The introduction of checkpoint immunotherapeutic agents in the last decade has revolutionized cancer treatment. Although anti-PD-1, anti-PD-L1 and anti-CTLA4 are promising therapies, many patients fail to respond or relapse due to drug resistance potentially due to redundancy of immune checkpoints. One of the ways to improve the efficacy of this cancer treatment is to target two or even three immune checkpoints. To date, the benefit of combined anti-VISTA/anti-PD-L1 therapy has been confirmed, but no one has investigated the efficacy of blocking these negative immune checkpoints with a bispecific anti-VISTA/anti-PD-L1 antibody.
In this study, the bispecific antibodies (bsAbs) were produced in three formats: symmetric (IgG-HC-scFv), asymmetric (Fab-scFv-Fc(KIH)) and 2 x scFv. The binding and blocking properties of these bispecific antibodies (bsAbs) and their efficacy compared to monotherapy and combination therapy were then determined using endometrial (RL95-2), pancreatic (PANC-1) and breast (BT-20) cancer cell lines.
The bsAbs generated in this study showed weaker binding properties to PD-1 and VISTA in ELISA (EC50) than the parent antibodies (atezolizumab and onvatilimab). Blockade of VISTA/VSIG-3 binding was also weaker with bsAbs compared to onvatilimab, but the ability to block the PD-1/PD-L1 pathway was slightly better than with atezolizumab. The Fc-based bsAbs showed statistically significant higher levels of lysis of endometrial, breast and pancreatic cancer cells. The symmetric bsAbs (IgG-HC-scFv) showed the most promising therapeutic potential. Higher levels of cancer cell lysis were associated with higher levels of pro-inflammatory cytokines. Both the asymmetric and symmetric bsAbs resulted in higher secretion levels of IFN-γ, TNFα and Granzyme B than anti-VISTA, anti-PD-L1 monotherapy and anti-VISTA/anti-PD-L1 combination therapy.
The high level of tumor cell lysis and increased expression of pro-inflammatory cytokines induced by the Fc-based bsAbs suggest a novel approach for the treatment of pancreatic, endometrial and breast cancer.
Copyright © 2025 Bielski, Barczyński, Mikitiuk, Myrcha, Rykała, Boon, Gąsior, Hec-Gałązka, Holak and Sitar.

The poor prognosis of pancreatic ductal adenocarcinoma (PDAC) is largely due to several challenges, such as late diagnosis, early metastasis, limited response to chemotherapy, aggressive tumor biology, and high rates of tumor recurrence. Therefore, the development of a non-invasive and effective method for early detection of PDAC is crucial to improving patient outcomes. Continued research and exploration in this area are essential to enhance early detection methods and ultimately improve the prognosis for individuals with PDAC. In this study, we examined 37 exosomal surface proteins through a multiplex flow cytometry test on peripheral plasma samples from a group of 51 clinical control individuals (including healthy volunteers and non-cancer patients (Cholecystectomy, Hernia, healthy volunteers)), 21 pancreatitis, and 48 patients diagnosed with PDAC. Our research findings revealed that the level of exosomal CD40 expression is significantly lower in patients with PDAC and pancreatitis compared to non-cancer patients (p < 0.0001). Additionally, pancreatitis patients exhibited higher levels of exosomal CD25 expression than PDAC patients (p = 0.0104). PDAC patients with higher exo-CD40 had worse survival than patients with lower exo-CD40 (p = 0.0035). Similarly, PDAC patients with higher exo-CD25 showed worse survival in comparison to patients with lower exo-CD25 (p = 0.04). Statistical analysis revealed that exosomal CD40 achieved an AUC of 0.827 in distinguishing PDAC from clinical controls. Combining exo-CD40 along with exo-CD25 and CA19-9 discriminated PDAC patients from clinical controls with an AUC of 0.92. Exo-CD40 and exo-CD25 proteins found in exosomes isolated from plasma can serve as excellent non-invasive biomarkers for the early diagnosis of PDAC. Further larger scale studies are needed to validate combined exo-CD40 and exo-CD25 as a diagnostic tool for the identification of PDAC patients through non-invasive liquid biopsy.

The primary outcome was the evaluation of the T-cell phenotype in autoimmune primary adrenal insufficiency (PAI). Secondary outcomes included the evaluation of the CD4+CD25+Foxp3+ Treg population and the gene expression levels of IL-6, IL-17A, cyclooxygenase (COX)-2, heat shock proteins (HSP)-70, indoleamine-2,3-dioxygenase (IDO), programmed death-ligand 1 (PD-L1), inducible nitric oxide synthase (iNOS), and thioredoxin (TXN)-1.
We prospectively included 15 patients with PAI on conventional glucocorticoid (GC) replacement therapy, 15 switched to dual-release hydrocortisone (DR-HC), and 20 healthy controls. Serum inflammatory parameters and peripheral blood mononuclear cells (PBMCs) were evaluated at baseline and after 12 months of treatment.
At baseline, significantly higher CD4+ and CD8+ (both p < 0.001) T-cell percentages, a lower CD4+/CD8+ ratio (p < 0.05), and higher CD25+ and CD4+/CD25+ T cells (both p < 0.001) were observed in PAI compared to controls. After 12 months of DR-HC treatment, we found significantly lower IL-6 (p = 0.019), IL-17A (p = 0.046), COX-2 (p < 0.001), HSP-70 (p = 0.006), and TXN-1 (p = 0.008) and higher PD-L1 (p < 0.001) and IDO (p < 0.001) mRNA values compared to baseline. After 12 months of DR-HC treatment, a significant increase in CD4+ T cells (p = 0.012), PD-L1 (p = 0.003), and IDO (p < 0.001) and a decrease in CD8+ T cells (p < 0.001), IL-6 (p = 0.003), IL-17A (p = 0.0014), COX-2 (p < 0.001), HSP-70 (p = 0.005), and TXN-1 (p = 0.0008), as well as a significantly higher conversion in the CD4+/CD8+ ratio (p = 0.033), were observed compared to conventional GCs.
The switch from conventional GCs to DR-HC treatment altered the T lymphocyte phenotype and CD4+/CD8+ ratio in a Treg-independent manner, inducing significant improvements in the immune and inflammatory profile in PAI.
Copyright © 2025 Tomasello, Coppola, Pizzolanti, Giordano, Arnaldi and Guarnotta.

Histone methyltransferase KMT2D is one of the most frequently mutated genes in diffuse large B-cell lymphoma (DLBCL) and has been identified as an important pathogenic factor and prognostic marker. However, the biological relevance of KMT2D mutations on tumor microenvironment remains to be determined. KMT2D mutations were assessed by whole-genome/exome sequencing (WGS/WES) in 334 patients and by targeted sequencing in 427 patients with newly diagnosed DLBCL. Among all 761 DLBCL patients, somatic mutations in KMT2D were observed in 143 (18.79%) patients and significantly associated with advanced Ann Arbor stage and MYC expression ≥ 40%, as well as inferior progression-free survival and overall survival. In B-lymphoma cells, the mutation or knockdown of KMT2D inhibited methylation of lysine 4 on histone H3 (H3K4), downregulated FBXW7 expression, activated NOTCH signaling pathway and downstream MYC/TGF-β1, resulting in alterations of tumor-induced regulatory T cell trafficking. In B-lymphoma murine models established with subcutaneous injection of SU-DHL-4 cells, xenografted tumors bearing KMT2D mutation presented lower H3K4 methylation, higher regulatory T cell recruitment, thereby provoking rapid tumor growth compared with wild-type KMT2D via FBXW7-NOTCH-MYC/TGF-β1 axis.
© The author(s).

Chronic active Epstein-Barr virus disease (CAEBV) is a proliferative disease of EBV+ T or natural killer (NK) cells with an unclear pathogenesis. This study aimed to examine the frequency and exhaustion levels of lymphocyte subsets in patients with CAEBV to further investigate the pathogenesis.
Using flow cytometry, we detected the frequency, expression levels of programmed cell death 1 (PD-1) and programmed death ligand 1 (PD-L1), and EBV infection status of peripheral T subsets and NK cells in patients with CAEBV and healthy individuals.
24 patients and 15 healthy individuals were enrolled in this study. Patients showed notably higher expression levels of PD-1 and PD-L1 in peripheral T subsets and NK cells compared to healthy individuals (P < 0.05). EBV+ lymphocytes exhibited significantly higher PD-L1 expression levels than EBV- lymphocytes. Additionally, the frequency of effector memory T (Tem) cells was significantly increased in patients, and the PD-L1 expression level was positively correlated with the EBV load. Besides, helper T cell 2 (Th2) immune bias, also favoring EBV amplification, was found in patients, including increased Th2 cell frequency, enhanced response capacity, and elevated serum levels of associated cytokines. The distribution and PD-1 expression levels of peripheral T subsets returned to normal in patients who responded to PD-1 blockade therapy.
The up-regulation of the PD-1/PD-L1 pathway of peripheral T and NK cells and Th2 immune predominance jointly promoted EBV replication and the development of CAEBV. PD-1 blockade therapy reduced the PD-1 expression level of lymphocytes and helped normalize the distribution of the T subsets.