Expression Of CD86 Research Articles

Circulating mucosal-associated invariant T cell alterations in adults with recent-onset and long-term oral lichen planus

BackgroundMucosal-associated invariant T (MAIT) cells play key roles in many inflammatory diseases. However, their effects on the long-term course of oral lichen planus (OLP) and recent-onset OLP remain unclear. In this study, we aimed to investigate the function of MAIT cells in the different processes of OLP and to explore the immunological background of this disease.MethodsThe frequency, phenotype, cytokine secretion, and clinical relevance of MAIT cells were investigated. MAIT cells were collected from the peripheral blood of 14 adults with recent-onset OLP (7–120 days after disease onset) and 16 adults with long-term course OLP (>2 years after diagnosis) using flow cytometry and compared with 15 healthy blood donors. Statistical analyses were performed using the GraphPad Prism software.ResultsMAIT cells from adults with recent-onset OLP exhibited an activated phenotype, as indicated by an increased frequency of CD69+ (p < 0.05) and CD38+MAIT cells (p < 0.01) and elevated production of the proinflammatory cytokine IL-17 A (p < 0.01), compared with healthy adult donors. In adults with long-term OLP, MAIT cells exhibited an activated and exhausted phenotype, characterized by high expression of CD69 (p < 0.01) and PD-1 (p < 0.001) and increased production of granzyme B released (p < 0.01). Compared with recent-onset OLP patients, long-term OLP patients showed a decreased production of CD8+, and CD4−CD8− cells, but an increase in PD-1+ production (p < 0.05).ConclusionsCirculating MAIT cells exhibited activation in OLP patients across varying disease durations. Given that PD-1 expression is elevated in adults with long-term OLP, it is reasonable to infer that circulating MAIT cells in long-term OLP may exhibit a more exhausted state than those in recent-onset OLP.

Recombinant ISRAA ameliorates imiquimod-induced psoriasis and knocking out Israa delays its onset.

Psoriasis, an inflammatory disease, is largely mediated by T-helper 17 cytokines. We have previously identified the immune system-released activating agent (Israa) as a novel gene that connects the nervous and immune systems. This research aims to investigate the role of the Israa gene in psoriasis in vivo using the imiquimod-induced psoriasis model. We established the model in C57BL/6 wildtype mice, which were then treated with 200 pg/mouse, 400 pg/mouse, or 800 pg/mouse of recombinant ISRAA compared to methotrexate. Subsequently, we also induced psoriasis in Israa-knockout mice to confirm the effect of Israa. Results consistently showed improvement in psoriasis in all groups receiving recombinant ISRAA. The 200 pg/mouse dose eliminated the disease, reduced the cutaneous release of IL-17 to one-third and TNF-α to one-sixth, increased IL-10 release to over 500 pg, completely resolved parakeratosis, decreased epidermal thickness to one-half, and reduced the expression of CD4 and neutrophil elastase in the skin (all p < 0.05). Israa-knockout mice exhibited less severe psoriasis in all scoring, biochemical, and histological parameters compared to wild-type mice (p < 0.05). This study highlights Israa as a crucial molecule in psoriasis and confirms its immunomodulatory role in inflammatory diseases.

4-aminopyridine attenuates inflammation and apoptosis and increases angiogenesis to promote skin regeneration following a burn injury in mice

Severe thermal skin burns are complicated by inflammation and apoptosis, which delays wound healing and contributes to significant morbidity. Diverse treatments demonstrate limited success in mitigating these processes to accelerate healing. Agents that alter cell behavior to improve healing would alter treatment paradigms. We repurposed 4-aminopyridine (4-AP), a drug approved by the US FDA for multiple sclerosis, to treat severe burns in mice (10-week-old C57BL/6 J male mice weighing 25 ± 3 g). We found that 4-AP, in the early stages of burn healing, significantly reduced the expression of pro-inflammatory cytokines IL1β and TNFα while increasing the expression of anti-inflammatory markers CD206, ARG-1, and IL10. We demonstrated increased intracellular calcium effects of 4-AP through Orai1-pSTAT6 signaling, where 4-AP significantly mitigated inflammatory effects by promoting M2 macrophage differentiation in in-vitro macrophages and post-skin burn tissues. 4-AP attenuated apoptosis, with decreases in apoptotic markers BAX, caspase-9, and caspase-3 and increases in anti-apoptotic markers BCL2 and BCL-XL. Furthermore, 4-AP promoted angiogenesis through increases in the expression of CD31, VEGF, and eNOS. Together, these likely contributed to accelerated burn wound closure, as demonstrated in increased keratinocyte proliferation (K14) and differentiation (K10) markers. In the later stages of burn healing, 4-AP increased TGFβ and FGF levels, which are known to mark the transformation of fibroblasts to myofibroblasts. This was further demonstrated by an increased expression of α-SMA and vimentin, as well as higher levels of collagen I and III, MMP 3, and 9 in mice treated with 4-AP. Our findings support the idea that 4-AP may have a novel, clinically relevant therapeutic use in promoting burn wound healing.

The Effect of Ginkgo Biloba Extract on Oxidative Stress, Anti-Inflammatory and Antiapoptotic in Vitiligo Treated with Topical Desoxymethasone

Vitiligo is a progressive and multifactorial condition of skin, mucosa, and hair depigmentation. Loss of functional melanocytes leads to the appearance of white macules on the skin, often affecting the lips and genitalia. The impact extends to psychological stress, decreased quality of life, and risk of psychiatric morbidity. Various therapeutic strategies have been designed to inhibit the immune response in vitiligo to reduce melanocyte damage while increasing melanocyte repopulation. There is no standardized method of evaluating treatment outcomes in vitiligo patients. Herbal medicines several studies have shown that GB also has many benefits for health and healing skin diseases, one of which is used in treating vitiligo. Therefore, this study aimed to evaluate the potential of Ginkgo biloba extract as a therapeutic supplement in enhancing the positive effects of desoxymethasone on vitiligo. Using a double-blind randomized control trial clinical trial research design, 26 subjects were divided into treatment and control groups. The control group received standard vitiligo therapy with topical desoxymethasone and placebo, while the treatment group received topical desoxymethasone plus Ginkgo biloba extract. Melanin index, VASI score, MDA, TNF-α, CD8 expression, and caspase 3 were measured before and after treatment. The results showed that the administration of Ginkgo biloba extract had a significant effect in reducing MDA, TNF-α, CD8 expression, and caspase 3 levels, and improving melanin index and erythema in vitiligo patients who received topical desoxymethasone therapy. These findings demonstrate the positive potential of Ginkgo biloba extract as an adjunct to vitiligo therapy, resulting in favorable clinical and molecular impacts in patients undergoing combination treatments.

Effects of PACAP Deficiency on Immune Dysfunction and Peyer's Patch Integrity in Adult Mice.

PACAP (pituitary adenylate cyclase activating polypeptide) is a widespread neuropeptide with cytoprotective and anti-inflammatory effects. It plays a role in innate and adaptive immunity, but data are limited about gut-associated lymphoid tissue. We aimed to reveal differences in Peyer's patches between wild-type (WT) and PACAP-deficient (KO) mice. Peyer's patch morphology from young (3-months-old) and aging (12-15-months-old) mice was examined, along with flow cytometry to assess immune cell populations, expression of checkpoint molecules (PD-1, PD-L1, TIM-3, Gal-9) and functional markers (CD69, granzyme B, perforin) in CD3+, CD4+, and CD8+ T cells. We found slight differences between aging, but not in young, WT, and KO mice. In WT mice, aging reduced CD8+ T cell numbers frequency and altered checkpoint molecule expression (higher TIM-3, granzyme B; lower Gal-9, CD69). CD4+ T cell frequency was higher with similar checkpoint alterations, indicating a regulatory shift. In PACAP KO mice, aging did not change cell population frequencies but led to higher TIM-3, granzyme B and lower PD-1, PD-L1, Gal-9, and CD69 expression in CD4+ and CD8+ T cells, with reduced overall T cell activity. Thus, PACAP deficiency impacts immune dysfunction by altering checkpoint molecules and T cell functionality, particularly in CD8+ T cells, suggesting complex immune responses by PACAP, highlighting its role in intestinal homeostasis and potential implications for inflammatory bowel diseases.

Design of a Robust Flow Cytometric Approach for Phenotypical and Functional Analysis of Human Monocyte Subsets in Health and Disease.

Human monocytes can be subdivided into phenotypically and functionally different classical, intermediate and non-classical monocytes according to the cell surface expression of CD14 and CD16. A precise identification and characterisation of monocyte subsets is necessary to unravel their role in inflammatory diseases. Here, we compared three different flow cytometric strategies (A-C) and found that strategy C, which included staining against CD11b, HLA-DR, CD14 and CD16, followed by several gating steps, most reliably identified monocyte subtypes in blood samples from healthy volunteers and from patients with stable coronary heart disease (CHD) or ST-elevation myocardial infarction (STEMI). Additionally, we established a fixation and permeabilisation protocol to enable the analysis of intracellular markers. We investigated the phagocytosis of lipid nanoparticles, the uptake of 2-NBD-glucose and the intracellular levels of CD74 and HLA-DM. This revealed that classical and intermediate monocytes from patients with STEMI showed the highest uptake of 2-NBD-glucose, whereas classical and intermediate monocytes from patients with CHD took up the largest amounts of lipid nanoparticles. Interestingly, intermediate monocytes had the highest expression level of HLA-DM. Taken together, we present a robust flow cytometric approach for the identification and functional characterisation of monocyte subtypes in healthy humans and patients with diseases.

Integrative analysis of H&E and IHC identifies prognostic immune subtypes in HPV related oropharyngeal cancer

BackgroundDeep learning techniques excel at identifying tumor-infiltrating lymphocytes (TILs) and immune phenotypes in hematoxylin and eosin (H&E)-stained slides. However, their ability to elucidate detailed functional characteristics of diverse cellular phenotypes within tumor immune microenvironment (TME) is limited. We aimed to enhance our understanding of cellular composition and functional characteristics across TME regions and improve patient stratification by integrating H&E with adjacent immunohistochemistry (IHC) images.MethodsA retrospective study was conducted on patients with Human Papillomavirus-positive oropharyngeal squamous cell carcinoma (OPSCC). Using paired H&E and IHC slides for 11 proteins, a deep learning pipeline was used to quantify tumor, stroma, and TILs in the TME. Patients were classified into immune inflamed (IN), immune excluded (IE), or immune desert (ID) phenotypes. By registering the IHC and H&E slides, we integrated IHC data to capture protein expression in the corresponding tumor regions. We further stratified patients into specific immune subtypes, such as IN, with increased or reduced CD8+ cells, based on the abundance of these proteins. This characterization provided functional insight into the H&E-based subtypes.ResultsAnalysis of 88 primary tumors and 70 involved lymph node tissue images reveals an improved prognosis in patients classified as IN in primary tumors with high CD8 and low CD163 expression (p = 0.007). Multivariate Cox regression analysis confirms a significantly better prognosis for these subtypes.ConclusionsIntegrating H&E and IHC data enhances the functional characterization of immune phenotypes of the TME with biological interpretability, and improves patient stratification in HPV( + ) OPSCC.

Bothrops atrox snake venom decreased MHC-II and CD86 expression in bone marrow-derived dendritic cells

The effect of Bothrops atrox venom (BaV) on the maturation of bone marrow-derived dendritic cells (BMDCs) from mice was investigated, with a focus on selected cell markers, TAP1 expression, and the release of pro-inflammatory cytokines during this process. The objective was to evaluate BaV's impact on dendritic cell (DC) function, as DCs are pivotal in antigen presentation and responsible for initiating the immune response mediated by naïve T cells, as well as regulating the immune system. Bone marrow cells were obtained from Swiss mice, and hematopoietic precursors were differentiated into BMDCs using GM-CSF and IL-4. On the 7th day, BaV and LPS were introduced into the culture, and the cells were analyzed 24 h later. BaV's ability to stimulate BMDC maturation was assessed through the analysis of surface marker expression. The findings demonstrated that BMDCs are highly influenced by culture environment factors, such as GM-CSF and IL-4, and are sensitive to additional stimuli like LPS and BaV. Mature DCs exhibited elevated levels of critical markers for T cell activation, such as MHC-II, CD80, and CD86, displaying specific phenotypic characteristics. However, the observed reduction in MHC-II and CD86 expression following BaV exposure suggests a substantial impact on the immunological activation capacity of these cells, potentially interfering with the adaptive immune response. Furthermore, the selective release of cytokines, such as IL-6, but not TNF-α or IL-1β, indicates differentiated modulation of inflammatory responses by DCs under various stimulation conditions.

Concomitant Expression of CD39, CD69, and CD103 Identifies Antitumor CD8+ T Cells in Breast Cancer Implications for Adoptive Cell Therapy.

In cancer, an effective immune response involves the action of several different cell types, among which CD8 T cells play a major role as they can specifically recognize and kill cancer cells via the release of cytotoxic molecules and cytokines, being of major importance for adoptive cell transfer (ACT) of ex vivo expanded tumor-infiltrating lymphocytes (TILs). The inflammation resulting from the tumor growth attracts both activated and bystander T cells. For an effective antitumor response, the T cell must express a specific group of chemokine receptors and integrins which include CD103, CD39, CD69, and CD25. These markers had already been analyzed in various cancers, not including breast cancer and their subsequent subtypes, until now. To analyze, the key receptors on ex vivo expanded tumor-infiltrating lymphocytes in luminal A and luminal B breast cancer (BC) subtypes. We were successful in expanding TILs ex vivo using a standard TIL culture condition from a cohort study of 15 primary luminal A and luminal B breast cancer patients. Furthermore, we examined the expression of CD103, CD39, CD69, and CD25 biomarkers after the expansion by flow cytometry. We found that the information about the percentage of TILs obtainable after the ex vivo expansion is not associated to nor it is dependent on the heterogeneity of the TIL population before the expansion and does not differ by the molecular subtype (p>0.05). We also found that there is a major population of memory-resident antitumor CD8+CD103+CD39+ and CD8+CD103+ CD69+ TILs present in the stroma after the expansion when compared to CD4 immunosubtypes (p<0.0001). Only the CD8+CD103+CD39+ subpopulation was related to BC subtype (0.0009). Evidence from our study suggests that CD8 TILs present in the stroma of luminal A and luminal B breast cancer patients can be quantified and phenotyped by flow cytometry and be further expanded ex vivo. The immuno-phenotyping of these markers may be targeted to improve the success of immunotherapeutic approaches, such as adoptive cellular therapy (ACT) in patients with BC.

Promotion of mandibular distraction osteogenesis by parathyroid hormone via macrophage polarization induced through iNOS downregulation

ObjectiveTo investigate whether Parathyroid hormone (PTH) can promote mandibular distraction osteogenesis by regulating macrophage polarization and the underlying mechanisms of this phenomenon. MethodsForty-eight Rabbits were used to establish the mandibular distraction osteogenesis experimental model, randomly divided into 2 groups. Intermittent post-operative injections of 20 μg/kg PTH and normal saline were administered to the experimental and control groups, respectively. Regenerated new bone was examined using HE staining, osteoclast numbers were determined through tartrate-resistant acid phosphatase (TRAP) staining, and macrophage polarization markers arginase 1 (Arg1) and inducible nitric oxide synthase (iNOS) expressions were elucidated using immunohistochemistry (IHC), the mRNA expression of CD206, CD11C, Arg1 and iNOS were detected using qPCR. ResultsThe bone trabeculae in the experimental group were thicker, with a more homogeneous structure and more new osteoid than in the control group. In the area of distraction osteogenesis, the osteoclast count in the experimental group was higher than in the control group (P < 0.05). IHC results indicated differential expressions of Arg1 and iNOS in the experimental group compared to the control group (P < 0.05). Relative mRNA expressions of CD11c and iNOS were lower in the experimental group than in the control group (P < 0.05), whereas the expressions of CD206 and Arg1 mRNA were higher in the experimental group compared to the control group (P < 0.05). ConclusionIntermittent PTH injections increased macrophage quantity in the mandible generated by distraction osteogenesis, downregulated iNOS, upregulated Arg1, and promoted macrophage polarization from M1 to M2 phenotype, thereby promoting mandibular distraction osteogenesis.

MiR-132-3p downregulates FOXO1 in CD4+ T cells and is associated with disease manifestations in patients with lupus.

This study aimed to evaluate the expression status of miR-132-3p in CD4+ T cells in patients with systemic lupus erythematosus (SLE) and explore its potential role in SLE development. The study included 60 patients with SLE and 30 healthy controls. miR-132-3p expression in CD4+ T cells was detected by real-time quantitative reverse transcription polymerase chain. Bioinformatics analyses were employed to predict target genes and explore the potential role of miR-132-3p. The associations between miR-132-3p levels and SLE Disease Activity Index (SLEDAI) score, as well as laboratory characteristics, were analyzed. miR-132-3p levels in CD4+ T cells were significantly higher in patients with SLE compared with healthy controls. Bioinformatics analysis identified FOXO1 as a potential target gene of miR-132-3p, with a particular emphasis on the FOXO signaling pathway. miR-132-3p up-regulation in CD4+ T cells was associated with high SLEDAI score, high anti-double-stranded DNA levels, low C3 and C4 levels, positive anti-ribosomal P, and high 24-hour urinary protein levels in patients with SLE. miR-132-3p may contribute to CD4+ T cell dysregulation during SLE by targeting FOXO1 and could potentially be used to assess disease severity.

Endometrial regeneration cell-derived exosomes loaded with siSLAMF6 inhibit cardiac allograft rejection through the suppression of desialylation modification

BackgroundsAcute transplant rejection is a major component of poor prognoses for organ transplantation. Owing to the multiple complex mechanisms involved, new treatments are still under exploration. Endometrial regenerative cells (ERCs) have been widely used in various refractory immune-related diseases, but the role of ERC-derived exosomes (ERC-Exos) in alleviating transplant rejection has not been extensively studied. Signaling lymphocyte activation molecule family 6 (SLAMF6) plays an important role in regulating immune responses. In this study, we explored the main mechanism by which ERC-Exos loaded with siSLAMF6 can alleviate allogeneic transplant rejection.MethodsC57BL/6 mouse recipients of BALB/c mouse kidney transplants were randomly divided into four groups and treated with exosomes. The graft pathology was evaluated by H&E staining. Splenic and transplanted heart immune cell populations were analyzed by flow cytometry. Recipient serum cytokine profiles were determined by enzyme-linked immunosorbent assay (ELISA). The proliferation and differentiation capacity of CD4+ T cell populations were evaluated in vitro. The α-2,6-sialylation levels in the CD4+ T cells were determined by SNA blotting.ResultsIn vivo, mice treated with ERC-siSLAMF6 Exo achieved significantly prolonged allograft survival. The serum cytokine profiles of the recipients were significantly altered in the ERC-siSLAMF6 Exo-treated recipients. In vitro, we found that ERC-siSLAMF6-Exo considerably downregulated α-2,6-sialyltransferase (ST6GAL1) expression in CD4+ T cells, and significantly reduced α-2,6-sialylation levels. Through desialylation, ERC-siSLAMF6 Exo therapy significantly decreased CD4+ T cell proliferation and inhibited CD4+ T cell differentiation into Th1 and Th17 cells while promoting regulatory T cell (Treg) differentiation.ConclusionsOur study indicated that ERC-Exos loaded with siSLAMF6 reduce the amount of sialic acid connected to α-2,6 at the end of the N-glycan chain on the CD4+ T cell surface, increase the number of therapeutic exosomes endocytosed into CD4+ T cells, and inhibit the activation of T cell receptor signaling pathways, which prolongs allograft survival. This study confirms the feasibility of using ERC-Exos as natural carriers combined with gene therapy, which could be used as a potential therapeutic strategy to alleviate allograft rejection.

The Expression pattern of NK cells in systemic lupus erythematosus patients with different disease activities

Data demonstrated the role of natural killer (NK) cells in systemic lupus erythematosus (SLE). We aimed to determine the immunophenotype and frequency of NK cells and their subsets, CXCR3, CD161 expression in blood and renal tissue of SLE patients with and without lupus nephritis and their relationship with disease activity. The study included 31 SLE patients and 11 controls. Study participants underwent full history and thorough clinical examination. SLE patients underwent routine laboratory investigations. Renal tissue biopsies were taken from patients with lupus nephritis. The frequency of NK cell subsets in blood of patients and controls and renal tissue from patients was performed by flow cytometry. An increase in circulatory CD56bright NK cells and its CD56bright CD16dim subtype was associated with the severity of systemic manifestations in SLE patients. Total CD56bright NK cells and its CD56bright CD16bright subtypes in renal tissues were related to renal damage. We detected decreased CD161 expression on NK cells related to renal damage and severity of systemic manifestations in SLE patients. Decreased expression of CXCR3 on NK cell surface in renal tissues causes misdirected trafficking of NK cells that seems to reduce the severity of lupus nephritis. In conclusion, our results paved the way to understanding the role of NK cells in the pathogenesis of SLE which may represent a future target for immune therapy of SLE. NK and CD56dim subset were decreased in the blood and increased in renal tissue of SLE patients, while the CD56bright subset was increased in blood and decreased in renal tissue reflecting their effects on renal damage and severity of manifestations in SLE.

Targeted immune cell therapy for hepatocellular carcinoma using expanded liver mononuclear cell-derived natural killer cells

Natural killer (NK) cells are a promising cellular therapy for T cell-refractory cancers but are frequently deficient or dysfunctional in patients with hepatocellular carcinoma (HCC). In the present study, we explored a novel therapy for HCC using NK cells derived from donor liver graft perfusate. These liver-derived NK cells, named LMNC-NK cells, are more abundant in liver mononuclear cells (LMNCs) than in peripheral blood mononuclear cells (PBMCs) from the same donor. We developed a method to expand LMNC-NK cells by 33.8±54.4-fold, enhancing their cytotoxic properties and cytokine production, including granzyme B, CD107a, TNF-α, and IFN-γ. These cells also showed an increased expression of cytotoxicity receptors. An RNA-seq analysis revealed considerable differences in gene expression between LMNC-NK and PBMC-NK cells, with 453 genes upregulated and 449 downregulated in LMNC-NK cells. These genes are involved in the mitogen-activated protein kinase cascade and cell differentiation, explaining the increased activity of LMNC-NK cells. Quantitative reverse transcription polymerase chain reaction confirmed the significant upregulation of TLR6, KIT, MMP14, IRF8, TCF7, FCERIG, LEF1, NLRp3, and IL16 in LMNC-NK cells. LMNC-NK cells effectively eliminated HepG-2-Luc cells in vitro, and in an orthotopic murine model of HCC, they exhibited a potent anti-tumor effect, outperforming PBMC-NK cells. The expression of the activation marker CD69+ in LMNC-NK cells was also significantly higher among tumor-infiltrating lymphocytes compared to PBMC-NK cells. Our research suggests that the adoptive transfer of LMNC-NK cells could be a promising treatment for HCC, offering a novel and effective source of NK cells with superior cytotoxic functions.

Are immunosenescent T cells really senescent?

Loss of proper T-cell functioning is a feature of aging that increases the risk of developing chronic diseases. In aged individuals, highly differentiated T cells arise with a reduced expression of CD28 and CD27 and an increased expression of KLRG-1 or CD57. These cells are often referred to as immunosenescent T cells but may still be highly active and contribute to autoimmunity. Another population of T cells known as exhausted T cells arises after chronic antigen stimulation and loses its effector functions, leading to a failure to combat malignancies and viral infections. A process called cellular senescence also increases during aging, and targeting this process has proven to be fruitful against a range of age-related pathologies in animal models. Cellular senescence occurs in cells that are irreparably damaged, limiting their proliferation and typically leading to chronic secretion of pro-inflammatory factors. To develop therapies against pathologies caused by defective T-cell function, it is important to understand the differences and similarities between immunosenescence and cellular senescence. Here, we review the hallmarks of cellular senescence versus senescent and exhausted T cells and provide considerations for the development of specific therapies against age-related diseases.

Clinical prognosticators and targets in the immune microenvironment of intrahepatic cholangiocarcinoma

ABSTRACT Background Intrahepatic cholangiocarcinoma (ICC) is a disease with poor prognosis and limited therapeutic options. We investigated the tumor immune microenvironment (TIME) to identify predictors of disease outcome and to explore targets for therapeutic modulation. Methods Liver tissue samples were collected during 2008–2019 from patients (n = 139) diagnosed with ICC who underwent curative intent surgery without neoadjuvant chemotherapy. Samples from the discovery cohort (n = 86) were immunohistochemically analyzed on tissue microarrays (TMAs) for the expression of CD68, CD3, CD4, CD8, Foxp3, PD-L1, STAT1, and p-STAT1 in tumor core and stroma areas. Results were digitally analyzed using QuPath software and correlated with clinicopathological characteristics. For validation of TIME-related biomarkers, we performed multiplex imaging mass cytometry (IMC) in a validation cohort (n = 53). Results CD68+ cells were the predominant immune cell type in the TIME of ICC. CD4+high T cell density correlated with better overall survival (OS). Prediction modeling together with validation cohort confirmed relevance of CD4+ cells, PD-L1 expression by immune cells in the stroma and N-stage on overall disease outcome. In turn, IMC analyses revealed that silent CD3+CD4+ clusters inversely impacted survival. Among annotated immune cell clusters, PD-L1 was most relevantly expressed by CD4+FoxP3+ cells. A subset of tumors with high density of immune cells (“hot” cluster) correlated with PD-L1 expression and could identify a group of candidates for immune checkpoint inhibition (ICI). Ultimately, higher levels of STAT1 expression were associated with higher lymphocyte infiltration and PD-L1 expression. Conclusions These results highlight the importance of CD4+ T cells in immune response against ICC. Secondly, a subset of tumors with “hot” TIME represents potential candidates for ICI, while stimulation of STAT1 pathway could be a potential target to turn “cold” into “hot” TIME in ICC.

Transcriptional and methylation outcomes of didehydro-cortistatin A use in HIV-1-infected CD4+ T cells.

Ongoing viral transcription from the reservoir of HIV-1 infected long-lived memory CD4+ T cells presents a barrier to cure and associates with poorer health outcomes for people living with HIV, including chronic immune activation and inflammation. We previously reported that didehydro-cortistatin A (dCA), an HIV-1 Tat inhibitor, blocks HIV-1 transcription. Here, we examine the impact of dCA on host immune CD4+ T-cell transcriptional and epigenetic states. We performed a comprehensive analysis of genome-wide transcriptomic and DNA methylation profiles upon long-term dCA treatment of primary human memory CD4+ T cells. dCA prompted specific transcriptional and DNA methylation changes in cell cycle, histone, interferon-response, and T-cell lineage transcription factor genes, through inhibition of both HIV-1 and Mediator kinases. These alterations establish a tolerogenic Treg/Th2 phenotype, reducing viral gene expression and mitigating inflammation in primary CD4+ T cells during HIV-1 infection. In addition, dCA suppresses the expression of lineage-defining transcription factors for Th17 and Th1 cells, critical HIV-1 targets, and reservoirs. dCA's benefits thus extend beyond viral transcription inhibition, modulating the immune cell landscape to limit HIV-1 acquisition and inflammatory environment linked to HIV infection.

A-136 Effects of Mogamulizumab on CD39, CD73 and CD38 ectonucleotidases expression in T-cells of Sézary syndrome patients

A-136 Effects of Mogamulizumab on CD39, CD73 and CD38 ectonucleotidases expression in T-cells of Sézary syndrome patients

Immunoprotective effect of recombinant Lactobacillus plantarum expressing largemouth bass virus MCP on largemouth bass

Largemouth bass virus (LMBV) is an infectious pathogen that causes high mortality rates in largemouth bass, and outbreaks of this virus can significantly harm the aquaculture industry. Currently, no vaccine has been developed that can effectively prevent the transmission of LMBV. In this study, we constructed a recombinant Lactobacillus plantarum (L. plantarum) strain capable of expressing the MCP gene of LMBV and displaying this protein on its surface; then, we evaluated the immunoprotective effect of this recombinant bacterium on largemouth bass. Western blotting, immunofluorescence, and flow cytometry confirmed that MCP was successfully expressed and anchored on the surfaces of NC8 cells. Immunization of largemouth bass with NC8-pSIP409-pgsA'-MCP via the oral feeding route induced CD4, CD8, IL-1β, and IL-6 gene expression. In addition, NC8-pSIP409-pgsA'-MCP at different CFUs increased the survival of largemouth bass after LMBV infection; in particular, NC8-pSIP409-pgsA'-MCP (109 CFU) resulted in approximately 30% survival. NC8-pSIP409-pgsA'-MCP immunization alleviated the pathological changes in the liver and spleen, exerting a more advantageous protective effect. These data suggest that the recombinant L. plantarum strain NC8-pSIP409-pgsA'-MCP can increase the resistance of largemouth bass to LMBV infection and that this strain is a promising candidate oral vaccine for the prevention of LMBV infection.

Characterisation of the tumour microenvironment in primary and recurrent glioblastomas.

Glioblastoma patients have a dismal prognosis, due to inevitable tumour recurrence and respond poorly to immunotherapy. Tumour-associated microglia/macrophages (TAMs) dominate the glioblastoma tumour microenvironment and have been implicated in tumour progression and immune evasion. Early recurrent glioblastomas contain focal reactive regions with occasional fibrosis, chronic inflammation, TAMs and tumour cells. Surgical specimens from these tumours are rare and provide crucial insights into glioblastoma recurrence biology. This study aimed to characterise TAM- and lymphocyte phenotypes in primary vs early- and late-recurrent glioblastomas. Patient-matched primary and recurrent glioblastomas were compared between patients with early recurrences (n = 11, recurrence ≤6months) and late recurrences (n = 12, recurrence after 12-19 months). Double-immunofluorescence stains combining Iba1 with HLA-DR, CD14, CD68, CD74, CD86, CD163, CD204 and CD206 along with stains for CD20, CD3, CD8 and FOXP3 were quantified with software-based classifiers. Reactive regions in early recurrent tumours contained more TAMs (31.4% vs 21.7%, P = 0.01), which showed increased expression of CD86 (59.4% vs 38.4%, P = 0.04), CD204 (48.5% vs 28.4%, P = 0.03), CD206 (25.5% vs 14.4%, P = 0.04) and increased staining intensity for CD163 (86.4 vs 57.7 arbitrary units, P = 0.02), compared to late recurring tumours. Reactive regions contained more B-lymphocytes compared to patient-matched primary tumours (0.71% vs 0.40%, P = 0.04). Fractions of total, cytotoxic and regulatory T-lymphocytes did not differ. Early recurrent glioblastomas showed enrichment for TAMs, expressing both pro- and anti-inflammatory markers and B-lymphocytes. This may indicate a time-dependent response to immunotherapy explained by time-dependent alterations in the immune-microenvironment in recurrent glioblastomas.