Adoptive immunotherapy with third-party virus-specific T lymphocytes (VSTs) is effective against refractory viral infections. However, its long-term efficacy and persistence must be enhanced. T memory stem cells (TSCMs) with superior self-renewal and multilineage differentiation potential may enhance VSTs durability, although their antiviral capacity is underexplored. Cytomegalovirus (CMV)-and Epstein-Barr virus (EBV)-specific T cells are enriched with CD8⁺ TSCM through cytokine and peptide stimulation. Comprehensive preclinical evaluations show that purified TSCM-VSTs exhibit reduced exhaustion, enhanced expansion, and stronger antiviral activity than central or effector memory VSTs (TCM or TEM). Transcriptomic and epigenetic analyses show significant enrichment of the MAPK and Wnt signaling pathways, consistent with stem-like characteristics. In a murine model, CD8⁺ TSCM VSTs provide more effective protection against Raji-pp65 tumors than TCM or TEM VSTs. In a phase I clinical trial, 10 patients with refractory CMV or EBV infections post-transplant who received third-party, off-the-shelf TSCM-enriched VSTs show a 100% overall response rate and 70% complete response, with persistence up to 12 weeks and no severe adverse events. These findings support TSCM-enriched VSTs as a potent, scalable antiviral immunotherapy and highlight TSCM proportion as a critical determinant of VSTs efficacy.

Cerebral amyloid angiopathy (CAA) is a common yet underdiagnosed disease of the small brain vessels, resulting in acute vascular events as well as subcortical neurodegeneration. Currently, it is identified only at advanced stages due to the limitations of non-invasive diagnostic tools. Our pilot study evaluates metabolic and immune alterations in stroke patients with imaging-confirmed CAA. This prospective cohort study included stroke patients admitted to the University Hospital Erlangen with CAA diagnosis based on MRI findings. Metabolomic analysis of cerebrospinal fluid and serum samples was performed using liquid chromatography/mass spectrometry, focusing on caffeine metabolism and amino acid pathways. Immunophenotyping of leukocytes was conducted via flow cytometry. The study included 22 stroke patients, of whom 10 had an MRI-based diagnosis of CAA. Metabolomic analysis revealed consistently lower levels of caffeine-related metabolites in CAA patients with significant differences in 5-acetylamino-6-amino-3-methyluracil, paraxanthine, theobromine, 3,7-dimethyluric acid, 3-methylxanthine and 1-methylxanthine. Amino acid levels showed no significant differences. Immunophenotyping revealed a reduction in CD4+ T cell subsets, including effector and central memory T cells, alongside an increase in cytotoxic NK cells in CAA patients. These results suggest that specific metabolic and immune signatures could contribute to the development of diagnostic tools for the detection of CAA.

Child maltreatment (MALT) is a devastating form of early-life adversity (ELA) and a primary risk for mental and physical illness. It is difficult to disentangle postnatal caregiving effects from heritable factors. Here we investigated the long-term effects of maternal care using a cross-fostering design to control for biological/heritable factors on immune function and inflammation during adolescence in a translational and naturalistic macaque model of MALT. We studied the impact of MALT on the immunophenotype of peripheral blood mononuclear cells (PBMCs) and assessed glucocorticoid receptor expression and function during adolescence. MALT was associated with elevated expression of NR3C1, the gene that encodes for the glucocorticoid receptor, in PBMCs. Glucocorticoid receptor function was not altered by MALT when examined for response to dexamethasone (DEX). In addition, MALT led to a reduction in the percentage of naïve CD4+ T cells and an increase in the percentage of central memory (Tcm) CD4+ T cells. These results suggest that MALT-exposed adolescents show residual effects of MALT on CD4+ T cells and increased expression of NR3C1 without demonstration of increased function of the glucocorticoid receptor. Taken together, these results suggest that ELA has enduring implications for cellular glucocorticoid receptor biology and CD4+ T cells.

Amniotic fluid embolism (AFE) represents an uncommon yet life-threatening obstetric emergency characterized by its sudden onset and significant contribution to maternal death. Immune dysregulation has been implicated as a key pathological mechanism in AFE progression. This investigation employed through comprehensive bioinformatics approaches transcriptomic profiling of clinical specimens to systematically identify immune-associated genetic alterations that can distinguish AFE - induced pulmonary embolism from normal controls. We conducted RNA sequencing on clinical specimens to delineate differentially expressed genes associated with AFE pathogenesis and immune responses. Functional annotation and protein-protein interaction (PPI) networks were generated using specialized R packages. Machine learning algorithms facilitated the selection of candidate biomarkers, whose expression patterns were quantitatively assessed. Diagnostic performance was evaluated through nomogram construction, while immune cell infiltration patterns were characterized using computational deconvolution methods. Potential N6-methyladenosine (m6A) sites were predicted via established databases, and regulatory networks were reconstructed. Future validation will include quantitative Polymerase Chain Reaction (PCR) verification of critical gene expression. Our analysis identified two upregulated biomarkers (MMP9 and PPBP) in AFE samples. The constructed nomogram demonstrated that elevated biomarker scores correlated with increased AFE probability. Validation through calibration curves, decision curve analysis, and receiver operating characteristic curves confirmed robust predictive accuracy and clinical applicability. Immunological assessment revealed significant negative correlations between biomarker expression and central memory CD4 + T cells, activated CD8 + T cells, and activated dendritic cells. Both biomarkers exhibited moderate to high-confidence RNA methylation sites. We have provided valuable peripheral blood sequencing data from AFE patients and healthy controls. Through bioinformatics analysis, we identified two immune-related biomarkers: MMP9 and PPBP and systematically examines their biological significance through immune infiltration profiling, gene set enrichment, regulatory network construction, and pharmacological association studies. This study provides preliminary evidence for the diagnostic potential of these two markers for AFE and offers new insights for research into the immune response associated with AFE.

Depression in people with multiple sclerosis (MS) is two to three times more frequent than in demographically matched people without MS. The MS-depression literature is large and has expanded exponentially over the past few years. This increase in new knowledge is the impetus for assessing whether there is now sufficient evidence to differentiate depression linked to multiple sclerosis from depression alone. Establishing the validity of MS-depression as a distinct diagnosis is important because it would enhance our understanding of the pathogenesis of depression in general, shed light on a clinical course that might diverge from what is expected from depression without MS, and suggest management strategies that may differ from those followed for people with depression alone. A review of the MS-depression literature from January 2018 to December 2024 (generating 114 papers for inclusion in the manuscript) reveals no unique, distinct MS-depression phenomenology. The factors encompassing predictive validity, namely the course of depression, employment, suicide, cognitive impairment and quality of life, are similar in kind but not severity between depressed people with and without MS. The paucity of randomized controlled trial psychotropic data in MS-related depression means it is unclear whether medication plus psychotherapy is the best treatment option for people with MS who are depressed, as it is in general population samples. In terms of construct validity, the posited immune signature of MS depression, namely an increased frequency of circulating CD4+CCR7low central memory T cells with a Th1 predilection, does not appear to be distinct from depression in the general population. There is considerable neuroimaging commonality, particularly in limbic regional involvement. The potential importance of the dopamine-rich ventral tegmental area in a putative MS depression neural circuit suggests a degree of specificity, but the absence of direct comparison between depressed people with and without MS hinders a more definite conclusion. As for personality factors and socio-economic status in depressed people with MS, the findings essentially overlap with the depression literature in the general population. There are, however, a couple of standout constructs suggesting the possibility of two distinct disorders: the equivocal data pertaining to a specific MS genetic diathesis to depression and the absence of a clear sex difference in depressed people with MS. Until these conundrums are explained, one cannot conclude with certainty that depression in people with and without MS is the same disorder. Further research comparing depressed people with and without MS is needed to understand why this difference may exist.

The anatomical source of infection is a major determinant of sepsis outcomes; however, how distinct sites shape immunity remains unclear. Here we applied multi-omic profiling, integrating single-cell transcriptomics, single-cell T cell receptor and B cell receptor sequencing, CITE-seq, bulk RNA sequencing and plasma proteomics, to analyze peripheral blood mononuclear cells and plasma from 281 adult and pediatric individuals with sepsis and controls. We identified an NR4A2+ central memory CD4+ T cell subset enriched in abdominal, pulmonary and skin sepsis, with features of exhaustion; genetic perturbations showed Nr4a2 loss improved survival, while overexpression worsened it. Proinflammatory CD8+ T, natural killer and natural killer T subsets expressing CCL4, CCL3 and tumor necrosis factor expanded in adult abdominal and pulmonary sepsis, while pediatric pulmonary sepsis featured proliferative CD14+ monocytes, findings validated in external single-cell cohorts and confirmed in 164 independent individuals. Plasma proteomics revealed shared mediators including interleukin-6 and EN-RAGE across anatomical sites and ages. Together, our findings delineate anatomical-specific and age-specific immune programs in sepsis, highlighting candidate targets for precision immunotherapy.

Glucocorticoid-Induced TNFR-related protein (GITR) is a costimulatory molecule involved in the proliferation and effector functions of CD8 + and CD4 + T-cells. Recently, it has gained attention as a novel target for HIV immunotherapy. However, reports on its expression in people living with HIV (PLWH), as well as functional studies of GITR ligands effects in the context of HIV remain scarce. Here, we performed a thorough immune characterization of GITR expression in PLWH following HIV peptide stimulation. We found a prevalence of an effector memory phenotype on HIV-specific GITR-expressing CD8 + T-cells that correlated with viral control. Costimulation with a hexameric GITR ligand (GITRL) showed a modest improvement on antiviral function. Characterization of CD4 T-cells revealed an association between GITR expression among regulatory T-cells and viral control, as well as the prevalence of a central and effector memory T-cell phenotype. Remarkably, GITRL costimulation enhanced viral transcription without increasing the reservoir size, positioning GITR as an interesting target for the development of novel latency reversal agents.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-26952-8.

Altered immune cell profiles in maternal and umbilical cord blood and fetal growth restriction: A flow cytometric analysis.

Dual targeting of BCMA and B7-H3 with CAR T cells and bispecific protein engagers enhances anti-myeloma activity.

Abstract Introduction CASPASE8 promotes both cell death and survival by acting as a trigger of apoptosis and a repressor of necroptosis. In T cells, the function and mechanisms of CASPASE8 is incompletely understood. Methods Here, we analysed mice in which Casp8 was conditionally deleted in T cells at different stages of development. Results In mice with deletion early in T cell development, we observed a modest reduction in early thymic progenitors and a striking absence of NKT cells in the thymus. Amongst mature peripheral T cells, there was a substantial and specific reduction in the CD8 T cell compartment, that included naive, central memory and virtual memory subsets. Using a tamoxifen inducible CD8CreERT to delete Casp8 revealed an acute requirement for continued CASPASE8 expression for survival of a fraction of mature CD8 T cells. Analysing Casp8 deficient mice that express a kinase dead RIPK1 suggested that in vivo, necroptosis contributed to death of thymic progenitors and CD8EM and CD8CM subsets. However, kinase dead RIPK1 failed to restore NKT cell development, or rescue the loss of CD4EM and CD4CM in mixed bone marrow chimeras, and only partially rescued CD8 VM T cell. Conclusion Together, these observations suggest that CASPASE8 promotes T cell survival independent of its established role in repressing RIPK1 dependent necroptosis.

The antigenic landscape of autoimmune diabetes reflects a failure to preserve self-tolerance, yet how novel neoantigens emerge in humans remains incompletely understood. Here we designed an immunopeptidomics-based approach to probe HLA-II-bound, islet-derived neoepitopes in patients with type 1 diabetes. We uncovered a Cys→Ser transformation, conserved between mice and humans, that reshapes autoreactivity to insulin at the single-residue level. This transformation, which we call C19S, arises from oxidative remodeling of insulin in stressed pancreatic islets and also occurs in cytokine-activated antigen-presenting cells, contributing to a feed-forward loop of neoepitope formation and presentation. Despite involving just one amino acid, C19S is recognized by HLA-DQ8-restricted, register-specific CD4+ T cells that expand at diabetes onset. These neoepitope-specific CD4+ T cells lack regulatory potential but acquire a poised central memory phenotype that persists throughout disease progression. These findings reveal a distinct, microenvironment-driven route of neoantigen formation that fuels sustained autoreactivity in diabetes.

Background. The aim of this project is to perform immunophenotyping of patients with JLS and healthy controls to assess dysregulated immunological pathways in JLS, and to identify biomarkers able to predict clinical features. Methods. We included in this study pediatric patients diagnosed with linear and mixed JLS. Peripheral blood mononuclear cells (PBMCs) and plasma samples at diagnosis or treatment escalation were identified retrospectively and retrieved from our biobank. We included only patients whose first available blood sample was obtained at time of diagnosis and before the initiation of any treatment. Levels of 50 cytokines were measured in the plasma by protein array (RayBiotech). Lymphocyte and monocyte subsets were analysed with a 26-colour flow cytometry panel and acquired on a FACSymphony (BD). Results. We identified samples at baseline from 11 patients with JLS and 10 controls. Cytokine array identified 15 cytokines that were significantly elevated in JLS patients compared to controls. Using Principal Component Analysis (PCA), we observed a distinct distribution between patients and controls, with approximately 50% of patients clustering away from controls, and the remaining 50% aligning closely with them (Figure 1A). Patients clustering near controls exhibited the lowest cytokine levels and were designated as non-inflammatory JLS (no_inf JLS), whereas those clustering away had the highest cytokine levels and were classified as inflammatory JLS (inf_JLS) (Figure 1A). We then validated the results of the cytokine array in ELISA: CXCL9, CXCL10, and CXCL13 were consistently elevated in all patients with inf_JLS, at baseline and during longitudinal follow-up, while cytokine levels in patients with no_inf JLS remained within normal ranges (Figure 1B). We then performed a 26-parameter staining of PBMCs, allowing comprehensive evaluation of major lymphocytes and monocytes subsets by flow cytometry. Our analysis identified 81 subpopulations of interest; a PCA analysis highlighted significant differences in the distribution of lympho-monocytic subsets between JLS patients and controls (Figure 1C). Within lymphocyte subsets, significant differences were observed in the frequency of activated CD4+ T cells, predominantly within the central memory (CM) subset. Patients stratified into inf_JLS and no_inf JLS groups based on cytokine levels showed minimal differences in cellular subpopulations upon PCA. However, two subpopulations demonstrated significant variability: activated CD4+ T cells (CD38+ HLA-DR+) and terminally differentiated effector memory CD4+ T cells (TEMRA) expressing PD1, were significantly expanded in patients with inf_JLS (Figure 1D). Conclusions. This study identified two distinct groups of patients with JLS: a non-inflammatory group, characterized by normal levels of circulating proinflammatory cytokines, and an inflammatory group, marked by elevated levels of circulating cytokines. Our data showed a higher frequency of activated CD4+ T cells, suggesting a central role of activated T cells in JLS pathogenesis, particularly in patients within the inflammatory group.

Early diagnosis and prognostic evaluation of hepatocellular carcinoma (HCC) remain significant challenges in clinical management. This study aims to identify prognostic biomarkers in HCC and to explore their implications in immune microenvironment infiltration. In this study, we constructed a single-cell transcriptomic atlas of HCC, focusing on the expression profiles of T cell-related genes. Analytical approaches included cell-cell communication analysis and pseudotime trajectory analysis. To further predict and validate T cell-associated prognostic genes, we integrated transcriptomic data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. Patients were stratified into high- and low-risk groups based on a prognostic model derived from these biomarkers. Immune infiltration levels in the tumor microenvironment were then evaluated across risk groups. A total of eight primary tumor samples and seven normal tissue samples were included as raw data in this study. Following stringent quality control and filtering, 53,477 cells were retained for downstream analysis. From these, we isolated 12,333 T cells, which were subjected to further clustering and annotation. The T cell subpopulations identified included 6314 natural killer T cells (NK T cells), 5199 effector memory CD4+ T cells, and 820 central memory CD8+ T cells. By integrating transcriptomic data from TCGA-LIHC and GEO datasets, we identified six prognostic biomarkers: LYZ, SPP1, EGR1, MARCO, FCN3, and PTTG1. A prognostic model was developed based on these biomarkers, enabling risk stratification into high- and low-risk groups. The model demonstrated robust predictive performance in estimating patient survival rates and immune cell infiltration levels within the tumor microenvironment. This study identified and validated prognostic biomarkers in HCC that effectively predict patient survival rates and immune infiltration characteristics. These findings provide a potential foundation for precision medicine strategies in HCC, offering novel insights into the tumor-immune microenvironment and its clinical implications.

Chimeric antigen receptor T (CAR-T) cell therapy has shown promise in treating solid tumors, but the clinical success is often limited by insufficient tumor infiltration. In this study, we sought to engineered two MSLN-targeted CAR-T cell variants, Intra2 and Intra6, expressing CCR2 and CXCR6, respectively, to improve their migration toward the tumor microenvironment. Flow cytometry confirmed stable receptor expression. In vitro assays demonstrated that both Intra2 and Intra6 CAR-T cells exhibited significantly improved functional phenotype, migration and invasion, as well as persistence in killing target cells compared to conventional MSLN CAR-T cells. Notably, in vivo, Intra6 CAR-T cells displayed superior antitumor effects, showing enhanced tumor suppression and reduced exhaustion. RNA sequencing analysis revealed that CXCR6 expression upregulated genes related to immune activation, migration, adhesion, and cytoskeletal remodeling, such as LFA-1, PAK1, and FSCN1, suggesting improved migration and transendothelial infiltration. RT-qPCR and flow cytometry confirmed higher LFA-1 expression and enhanced migratory capacity in Intra6 CAR-T cells. Importantly, LFA-1 was crucial for CXCR6-driven migration. These results suggest that chemokine receptor modification of MSLN-targeted CAR-T cells can significantly improve their tumor infiltration and therapeutic efficacy, offering a potential strategy to optimize CAR-T cell therapy for mesothelin-expressing cancers. METHODS: We engineered two CAR-T cell variants, Intra2 and Intra6, by introducing chemokine receptors CCR2 and CXCR6, respectively. The migration and tumor infiltration capabilities of these modified cells were evaluated in vitro through migration assays and in vivo using tumor-bearing mouse models. The phenotypic characteristics of the CAR-T cells, including memory T cell subsets (stem cell memory and central memory), were analyzed by flow cytometry. Tumor growth inhibition was assessed, and markers of immune exhaustion and evasion were quantified.

Cisplatin is a cornerstone chemotherapeutic agent frequently associated with dose-limiting ototoxicity. Increasing evidence suggests that immune-mediated mechanisms may influence interindividual susceptibility to this adverse effect; however, the role of inherited immune traits remains poorly understood. This study aimed to evaluate the causal relationship between 33 inherited immune traits and cisplatin-induced ototoxicity using Mendelian randomization (MR), and to identify age-stratified susceptibility markers in pediatric and adult cancer survivors. MR was used to assess the causal effects of genetically predicted immune traits on cisplatin-induced ototoxicity. Single-nucleotide polymorphisms associated with immune traits were selected from large-scale genome-wide association study datasets. The primary analysis used the inverse variance weighted method with MR-Egger, weighted median, weighted mode, and MR-PRESSO as the sensitivity approaches. Bidirectional MR and sensitivity analyses were conducted to assess robustness and rule out reverse causation. Bonferroni correctionwas employed to minimize potential false-positive findings (P < 0.05/165 ≈ 0.0003). Transforming Growth Factor-beta principal component analysis (TGF-β PCA) showed an age-stratified effect: it was associated with increased risk of hearing loss in pediatric patients (OR (95% CI): 1.0 × 101 (1.6 × 100-6.6 × 101), P = 0.014) but conferred strong protection against Speech Recognition Threshold (SRT) impairment (OR (95% CI): 2.8 × 10⁻1 (1.4 × 10⁻1-5.3 × 10⁻1), P = 0.00012) and hearing loss (OR (95% CI): 4.7 × 10⁻1 (2.7 × 10⁻1-8.1 × 10⁻1), P = 0.006) in adults. Additional protective associations have been identified for T Central Memory (TCM) cells and Programmed Cell Death Protein-1 (PD-1) in adults. Reverse MR analysis excluded significant reverse causation. Following Bonferroni correction, the association between TGF-β PCA and SRT remained statistically significant (P < 0.0003) in the adult cohort. However, all other associations in adults and the entire pediatric cohort demonstrated only nominal significance (0.0003 ≤ P < 0.05). Inherited immune traits, particularly TGF-β PCA, PD-1, and TCM cells, exhibit age-stratified causal effects on cisplatin-induced ototoxicity. These findings suggest the use of immunogenetic profiling for risk prediction and personalized strategies in oncology pharmacy practice.

Checkpoint inhibitor-associated autoimmune diabetes (CIADM) is a rare but life-altering complication of immune checkpoint inhibitor (ICI) therapy. Biomarkers that predict type 1 diabetes (T1D) are unreliable for CIADM. To identify biomarkers for prediction of CIADM. From our prospective biobank, 14 CIADM patients who had metastatic melanoma treated with anti-PD-1 ± anti-CTLA4 were identified. Controls were selected from the same biobank, matched 2:1. Pre-treatment, on-ICI and post-CIADM serum and peripheral blood mononuclear cells (PBMCs) were analysed. Serum was analysed for T1D autoantibodies, C-peptide, glucose and cytokines. PBMCs were profiled using flow cytometry. Pancreatic volume was measured using CT volumetry. Before treatment, CIADM patients had smaller pancreatic volume (27% reduction, p=0.044) and higher anti-GAD antibody titres (median 2.9 versus 0, p=0.01). They had significantly higher baseline proportions of Th17 helper cells (p=0.03), higher CD4+ central memory cells (p=0.04) and lower naïve CD4+ cells (p=0.01). With ICI treatment, greater declines in pancreatic volume were seen in CIADM patients (p<0.0001). Activated CD4+ subsets increased significantly in CIADM and controls with immune-related adverse effects (IRAE) but not controls without IRAE. Using only pre-treatment results, pancreatic volume, anti-GAD antibody titre and baseline immune flow profile were highly predictive of CIADM development, with an area under the curve (AUC) of >0.96. People who develop CIADM are immunologically predisposed and have antecedent pancreatic and immunological changes that accurately predict disease with excellent sensitivity. These biomarkers could be used to guide ICI use, particularly when planning treatment for low-risk tumours. JEG is supported by NHMRC Investigator grant 2033228. AMM by NHMRC Investigator grant2009476 and GVL by NHMRC Investigator grant 2007839.

Objectives: Vaccination by a nonmucosal route to elicit CD8+ T cell-mediated mucosal immunity against respiratory infections presents a great challenge for the development of an effective vaccine or immunization strategy. This study aimed to explore a new strategy to address the challenge. Methods: To test this strategy, s.c. vaccinated mice were administered i.p. with tanshinone I (TSN1), a main bioactive compound found in the root of Salvia miltiorrhiza, and CD8+ T cell responses were analyzed using flow cytometry. The differentiating effects of TSN1 on CD8+ T cells from naïve mice were also evaluated in an in vitro setting. Results: Nonmucosal vaccination and administration of TSN1 induce pulmonary-resident vaccine-specific memory CD8+ T cells through increased lung-specific recruitment and retention. The improved memory response appears to result from the impact of TSN1 introduced during the primary immunization phase. Given a specific range of varying concentrations of this natural compound, it exhibits a differential effect on the memory differentiation of CD8+ T cells in the process of being activated. Effector memory T cells expand robustly relative to central memory T cells, and both memory subsets have additionally increased expression of CD44 and CD69. With more potent cytolytic activity, CD8+ Trm expressing CD69 particularly predominate the population lacking the CD69 expression in the lungs of TSN1-treated mice. Conclusions: Our study suggests that TSN1 as an important natural compound may hold great promise for novel approaches to the design and development of a more practical and efficient vaccination strategy to generate effective respiratory mucosal immunity.

Occupational exposure to volatile organic solvents has been reported to lead to hazardous effects. Ethyl acetate is a volatile organic compound used commonly in industry and found in many commercial products. The present study aimed to investigate the acute behavioural effects of ethyl acetate exposure in rats. The mechanism of its effects was further investigated by focusing on the possible involvement of L-type calcium channels. For this purpose, ethyl acetate (0.3g/kg, i.p.) alone or concurrently with nimodipine (3 and 5mg/kg, i.p.), a dihydropyridine calcium channel antagonist selective to L-type calcium channels, was administered to male Wistar albino rats. When compared to the saline control group, ethyl acetate significantly decreased the number of square-crossing, rearing, and sniffing in the open-field and impaired the reference memory performance in the three-panel runway. However, administration of nimodipine at the given doses did not block these effects of ethyl acetate. The findings suggest that L-type calcium channels may not contribute to the mechanism(s) responsible for the acute toxicity of ethyl acetate in rats regarding their central nervous system depression and memory disturbances although it should be more thoroughly investigated in further studies.

Aim: This study aimed to characterize and compare the composition of central (TCM), effector (TEM), tissue-resident (TRM), and terminally differentiated (TEMRA) memory T cells in mid-luteal endometrium during the window of implantation (WOI) in women with and without a previous miscarriage. Methods: Stromal lymphocytes from endometrial samples (P + 5) were analyzed by multicolor flow cytometry to quantify total, CD4+ and CD8+ TCM (CD45RA−CCR7+), TEM (CD45RA−CCR7−), TRM (CD69+), and TEMRA (CD45RA+CCR7−) subsets. Participants were grouped as having no previous miscarriage (n = 38) or ≥1 previous miscarriage (n = 33), and the relative distribution of these memory subsets was compared between groups. Correlations, PCA and logistic regression were used to assess global memory network organization. Results: Women with prior miscarriage exhibited higher TCM proportions among total and CD8+ lymphocytes (p < 0.01), alongside lower CD8+ TEM (p = 0.02) and higher CD4+ TEM (p = 0.01). TRM showed a mild, non-significant increase (p = 0.18), while TEMRA remained stable. TRM correlated positively with both TCM (r = 0.51) and CD4+ TEM (r = 0.40), indicating coordinated organization among memory subsets. Multivariate analyses (PCA and logistic regression) confirmed these trends and identified the TCM/TEM ratio as the most discriminative parameter. Conclusions: Endometrial memory T-cell composition during the WOI differs in women with miscarriage history, characterized by central memory expansion and reduced effector memory proportions, with parallel increases in tissue-resident cells. These changes suggest persistent remodeling of the local immune memory network toward a long-lived, less differentiated phenotype that may influence implantation readiness in subsequent cycles.

Single-cell gene expression data can provide insights into cell-cell communication, enabling us to understand the interaction between cancer cells and microenvironmental cells. Here, our goal was to unravel how intercellular communication influences terminally exhausted CD8 + T cells in the ovarian tumor microenvironment. We processed and integrated ovarian cancer scRNA-Seq samples and delineated distinct cellular populations based on the expression patterns of established canonical marker genes. We performed a pseudotime trajectory analysis of CD8 + T cells and analyzed the communication of ovarian cancer cells with terminally exhausted CD8 + T cells. Investigating cell lineage and inferring pseudotimes revealed the transition of the CD8 + T cells from naïve-like to six different end-states, with central memory (35%), effector memory (31%), and terminally exhausted (25%) CD8 + T cells being the most abundant CD8 + T cell subtypes. Cell-cell communication analysis identified the HMGB1-HAVCR2 ligand-receptor pair mediating communication from ovarian cancer cells to terminally exhausted CD8 + T cells. High Mobility Group Box 1 (HMGB1) was identified as a key ligand expressed in ovarian cancer cells influencing the IL32 expression in terminally exhausted CD8 + T cells. The signaling path from HMGB1 to IL32 revealed NFKB1 as the most significant signaling mediator and TP53 as the most significant transcriptional regulator via which HMGB1 influenced IL32 expression in CD8 + T cells. The HMGB1-IL32 signaling pathway identified in our analysis can serve as a therapy target for a new generation of adjuvant therapy designed to suppress and disrupt tumor cells' influence on the microenvironment and enhance immunotherapy efficiency.