNT cell viability and immunoregulatory function. However, the mechanisms of MASLD-induced CD39 dysregulation remain unknown. In this study, a transcriptomic analysis of hepatic DNT cells from MASLD mice revealed reduced ATP hydrolysis, as confirmed by flow cytometry, and lower CD39 level compared to normal control diet-fed mice. Similarly, single-cell transcriptomic data showed that CD39 was downregulated in hepatic DNT cells from MASLD patients, and higher CD39 expression was associated with enhanced cytotoxicity and viability. Furthermore, in vitro experiments confirmed that CD39 maintained the immunosuppressive function of DNT cells by hydrolyzing ATP and suppressing monocyte activation. Mechanistically, arachidonic acid (AA), which is enriched in the livers of MASLD, suppressed CD39 via hypoxia-inducible factor-1α (HIF-1α). This AA-HIF-1α-CD39 axis impaired DNT cell-mediated monocyte immunosuppression, accelerating immune imbalance and MASLD progression. These findings establish the AA-HIF-1α-CD39 axis as a key pathway driving DNT cell dysfunction in MASLD, highlighting potential therapeutic strategies targeting metabolic-immune crosstalk to restore hepatic immune balance.

BACKGROUND: Hepatic ischemia-reperfusion injury (HIRI) is a critical contributor to adverse clinical outcomes and increased mortality after liver surgery or transplantation. Lactate, formerly regarded as a metabolic waste product, has emerged as a critical immunomodulatory molecule. This study investigated how lactate modulates HIRI by regulating intrahepatic inflammation and intercellular communication. METHODS: A mouse model of HIRI was constructed, along with in vitro hypoxia/reoxygenation (H/R) of primary hepatocytes and LPS-stimulated bone marrow-derived neutrophils, to evaluate the protective effects of sodium lactate. RNA sequencing was performed to identify changes in transcriptional profiles induced by lactate pretreatment and to explore regulatory mechanisms involving hepatocytes and neutrophils during HIRI. Cell-cell interaction analysis was performed to identify key interactions by which lactate regulates hepatocyte-neutrophil communication. Furthermore, analysis of transcription factor binding motifs using the JASPAR database was conducted to elucidate potential molecular mechanisms underlying the previously identified key interactions. RESULTS: Lactate administration significantly alleviated hepatic inflammation and tissue injury in HIRI mice. Transcriptomic analysis revealed that lactate attenuated hepatocyte-mediated inflammatory responses, chemotaxis, and apoptosis, while also suppressing neutrophil-driven inflammation, migration, oxidative stress, and NET formation. Integrated digital spatial profiling (DSP) combined with cell-cell interaction analysis revealed IL-33/ST2 as a critical ligand-receptor pair mediating hepatocyte-neutrophil crosstalk, which was significantly downregulated by lactate. IL-33 has been demonstrated to stimulate neutrophil activation and exacerbate liver inflammation via its receptor ST2 in HIRI. Consistently, lactate pretreatment suppressed this pathway, thereby mitigating hepatic injury and intrahepatic inflammation. Mechanistically, lactate inhibited IL-33 expression in hepatocytes via the AKT/c-Fos signaling axis and reduced ST2 expression in neutrophils through the JAK/STAT3 pathway. CONCLUSIONS: Lactate alleviates HIRI by protecting hepatocytes, suppressing neutrophil-driven inflammation, and disrupting hepatocyte-neutrophil communication through the IL-33/ST2 axis.

Retrospective studies suggested that autologous stem cell transplantation (auto-SCT) might achieve comparable or even better outcomes compared with allogeneic SCT for patients with genetic intermediate-risk acute myeloid leukemia (IR-AML) and no detectable measurable residual disease (MRD-). However, prospective studies are lacking comparing auto-SCT and matched sibling donor SCT (MSD-SCT). We initiated a biological assignment comparative study at fourteen institutions in China. Patients with newly diagnosed de novo IR-AML and MRD- within 3 cycles of intensive chemotherapy were eligible for the study if they were 14 to 60 years old and received auto-SCT or MSD-SCT. Patients were assigned to auto-SCT or MSD-SCT based on donor availability. The primary endpoint was the 3-year disease-free survival (DFS). The trial was registered with Clinicaltrials.gov, NCT03620955 and completed. Between November 15, 2018, and March 31, 2022, 708 patients were screened and 518 eligible for analysis, including 259 in the auto-SCT and 259 in the MSD-SCT groups. With a median follow-up time of 55.2 (IQR 47.4–65.2) months, the 3-year DFS was 82.2

Double-negative T (DNT) cells (TCRαβ+CD4-CD8-NK1.1-/CD56-) exhibit strong tumor-killing capabilities. Our single-cell transcriptome analysis has revealed high Fcer1g expression in DNT cells, but its role in tumor immunity remains unclear. In this study, we demonstrated that IgG1 stimulation significantly upregulated IgG Fc receptors and cytotoxic molecules in DNT cells, enhancing their cytotoxicity against MC38 tumor cells in vitro. FcεRIγ-deficient DNT cells failed to respond effectively to IgG1 stimulation. Inhibiting the downstream spleen tyrosine kinase (Syk) of FcεRIγ reduced cytotoxicity of DNT cells and phosphorylation levels of molecules such as AKT and NF-κB. In a subcutaneous tumor model, combined treatment with DNT cells and tumor-specific antibodies more effectively inhibited tumor growth compared to DNT cells alone, while FcεRIγ-deficient DNT cells combined with antibodies showed no significant difference in efficacy compared to DNT cells alone, suggesting that DNT cells enhance tumor cell killing via FcεRIγ-mediated antibody-dependent cellular cytotoxicity (ADCC). These results indicate that DNT cells mediate antitumor ADCC effects through high FcεRIγ expression. Binding of IgG1 to FcεRIγ activates the FcεRIγ/Syk/AKT/NF-κB pathway, consequently enhancing tumor cell killing. Thus, DNT cells may play a significant role in cancer immunity, providing a basis for novel immune cell and antibody combination therapies.

Humoral immunological memory mediated by memory B cells (MBCs) and long-lived plasma cells (LLPCs) is critical for sustained protection following infection or vaccination. LLPCs protect the hosts by secreting protective neutralizing antibodies over extended periods. However, the mechanism regulating their survival and thus the durability of protective antibodies remains unclear. Here, we showed in human and mouse models that intermittent fasting impaired humoral immunological memory by accelerating antibody decay. Fasting selectively depleted LLPCs while sparing MBCs in mice. Mechanistically, this effect was mediated by increased extracellular β-hydroxybutyrate, a ketone body produced during fasting, which acted through the hydroxycarboxylic acid receptor 2 (HCAR2) on plasma cells. Activation of the HCAR2-Gαi-adenylate cyclase-cAMP axis by β-hydroxybutyrate downregulated CXCR4, leading plasma cells to exit their bone marrow niche and undergo apoptosis in the periphery. These findings reveal that fasting-induced metabolic signals regulate humoral immunity duration and suggest that diet and lifestyle could influence vaccine effectiveness.

Supplementary Figure S5. Incorporating HHT to overcome VEN resistance.

Supplementary Figure S2. Treatment response rates by co-mutations.

Supplementary Table S2. Univariable and Multivariable analyses for the survival of the whole corhot.

Primary biliary cholangitis (PBC) is a liver-specific autoimmune disease. Treatment of PBC with ursodeoxycholic acid (UDCA) is not sufficient to prevent disease progression. Our previous study revealed that the number of hepatic double-negative T cells (DNT), which are unique regulatory T cells, was reduced in PBC patients. However, whether replenishment of DNT can prevent the progression of PBC remains unclear. DnTGFβRII (Tg) mice and 2OA-BSA-immunized mice received DNT alone or in combination with oral UDCA. After 6-12 weeks of treatment, these mice were assessed for serological changes, liver pathological manifestations and intrahepatic immune responses. Adoptive transfer of DNT alone significantly decreased serum levels of alanine transaminase (ALT), aspartate transaminase (AST), antimitochondrial antibody M2 (AMA-M2) and immunoglobulin M (IgM) in both Tg and 2OA-BSA-immunized PBC mouse models. In addition, DNT exhibited a strong killing effect on liver T cells and strong inhibition of their proliferation, but did not significantly improve the histology of PBC liver. However, combination therapy with DNT and oral UDCA predominantly ameliorated liver inflammation and significantly inhibited hepatic T and B cells. In vitro further study revealed that UDCA up-regulated the proliferation of DNT, increased the expression of the functional molecule perforin, and reduced the expression of NKG2A and endothelial cell protein C receptor (EPCR) through the farnesoid X receptor (FXR)/JNK signaling pathway in both mice and human DNT. A single transfer of DNT ameliorated PBC in mice, while combination therapy of DNT with oral UDCA displayed a better efficacy, with stronger inhibition of hepatic T and B cells. This study highlights the potential application of DNT-based combination therapy for PBC, especially for UDCA non-responders.

Abstract Introduction Treating acute myeloid leukemia (AML), a diverse group of hematological cancers affecting bone marrow and blood, remains difficult, with a five-year survival rate of about 30%. Although screening for genetic mutations and fusion genes has become essential in AML diagnosis and risk stratification, the clinical significance of copy number aberrations (CNAs) remains poorly understood. To this end, we conducted a multicenter prospective clinical trial (ChiCTR2300077695) to characterize the genomic landscape of CNAs using shallow whole-genome sequencing (sWGS), termed LeukoPrint, in AML. Interim results from the first 205 patients (pts) are reported here. Methods In this prospective multicenter clinical trial, we aim to enroll 600 newly diagnosed AML pts (excluding APL) across 13 participating hospitals in China. Bone marrow samples were collected and underwent CNA profiling via LeukoPrint (1× sWGS) at three stages: pretreatment, post-induction, and relapse. CNAs only those greater than 5 Mbp were analyzed. Conventional karyotyping analysis was performed in parallel for this cohort, and its diagnostic performance was compared with LeukoPrint. European Leukemia Net (ELN)-defined CNAs were used for risk stratification, consistent with standard cytogenetic analysis protocols. The trial received approval from the Ethics Committees of all participating hospitals, and all pts provided informed consent. By April 9, 2025, 205 pts were enrolled in this study, with 136 in the younger group (aged 18–59) and 69 in the older group (aged ≥60). All pts underwent CNA profiling using LeukoPrint at least twice, at pretreatment and post-induction phases. Results Using LeukoPrint, we detected 193 CNAs in 87 pts (42.4% of the cohort). No notable difference in detection rates was observed between younger and older groups (41.9% vs. 43.5%). CNAs were frequently identified in chromosomes 7, 8, 11, and Y (each with >5% prevalence), with the most common recurrent CNAs occurring at 8q24.21 (13% prevalence), the locus containing the MYC oncogene. Recurrent deletions were predominantly observed in 5q31.3 and 7q36.1, the genomic regions harboring the oncogenes ACSL6, CD74, and EZH2. LeukoPrint outperformed karyotyping in CNA detection (42.4% vs. 27.3%), enhancing results for 51 pts (24.9% of the cohort). Notably, 38 pts, initially classified with normal karyotypes or failed karyotyping, were reclassified as carrying CNAs. Applying the 2022 ELN criteria, three pts initially classified as low- or intermediate-risk based on genetic mutations, fusions, and karyotypes were reclassified as high-risk following LeukoPrint analysis in place of karyotyping. Clinical follow-up in two cases confirmed poor outcomes in one, supporting LeukoPrint’s prognostic value. LeukoPrint demonstrates strong potential for prognostic prediction by monitoring dynamic change of molecular response in 115 pts. A significantly higher proportion of pts with post-induction CNAs were identified as non-responders than those without CNAs (6 vs. 3). Similarly, the proportion of pts without detectable post-induction CNAs was 95.5% in complete remission (CR, n=22), 83.3% in CR with partial hematologic recovery (CRh, n=6), 71.4% in CR with incomplete hematologic recovery (CRi, n=7), 0% in partial remission (PR, n=3), and 0% in non-responders (NR, n=3). Copy-neutral loss of heterozygosity (CN-LOH) occurs when one allele is lost and the remaining allele is duplicated, resulting in no net change in copy number. This genomic alteration, such as TP53 CN-LOH, can have significant clinical impact but is undetectable by traditional karyotyping or fluorescence in situ hybridization (FISH). LeukoPrint identified CN-LOH in 43 (20.9%) of 205 pts in this cohort. Notably, CN-LOH was detected in 25 out of 114 pts without detectable CNAs, revealing that LeukoPrint provides additional insights into chromosomal aberrations in 12% (25/205) of pts. Combined with 24.9% from standard LeukoPrint analysis, LeukoPrint enhanced CNA detection in at least 36.9% of pts. Conclusions LeukoPrint outperforms traditional karyotyping in detecting CNAs, improving data accuracy in over one-third of pts in this cohort. It enables dynamic monitoring of treatment response and disease progression, demonstrating strong correlation with clinical outcomes. These findings suggest that LeukoPrint holds significant promise as a complementary or alternative tool for conventional cytogenetic methods in AML.

Background:Letermovir (LTV) is an effective strategy for cytomegalovirus (CMV) reactivation prophylaxis and is increasingly used for allogeneic hematopoietic stem cell transplantation. However, it carries the risk of delayed immune reconstitution. This retrospective study assessed the impact of primary LTV prophylaxis on viral infections, disease relapse, and immune reconstitution in haploidentical hematopoietic stem cell transplantation (haplo-HSCT) recipients. Methods:Among 462 patients from Nanfang Hospital, propensity score matching created two cohorts: 106 with LTV prophylaxis and 212 without LTV prophylaxis. EBV/CMV infection, relapse, and survival were analyzed by competing risk models and Cox regression. Immune reconstitution and function were assessed by flow cytometry. Results:LTV prophylaxis had protective effects against CMV viremia, with a 1-year incidence of 32.1% in the LTV group compared with 46.2% in the non-LTV group (P = 0.009). However, the 1-year cumulative incidence of EBV viremia was significantly higher in the LTV group than in the non-LTV group (38.7% vs.13.7%, P<0.001). On multivariate analysis, LTV prophylaxis was a protective factor for CMV viremia (HR = 0.54, P = 0.014) but a risk factor for EBV viremia (HR = 2.69, P<0.001). Additionally, the 1-year cumulative incidence of relapse post-HSCT was notably higher in the LTV group than in the non-LTV group (13.2% vs. 6.1%, P = 0.032). In multivariate analysis, LTV prophylaxis was an independent risk factor for relapse (HR = 2.56, P = 0.024). Lymphocyte subset counts and functions post-transplantation were significantly lower in the LTV group than in the non-LTV group. Conclusion:LTV prophylaxis might play a dual role in haplo-HSCT recipients, reducing CMV infection but increasing EBV infection and relapse.

Whether adding anthracycline to intermediate- or high-dose cytarabine as consolidation is beneficial remains unclear in acute myeloid leukemia (AML). Eligible AML patients in first complete remission were randomly assigned (1:1) to receive either high-dose cytarabine with idarubicin (IA3 + 3) (idarubicin 10 mg/m2, d1-3 and cytarabine 2 g/m2, every 12 h, d1-3) or high-dose cytarabine (HDAC) (cytarabine 3 g/m2, every 12 h, d1-3) regimens as first consolidation. The primary endpoint was the rate of negative measurable residual disease (MRD−) after first consolidation. Between November 2018 and December 2021, 407 patients were assigned to IA3 + 3 (n = 204) or HDAC (n = 203) groups. MRD− after first consolidation for IA3 + 3 and HDAC groups was 65.2

Supplementary Table S5. The 167-gene next generation sequencing panel.

Rovadicitinib (TQ05105) is a novel, oral dual Janus kinase 1/2 and rho-associated coiledcoil-containing protein kinase-1/2 inhibitor targeting inflammatory and fibrotic components of chronic graft-versus-host disease (cGVHD). This phase 1b/2a, multicenter, open-label study enrolled patients with moderate or severe glucocorticoid-refractory or-dependent cGVHD to evaluate the safety and efficacy of rovadicitinib. The study followed a 3+3 design with 2 escalating doses (rovadicitinib 10 and 15 mg twice daily) and a dose expansion cohort. Primary end points included safety and recommended phase 2 dose (RP2D); the best overall response (BOR) was the key secondary end point. A total of 44 patients were enrolled (29 at 10 mg, 15 at 15 mg twice daily). Rovadicitinib was well tolerated without dose-limiting toxicity at both dosages, and no rovadicitinib-related adverse events (AEs) led to discontinuation. The most prevalent hematological AE was anemia (38.6%), with grade >= 3 of 4.6%. The RP2D was 10 mg twice daily. The BOR was 86.4% (95% confidence interval [CI], 72.6-94.8), with no difference between the 2 dosage cohorts. Besides, BOR was 72.7% in the steroid-refractory cohort and 90.9% in the steroid-dependent cohort. All affected organs exhibited responses regardless of prior therapy. The failure-free survival rate was 85.2% (95% CI, 64.5-94.3) at 12 months. Rovadicitinib reduced corticosteroid doses in 88.6% of patients and improved cGVHD symptoms in 59.1%. Rovadicitinib has favorable tolerability and notable clinical response rates, ameliorating the quality of life and reducing corticosteroid dose requirements in patients with glucocorticoidrefractory or-dependent cGVHD. This trial was registered at www.ClinicalTrials.gov as #NCT04944043.