Search Results (7,190)

Background/Objectives: Penile squamous cell carcinoma (PSCC) is a rare malignancy with poor prognosis in advanced and recurrent disease, and therapeutic options remain limited. Increasing evidence suggests that the tumor immune microenvironment (TIME), including immune cell composition and spatial organization, plays a critical role in tumor progression and survival outcomes. This study aimed to characterize immune cell density and geospatial clustering patterns within the TIME of PSCC and to evaluate their associations with clinical outcomes. Methods: Multiplex immunofluorescence (mIF) was performed on tumor samples from 57 patients with PSCC using a panel of immune markers to identify lymphoid and myeloid cell populations. Immune cell densities were quantified within tumoral and stromal compartments. Spatial relationships among immune cells and between immune cells and tumor cells were analyzed using point pattern analysis. Survival outcomes, including overall survival (OS), recurrence-free survival (RFS), and cancer-specific survival (CSS), were assessed using Kaplan–Meier methods and Cox proportional hazards models, with analyses stratified by nodal and human papillomavirus (HPV) status. Results: Higher intratumoral and stromal densities of pro-immunogenic M1 macrophages were associated with improved OS. Increased densities of CD3⁺CD4⁺ helper T cells in both compartments were also associated with favorable survival outcomes. In contrast, close clustering of pro-tumorigenic M2 macrophages with tumor cells and with one another was associated with worse OS, RFS, and CSS. Bivariate clustering of helper T cells with tumor cells was associated with improved OS, including among patients with node-positive disease. Survival outcomes did not differ significantly by HPV status in patients with high helper T cell clustering. Conclusions: Immune cell density and spatial organization within the TIME are associated with survival outcomes in PSCC. Favorable patterns involving helper T cells and M1 macrophages correlate with improved survival, whereas clustering of M2 macrophages is associated with poorer outcomes, supporting the relevance of spatial immune profiling in this disease. Full article

Background: Axillary lymph node (ALN) metastasis is a critical prognostic determinant in breast cancer (BC) that informs surgical management. However, surgically clearing the axilla carries morbidity, so less invasive methods of risk-stratifying patients are needed. ALN fine needle aspiration (FNA) is currently used to detect BC metastases, but these samples also contain immune cells. Methods: Cells obtained via FNA from BC-patient-derived ALNs were analysed using flow cytometry. Results: FNA acquires sufficient leukocytes for comprehensive immunophenotyping of reactive, patient-derived ALNs. All CD4⁺ and CD8⁺ T-cell subsets (naïve, terminal effector, central memory, and effector memory) and rarer (<2%) natural killer (NK) and plasmacytoid dendritic cell (pDC) populations are represented. Importantly, the immune-cell profile of one reactive ALN appears to reflect the immune status of the patient’s axilla. Furthermore, FNA captures immune differences between patients with ≤1 or ≥2 metastatic ALNs. Increased numbers of naïve CD4⁺ T cells, but fewer terminal effector, central memory, and effector memory subpopulations, were obtained from patients with ≥2 metastatic ALNs. Moreover, despite their sparse distribution pattern on whole-section immunohistochemistry (WSI), FNA revealed that CD56⁺ NK cell activation receptors were decreased in patients with ≥2 metastatic ALNs. Finally, FNA captured a decrease in pDCs in patients with ≤1 metastatic ALNs, despite their clustered distribution pattern on WSI. Conclusions: FNA is not only feasible for sampling leukocytes from reactive, patient-derived ALNs, but also identifies immune-cell profiles that reflect axillary tumour burden in BC. Thus, this technique could be used to risk-stratify BC patients in the future. Full article

Background: Depression and Metabolic Dysfunction-Associated Steatotic Fatty Liver Disease (MASLD) are common chronic diseases, respectively. However, the causal and molecular links between them remain unclear. In order to explore whether depression contributes to an increased risk of MASLD and whether inflammation mediates this effect, we integrated multi-level evidence from the epidemiology of the National Health and Nutrition Examination Survey (NHANES), the genetics of GWAS, the transcriptomes of GEO, and single-cell RNA sequencing datasets. Methods: A multi-level integrative analysis strategy was used to validate this pathway. First, a cross-sectional epidemiological analysis based on NHANES data was used to reveal the association between depression and MASLD, and to explore the mediating role of inflammation and liver injury markers. Secondly, a two-sample Mendelian randomization analysis was used to infer the causal direction of depression and MASLD, and to verify the mediating effect of systemic inflammation and liver injury indicators at the genetic level. Then, the transcriptome co-expression network analysis and machine learning were used to screen the common hub genes connecting the two diseases. Finally, single-cell transcriptome data were used to characterize the dynamic expression of potential key genes during disease progression at cellular resolution. Results: Depression significantly increased the risk of MASLD, especially in women (OR = 1.39, 95%CI [1.17–1.65]). Parallel mediation analysis showed that high-sensitivity C-reactive protein (hs-CRP) (p < 0.001), γ-glutamyltransferase (GGT) (p < 0.001), and alkaline phosphatase (ALP) (p < 0.001) mediated this relationship. Mendelian randomization analysis confirmed the unidirectional causal effect of depression on MASLD, and there was no reverse association (β = 0.483, SE = 0.146, p = 0.001). Weighted gene co-expression network analysis and machine learning identified CD40LG as a potential molecular bridge between depression-associated immune modules and MASLD. In addition, single-cell data analysis revealed a stage-specific trend of CD40LG expression in CD4⁺ T cells during MASLD progression, while its receptor CD40 was also activated in B cells. In the female sample, CD40LG maintained an upward trend. However, the stability of this result is limited by the limited sample size. Conclusions: This study provides converging multi-omics evidence that depression plays a causal role in MASLD through inflammation-mediated immune signaling. The CD40LG-CD40 axis has emerged as an immune mechanism that transposes depression into the pathogenesis of MASLD, providing a potential target for the intervention of gender-specific metabolic liver disease. Full article

Chimeric antigen receptor (CAR)-T-cell therapy is a revolutionary approach in immunotherapy that has shown remarkable success in the treatment of blood cancers. Many preclinical studies are currently underway worldwide to extend the CAR-T-cell therapy benefits to a broad spectrum of cancers, using rodent models. Alternative in vivo platforms are essential for overcoming the drawbacks associated with rodent models, including immunodeficiency in humanized models, ethical concerns, extended time requirements, and cost. In this work, we used the chicken chorioallantoic membrane (CAM) assay to evaluate the in vivo efficacy of cluster-of-differentiation 19 (CD19)-targeting CAR-T cells expressing a second-generation CAR construct against human lymphoma derived from the Raji cell line. Our results confirm the efficacy of selected CAR-T cells on tumor growth, metastasis, and angiogenesis. Further, the chicken embryo has an intrinsic active immune system. Therefore, the dialog between CAR-T cells and endogenous immune cells, as well as their participation in the tumor challenge, has also been studied. In conclusion, our study demonstrates that the chicken CAM assay provides a relevant in vivo, 3Rs (Replacement, Reduction and Refinement)-compliant new approach methodology (NAM), which is well-suited for the current needs of preclinical research on CAR-T-cell therapy. Full article

Lentiviral transduction remains the gold standard in adoptive modified cellular therapy, such as CAR-T; however, genome integration is not always desirable, such as when treating non-fatal autoimmune disease or for additional editing steps using CRISPR to produce allogeneic CAR-modified cells. Delivering in vitro-transcribed (IVT) mRNA represents an alternative solution but the labile nature of mRNA has led to efforts to improve half-life and translation efficiencies using a range of approaches including chemical and structural modifications. In this study, we explore the role of N⁶–methyladenosine (m6A) in a CD19-CAR sequence when delivered to T cells as an IVT mRNA. In silico analysis predicted the presence of four m6A consensus (DRACH) motifs in the CAR coding sequence and treating T cells with an inhibitor of the m6A methyltransferase (METTL3) resulted in a significant reduction in CAR protein expression. RNA analysis confirmed m6A bases at three of the predicted sites, indicating that the modification occurs independently of nuclear transcription. Synonymous mutation of the DRACH sites reduced the levels of CAR protein from 15 to >50% depending on the T cell donor. We also tested a panel of CAR transcripts with different UTRs, some containing m6A consensus motifs, and identified those which further improved protein expression. Furthermore, we found that the methylation of consensus m6A sites seems to be somewhat sequence-context-dependent. These findings demonstrate the importance of the m6A modification in stabilising and enhancing expression from IVT-derived mRNA and that this occurs within the cell, meaning targeted in vitro chemical modification during mRNA manufacturing may not be necessary. Full article

In type 1 diabetes (T1D) in non-obese diabetic (NOD) mice, dendritic cells (DCs) exhibit a Stat5b mutation that impairs regulatory T cell (Tregs) numbers and suppressive function. To correct this defect, we generated transgenic NOD mice expressing constitutively active Stat5b (NOD.Stat5b-CA) in DCs, which conferred protection from diabetes that was associated with an expanded Treg population and a marked reduction in CD8⁺ T cell frequencies in secondary lymphoid organs. However, the phenotypic characteristics and underlying mechanisms to eliminate CD8⁺ T cells in NOD.Stat5b-CA mice are unknown. In this study, we found that the frequency of Tregs was significantly higher in the thymus and peripheral lymphoid organs of NOD.Stat5b-CA mice compared with NOD mice. Tregs in the peripheral lymphoid organs exhibited increased expression of activation markers CD69 and OX40, alongside reduced CD62L. We also found that CD8⁺ T cell frequencies were reduced in the peripheral organs but not in the thymus of NOD.Stat5b-CA mice, while CD4⁺ T cell frequencies remained unchanged across all organs. Furthermore, NOD.Stat5b-CA mice exhibited a reduced frequency of central Tregs (CD62L^high CD44^low) and increased frequency of effector Tregs (CD62L^low CD44^high) under steady-state conditions compared to NOD mice. Notably, Tregs from NOD.Stat5b-CA mice displayed enhanced cytotoxic activity, evidenced by increased expression of perforin, granzyme B, and Fas ligand, potentially mediating CD8⁺ T cell frequency reduction. Collectively, these findings highlight a novel role for Stat5b-CA.DC-educated Tregs in modulating immune responses by eliminating peripheral pathogenic CD8⁺ T cells via cytotoxic pathways, thereby contributing to immune regulation in NOD.Stat5b-CA mice. Full article

This study successfully developed an oral vaccine for Type 1 Diabetes utilizing recombinant Lactococcus lactis expressing the GAD65 autoantigen. We conducted an in-depth investigation into its protective mechanisms in NOD mice, with a particular focus on its effects on the gut microbiota and metabolome. The administration of the GAD65-L. lactis vaccine resulted in a significant delay in diabetes onset and the preservation of pancreatic function. Our analyses revealed notable alterations in the gut microbial ecosystem, enhancing its diversity and the abundance of beneficial bacteria. Metabolomic profiling indicated time-dependent changes in metabolic pathways, with a marked enrichment of pyrimidine metabolism at 16 weeks and arachidonic acid metabolism at 24 weeks after vaccination by both GAD65-L. lactis and NZ9000-L. lactis. Integrated correlation analysis identified specific microbiota–metabolite interactions, including associations between Ruminiclostridium and lipid species in the GAD65-L. lactis group. These modifications in the microbial community and metabolic landscape were accompanied by enhanced immunoregulatory responses in intestinal LPLs, including expanded Treg populations and suppressed CD8⁺ T cells, a rising trend in IL-10-producing naive dendritic cells, and increased concentrations of TGF-β. Full article

Background: Ovarian cancer remains the deadliest gynecologic malignancy, with its progression closely tied to age-associated remodeling of the tumor immune microenvironment. The laying hen serves as a valuable spontaneous model for human ovarian cancer. Its single-cell analyses may provide valuable insights into the immune-related axis linking ovarian aging to carcinogenesis. Methods: This study applied single-cell RNA sequencing to profile ovaries from three laying hen groups, including 35-week-old normal ovaries (A35w), 110-week-old normal ovaries (B110w), and 110-week-old ovarian cancer tissues (C110w). Key analyses had UCell-based scoring of senescence-related pathways and cancer hallmarks, differential expression analysis for overlapping dysregulated genes, LASSO regression-based prognostic model construction, and assessment of chemotherapy sensitivity and immune infiltration. Results: A comprehensive cellular landscape of chicken ovaries was established, identifying major immune populations including B cells, CD4+ T cells, CD8+ T cells, macrophages, and plasma cells. Senescence-related pathways and cancer hallmarks showed progressive activation in immune cells from A35w to B110w to C110w. A total of 216 genes commonly dysregulated in aging and carcinogenesis, reveal core links between immune dysfunction and malignant transformation. The 20-gene prognostic model derived from these genes stratified human ovarian cancer patients into high-risk and low-risk groups with significant overall survival differences, exhibited robust predictive performance across TCGA, GSE32063, and GSE140082. The model also predicted the differential chemotherapy sensitivity in high-risk and low-risk patients and correlated with specific immune infiltration patterns in the tumor microenvironment. Conclusions: Notably, this is the first single-cell RNA sequencing study of chicken ovarian cancer, and we constructed the 20-gene prognostic model for human ovarian cancer using 216 genes that change significantly in immune cells during both ovarian aging and carcinogenesis. This work provides support to establish the hen as a potential preclinical animal model and a translational tool to guide personalized therapy. Full article

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous malignancy characterized by the proliferation of skin-homing CD4⁺ T cells and profound immune dysregulation within the tumor microenvironment (TME). This review synthesizes evidence on chemokine–receptor networks that govern malignant T-cell trafficking among blood, skin, and lymph nodes, the formation of immunosuppressive niches, and clinically actionable biomarker candidates. Among the best-supported axes, CCL17/CCL22–CCR4 and CCL27/CCL28–CCR10 mediate skin tropism, CCL19/CCL21–CCR7 contributes to lymph node homing, and CXCL12–CXCR4 supports skin trafficking and is associated with disease progression. In contrast, CCR2/CCR5/CCR6/CCR8-centered circuits and CXCR3/CXCR5 pathways are emerging regulators of myeloid recruitment, regulatory T-cell accumulation, and context-dependent immune activation. Therapeutically, agents targeting chemokine pathways, most notably the CCR4 monoclonal antibody Mogamulizumab, have demonstrated clinical efficacy, while emerging inhibitors of CCR6, CCR5, and CXCR4 offer promising avenues for intervention. We further highlight how recent single-cell and other high-dimensional omics studies refine cell-type–specific chemokine sources and receptor expression, enabling more precise mapping of chemokine-driven intercellular communication programs in CTCL TME remodeling and better prioritization of therapeutic targets and biomarkers. Full article

Bone metastases mark a critical and often terminal phase in cancer progression, where disseminated tumor cells (DTCs) manage to infiltrate and exploit the complex microenvironments of the bone marrow. While most current therapies focus on the well-known late-stage “vicious cycle” of osteolysis, they often overlook the earlier stages, namely, tumor cell colonization and dormancy. During these early phases, cancer cells co-opt hematopoietic stem cell (HSC) niches, using them as sanctuaries for long-term survival. In this review, we bring together emerging insights that highlight a trio of underappreciated cellular players in this metastatic takeover: megakaryocytes, erythroid lineage cells, and perivascular stromal subsets. Far from being passive bystanders, these cells actively shape the metastatic niche. For instance, megakaryocytes and platelets go beyond their role in transport; they orchestrate immune evasion and dormancy through mechanisms such as transforming growth factor-β1 (TGF-β1) signaling and the physical shielding of tumor cells. In parallel, we uncover a distinct “erythroid-immune” axis: here, stress-induced CD71⁺ erythroid progenitors suppress T-cell responses via arginase-mediated nutrient depletion and checkpoint engagement, forming a potent metabolic barrier against immune attack. Furthermore, leptin receptor–positive (LepR⁺) perivascular stromal cells emerge as key structural players. These stromal subsets not only act as anchoring points for DTCs but also maintain them in protective vascular zones via CXCL12 chemokine gradients. Altogether, these findings reveal that the metastatic bone marrow niche is not static; it is a highly dynamic, multi-lineage ecosystem. By mapping these intricate cellular interactions, we argue for a paradigm shift: targeting these early and cooperative crosstalk, whether through glycoprotein-A repetitions predominant (GARP) blockade, metabolic reprogramming, or other niche-disruptive strategies, could unlock new therapeutic avenues and prevent metastatic relapse at its root. Full article

C. perfringens is a Gram-positive, anaerobic, spore-forming bacterium that causes serious diseases in humans and animals. In this study, C. perfringens was isolated from the intestinal content of an alpaca, cultured, and then identified using laboratory methods including Gram staining, biochemical tests, and PCR for 16S rRNA of six toxins. Furthermore, the pathogenicity of different strains was analyzed in mice. The results showed that C. perfringens was identified as type A and caused severe pathology of the spleen, lungs, and duodenum in mice through CD4⁺ and CD8⁺ T cells. Also, the mRNA expression levels of ZO-1 and Occludin were further quantified by qRT-PCR with normalization to β-actin, which showed decreased expression levels in the duodenum of mice in the gavage group compared to those in the NC groups, with significant differences (n = 3; * p < 0.05, ** p < 0.01). The results could inform the development of drugs and vaccines resistant to C. perfringens in alpaca. Full article

Background/Objectives: This study examined the metabolic, oxidative, immunological, and histomorphological effects of the multicomponent fermented biological product derived from camel milk, Inullact-Fito, in comparison to metformin in a rat model of alloxan-induced diabetes resulting from insulin insufficiency. The model was chosen as an experimental system that replicates pancreatic β-cell damage induced by oxidative stress rather than insulin resistance. Methods: Alloxan-induced diabetes was used to evaluate metabolic, oxidative, immunological, and histomorphological alterations. Metformin was utilized as a pharmacological comparator. Blood glucose levels, circulating insulin concentrations, markers of oxidative stress and lipid peroxidation, immunoglobulin levels, CD4⁺/CD8⁺ T cell balance, and pancreatic histostructure were assessed. Results: Alloxan administration led to substantial hyperglycemia, oxidative stress, immunological imbalance, and structural damage to pancreatic tissue. Following therapy with Inullact-Fito, blood glucose levels reduced dramatically (from 21.9 ± 0.22 to 9.85 ± 0.10 mmol/L, p < 0.05), circulating insulin concentrations were largely corrected, oxidative stress and lipid peroxidation markers decreased. Immunological evaluation revealed decreased serum immunoglobulin M and IgG levels (p < 0.05) and partial normalization of the CD4⁺/CD8⁺ T cell balance. Metformin showed comparative effects; however, its activity in this model is limited by its primary mechanism related to insulin resistance. Conclusions: Overall, the data reveal that Inullact-Fito combines metabolic, antioxidant, and immunomodulatory actions under experimental oxidative and metabolic stress conditions. Further research using models of insulin resistance and type 2 diabetes, as well as long-term clinical trials, is needed to fully evaluate the therapeutic potential, safety profile, and translational importance of this fermented dairy product as a functional nutritional intervention. Full article

Alpha-1 antitrypsin (AAT) is a serine protease inhibitor with potent anti-inflammatory and immunomodulatory properties, but its role in cancer is context-dependent across tumor types. We integrated transcriptomic analyses of human melanoma cohorts, in vivo studies using AAT-transgenic (hAAT-TG) mice, and in vitro assays in murine and human melanoma cells to define the biological functions of AAT in melanoma. SERPINA1 expression increased progressively from normal skin to nevi and metastatic melanoma, yet higher intratumoral levels correlated with improved overall survival in metastatic disease. In hAAT-TG mice, melanoma growth was markedly inhibited compared with wild-type controls, and the inhibitory effect required CD8⁺ T cells and was enhanced by CD4⁺ T-cell depletion, demonstrating that AAT promotes cytotoxic T-cell activity while attenuating regulatory T-cell suppression. Histologic analysis showed heavily pigmented tumors in hAAT-TG mice. In vitro, hAAT upregulated melanocytic differentiation markers (MITF, TYR, PMEL, MART-1) and increased melanin production in murine and human melanoma lines, suggesting enhanced tumor immunogenicity. In conclusion, hAAT exerts antitumor effects in melanoma indirectly by reprogramming the tumor microenvironment toward differentiation and immune activation. These findings highlight a previously unrecognized role for AAT as a dual immunoregulatory and differentiation-promoting factor and support AAT as a potential immunoregulatory adjuvant in melanoma. Full article

Background: Liver X receptors (LXRs) are critical regulators of cholesterol homeostasis that modulate T cell function with anti-inflammatory effects. LXR downregulation has been implicated in the pathogenesis of inflammatory bowel disease (IBD), although its underlying mechanisms remain to be fully elucidated. Recent evidence has confirmed the link between T cell senescence and autoimmune diseases. Here, we sought to investigate whether and how LXRs regulate T cell senescence in controlling intestinal inflammation. Methods and Results: We found that LXRβ expression was decreased in the colons of mice with experimental colitis, and LXRβ deficiency (Lxrβ^−/−) significantly aggravated their colitis. Intriguingly, this finding was accompanied by enhanced CD4⁺ T cell senescence both in the colons and spleens of Lxrβ^−/− mice, evidenced by upregulation of SA-β-gal levels and the remarkable expansion of effector memory subclusters in CD4⁺ T cells. Moreover, senescent Lxrβ^−/− CD4⁺ T cells secreted elevated levels of proinflammatory cytokines, especially in effector memory populations, exhibiting a pronounced proinflammatory phenotype. RNA-sequencing further confirmed the role of LXRβ in restricting CD4⁺ T cell senescence. Mechanistically, the absence of LXRβ in CD4⁺ T cells directly enhanced senescence by promoting the cGAS/STING pathway. Blocking STING signaling with a targeted inhibitor significantly alleviated senescence in Lxrβ^−/− CD4⁺ T cells. Conclusions: Our findings demonstrate the role of LXRβ in regulating intestinal CD4⁺ T cell senescence to inhibit colitis development, identifying LXRβ as a potential therapeutic target for treating IBD. Full article

Glaesserella parasuis (G. parasuis) is a key pathogen responsible for swine respiratory disease, and the development of broadly protective vaccines is hampered by its high antigenic diversity. The iron-acquisition protein TbpB is a conserved vaccine candidate, but the cellular immune responses it elicits, particularly T-cell subset dynamics during immunization and challenge, remain insufficiently defined. This study characterized these responses after oral immunization of colostrum-deprived piglets with the TbpB^Y167A mutant. Ten colostrum-deprived piglets were allocated to immunized and non-immunized (PBS) groups, immunized at days 15 and 30 of life and subsequently challenged with G. parasuis (45 days old); peripheral blood mononuclear cells were collected at baseline, after each immunization, and at 1 and 3 days post-infection. Multiparametric flow cytometry was used to quantify major leukocyte subsets and T-cell phenotypes defined by sIgM, CD172a, CD3, TCRγδ, CD8α/β, CD4 and CD27 expression. Booster immunization induced significant expansion of B cells (p < 0.01), TCRγδ T cells (p < 0.01), CD8⁺ αβ T cells (p < 0.001) and CD4⁺ memory T cells (p < 0.01) in immunized piglets compared with controls. After challenge, CD8⁺ cytotoxic T cells in immunized animals rapidly shifted from naïve to memory phenotypes, peaking at 48–72 h (p < 0.01). These biphasic T-cell dynamics are consistent with the protective efficacy previously demonstrated for this vaccine in colostrum-deprived piglets, and support a key contribution of TCRγδ, CD8⁺ cytotoxic and CD4⁺ memory T cells to immunity against G. parasuis and to the design of next-generation vaccines. Full article