Levels Of CD4 Research Articles

Abstract TP341: Longitudinal Analysis of Peripheral Blood Dynamics During the First Week after Ischemic Stroke: Biomarkers and Correlation to Functional Outcome

Introduction: Peripheral blood-based biomarker development for neurological diseases such as ischemic stroke has traditionally focused on measuring alterations in leukocyte number and composition. Deeper immunophenotyping of cells using fluorescent probes that detect alterations among organelles and structures can provide a better understanding of the biological function of leukocytes after ischemic stroke. Methods: We evaluated the utility of BODIPY 493/503 and LysoTracker as fluorogenic probes to assess cellular changes in lipid content and lysosomal burden. Flow cytometric assessments were performed on peripheral mononuclear blood cells from healthy volunteers (HV, N=31), and from ischemic stroke patients (N=43) at 3d and 7d to capture the temporal profile of these biomarkers. Results: We found decreased frequency of CD3+ T cells 3d after stroke, which was also seen at 7d after stroke. We found CD4:CD8 ratios were significantly and persistently increased in stroke patients compared to HV controls. Within the CD4 subset, we found a persistent increase in the frequency of effector CD4 T cells after stroke, this finding is consistent with previous literature. Interestingly, we noted a delayed, but profound increase in lipid content of all cell types at 7d after stroke compared controls. This difference was more pronounced among females. Lysosomal content was differentially altered by cell type, with increased levels in CD4 naïve T cells, but decreased levels in CD4 central memory, CD8 naïve, CD8 effector, CD8 central and effector memory T cells after stroke. While we did not see an association with increase in severity at admission, high lipid content at the acute phase was significantly associated with increased disability at 3 months after hospitalization. Conclusion: Taken together, our findings provide greater context to the previously documented compositional changes in the blood leukocyte compartment after stroke. Ischemic stroke causes a pervasive increase in cellular lipidosis. Lysosomal content has also been significantly positively correlated to functional disability among stroke patients. Using cell-based functional assays with flow cytometric immunophenotyping may provide meaningful insights into biological health and function of circulating leukocytes after brain injury. Understanding the effects of these compositional changes seen in the acute phase in respect to functional outcomes should be a priority for researchers moving forward.

SHMT2 regulates CD8+ T cell senescence via the reactive oxygen species axis in HIV-1 infected patients on antiretroviral therapy.

Although antiretroviral therapy (ART) effectively inhibits viral replication, it does not fully mitigate the immunosenescence instigated by HIV infection. Cellular metabolism regulates cellular differentiation, survival, and senescence. Serine hydroxymethyltransferase 2 (SHMT2) is the first key enzyme for the entry of serine into the mitochondria from the de novo synthesis pathway that orchestrates its conversion glutathione (GSH), a key molecule in neutralising ROS and ensuring the stability of the immune system. It remains incompletely understood whether SHMT2 is involved in the senescence of CD8+ T cells, crucial for immune vigilance against HIV. HIV-infected individuals receiving antiretroviral therapy were enrolled in our study. SHMT2-siRNA was electroporated into T cells to disrupt the gene expression of SHMT2, followed by the quantification of mRNA levels of crucial serine metabolism enzymes using real-time PCR. Immunophenotyping, proliferation, cellular and mitochondrial function, and senescence-associated signalling pathways were examined using flow cytometry in CD8+ T cell subsets. Our findings revealed that CD8+ T cells in HIV-infected subjects are inclined towards senescence, and we identified that SHMT2, a key enzyme in serine metabolism, plays a role in CD8+ T cell senescence. SHMT2 can regulate glutathione (GSH) synthesis and protect mitochondrial function, thus effectively controlling intracellular reactive oxygen species (ROS) levels. Moreover, SHMT2 significantly contributes to averting immunosenescence and sustaining CD8+ T cell competence by modulating downstream DNA damage and phosphorylation cascades in pathways intricately linked to cellular senescence. Additionally, our study identified glycine can ameliorate CD8+ T cell senescence in HIV-infected individuals. Decreased SHMT2 levels in HIV-infected CD8+ T cells affect ROS levels by altering mitochondrial function and GSH content. Increased ROS levels activate senescence-related signalling pathways in the nucleus. However, glycine supplementation counteracts these effects and moderates senescence. This study was supported by grants from the National Key R&D Program of China (2021YFC2301900-2021YFC2301901), National Natural Science Foundation of China (82372240), and Department of Science and Technology of Liaoning Province Project for the High-Quality Scientific and Technological Development of China Medical University (2022JH2/20200074).

Immunosuppressive State May Lead to Brain Metastases in Lung Squamous Cell Carcinoma: Gene Expression and Immunohistochemical Analysis.

Brain metastases (BMs) are rare in lung squamous cell carcinoma (LUSC). Therefore, research on biomarkers or mechanisms that can be used to predict them is limited. To verify whether mRNA profiles can accurately predict BMs, we used a machine learning approach on 20 TNM-matched LUSC tissue samples. We conducted pathway and immunohistochemical analyses to investigate the underlying mechanisms of BM. A total of 15 mRNAs linked to BM were identified. The 15-mRNA signature was highly accurate in predicting BM, with an area under the curve and accuracy of 0.940 and 0.9, respectively. The pathway analysis revealed that immune-related pathways (leukocyte transendothelial migration, Fc gamma R-mediated phagocytosis, and natural killer cell-mediated cytotoxicity) were suppressed, suggesting that an immunosuppressive state may be involved in the development of BM. In the validation set confirmed by immunohistochemical staining, the BM group exhibited significantly lower levels of CD4+ T cells or CD8+ T cells than the group without BM. BM in patients with LUSC was associated with an immunosuppressive state. Immunotherapy may be effective in preventing BM in patients with LUSC.

Diagnostic and predictive values of m5C‑associated genes in idiopathic pulmonary fibrosis.

In patients with idiopathic pulmonary fibrosis (IPF), the role of 5‑methylcytosine (m5C)‑associated genes in the pathogenesis and development of the disease remains unclear. The present study aimed to identify reliable diagnostic markers based on the expression of m5C‑associated genes for the early detection of IPF. Count data were obtained by screening the IPF genome‑wide assay in the Gene Expression Omnibus database, followed by a comparison of m5C gene expression in patients with IPF and controls. The GSE150910 and GSE173355 datasets yielded a total of 23 differentially expressed m5C‑associated genes, which were then investigated for their functions. A diagnostic model was built using eight m5C genes and validated with training sets and the GSE124685 dataset. IPF subtypes were identified based on expression of m5C‑related genes as well as clinical and immunological characteristics. Furthermore, a pulmonary fibrosis model was established in mice by administering bleomycin into the trachea. Lungs were harvested and analyzed using quantitative PCR to determine the expression of m5C‑related genes. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that these genes were significantly enriched in 'base excision repair'. Immunoassay results revealed that 13 immune cell markers (naive, memory and B cell plasma, T cell CD4 naive, T cell CD4 memory resting, T cell follicular helper, T cell regulatory Tregs, NK cell resting, Monocyte, Macrophage M0, Mast cell activated, Eosinophil, and Neutrophil) were significantly associated with IPF. Patients with IPF had lower levels of resting memory CD4+ T cells, which were positively associated with Tet methylcytosine dioxygenase2 (TET2) and Thymine‑DNA glycosylase (TDG) but negatively correlated with NOP2/Sun RNA methyltransferase5 (NSUN5) expression. All samples were classified into based on the levels of the eight diagnostic m5C genes. Samples with high m5C scores are subtype 1, and those with low m5C scores are subtype 2. In subtype 2, male patients had lower levels of CD27 and CD70 but higher levels of CD274, CD86, Cytotoxic T‑lymphocyte‑associated protein4 and Hepatitis A virus cellular receptor2 (HAVCR2). When compared with normal mouse lung tissue samples, expression levels of NOP2/Sun RNA methyltransferase6 (NSUN6), Ubiquitin‑like with PHD and RING Finger Domains1, TDG and TET2 in lung fibrosis tissue samples were significantly higher, while expression levels of NSUN5, NTH‑like DNA glycosylase1, DNA (cytosine‑5‑)‑methyltransferase3 β and Methyl‑CpG binding domain protein 3) were lower. It is possible that m5C‑associated genes play an important role in the diagnosis and typing of IPF. These genes may facilitate investigation of the pathophysiology of IPF and identification of potential treatment targets.

CD16 and CD57 expressing gamma delta T cells in acute HIV-1 infection are associated with the development of neutralization breadth.

New HIV vaccine approaches are focused on eliciting broadly neutralizing antibodies. We characterized early gamma-delta (γδ) T cell responses starting from pre-acquisition and during acute HIV infection (AHI) in participants previously characterized for neutralization breadth development. We found significant differences in γδ T cell surface marker expression in participants that developed neutralization breadth compared to those that did not. Activation of γδ T cells occurred within the first weeks of HIV acquisition and associated with viral load. Expression of CD16 on Vδ1 T cells and CD57 on Vδ2 T cells were found to be significantly higher in broad neutralizers during AHI, and associated with the development of neutralization breadth years later. In addition, the levels of CD16 on Vδ1 T cells was associated with early production of founder virus Env-specific IgM. Thus, γδ T cells may promote development of neutralization breadth, which has implications for HIV vaccine strategies.

P-2067. Antibody Response in People Living with HIV on Effective ART: A Comparison of CD4 T-cell counts >200 versus ≤200 cells/mm³ Following a Bivalent mRNA COVID-19 Vaccine Booster, with Evaluation of the Correlation Between Anti-RBD IgG (Anti-Receptor Binding Domain Immunoglobulin) and sVNT (SARS-CoV-2 Surrogate Virus Neutralization Test)

Abstract Background Despite protection against severe COVID-19 and SARS-CoV-2 variants in the post-pandemic era, the importance of mRNA booster doses may be underestimated. We assessed the immunogenicity of bivalent original/Omicron BA.4-5 COVID-19 vaccine booster in PLWH by immunosuppression level. Table 1: Baseline Characteristics of People Living With Human Immunodeficiency Virus (n = 74) at the time of administration of a bivalent original/omicron BA.4-5 mRNA vaccine booster dose divided into the low CD4 group (CD4 T-cell levels ≤200 cells/mm³) and high CD4 group (CD4 T-cell levels >200 cells/mm³). Abbreviations: HIV, human immunodeficiency virus; IQR, interquartile range; BMI, Body mass Index; ARV, antiretroviral; NNRTI, Non-Nucleoside Reverse Transcriptase Inhibitor Methods This prospective cohort study enrolled participants aged ≥18 years, on stable ART, with ≥2 doses of COVID-19 vaccine, at King Chulalongkorn Memorial Hospital and HIV Netherlands Australia Thailand Research Collaboration. Exclusion criteria were virologic failure, previous immunosuppressant or monoclonal antibody use, recent vaccination, or SARS-CoV-2 infection. We evaluated anti-RBD IgG and sVNT against SARS-CoV-2 variants, before and 4 weeks after bivalent original/Omicron BA.4-5 mRNA vaccine booster. Primary outcomes were pre- and post-vaccination geometric mean titres (GMT) and post-vaccination GM ratios (GMR) of anti-RBD IgG in PLWH with CD4 T-cell levels ≤200 vs. >200 cells/mm³. Secondary outcome was the relationship between variant-specific sVNT and anti-RBD IgG.Figure 1:Kinetic of anti RBD Ig response after bivalent covid vaccine booster in patients with CD4 ≤ 200 cells/mm3 versus CD4 > 200 cells/mm³Geometric mean titres (GMT) with 95% confidence interval (CI) of anti-RBD total Ig levels in CD4 ≤ 200 cells/mm3 and CD4 > 200 cells/mm³ group., Time points preboost: the day of vaccination; post boost, 4-week post vaccination; *** p<0.001; ** p<0.05The cut off for seropositive from The SARS-CoV-2 IgG II Quant assay is log Anti RBD Ig 1.7 AU/mL. (50 AU/mL) Results Of 74 participants, 22 (29.7%) had CD4 ≤200 and 52 (70.3%) had CD4 >200 cells/mm³. In the low CD4 group, median time since primary vaccination was 678 days; pre-and post-boost anti-RBD IgG GMT was 687 AU/mL and 13,959 AU/mL respectively (GMR increase = 20.3 (95%CI 7.3 to 56.2; P< 0.001). In the higher CD4 group, median time since primary vaccination was 587 days; pre- and post-boost GMTs were 3,479 and 35,238 AU/mL respectively (GMR increase =10.1 (95%CI 7.5 -13.7); P< 0.001) (Figure 1). Anti-RBD Ig titer correlated strongly with post-boost sVNT of Ancestral and BA.5 (vaccinated strain), XBB1.16, and XBB1.5 (circulating strains). Predicted sVNT inhibition was >40% for all strains at 4 log10 antiRBD IgG AU/mL (Figure 3).Figure 2A-C: Dynamic changes of sVNT (% inhibition) with different SARS-CoV-2 variants in high and low CD4 group.Figure 2A: Dynamic change of sVNT (% inhibition) against different strains comparing high CD4 group (HCD4; CD4 ≤ 200 cells/mm³ ) and low CD4 group (LCD4; CD4 > 200 cells/mm³ )Figure 2B: Pool kinetic response of sVNT (% inhibition) against concerning Omicron at the time of study, Delta, and Ancestral strainsFigure 2C : sVNT (% inhibition) of post booster samples in different strains comparing high CD4 group (HCD4; CD4 ≤ 200 cells/mm³ ) and low CD4 group (LCD4; CD4 > 200 cells/mm³ )Abbreviations: IQR, interquartile range.The cut off for seropositive from sVNT is 40% inhibition. Conclusion Participants in our low and higher CD4 groups had significant increases in anti-RBD IgG, and neutralization antibodies with cross-reactivity against Omicron variants post-boost. This supports the importance of annual mRNA vaccine boosters for PLWH, especially those with low CD4 counts. Further research is needed to determine the optimal anti-RBD IgG level for adequate neutralization and assess the time course of immune protection to refine vaccination schedules in PLWH.Figure 3:Correlation of predicted sVNT against XBB1.5, XBB1.16, BA.5, BA.2, Delta, Ancestral strain and log Anti RBD Ig titers using non-linear regression analysis. Sera samples from individuals from pre-boost (blue) and at 4 weeks post-boost (red) with bivalent covid vaccine were tested with anti-RBD IgG and sVNT against XBB1.5, XBB1.16, BA.5, BA.2, Delta, and Ancestral strain. Predicted sVNT level, using non-linear regression analysis with cluster-robust standard errors (N = 148) against different strains were based on the log anti RBD titer. The y axis represents percent inhibition of sVNT against different strains. The x axis represents log 10 scale of anti-RBD Ig titer (AU/mL). The purple lines indicating sVNT cut off at 40%. The r-square was calculated according to the non-linear equation using STATA v.17.0 software. (The minimum log AntiRBD Ig required to achieve 40% sVNT was 3.75 for the ancestral/BA.5 strain (vaccinated strain) and 4 for the XBB1.5, XBB1.16, BA.2, and Delta strains.) Disclosures All Authors: No reported disclosures

Efficacy and safety of pseudolaric acid B against Echinococcus multilocularis in vitro and in a murine infection model

IntroductionAlveolar echinococcosis (AE) is a chronic zoonotic disease caused by the larvae of the Echinococcus multilocularis (E. multilocularis). The current chemotherapy for AE relies on albendazole and mebendazole, which exhibit only parasitostatic rather than parasiticidal effects. Therefore, there is a need to find new anti-Echinococcosis drugs. Pseudolaric acid B (PAB) has been described to have strong antiparasitic effects. However, the in-depth mechanism by which PAB acts against E. multilocularis remains unclear.MethodsTo evaluate the effect of a PAB intervention on protoscoleces, metacestode vesicles and germinal cells in E. multilocularis in vitro. In addition, the effects of PAB on T lymphocyte and collagen synthesis were evaluated after PAB administration in a mouse model.ResultsMetacestode vesicles and germinal cells were successfully cultured, and specific genes were amplified via RT-PCR to identify the protoscoleces, vesicles, and germinal cells as the sources of E. multilocularis. In vitro studies have demonstrated that PAB exhibits dose- and concentration-dependent cytotoxicity against E. multilocularis protoscoleces. Scanning electron microscopy revealed that the microvilli structure of the protoscolex was destroyed and the rostellar hooks had fallen off. PAB induced. The release of PGI from the metacestode vesicles, leading to the structural destruction of the inner surfaces. PAB suppressed the proliferation of germinal cells. After PAB treatment, the serum and the host tissue surrounding the metacestodes IFN-γ levels were upregulated and the IL-4 and IL-10 levels was downregulated. After PAB treatment, the levels of CD4+ T lymphocytes increased and the levels of CD8+ T lymphocytes decreased in the host tissue surrounding the metacestodes and the spleen. The proportions of the Th1 and Th17 cell subpopulations were increased and the proportion of Th2 cell and Treg cell subpopulations was decreased in the host tissue surrounding the metacestodes. Additionally, collagen deposition was increased after PAB treatment. PAB suppressed the expression of matrix metalloproteinases (MMPs 1, 2, 3, 9, 13) and the activation of the PI3K/AKT signaling pathway in the host tissue surrounding the metacestodes.ConclusionPAB has a significant killing effect on E. multilocularis, suggesting that it is a potential candidate for the treatment of AE.

Tinosinenside A inhibits neuroinflammation and protects HT22 cells by suppressing the TLR4/NF-κB/NLRP3 signaling pathway in BV2 cells.

Microglia-mediated neuroinflammation plays a crucial role in Alzheimer's disease (AD). Tinosinenside A (Tis A) is a novel sesquiterpene glycoside isolated from the dried rattan stem of Tinospora sinensis (Lour.) Merr. Tis A exhibited anti-inflammatory and neuroprotective activities in vitro. However, the mechanism underlying the inhibition of neuroinflammation and protection of nerve cells remains obscure. This study used lipopolysaccharide (LPS)-induced inflammatory response in BV2 cells to simulate a neuroinflammatory model and used Aβ1-42-induced HT22 cells to establish an AD cell model, aiming to investigate the efficacy and mechanism of Tis A through anti-neuroinflammation to protect nerve cells. Tis A had no effect on the proliferation of BV2 and HT22 cells at the tested concentrations. The time- and dose-dependent effects of Tis A on the LPS-induced inflammatory response of BV2 cells demonstrated that the best anti-inflammatory efficacy appeared after 12h of pretreatment. Tis A inhibited the gene levels of TNF-α, IL-6, IL-1β, iNOS, and IL-10 while enhancing the gene levels of IL-4 and TGF-β. Additionally, Tis A reduced the gene expression levels of CD16 and CD32 and increased the CD36 and CD206 gene expression levels. It also downregulated the protein expression of Iba-1 and iNOS while upregulating CD206. Tis A obviously inhibited NLRP3 gene and protein expression in LPS-stimulated BV2 cells. The inhibitory effect of Tis A on NLRP3 was counteracted by the NLRP3 activator nigericin and overexpression plasmid GV358. Tis A inhibits NLRP3 protein expression to reduce the assembly of NLRP3/ASC/Caspase-1 inflammasome, then regulates the TLR4/NF-κB/NLRP3 signaling pathway. It regulates microglia activation and M1/M2 phenotypic polarization, then inhibits the production of inflammatory factors, and reduces the apoptosis rate of HT22 cells under inflammatory conditions, improving the survival rate of nerve cells to protect neurons.

Formin protein DAAM1 positively regulates PD-L1 expression via mediating the JAK1/STAT1 axis in pancreatic cancer

BackgroundDishevelled-associated activator of morphogenesis1 (DAAM1) is a member of the evolutionarily conserved Formin family and plays a significant role in the malignant progression of various human cancers. This study aims to explore the clinical and biological significance of DAAM1 in pancreatic cancer.MethodsMultiple public datasets and an in-house cohort were utilized to assess the clinical relevance of DAAM1 in pancreatic cancer. The LinkedOmics platform was employed to perform enrichment analysis of DAAM1-associated molecular pathways in pancreatic cancer. Subsequently, a series of in vitro and in vivo experiments were conducted to evaluate the biological roles of DAAM1 in pancreatic cancer cells and its effects on intratumoral T cells.ResultsDAAM1 was found to be upregulated in pancreatic cancer tissues, with higher expression levels observed in tumor cells. Additionally, high expression of DAAM1 was associated with poor prognosis. DAAM1 acted as an oncogene in pancreatic cancer, and its inhibition suppressed tumor cell proliferation, migration, and invasion, while promoted apoptosis. Furthermore, DAAM1 was involved in the JAK1/STAT1 signaling pathway and regulated PD-L1 expression in pancreatic cancer cells. The inhibition of DAAM1 also significantly reduced the exhaustion levels of CD8+ T cells.ConclusionIn conclusion, DAAM1 functions as an oncogene and is immunologically implicated in pancreatic cancer, these findings suggest that DAAM1 may serve as a promising therapeutic target for the clinical management of pancreatic cancer.

P-1274. Mpox: Characterization and Clinical Outcomes of Patients Attended in Health Service Providing Institutions in Colombia

Abstract Background The monkeypox virus, part of the Orthopoxvirus genus, triggered a worldwide outbreak in 2022. While this outbreak had widespread effects, there's limited information on mpox's specific impact in Colombia, particularly in terms of how it is managed, its burden, and its epidemiology. This research seeks to examine the medical context, clinical variations, and health consequences in individuals diagnosed with mpox, especially those with HIV in Colombian health institutions Methods This retrospective study was conducted in health institutions in Colombia based on clinical records from Jan 2022 to Dec 2023. . Participants in the study were diagnosed through molecular methods and their clinical evolution was tracked through medical records. CD4 groups, BMI groups, viral suppression status, and HIV status were used to analyse the results. Results One thousand four hundred thirteen (1,413, 97.2% male) individuals, including 2.6% identified as healthcare workers were included in this study. Concomitant sexually transmitted diseases were common, affecting 30.1%, mainly syphilis (80%) and Neisseria gonorrhoeae (16.4%). 54% of the population (764/1413 individuals, 99.3% male) were persons living with HIV (PWH), and almost one-third (31%, n=284) of participants had concomitant sexually transmitted diseases and HIV PWH also had a higher proportion of gastrointestinal symptoms and genital symptoms. An important difference between HIV-positive and negative patients is that the former had a higher proportion of syphilis (p-value 0.5909) and other STIs (p-value 0.0026). Although PWH were younger (34.3±7.7 vs. 37.7±7.5), the difference in mean age was not statistically significant, although the differences in proportion were (15 to 44 years group, p-value 0.0097). Conclusion The evidence presented shows that half of the population were people living with HIV, and the presence of mpox was not statistically significant in people with unsuppressed viral load or with low CD4 levels. The evidence provided by this study, whose results were standardized by the CRFs, and emphasizes the importance of interdisciplinary attention for patients suffering from mpox. Special emphasis should be placed on individuals support and follow-up, focusing on detecting concomitant STIs. Disclosures All Authors: No reported disclosures

NK Count and Natural Cytotoxicity in Immune Nonresponders Versus Responders Living With HIV.

Despite viral suppression with antiretroviral therapy, immune nonresponders (INR) among people living with HIV (PLWH) still have a higher risk of developing AIDS-related and non-AIDS-related complications. Our study aimed to investigate the phenotype and functions of Natural Killer (NK) cells in INR, to better understand underlying mechanisms of immune nonresponse. Our cross-sectional study included PLWH aged over 45 with an undetectable HIV viral load sustained for at least 2 years. Participant CD4+ T-cell counts exceeded 500 cells/mm3 for IR or below 350 cells/mm3 for INR. We characterized NK cell subsets, performed natural cytotoxicity and ADCC assays, and phenotyped NK cells before and after functional assays. Median CD4 levels were 247.5 (IQR, 208.8-286.3) cells/mm3 and 768.5 (IQR, 673.3-957.8) cells/mm3 in the INR (n = 20) and IR (n = 40), respectively. NK cell counts were lower in INR (p = 0.00066), but the percentages were similar between IR and INR. NKG2A was the only differentially expressed marker between groups, showing higher expression in INR. Additionally, NK cell natural cytotoxicity was elevated in the INR group. Multivariate analyses associated CD4 nadir and low NK cell count with immune nonresponse. Our results do not indicate a clear role for NK-mediated ADCC in immune nonresponse. We did not objectify an association between CD8 hyperactivation and INR.

Gastric cancer mesenchymal stem cells upregulate PD-1 expression on the CD8+ T cells by regulating the PI3K/AKT pathway.

Gastric cancer mesenchymal stem cells (GC-MSCs) are a crucial component of the gastric cancer microenvironment, exerting a pivotal influence on the formation of a suppressive immune microenvironment and the progression of gastric cancer. In this study, we utilized GC-MSCs to co-culture peripheral blood mononuclear cells (PBMCs) obtained from both gastric cancer patients and healthy individuals in a proportionate manner by direct cell-to-cell contact. Our findings reveal that co-culture of GC-MSCs with PBMCs led to a notable reduction in CD8+ T cells percentages and an increase in surface PD-1 expression levels on CD8+ T cells. However, flow cytometry analysis demonstrated no significant changes following pretreatment with GC-MSC-conditioned medium (GC-MSC-CM). Moreover, western blotting analysis demonstrated a notable reduction in phosphorylated AKT levels in co-cultured PBMCs. Collectively, our results suggest that GC-MSCs impair the anti-tumor immune response of PBMCs by elevating the PD-1 levels of CD8+ T cells and impairing the killing of CD8+ T cells. These findings provide new evidence that GC-MSCs contribute to immunosuppression within the tumor microenvironment (TME).

Estrogen deficiency promotes neurodegeneration in female hemi-parkinsonian mice: The role of regulatory T cells.

Circulating levels of the female hormone estrogen has been associated with the development of Parkinson's disease (PD), although the underlying mechanism remains unclear. Immune homeostasis mediated by peripheral regulatory T cells (Treg) is a crucial factor in PD. The aim of this study was to explore the effects of estrogen deficiency on neuroinflammation and neurodegeneration in a rodent model of PD, with particular reference to Treg. Estrogen deficiency was established in a mouse model by bilateral ovariectomy (OVX). PD was modeled by the injection of LPS into the striatum. Motor performance was assessed in each experimental group. Dopaminergic degeneration was evaluated using tyrosine-hydroxylase (Th) immunohistochemical staining of the substantia nigra (SN) and striatum. Dopamine and dopamine metabolite levels in the striatum were also evaluated, together with the infiltration of CD4 T cells into the SN. Neuroinflammation was assessed by evaluating the mRNA level of microglial and M1/M2 phenotype markers, as well as the abundance of pro-inflammatory cytokines in the midbrain. The frequency of peripheral Treg cells was evaluated using flow cytometry. OVX prior to LPS injection markedly aggravated neurodegeneration, neuroinflammation, motor performance, and CD4 T cell infiltration compared to the LPS-only group. Estradiol treatment or activation of the G protein-coupled estrogen receptor (GPER) in OVX mice prior to LPS injection induced functional improvement and reduced the levels of neurodegeneration, neuroinflammation, and CD4 T cell infiltration. OVX prior to LPS injection also led to a decreased frequency of Treg compared to the LPS-only group. Moreover, estradiol treatment or GPER activation of OVX mice prior to LPS injection significantly increased the Treg frequency. Antibody-mediated depletion of Treg after GPER activation counteracted the ability of GPER to alleviate neurodegeneration, CD4 T cell infiltration, the release of pro-inflammatory cytokines, and motor performance in the PD model. Estrogen deficiency can disrupt Treg-mediated immune homeostasis and thus further aggravate microglial inflammation and dopaminergic degeneration in PD model. The effects of estrogen on Treg may be partially mediated by GPER signaling, and thus GPER is a promising target for PD, especially in estrogen-deficient women.

P1002 Changes in circulating lymphocyte subsets and CCR9 transcripts as mechanistic biomarkers of the small molecule α4β7 inhibitor MORF-057 in patients with Ulcerative Colitis

Abstract Background MORF-057 is an orally administered, selective, and potent small molecule inhibitor of the cell surface receptor integrin a4b7. Upon binding to the a4b7 heterodimer, MORF-057 blocks the interaction of this integrin with the endothelial ligand mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) which, in turn, prevents the extravasation of circulating lymphocytes into the intestinal mucosa. In this study, the pharmacodynamic effects of MORF-057 on immune cell trafficking were evaluated in UC patients using a flow cytometry-based assay and by assessing the expression of CCR9 gene in blood. Methods As part of the induction phase of an open-label, single-arm, Phase 2a study (NCT05291689) the efficacy, safety, and tolerability of MORF-057 in 35 patients with moderately to severely active UC disease were evaluated. Patients were treated with MORF-057 at a dose of 100 mg BID orally for an induction period of 12 weeks followed by a 40-week maintenance period. Blood was collected for peripheral blood mononuclear cell (PBMC) isolation and RNA extraction at baseline and post-treatment at Week 2, 6, 12, 20, 28, 36, 44, and 52. Using flow cytometry, levels of CD4+β7+ naïve, CD4+β7+ central memory (TCM), CD4+β7 Hi effector memory (TEM) T cells, and CD19+β7+ switched memory B cells (BSM) subpopulations were quantified as a percentage of the parent populations CD4 naive, TCM, TEM and BSM, respectively, then normalized to pre-treatment baseline. Expression of CCR9 mRNA in the blood was quantified as a fold ratio relative to baseline for the same timepoints. Statistical analyses were performed using the Wilcoxon Signed Rank test. Results The percentages of circulating lymphocyte subsets CD4+β7 Hi TEM cells, CD4+β7+ TCM and CD19+β7+ BSM were significantly elevated relative to baseline as early as Week 2 and up to Week 52 (Table 1). These changes were not observed in the overall lymphocyte counts nor in the CD4+β7+Naïve T cells (except at Week 52). The baseline-normalized expression levels of CCR9 at Weeks 12 and 52 were 1.6 [0.6] (mean [SD]) and 1.7 [0.8], respectively (Figure 1). Conclusion Patients receiving MORF-057 for a period of up to 52 weeks achieved statistically significant increases in specific β7+ lymphocyte subsets expressing ITGβ7. Elevated levels of the CCR9 transcript were observed in blood over the course of treatment. Effects observed were consistent with those reported for patients receiving biologic inhibitors of the same pathway as well as in healthy volunteers receiving MORF-0571,2. These changes in circulating lymphocyte populations and CCR9 transcript levels may be used as mechanistic biomarkers for MORF-057 in UC patients.

Multiplex immunohistochemistry to explore the tumor immune microenvironment in HCC patients with different GPC3 expression

ObjectivesGPC3 has been recognized as a promising target for immunotherapy in hepatocellular carcinoma (HCC). However, the GPC3-targeted immunotherapies have shown limited therapeutic efficacy. The use of anti-PD-1/PD-L1 monoclonal antibodies in HCC treatment is considerably constrained. Furthermore, there is still a notable lack of understanding concerning the immune landscape in HCC, especially regarding varied GPC3 expression levels. Therefore, thorough exploration of the intricate tumor immune microenvironment at different GPC3 expression levels is essential for guiding and improving HCC treatment strategies.MethodsSixty patients with HCC were enrolled in this study, receiving a first-line treatment that combined anti-angiogenesis targeted drugs and immunotherapy. Immunohistochemistry was used to assess the levels of GPC3 expression. Multiple immunohistochemical markers, such as CD8, PD-1, LAG3, TIGIT, TIM-3, CD103, Claudin18.2, PD-L1, CD4, Foxp3, CD68, CD163, GPC3, CD11C, CD14, CD66b, and HLA-DR, were used to characterize the immune microenvironment and spatial distribution of immune cells in HCC tumors with different levels of GPC3 expression. Cell expression levels and spatial distribution were determined by fluorescence staining and subsequent analysis of fluorescence intensity using the Panoramic Pathology Workstation (Pano ATLAS). This approach facilitated a detailed examination of cell characteristics and spatial information within the samples.ResultsBased on the result of GPC3 immunohistochemical analysis, patients with strong positive GPC3 expression were classified as high GPC3 expression, while the others were classified as low GPC3 expression. Patients in the low GPC3 expression group had longer overall survival (OS) than in the high group (P = 0.003, HR = 2.9240). Further exploration of the immune microenvironment based on different GPC3 expression levels revealed that in high GPC3 expression group, the proportions of CD8+ T cells(P = 0.0435), TIM-3+ T cells(P = 0.0447), CD103+CD8+ tissue-resident T cells(P = 0.0410), CD11C+CD14−DC cells(P = 0.0497), CD11C+HLA-DR−DC cells(P = 0.0309), CD11C+CD14−HLA-DR−DC cells(P = 0.0233), and CD11C+CD14−CD66b−DC cells(P = 0.0474) were all higher compared to low expression group. At spatial distances of 10 μm, 20 μm, and 30 μm, the levels of CD8+ T cells were higher in the high expression group compared to the low expression group (high vs. low: P = 0.0281, P = 0.0236, P = 0.0220).ConclusionsMultiple immunohistochemistry is a powerful technique for exploring the intricate immune microenvironment of hepatocellular carcinoma, enabling the precise identification of diverse cell subsets and their spatial distribution within the tumor microenvironment. This methodology provides valuable insights into the complex interactions and spatial organization of immune cells in the context of hepatocellular carcinoma progression. Low GPC3 expression in HCC patients indicates potential benefits from combined targeted and immunotherapy. Different levels of GPC3 expression levels can predict the effectiveness of targeted combination immunotherapy in HCC patients. Additionally, different GPC3 expression patterns in HCC patients correspond to unique tumor immune microenvironments, which have implications for guiding HCC treatment approaches.

TYK2:p.Pro1104Ala Variant Protects Against Autoimmunity by Modulating Immune Cell Levels.

The TYK2:p.Pro1104Ala (rs34536443) hypomorph variant has been associated with protection against numerous autoimmune disorders. Thus, its mechanism of action becomes of great interest. Here, consistent with the participation of activated immune cells in autoimmunity, we show that the variant regulates the levels of immune cells at a human, general population level and is associated particularly with higher levels of T and B lymphocytes, especially the naïve (non-activated) compartment. Also, consistent with a protective function in autoimmunity, the level of regulatory CD4+ T cells was increased. Thus, this variant decreases immune activation thereby protecting from autoimmunity. Our work links the cellular mechanism regulated by the TYK2:p.Pro1104Ala variant to autoimmunity protection and supports TYK2 as a therapeutic target in autoimmunity.

The effect of three types of water-based training protocols on thymus atrophy and specific indicators of cellular immune senescence in aged male rats

The collective detrimental impact of aged naive lymphocytes and thymus atrophy on the aging of the immune system can be mitigated by exercise. Hence, this research aims to explore the effects of three methods of water-based exercises on immune system aging and thymus atrophy in elderly rats. Thirty-two 24-month-old rats, with an average weight of 320 ± 5 g, were randomly allocated into four groups of endurance training (n = 8), resistance training (n = 8), combined training (n = 8), and control (n = 8).The training protocols (10 weeks) were conducted four times a week in a container measuring 50 × 50x100 cm filled with water at 30 ± 1 °C. The evaluation of naïve and memory T lymphocytes was conducted for the intervention groups based on the expression or lack of expression of the CD28 and CD57 markers in the subsets of CD4 + and CD8 + T cells. Naïve T cells were represented by CD28 + CD57- T lymphocytes, memory T cells were represented by CD28- CD57- T lymphocytes, aged naïve T cells were indicated by CD28 + CD57 + lymphocytes, and aged memory T cells were represented by CD28- CD57 + lymphocytes. The findings of the study showed that all three exercise protocols resulted in a significant decrease in levels of memory CD8, aged CD8, naive and naive CD4 and CD8, and aged memory, as well as an increase in levels of CD4, CD8, CD4 + , and naive CD8 when compared to the control group. It was observed that thymus atrophy, memory CD4, and aged CD4 had a significant decrease only in the combined exercise group compared to the control group, with no significant differences observed in these indicators for the resistance and endurance groups. Furthermore, the ratio of CD4 to CD8 remained unchanged across all groups. The findings of this study suggest greater efficacy of combined training in enhancing specific health indicators of cell immunity among elderly populations. Moreover, engaging in water exercises of all three types of combined, resistance, and endurance training are deemed safe activities for older individuals to bolster their immune system and mitigate the aging process of T cells.

A powerful organic photosensitizer for effective treatment of malignant tumor via activating antitumor immune response.

Photodynamic therapy (PDT) has emerged as an innovative approach in cancer treatment, effectively inducing tumor cell death through light-triggered reactive oxygen species (ROS) generation. Additionally, PDT can also trigger antitumor immune responses, thereby reducing the risk of postoperative tumor recurrence. However, the development of highly efficient photosensitizers aimed at activating immune responses for comprehensive tumor eradication remains at an early stage. In this study, we developed a new organic photosensitizer, ThC, which exhibits excellent mitochondrial-targeting effect and significantly enhanced ROS production compared to traditional photosensitizers, such as Chlorin e6 (Ce6). Our findings demonstrate that ThC robustly induces immunogenic cell death (ICD) process in hepatic cancer cells, which could effectively transform immunologically "cold" tumors into "hot" tumors. Through in situ injection and subsequent white light irradiation, ThC achieved superior efficacy in eliminating subcutaneous hepatic tumors compared to Ce6 treatment. Immunological analyses revealed that ThC treatment led to elevated levels of CD4+ and CD8+ T cells, along with a reduction in immunosuppressive cell populations (Tregs and tumor-associated macrophages) within the tumor microenvironment. This study provides a novel therapeutic agent with significant potential for clinical translation in the treatment of malignant tumors.

Dipyridamole Attenuates Experimental Periodontitis by Regulating M1 Macrophage Polarization via PKA/PKG Pathways.

Periodontitis is a chronic inflammatory disease initiated by dysbiosis of the local microbial community. As a non-specific phosphodiesterase inhibitor, dipyridamole features anti-oxidant and anti-inflammatory properties. This study aimed to investigate the effects of dipyridamole in an experimental rat model of periodontitis. Thirty rats were divided randomly into three groups (n = 10): non-ligature group (NL), ligature-induced periodontitis group (L), and ligature-induced periodontitis with dipyridamole administered group (L + D). All rats were euthanized on Day 14. Alveolar bone resorption was analyzed by microcomputed tomography. The mRNA levels of Il1b, Il6, tumor necrosis factor alpha (Tnfa), and inducible nitric oxide synthase (iNos) in gingival tissue were assessed by real-time quantitative polymerase chain reaction (qRT-PCR). Inflammation level, osteoclasts, and macrophages infiltration were analyzed histologically. RAW264.7 macrophages were stimulated with Porphyromonas gingivalis lipopolysaccharide (P.g. LPS) to induce M1 polarization. Different concentration of dipyridamole (0/2/10 μM) was added simultaneously. To explore the role of PKA/PKG pathways, RAW 264.7 macrophages were pretreated with 10 μM H-89 (PKA inhibitor) or 1 μM KT-5823 (PKG inhibitor), respectively. Expression of pro-inflammatory cytokines and M1 markers were detected by qRT-PCR, ELISA, and flow cytometry. Dipyridamole administration reduced alveolar bone loss, protein levels of inflammatory cytokines, and osteoclastogenesis in rats with experimental periodontitis. It also showed a tendency to decrease mRNA levels of Il1b, Il6, and Tnfa but without significant differencesin gingival tissues. Moreover, the infiltration of macrophage and M1 macrophage polarization in gingival tissue of periodontitis rats were inhibited by dipyridamole administration. In addition, dipyridamole could downregulate the gene expression of Il1b and Tnfa, as well as the protein level of TNF-α, CD86, and iNOS in RAW264.7 treated with P.g. LPS. When PKA/PKG pathways were blocked, the suppression of TNF-α, CD86, and iNOS was reversed significantly. Dipyridamole alleviated experimental periodontitis in rat models by regulating M1 polarization via activation of PKA/PKG pathways and emerges as a hopeful remedy for periodontitis.

ADC histogram analysis of tumor-infiltrating CD8+ T cell levels in meningioma.

To investigate the value of preoperative MRI features and ADC histogram analysis for evaluating tumor-infiltrating CD8+ T cells in meningiomas. In this single-center cross-sectional study, we conducted a retrospective analysis of clinical, imaging, and pathological data from 84 patients with meningioma and performed immunohistochemical staining to quantitatively evaluate CD8+ T cells. Using X-Tile software, we divided the patients into high-and low-CD8+ T cells groups based on cut-off values. Furthermore, we compared the clinical and MRI features between the two groups and assessed the predictive value of significant parameters by plotting ROC curves. Additionally, Spearman's analysis was used to examine the association between ADC histogram parameters and CD8+ T cells. The level of tumor-infiltrating CD8+ T cells was found to have a negative correlation with recurrence in patients with meningiomas (r=-0.235, p = 0.031). No statistically significant differences were found in clinical and conventional MRI features between the two groups (all p > 0.05). Conversely, among the ADC histogram parameters, the coefficient of variation (CV), Perc.01, Perc.05, Perc.10, and Perc.25 showed statistically significant differences between the two groups (all p < 0.05) and combined ADC histogram parameters had the highest AUC (0.791; 95%CI (0.689-0.872)). Additionally, we observed a positive correlation between Perc.01, Perc.05, Perc.10 and CD8+ T cells (p < 0.05), the CV and variance was negatively correlated with the levels of CD8+ T cells (p < 0.05). ADC histogram analysis can be used as an imaging tool to preoperatively assess CD8+ T cells in patients with meningioma, and found a certain correlation between them.