Mistletoe extract is a widespread complementary therapy mainly used for quality-of-life improvement in cancer patients. Advanced pancreatic cancer is associated with poor quality of life and better therapies for symptomatic relief are highly needed. MISTRAL aimed to assess the impact of mistletoe extract on quality of life, body weight, observed costs and blood biomarkers in patients with advanced pancreatic cancer. MISTRAL was an investigator-initiated, phase III, randomized, double-blind, placebo-controlled, parallel-group, superiority, multicenter, clinical trial with a nested biomarker study. Registration EudraCT 2014-004552-64, NCT02948309. At 9 oncology centers, 290 participants were randomized to standard treatment (palliative chemotherapy or best supportive care) plus subcutaneous mistletoe extract or placebo. Main inclusion criteria were advanced pancreatic cancer, performance status 0-2, main exclusion criteria neuroendocrine pancreatic tumor. EORTC-QLQ-C30, EORTC-QLQ-PAN26, body weight, cost parameters and biomarkers were assessed from baseline up until 9 months. No statistically significant differences for quality of life and weight were evident between treatment arms. Parameters for observed costs for supportive and inpatient care (days at hospital, parenteral nutrition infusions, nutritional supplement drinks, number of visits of palliative home care teams, symptom-relieving medication) were similar in both arms. Thus, calculation of costs was not performed. No effect on explored biomarkers (differential blood count, lymphocyte subpopulations, C-reactive protein, albumin and Ca19-9) was found except for a statistically significant increase of eosinophils in the mistletoe arm without association to clinical effect. Since no benefit was observed, there is no clinical reason to recommend mistletoe extract in patients with advanced pancreatic cancer.

Horse-derived anti-thymocyte globulin (ATGAM) in combination with long-term ciclosporin is the first-line treatment for most immune-mediated aplastic anemia (AA) patients. The exact impact of this immunosuppressive therapy (IST) on hematologic recovery and the immune landscape, however, remains poorly understood. We report a longitudinal analysis of the pharmacodynamic effects of ATGAM-based IST in a cohort of 44 AA patients. We used flow cytometry to quantify plasma levels of lymphocyte-binding ATGAM, which is believed to mediate the therapeutic effect. Population pharmacokinetic modeling revealed substantial between-patient variability in ATGAM exposure, with higher exposure levels associating with earlier hematologic recovery. ATGAM bound all lymphoid lineages and profoundly depleted T and natural killer cells at high plasma concentrations. Strikingly, ATGAM did not deplete B cells but instead induced an increase in CD27+ B cells. Deep immunophenotyping on series of peripheral blood samples collected up to three years after start of IST demonstrated that ATGAM induced rapid depletion of T cells, including KLRG1+ terminally differentiated CD8+ T cells and Th17-like CCR6+CD4+ T cells. Although naïve and pathogen-specific T cells were also depleted, they recovered quickly, indicating preservation of protective immunity. Notably, CCR6++ B cells, implicated in AA pathogenesis, escaped ATGAM depletion but reduced gradually over time along with residual potentially pathogenic T cells, including the CCR6+CD4+ T cells. This could explain the crucial contribution of long-term ciclosporin to successful IST. Collectively, our results identify ATGAM exposure as a factor influencing hematologic recovery and indicate that the therapeutic effect of IST goes beyond total lymphodepletion but is rather the result of selective depletion and suppression of key lymphocyte subpopulations.

Despite the proven cardiovascular efficacy and safety of statins in patients with MASLD, their effect on short-term disease progression remains poorly understood. A prospective observational cohort study was conducted in 54 patients with metabolic syndrome and MASLD, with a 6-month follow-up. Multivariate logistic regression was used to assess the association between statin use and progression of MASLD, adjusting for waist-to-hip ratio (WHR) and glycated hemoglobin (HbA1c). Noninvasive liver injury markers, including CAP, FIB-4, and NAFLD Fibrosis Score, were beneficial, with no evidence of clinically significant hepatotoxicity. In the adjusted logistic model, statin use was independently associated with a reduced risk of adverse progression of MASLD (adjusted odds ratio 0.16; 95% CI 0.03-0.75; p = 0.020), whereas higher baseline WHR and HbA1c were associated with an increased risk. The model had good discriminatory power (AUC = 0.83). Exploratory immunoassays revealed changes in lymphocyte subpopulations. In patients with metabolic syndrome and MASLD, short-term statin therapy was associated with a lower risk of disease progression after adjustment for WHR and HbA1c. The findings support the metabolic and hepatic safety of statins and emphasize the importance of abdominal obesity and glycemic control in determining MASLD progression.

Peripheral lymphocyte remodeling following sound-based anxiety intervention: prominent alterations in γδ T-cell subsets.

Lymphopenia has been associated with poor outcomes in metastatic breast cancer (BC), but its prognostic relevance in early-stage disease remains unclear. This study aimed to evaluate the prognostic value of baseline lymphopenia in patients with non-metastatic BC using data from the prospective French CANTO cohort (NCT01993498). 10,854 patients with baseline absolute lymphocyte count (ALC) were analysed. Lymphopenia was defined as ALC < 1.0 × 10⁹ cells/L, measured before any cancer treatment. Relapse-free survival (RFS), overall survival (OS), and treatment-related toxicities were assessed using Kaplan-Meier estimates, piecewise-Cox regression models, and cause-specific hazard analyses considering pre-specified time periods. Baseline lymphopenia was observed in 342 patients (3.1%). It was associated with ECOG performance status > 0, hypoalbuminemia, and prior neoplasia. Lymphopenia correlated with inferior 5-year RFS (89.1% vs. 92.9%) and OS (92.5% vs. 96.4%). In multivariable analyses, lymphopenia was associated with increased risk of early relapse during the first 24 months (HR = 2.11; 95%CI: 1.28-3.48; p = 0.003), but not beyond, and no independent prognostic impact on OS was observed. No significant differences were found in surgical complications or chemotherapy dose intensity, though granulocyte colony-stimulating factor use was higher among lymphopenic patients. Baseline lymphopenia is uncommon in early BC but may serve as a marker of early relapse risk. Although it does not independently predict long-term survival, its presence could reflect underlying tumour aggressiveness or patient frailty. These findings support further investigation of lymphocyte subpopulations to refine prognostic stratification in early BC. Approved by French ethics committee in 2011 (ID-RCB:2011-A01095- 36,11-039 /NCT01993498 [20FEB2012]).

Background/Objectives: African Swine Fever (ASF) represents one of the most serious threats to animal health and global food security. The causative agent of ASF is the African swine fever virus (ASFV), a DNA virus belonging to the Asfarviridae family. Here, we describe ex vivo results for an original anti-ASFV vaccine approach based on the cellular immune response induced by extracellular vesicles (EVs) engineered to express four ASFV proteins. EV engineering was achieved by expressing a DNA vector encoding a biologically inactive HIV-1 Nef protein (Nefmut), which exhibits unusually high efficiency of incorporation into EVs, even when fused to foreign proteins. Previous studies have demonstrated that intramuscular injection of Nefmut-based vectors leads to the engineering of Evs, spontaneously released by muscle cells, and induction of antigen-specific CD8+ T cell immunity. Methods: We designed DNA vectors expressing the fusion products between Nefmut and each of the four ASFV structural proteins p30, p54, pp62, and p72. Engineered EVs were molecularly characterized by Western blot and nanotrack analysis, and their potential immunogenicity was assessed by priming and cross-presentation assays. Results: We assessed that the four fusion proteins were successfully expressed in transfected mammalian cells, with the release of valuable amounts of engineered EVs. When immature swine dendritic cells were challenged with the engineered EVs and then co-cultivated with autologous peripheral blood lymphocytes in priming assays, lymphocyte subpopulations specifically reacting against each ASFV antigen were elicited, as detected by an IFN-γ ELISpot assay. In addition, we provide evidence that the Nefmut-based fusion products incorporated into the engineered EVs can be cross-presented by professional antigen-presenting cells, leading to cross-priming of autologous lymphocytes. Conclusions: These results represent the best premise to go forward with experiments examining immunogenicity and antiviral efficiency in pigs.

Patients with lung cancer brain metastases can benefit from immune checkpoint inhibitors (ICI). However, intracranial responses are often limited and not always concordant with activity seen in extracranial disease. Defects in IFNγ signaling and HLA class-I antigen presentation machinery (APM) on malignant cells can drive immune evasion and ICI resistance, and have traditionally been viewed as interdependent. The possible role of these alterations in non-small cell lung cancer (NSCLC) brain progression remains poorly understood. Using multiplex quantitative immunofluorescence, we measured and spatially mapped IFNγ signaling markers (pSTAT1 and IRF1) and multiple HLA class-I APM components (β2M, PSMB8, PSMB9, PSMB10, TAP1, TAP2, Tapasin, Calreticulin, and ERp57) in cancer cells and neighboring non-malignant stromal cells from two patient cohorts, including primary tumors, intra- and extra-cranial NSCLC metastases. We also studied tumor-infiltrating lymphocyte (TILs) subpopulations in the cohorts, performed whole transcriptomic analysis of parental human NSCLC H2030 cells and their brain metastatic counterpart H2030-BrM3, and expanded the results using spatial transcriptomics of human tumors. We found comparable levels of IFNγ signaling markers in primary and metastatic lesions and marked downregulation of multiple APM components in metastases, some of which were restricted to the brain. Downregulation of HLA class-I APM components was associated with reduced effector TILs and worse survival. Analysis of human parental H2030 and brain metastatic H2030-BrM3 lung adenocarcinoma cells showed comparable signaling responses after IFNγ stimulation and reduced HLA class-I APM markers in metastatic cells. Transcriptomic analysis of primary/metastatic cells and human tumors identified differential expression of multiple genes associated with HLA class-I APM downregulation. Our results reveal that APM downregulation is a prominent feature of NSCLC brain metastases, is independent from local IFNγ signaling defects and is associated with unfavorable clinical features. We also identified candidate modulators of the APM pathway in brain metastases with potential translational significance.

Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure syndrome characterized by neutropenia and pancreatic insufficiency. The current understanding of immune function in SDS is limited. We performed a retrospective study of the US-based Shwachman-Diamond Syndrome Registry (SDSR) to characterize the immunological profile of patients with SDS. Data were obtained from chart review of patients with biallelic mutations in SBDS enrolled on the SDSR (n = 223) or followed clinically at participating institutions with local IRB approval (n = 6). Immunological data were available from 409 time points on 98 individuals with SDS at healthy time points. We identified limited immune deficits, largely quantitative changes in the B-cell compartment in a subset of patients with SDS including low absolute B cells in 31% of patients and low IgM in 30% of patients. Patients with SDS with low B cells may be at increased risk for sinopulmonary, skin or soft tissue infections particularly if they also have low IgG levels. These data support a baseline immunological evaluation, including a full blood count, lymphocyte subpopulations and immunoglobulin levels, for patients with SDS. However, it is reasonable to reserve more detailed assessments for patients with recurrent or severe infections.

Dengue fever is a mosquito - borne infectious disease caused by the dengue virus (DENV). Understanding its immune response mechanisms is crucial for developing effective treatments and vaccines, especially regarding the dynamic changes of peripheral blood lymphocyte subsets. Traditional immunological methods have limitations in accurately analyzing lymphocyte subsets and their functional dynamics. However, the combination of single - cell RNA sequencing and flow cytometry can explore the immune response of dengue patients more deeply. We integrated four public datasets from the Gene Expression Omnibus (GEO), comprising samples from acute dengue patients, healthy individuals, and convalescent patients, for single-cell RNA sequencing analysis. The analysis included data preprocessing, cell annotation, functional enrichment, T cell state scoring, intercellular communication analysis, and pseudotime analysis. Additionally, we collected peripheral blood samples from 81 DENV-1-infected individuals and 30 healthy controls to analyze lymphocyte subsets, activation status, and apoptosis using flow cytometry. Single-cell RNA sequencing identified 11 cell clusters (including T cells, B cells, and plasma cells). Functional analyses demonstrated diverse roles of lymphocyte subpopulations in immune responses and metabolic regulation, as well as frequent intercellular communication. Pseudotime analysis revealed the differentiation trajectory of CD4⁺ naïve T cells and related signaling changes. Flow cytometry showed significantly decreased absolute T cell counts, altered subpopulation distribution and activation, and increased apoptosis; immunological marker variations existed among T cell subpopulations, with correlations between absolute and relative counts.In conclusion, dengue virus infection significantly impairs lymphocyte composition and function, causing immune dysregulation. This study provides insights into the dengue immune regulatory network, with potential implications for targeted immunotherapy and vaccine development, though its small sample size limits the findings and requires further research. Dengue virus infection significantly affects the composition and function of lymphocyte subsets, leading to immune dysregulation. This study provides new insights into the immune regulatory network of dengue fever and holds potential implications for the development of targeted immunotherapies and vaccines. However, the study has limitations, such as small sample size, and further research is needed in the future.

CTLA-4 haploinsufficiency is caused by heterozygous variants in CTLA4. We report a cohort of five patients with a clinical presentation including immune dysregulation, hypogammaglobulinemia, lung damage, and gastrointestinal symptoms, consistent with CTLA-4 haploinsufficiency. Patients ranged from 10 to 23 years of age, with three presenting before 7 years. Immunoglobulin levels and lymphocyte subpopulations were evaluated. All the patients evaluated showed low levels of CTLA-4 and lymphoproliferation in unstimulated conditions. Treatment before genetic diagnosis consisted mainly of intravenous immunoglobulin replacement and pharmacological immunosuppression. Heterozygous variants in CTLA4 were detected by exome sequencing. Immunological evaluation revealed reduced CTLA-4 expression and spontaneous lymphoproliferation. The identified heterozygous CTLA4 variants included two novel variants, one previously classified as likely pathogenic, one as pathogenic, and one as a variant of uncertain significance. These findings expand the spectrum of CTLA4 variants and support their clinical relevance.

Neoadjuvant immunochemotherapy (NIC) has emerged as a promising strategy for esophageal squamous cell carcinoma (ESCC). However, its impact on tertiary lymphoid structures (TLSs) and the immune microenvironment remains to be fully elucidated. We retrospectively analyzed 50 ESCC patients treated with NIC (n = 25) or chemotherapy alone (n = 25) between 2021 and 2023. TLSs and lymphocyte subpopulations in paired tumor tissues were assessed via multiplex immunofluorescence. Disease-free survival (DFS) was evaluated using Cox regression, and correlations with peripheral blood lymphocytes were analyzed. NIC significantly increased the abundance and maturity of TLSs compared to chemotherapy alone. Specifically, NIC-induced TLSs were enriched with CD3 + CD8 + PD-1+ T cells and CD103 + CXCL13+ dendritic cells (DCs). Clinically, the NIC group demonstrated a significantly prolonged median DFS compared to the chemotherapy group (17.4 vs. 13.5 months, p = 0.009). High abundance of mature TLSs was an independent predictor of improved DFS (HR = 0.45). Furthermore, elevated proportions of CD3 + CD8 + PD-1+ T cells and CD103 + CXCL13+ DCs within TLSs were strongly associated with longer survival. These local TLS alterations also correlated with specific changes in peripheral blood lymphocyte subsets. Our preliminary data suggest that NIC promotes TLS maturation and is associated with enrichment of specific immune subsets within TLSs. CD3 + CD8 + PD-1+ T cells and CD103 + CXCL13+ DCs may serve as exploratory biomarkers associated with prognosis and immunotherapy response.

ABSTRACTCongenital heart disease (CHD) is a major global health problem. Although treatment and survival have impressively improved, many patients face comorbidities such as increased susceptibility to infection that may shorten their lives. As many children undergo cardiac surgery with concomitant thymectomy early in life, most studies of the immune system in patients with CHD have focused on the quantitative analysis of lymphocyte subpopulations, maturation, and T cell receptor repertoires, whereas knowledge of effector functions remains limited. We analysed αβ T cell phenotypes, transcriptomes, and functions in children with CHD who underwent cardiac surgery within a year of birth and were followed up to five to ten years after thymectomy, in comparison to age‐matched healthy controls.Children with CHD showed reduced T cell populations, a reduction of recent thymic emigrants (RTEs) and naive T cells, regulatory T cells with a higher suppressive phenotype, and, most importantly, high activation states of T cells, further reflected in higher granzyme and cytokine production. This reveals persistent alterations in T cell immunity years after early‐life thymectomy in children with CHD, highlighting the need for long‐term immune monitoring and providing a basis for understanding immune‐related comorbidities in this patient population.

IntroductionIdiopathic inflammatory myopathies (IIM) are a heterogeneous group of autoimmune diseases characterized by muscle inflammation and systemic involvement. The study aimed to evaluate the immunophenotypic profile of circulating lymphocytes in patients with IIM and investigate its association with clinical manifestations, serological profiles, treatment regimens, and disease outcomes.Material and methodsThis single-center, cross-sectional study included 40 patients with IIM and 5 age- and sex-matched healthy controls. Peripheral blood samples were collected and processed to isolate peripheral blood mononuclear cells using the density gradient centrifugation method. Flow cytometry analysis was performed to phenotype lymphocyte subpopulations, including T cells (CD3+CD4+ and CD3+CD8+), B cells (CD3–CD19+), and natural killer (NK) cells (CD3–CD16+CD56+). Statistical analysis was conducted to compare lymphocyte profiles between the study and control groups and to assess correlations with clinical features, serological markers, and treatment regimens.ResultsThe IIM group had a mean age of 58.55 ±12.70 years, with a predominance of females (60%). The most common IIM subtypes were antisynthetase syndrome (35%) and dermatomyositis (30%). No significant differences in the profiles of circulating lymphocytes were found between IIM patients and healthy controls. Patients with cardiac involvement had significantly higher proportions of double-positive T cells (p = 0.0035), while those with cutaneous ulcers and dysphagia showed significantly lower proportions of CD4+CD8+ T cells. Natural killer cells were significantly lower in patients with anti-Mi2 antibodies (p = 0.0155) but higher in anti-Ro52 positive patients (p = 0.0150). In contrast, B cells were notably higher in patients with anti-Mi2 (p = 0.0182) and lower in individuals with anti-Ro52 (p = 0.0124). Patients with anti-Ro52 positivity were also characterized by higher proportions of double-negative T cells (p = 0.0465). Treatment regimens did not impact the profiles of circulating lymphocytes.ConclusionPatients with IIM exhibit distinct alterations in lymphocyte subpopulations, with specific immune cell profiles associated with clinical phenotypes and serological profiles.

BackgroundFibulin-5 (FBLN5) is a key member of the extracellular matrix (ECM), which plays an important role in the occurrence and development of tumors. This study aimed to determine the role of FBLN5 in the diagnosis, prognosis, and immune regulation of tumors using a pan-cancer analysis.MethodsBy integrating and analyzing data from multiple public databases, including The Cancer Genome Atlas (TCGA), Firehose, University of California, Santa Cruz (UCSC) Xena, cBioPortal, Gene Expression Omnibus (GEO), Clinical Proteomic Tumor Analysis Consortium (CPTAC), Tumor Immune Estimation Resource 2.0 (TIMER2.0), Tumor Immune Single-cell Hub (TISCH), CellMiner, Genomics of Drug Sensitivity in Cancer (GDSC), and Cancer Therapeutics Response Portal (CTRP), we comprehensively investigated the diagnostic and prognostic potential of FBLN5 across multiple types. We further performed a gene set enrichment analysis (GSEA) to elucidate the biological functions associated with FBLN5 expression. Additionally, a survival analysis was conducted to assess its prognostic value, and genetic alteration, methylation, and pathway analyses were also performed. Finally, we examined the relationship between FBLN5 and immunity response, and identified potential drugs that may affect the efficacy of tumor treatment.ResultsThe analysis of TCGA data revealed that FBLN5 RNA was differentially expressed across multiple tumor types, and in 92% of tumor types, the expression level of FBLN5 protein is lower. Genetic and epigenetic alterations—including mutations, somatic copy number alterations, and DNA methylation—were associated with its dysregulated expression. FBLN5 expression was also correlated with several key clinical features. Further, functional analyses linked FBLN5 expression to various metabolism-, metastasis-, and immune-related pathways. Elevated FBLN5 expression had a controversial effect on prognosis across multiple tumors, and influenced sensitivity to numerous therapeutic agents. Additionally, FBLN5 expression showed significant correlations with immunoregulatory molecules and biomarkers indicative of lymphocyte subpopulation infiltration.ConclusionsThis study conducted a systematic analysis of FBLN5 and its regulatory network, demonstrating that FBLN5 expression is closely associated with DNA methylation and drug resistance across multiple tumor types. Further, FBLN5 was found to remodel the tumor microenvironment and cause resistance to immunotherapy.

Anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) is closely associated with factors such as genetic predisposition, environmental pollution, medications, and infections. Some bacteria have been definitively linked to AAV. Although gut microbiota dysbiosis has been confirmed in individuals with AAV, the specific role of this dysbiosis and the associated mechanisms contributing to AAV pathogenesis remain unexplored. The most common type of AAV in China is microscopic polyangiitis (MPA). We used propylthiouracil/phorbol 12-myristate 13-acetate (PMA/PTU) to establish a rat model of MPA. We then administered ceftriaxone sodium to induce dysbiosis of the rat intestinal microflora. We determined differences in gut microbiota using 16sRNA analysis, the degree of kidney and lung injury in rats using haematoxylin-eosin staining, the level of inflammatory factors in peripheral blood using an enzyme-linked immunosorbent assay, the distribution of splenic lymphocyte subpopulations using flow cytometry, the expression of Toll-like receptor 7 (TLR7) in the colon using Western blotting, and the release of neutrophil extracellular traps (NETs) from kidneys using immunofluorescence to assess the impact of gut dysbiosis on MPA. The results showed that the MPA rat model displayed characteristics consistent with those of human MPA patients. The gut microbiota in rats in the gut dysbiosis group significantly differed from that in rats in the MPA group. Gut microbiota dysbiosis triggered increased release of renal NETs through activation of TLR7 protein, exacerbated microinflammation, increased peripheral blood Interleukin-1β, interleukin-6, C-reactive protein, and tumour necrosis factor-α, disrupted immune homeostasis, and increased the number of CD4+ T cells, CD8+ T cells, and Th17 cells through TLR7 signalling. Our findings show that microbiota dysbiosis led to increased release of NETs, more severe microinflammation, disruption of immune homeostasis, and exacerbated organ damage in the kidneys of MPA rats through activation of TLR7.

Background and Objectives: The incidence of colorectal cancer (CRC) in developed Western countries is constantly growing. CRC represents the third most common cancer and the second leading cancer-related cause of death worldwide. Innate and adaptive immunity play a pivotal role in the tumor response, but many of these interactions are still not well understood. Granulysin (GNLY) is an effector, cytolytic molecule, present in human cytotoxic granules of different lymphocyte subpopulations, mainly in cytotoxic T cells and NK cells. Pore-forming proteins GNLY, perforin and granzymes play a key role in cell-mediated immune responses against tumors and infections. Materials and Methods: We aimed to analyze perforin and GNLY-mediated cytotoxicity in the peripheral blood of patients with CRC by flow cytometry. Simultaneously, the cells were labeled with monoclonal antibodies against perforin, GNLY and different surface antigens (CD3, CD4, CD8 and CD56). Phenotypes of lymphocyte subpopulation and expression of perforin and GNLY were analyzed using intracellular and surface immunofluorescence. Results: Total perforin and GNLY expressions in peripheral blood mononuclear cells (PBMC) were significantly lower than in the control group. Statistically significant differences were observed in the distribution of perforin and GNLY expression in different stages of tumors classified according to Dukes', indicating that the percentage of total perforin and GNLY was significantly diminished in accordance with tumor progression. Perforin and GNLY expression were significantly reduced in NK and NKT cells, accompanied by reduced cytolytic potential in patients with CRC and a consequent reduction in their ability to eliminate tumors and infected cells. Conclusions: The determination of cytotoxic potential may provide a valuable assessment of a patient's immune status and represent a novel therapeutic target. Patients with CRC exhibit markedly impaired perforin- and GNLY-mediated cytotoxicity that correlates with disease progression. Assessment and restoration of cytolytic potential may therefore serve as indicators of immune competence and promising therapeutic strategies to improve perioperative and oncologic outcomes.

Abstract Cancer arises within a complex cellular milieu, where the extent and composition of tumor-infiltrating immune cells strongly influence tumor progression and patient prognosis. Emerging research highlights the therapeutic potential of fasting mimicking diets (FMDs) in delaying cancer onset, providing cellular protection and regulating immunity. My project investigates immune profiles, before and after the transfer of donor-derived immune cells previously exposed to fasting, either independently or combined with therapeutic drugs. We aim to identify immune populations, enhanced by this combined treatment, and to recognize their impact on tumor advancement and immune activation. Using the syngeneic 4T1, triple negative breast cancer mouse model, we evaluated how fasting and chemotherapy affect tumor growth and immune competence. Flow cytometry and immunohistochemistry were used to characterize immune cells, recruited at tumors, spleens and bone marrows of treated hosts. A subpopulation of preconditioned splenocytes and tumor-infiltrating lymphocytes, recognized through immunophenotyping, were transplanted into tumor-bearing recipients to assess tumor control, immune priming and cytotoxicity. We observed reduced tumor volume, delayed progression and increased survival rate in the transplants exposed to FMD and doxorubicin. The treatment preserved the size, morphology, and cellularity of healthy, primary and secondary lymphoid organs, while expanded beneficial, antitumor immune signatures. Integrating FMD with standard therapeutic strategies could enhance antitumor immunity and broaden the spectrum of malignancies that respond effectively. Exploring the underlying mechanisms may reveal immune pathways that can be therapeutically leveraged to eradicate tumors, in a field that currently remains unexplored. Citation Format: Evangelia Pavlou, Olga Blazevits, Giulia Salvadori, Sara Martone, Valter Longo. Integrating a fasting mimicking diet to augment antitumor immunity following adoptive cell transfer in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB146.

Abdominal obesity is linked to an increased risk of non-communicable diseases (NCD) and may affect immune function, particularly lymphocyte subpopulations. Waist circumference (WC) is a common indicator of abdominal obesity, but its relationship with lymphocyte subsets is unclear. The role of triglycerides (TG) in this context remains uncertain. This study aims to investigate the relationship between WC and lymphocyte subpopulations, while also evaluating TG's potential mediating effect. A retrospective analysis was conducted on physical examination records from March 2021 to September 2023, excluding incomplete data or extreme values. A total of 1836 records with normal TG was analyzed using t-tests, linear regression, and mediation analysis via SPSS 29.0 software. After adjusting for confounders, WC was significantly positively correlated with CD3+ T cell count, CD4+ T cell count, and overall lymphocyte count, with TG demonstrating a full mediating effect (Beta [95% CI]: 2.11 [1.39-2.91], 1.39 [0.93-1.89], and 2.83 [1.86-3.87], respectively). WC were also correlated with B cell count, with TG showing a partial mediating effect (Beta [95% CI]: 0.65 [0.44-0.87], with a mediation effect ratio of 30.37%, and 0.02 [0.01-0.02], with a mediation effect ratio of 33.33%). In health checkups, individuals with normal TG levels, TG plays a significant mediating role in the changes of lymphocyte subpopulations related to abdominal obesity, particularly exerting a positive influence on CD4+ T cells and B cells, with no significant effect on NK cell numbers. These findings provide new insights into the relationship between abdominal obesity and immune dysregulation.

Disorders of lipid metabolism contribute to the occurrence and progression of hematological malignancies, including chronic lymp hocytic leukemia (CLL). The objective of this study was to investigate the association between serum lipid levels and prognosis in patients with CLL. We retrospectively analyzed data from 142 patients with newly diagnosed CLL (ND-CLL). Our survival analysis showed that low levels of high-density lipoprotein cholesterol/Apolipoprotein-A1 (HDL-C/ApoA1) were associated with poor overall survival (OS) and progression-free survival in patients with ND-CLL. Multivariate analysis identified HDL-C as an independent hazard element for OS. A nomogram combining HDL-C levels and classical prognostic factors increased the accuracy of predicting 5-year OS compared to prediction based solely on classical prognostic factors. The adverse effect of low levels of HDL-C on prognosis in CLL patients was further validated through external validation, wherein the functionality of HDL-C was simulated by assessing cholesterol efflux in a cohort comprising 448 CLL patients. Moreover, flow cytometry analysis of 194 peripheral blood lymphocyte subpopulations demonstrated that the levels of HDL-C/ApoA1 was significantly positively correlated with natnatural killer (NK) cells. Subsequent invitro cell experiments confirmed the ability of HDL to augment NK cell cytotoxicity, as evidenced by elevated expression levels of CD107a and tumor necrosis factor-α. In conclusion, low serum HDL-C levels are related to a poor prognosis in patients with ND-CLL, potentially due to impaired NK cell cytotoxicity, providing insight into the prognostic role of HDL-C and proposing a new potential therapeutic target in patients with ND-CLL.

Chronic active Epstein-Barr virus (EBV) disease (CAEBV) is an uncommon lethal infection involving EBV-infected T and/or NK-cells, often complicated by hemophagocytic lymphohistiocytosis (HLH). Patients need curative hematopoietic cell transplantation (HCT), but the indication and timing remain unclear. This study aimed to assess the prognostic value of EBV-infected lymphocyte subsets. We analyzed 52 pediatric/young-adult patients diagnosed with persistently EBV infection from 2003 to 2025 at a single tertiary center in Japan, excluding acute infectious mononucleosis and secondary immunodeficiencies. Dominant EBV-infected cell types (CD4+, CD8+, γδT-cells, CD19+B-cells, and CD56+NK-cells) were determined at diagnosis of CAEBV (n = 21), EBV-HLH (n = 19), or inborn errors of immunity (IEI, n = 12). The long-term outcomes were analyzed by major infected cell types and treatment. CAEBV included 12 T-cell (6 CD4+, 4 CD8+, and 2 γδ) and 9 NK-cell dominant infections. EBV-HLH and IEI exclusively involved CD8+T-cell and B-cell infections, respectively. Thirteen CAEBV patients (62%) underwent HCT to control progression including 5 patients who presented with HLH. The 3-year overall survival (OS) was 88%, although CD4+T-cell disease (all CAEBV) showed a significantly lower survival rate. Posttransplant deaths occurred in 3 of 13 CAEBV and none of EBV-HLH patients. Among 9 CAEBV patients with median 11-year progression-free survival without HCT, each one of CD4+T-cell or NK-cell case transformed to lymphoma or leukemia after being untreated for >10 years. EBV-infected lymphocyte profiling guided prolonged HCT-free control of CAEBV. CD4+T-cell CAEBV requires a prompt rather than elective HCT to prevent progression and transformation.