Fas Ligand Expression Research Articles

Efficacy and safety of hepatic arterial infusion chemotherapy combined with donafenib in the treatment of unresectable hepatocellular carcinoma.

This study aimed at ascertaining the efficacy and safety of hepatic arterial infusion chemotherapy (HAIC) combined with donafenib versus HAIC alone in the treatment of unresectable hepatocellular carcinoma (HCC). Seventy HCC patients were enrolled for our study, and they were randomized by simple randomization using computer-generated random numbers into two groups: control group and observation group. Regular follow-up reviews were conducted to assess the efficacy of treatments. The levels of apoptotic factors, the levels of hepatic fibrosis indices, the levels of serum tumor vascular factors and tumor markers, and the occurrence of adverse reactions in the two groups were recorded and compared. Disease control rate, objective response rate, and progression-free survival (PFS) of patients in the observation group were higher in contrast to the control group. After 12 weeks of treatment, lower mRNA expression of c-mesenchymal-epithelial transition factor, telomerase, and Fas Ligand and higher mRNA expression of Fas and Caspase-3 were observed in HCC tissues of the observation group versus the control group (p<0.05); lower detection values of serum laminin, hyaluronic acid, collage type IV, vascular endothelial growth factor receptor 2, and alpha-fetal protein (AFP) were noted in HCC patients of the observation group in comparison to the control group (p<0.05); there was no difference in the incidence of adverse reactions between the two groups. Donafenib combined with HAIC in the treatment of unresectable HCC patients can notably reduce serum AFP levels, improve hepatic fibrosis, enhance short-term efficacy, prolong PFS, and have a favorable safety profile.

Selenium nanoparticles enhance the anti-tumor immune responses of anti-4-1BB antibody and alleviate the adverse effects on mice

4-1BB agonists for cancer immunotherapy have shown good preliminary efficacy in clinical trials, but several of the first-generation 4-1BB agonistic antibodies entering the clinic have failed due to safety issues. Selenium nanoparticles (SeNPs) exhibit anti-inflammatory, anti-tumor, antioxidant, and immune-modulating properties. In addition, they have been shown to have detoxifying effects and prevent oxidative liver damage. In this study, we used an anti-4-1BB antibody in combination with SeNPs to evaluate the anti-lung cancer effects in in vitro and in vivo experiments and explore the underlying mechanisms by pathological analyses, quantitative PCR, and enzyme-linked immunoassay. We found that 5 μmol·L–1 anti-4-1BB antibody combined with 1 μmol·L–1 SeNPs increased the expression of IFN-γ and promoted the killing effects of peripheral blood mononuclear cells on Lewis lung carcinoma cells, with a lethality rate up to 56.88 %. Experiments in tumor-bearing mice showed that the tumor inhibition rate was 58.61 % after treatment with 3.5 mg/kg anti-4-1BB antibody combined with 0.25 mg/kg SeNPs, and the liver function index returned to normal. When the combined treatment was compared with the antibody treatment alone, detection of immune relevant factors demonstrated that the expression of FOXP3, IL-2, IL-12, and TNF-α in the spleen was downregulated, whereas the expression of IFN-γ in the spleen, serum, and tumor was upregulated, accompanied by increased Fas ligand expression in the tumor tissues. Based on these findings, we get the conclusion that anti-4-1BB antibody combined with SeNPs may alleviate the immunosuppression of regulatory T cells, promote the immune cell proliferation and metastasis to synergistically kill tumor cells. This combination also reduces the inflammatory damage to normal tissues and slows overstimulation of the splenic immune response.

Differential Impairment Mechanism of Sperm Production via Induction of miR-34c-Activated Apoptosis and Spermatogenesis Pathway in Diet-Induced Obesity and Resistant Mice and GC-1 Spg Cells.

Male reproductive dysfunction is a clinical disease, with a large number of cases being idiopathic. Reproductive disorders have been found in obese (diet-induced obesity and diet-induced obesity-resistant) mice, but the mechanism behind the male reproductive dysfunction between them may be different. The purpose of this study was to explore the possible role and mechanism of miR-34c on sperm production in high-fat-diet-induced obesity-resistant (DIO-R) mice and GC-1 spg cells, which may differ from those in high-fat-diet-induced obesity (DIO) mice. In vivo and in vitro experiments were performed. C57BL/6J mice were fed a high-fat diet for 10 weeks to establish the DIO and DIO-R mouse model. GC-1 spg cells were used to verify the mechanism of miR-34c on sperm production. During in vivo experiments, sperm production damage was found in both DIO and DIO-R male mice. Compared to the control mice, significantly decreased levels of testosterone, LH, activities of acrosome enzyme (ACE), HAse, and activating transcription factor 1 (ATF1) were found in both DIO and DIO-R male mice (p < 0.05). Compared with the control group, the ratio of B-cell lymphoma-2 (Bcl-2)/bcl-2-associated X protein (Bax) in the DIO group was significantly decreased, and the expression level of cleaved caspase-3 was significantly increased (p < 0.05). Compared with the control group, the Bcl-2 protein expression level in the testes of the DIO-R group significantly decreased (p < 0.05). However, the Bax expression level increased. Thus, the Bcl-2/Bax ratio significantly decreased (p < 0.01); however, the factor-related apoptosis (Fas), Fas ligand (FasLG), cleaved caspase-8, caspase-8, cleaved caspase-3, and caspase-3 protein expression levels significantly increased (p < 0.05). Compared with the DIO group, in DIO-R mice, the activities of ACE, ATF1, Bcl-2, and Bcl-2/Bax's spermatogenesis protein expression decreased, while the apoptosis-promoting protein expression significantly increased (p < 0.05). During the in vitro experiment, the late and early apoptotic ratio in the miR-34c over-expression group increased. MiR-34c over-expression enhanced the expression of apoptosis-related proteins Fas/FasLG and Bax/Bcl-2 while inhibiting the expression of ATF1 and the sperm-associated protein in GC-1 spg cells. DIO and DIO-R could harm sperm production. DIO-R could impair sperm production by inducing the miR-34c-activated apoptosis and spermatogenesis pathway, which may be different from that of DIO.

Immuno-protective vesicle-crosslinked hydrogel for allogenic transplantation

The longevity of grafts remains a major challenge in allogeneic transplantation due to immune rejection. Systemic immunosuppression can impair graft function and can also cause severe adverse effects. Here, we report a local immuno-protective strategy to enhance post-transplant persistence of allografts using a mesenchymal stem cell membrane-derived vesicle (MMV)-crosslinked hydrogel (MMV-Gel). MMVs are engineered to upregulate expression of Fas ligand (FasL) and programmed death ligand 1 (PD-L1). The MMVs are retained within the hydrogel by crosslinking. The immuno-protective microenvironment of the hydrogel protects allografts by presenting FasL and PD-L1. The binding of these ligands to T effector cells, the dominant contributors to graft destruction and rejection, results in apoptosis of T effector cells and generation of regulatory T cells. We demonstrate that implantation with MMV-Gel prolongs the survival and function of grafts in mouse models of allogeneic pancreatic islet cells and skin transplantation.

Mangiferin, A Naturally Occurring Glucosylxanthone, Induces Apoptosis in Caco-2 Cells In Vitro and Exerts Protective Effects on Acetic Acid-Induced Ulcerative Colitis in Mice through the Regulation of NLRP3.

Inflammatory bowel diseases (IBD), an inflammatory disease, include Crohn's disease and ulcerative colitis. Dysregulated autoimmune response to gut dysbiosis is mainly involved in the pathogenesis of IBD and is triggered by various inciting environmental factors. With its rising prevalence in every continent, IBD has evolved into a global disease, which is on the rise, affecting people of all ages. There is a growing incidence of IBD in the elderly population, as evidenced by epidemiological data. IBD is characterized by an inflammatory process that requires a lifelong treatment. The main challenge in IBD management is the adverse side effects associated with almost all of the currently available drugs. Hence, there is a search for drugs with more efficacy and fewer side effects. Natural products with great structural diversity and ease of modification chemically are being explored, as they were shown to control IBD by safely suppressing pro-inflammatory pathways. The present study aims at understanding the role of mangiferin, a COX-2 inhibitor isolated from tubers of Pueraria tuberosa in the treatment of IBD and colon cancer, in vitro on the Caco-2 human colon cancer cell line and in vivo in the acetic acid-induced IBD mouse model. In the acetic acid-induced colitis model, it prevented the decrease in length of the colon, mucosal erosion, and cellular infiltration in a dose-dependent manner. The expression levels of various pro-inflammatory markers like COX-2, IL1β, TNF-α, INF-γ, IL-6, NLRP3, and caspase-1 were downregulated in an acetic acid-induced mouse model on treatment with mangiferin in a dose dependent manner. Mangiferin also showed anticancer effects on Caco-2 cells by increasing the expression of Fas ligand, Fas receptor, FADD, caspase-8, and caspase-3 proteins, whereas Bid and Bcl-2 proteins showed decreased expression. These data suggest that mangiferin, an inhibitor of COX, induces apoptosis in colon cancer cells in vitro and protects mice from acetic acid-induced colitis in vivo.

Platelet-derived TGF-β1 induces functional reprogramming of myeloid-derived suppressor cells in immune thrombocytopenia

AbstractPlatelet α-granules are rich in transforming growth factor β1 (TGF-β1), which is associated with myeloid-derived suppressor cell (MDSC) biology. Responders to thrombopoietin receptor agonists (TPO-RAs) revealed a parallel increase in the number of both platelets and MDSCs. Here, anti-CD61 immune-sensitized splenocytes were transferred into severe combined immunodeficient mice to establish an active murine model of immune thrombocytopenia (ITP). Subsequently, we demonstrated that TPO-RAs augmented the inhibitory activities of MDSCs by arresting plasma cells differentiation, reducing Fas ligand expression on cytotoxic T cells, and rebalancing T-cell subsets. Mechanistically, transcriptome analysis confirmed the participation of TGF-β/Smad pathways in TPO-RA–corrected MDSCs, which was offset by Smad2/3 knockdown. In platelet TGF-β1–deficient mice, TPO-RA-induced amplification and enhanced suppressive capacity of MDSCs was waived. Furthermore, our retrospective data revealed that patients with ITP achieving complete platelet response showed superior long-term outcomes compared with those who only reach partial response. In conclusion, we demonstrate that platelet TGF-β1 induces the expansion and functional reprogramming of MDSCs via the TGF-β/Smad pathway. These data indicate that platelet recovery not only serves as an end point of treatment response but also paves the way for immune homeostasis in immune-mediated thrombocytopenia.

Abstract 1605: Oncolytic hsv-1(vc2) gm-csf regulates the proinflammatory profile suggestive of an immunomodulatory effect in PDAC cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease that is projected to be the second leading cause of cancer-associated deaths by 2030. Owing to its intrinsic immune evasive tumor microenvironment (TME), PDAC is regarded as an immunologically “cold” tumor with a significant therapeutic challenge. Although 5-fluorouracil or gemcitabine-based chemotherapy is considered as standard of care, harnessing immunological pathways may potentially convert PDAC to a “hot” tumor, thereby providing survival advantage to patients. Lately, the use of oncolytic virus has proven to be an alternative strategy to activate the innate immune response within the TME coupled with its cytotoxic effect against the cancer cells. The FDA approval of T-VEC for the treatment of stage IIIB/IV melanoma validates the preceding argument. In this study, we have investigated the effect of herpes simplex virus type-1 (HSV-1) (VC2) overexpressing granulocyte-macrophage colony-stimulating factor (GMCSF), HSV-1VC2GM-CSF in the murine KRAS, p53, and Cre (KPC), and human MIA PaCa-2 pancreatic carcinoma cells. Following a 24h infection of the KPC and MIA PaCa-2 cells with the HSV-1VC2GM-CSF, the observed cytopathic effects (CPE) included cell rounding, detachment, and aggregation as compared to the wild-type HSV-1VC2 treated or the uninfected control cells. The CPE effect of HSV-1VC2GM-CSF was proven to be a more direct and dynamic response against these cells. In the next steps, the viral titers were determined to be 9.6E7 pfu/ml, 1.26E7 pfu/ml for HSV-1VC2 and VC2GM-CSF respectively. The infection of VC2GM-CSF 0.1, 1.0 and 10 MOI in KPC cells resulted in a significant increase in the expression of proinflammatory markers including interleukin 10 (IL-10), cyclooxygenase -2 (COX-2) and iNOS at the gene level in contrast to the uninfected controls. This was coupled with an increase in the expression of Fas ligand (FasL) in the treated cells. Intriguingly, the treated cells showed a significant upregulation of programmed death ligand (PD-L1). Interestingly, in a 3D-coculture of KPC and RAW264.7 cells the expression of PD-L1 was significantly upregulated. Based on the evidence presented here, it is speculated that VC2GM-CSF-induced proinflammatory profile in KPC and Mia PaCa-2 cells converts to an immunomodulatory one characterized by the regulation of FasL and PD-L1. The effect of VC2GM-CSF in a PDAC organoid model to understand the interplay between the tumor and the stromal cells in response to the virus is currently underway. Citation Format: Naveen Chintala Ramulu, Shilpa Thota, Brent Stanfield, Konstantin Kousoulas, Joseph Francis. Oncolytic hsv-1(vc2) gm-csf regulates the proinflammatory profile suggestive of an immunomodulatory effect in PDAC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1605.

Abstract 40: Persistence but not antitumor efficacy of CAR-engineered lymphocytes is governed by a FAS/FAS ligand auto-regulatory circuit

Abstract Adoptive cell transfer of chimeric antigen receptor (CAR) engineered T and NK cells has revolutionized the treatment of many B-lymphoid malignancies; however, limited persistence of infused lymphocytes can restrain the full potential of this approach. Apoptosis induction through the FAS ligand/FAS pathway is a key negative regulator of lymphocyte survival. To define which cellular subsets express FASLG, the gene encoding FAS ligand, we generated a single-cell transcriptomic atlas from 37 patients with diverse cancer types. We discovered that FASLG expression is highly constrained and limited primarily to T and NK cells while stromal and tumor cells express minimal to no FASLG. Multiplex RNA in situ hybridization of clinical samples from patients treated with anti-CD19 CAR-T cells confirmed these results and revealed significantly higher FASLG content in CAR-expressing compared with non-CAR expressing T cells. Based on these findings, we hypothesized that FAS ligand expression by CAR+ T and NK cells will engage FAS+CAR+ cells to induce apoptosis and limit cellular persistence. To test this hypothesis, we performed competitive fitness assays using two phenotypically discernable populations of human CAR-T and CAK-NK cells that are either responsive or unresponsive to FAS-signaling through expression of a FAS dominant negative receptor (ΔFAS). ΔFAS+ and ΔFAS− CAR-expressing cells were co-infused 1:1 into tumor bearing hosts and persistence of each population was quantified. For both CAR-T and CAR-NK, we measured significant enrichment in ΔFAS-expressing cells across multiple tissues, demonstrating that FAS-signaling regulates CAR+ cell survival in a cell-intrinsic manner. Knock out (KO) of FASLG using CRISPR/Cas9 prevented population skewing while KO of the AAVS1 control locus had no effect, indicating that CAR-derived FASLG is sufficient to restrain the persistence of transferred cells. By contrast, we discovered that FASLG was dispensable for CAR-T and CAR-NK mediated tumor killing both in vitro and in vivo. Finally, using two different tumor models, we found that adoptive transfer of CAR-T or CAR-NK cells that co-express ΔFAS resulted in superior antitumor efficacy compared with control cells that express the CAR alone. Taken together, these findings reveal that CAR-engineered lymphocyte persistence is governed by a FAS/FAS ligand auto-regulatory circuit. Further, they indicate that the therapeutic benefit of a FAS-dominant negative receptor is broadly applicable and agnostic of whether FASLG is expressed by cells in the tumor microenvironment. Citation Format: Fei Yi, Tal Cohen, Paul Zumbo, Razan Eltilib, Hannah Arkin, Michael Gormally, Christopher S. Hackett, Korbinian Kropp, Smita Chandran, Jae H. Park, Doron Betel, Christopher A. Klebanoff. Persistence but not antitumor efficacy of CAR-engineered lymphocytes is governed by a FAS/FAS ligand auto-regulatory circuit [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 40.

The Polysaccharide from Dendrobium officinale Can Improve Mice with Sjögren’s Syndrome by Regulating BAFF and Fas Expressions

Exploring herbs and their compounds for potential drugs is a significant research focus due to the incurable nature of Sjögren’s syndrome (SS), a prevalent chronic inflammatory autoimmune disease. Dendrobium officinale polysaccharide (DOP) has demonstrated diverse biological effects, particularly in immunomodulation. Nonetheless, limited research has been conducted on DOP concerning the treatment of SS. This research aims to examine the impacts of DOP on experimental SS (ESS) mice and its possible mechanisms. An ESS animal model was established using the immune induction method. Besides the control group, ESS mice were randomly assigned to the model (distilled water), the HCQ (60 mg/kg), the DOP (43 mg/kg), the DOP (130 mg/kg), and the DOP (390 mg/kg) groups. Drugs were treated for three weeks. The findings indicated that DOP enhanced saliva production of ESS mice and diminished lymphocyte infiltration and foci in the submandibular gland (SMG). DOP also significantly downregulated the mRNA content of proinflammatory factors (TNF-α, IL-1β, and IL-17) and the expression of genes and proteins of apoptotic factors (Bax and Caspase-3). Furthermore, DOP downregulated the BAFF and BAFF receptor (BAFFR) as well as Fas and Fas ligand (Fas-L) expressions in the SMG. Our study demonstrates that DOP may reduce inflammation and apoptosis by inhibiting BAFF/BAFFR and Fas/Fas-L signaling, thus producing therapeutic effects in ESS mice.

Plasma Immune Proteins and Circulating Tumor DNA Predict the Clinical Outcome for Non-Small-Cell Lung Cancer Treated with an Immune Checkpoint Inhibitor

Immunotherapy has altered the therapeutic landscape for patients with non-small-cell lung cancer (NSCLC). The immune checkpoint inhibitor pembrolizumab targets the PD-1/PD-L1 signaling axis and produces durable clinical responses, but reliable biomarkers are lacking. Using 115 plasma samples from 42 pembrolizumab-treated patients with NSCLC, we were able to identify predictive biomarkers. In the plasma samples, we quantified the level of 92 proteins using the Olink proximity extension assay and circulating tumor DNA (ctDNA) using targeted next-generation sequencing. Patients with an above-median progression-free survival (PFS) had significantly higher expressions of Fas ligand (FASLG) and inducible T-cell co-stimulator ligand (ICOSLG) at baseline than patients with a PFS below the median. A Kaplan–Meier analysis demonstrated that high levels of FASLG and ICOSLG were predictive of longer PFS and overall survival (OS) (PFS: 10.83 vs. 4.49 months, OS: 27.13 vs. 18.0 months). Furthermore, we identified a subgroup with high expressions of FASLG and ICOSLG who also had no detectable ctDNA mutations after treatment initiation. This subgroup had significantly longer PFS and OS rates compared to the rest of the patients (PFS: 25.71 vs. 4.52 months, OS: 34.62 vs. 18.0 months). These findings suggest that the expressions of FASLG and ICOSLG at baseline and the absence of ctDNA mutations after the start of treatment have the potential to predict clinical outcomes.

Platelet-Derived TGF-β1 Induces Functional Reprogramming of Myeloid-Derived Suppressor Cells in Immune Thrombocytopenia

Introduction: Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by isolated thrombocytopenia and varying degrees of bleeding symptoms. Platelets are anucleate cytoplasmic cell fragments that play dual physiological roles as both cellular mediators of thrombosis and immune modulatory cells. Platelets are the largest storage pool of TGF-β1, a crucial enforcer of immune homeostasis and tolerance. Studies have shown that TGF-β1 released by platelets contributes to the pathogenesis of pulmonary arterial hypertension and cervical carcinoma. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of immature myeloid cells with potent immunosuppressive functions. Our previous studies have shown that MDSCs are impaired in number and function in patients with ITP. The objective of this study was to elucidate the underlying molecular mechanisms of MDSCs reprogramming by platelet-derived TGF-β1. Methods: A transwell assay was used to determine the impact of platelets on MDSCs induced from peripheral blood mononuclear cells (PBMCs) of ITP patients and healthy controls. The percentage of circulating MDSCs in ITP patients was detected before and after administration of thrombopoietin receptor agonists (TPO-RAs). Besides, anti-CD61 immune-sensitized splenocytes were transferred into SCID mice to establish an active murine model of ITP. Subsequently, MDSCs from TPO-RA-treated ITP mice were transplanted as cell therapy to rescue treatment-free ITP mice. In specific, adeno-associated viral (AAV) vectors efficiently infected MDSCs to enable cell-specific Smad2/3 knock-down. Finally, platelet specific TGF-β1-deficient mice were used to validate the effect of platelet-derived TGF-β1 on MDSCs. Results: TGF-β1 levels in the supernatant of MDSCs co-cultured with platelets of high concentration was significantly higher than that in MDSCs co-cultured with platelets of low concentration or alone. FACS analysis showed that platelet mass enhanced the inhibitory effects of MDSCs on the proliferation of CD4 + T cells, which could be offset by TGF-β1 inhibitor (TGF-βi). Besides, TPO-RAs treatment induced significantly increased percentage of circulating MDSCs in responders with chronic ITP. Likewise, TPO-RA significantly expanded MDSC population in active ITP murine models. Furthermore, after receiving donor MDSCs from TPO-RA-treated ITP mice, arrested differentiation of plasma cells, reduced Fas ligand expression on cytotoxic T cells, and re-balanced T cell subsets were found in the recipient mice, indicating augmented inhibitory functions of MDSCs. Mechanistically, transcriptome analysis confirmed the participation of TGF-β/Smad pathway in donor MDSCs from TPO-RA-treated ITP mice, which was offset by Smad2/3 knockdown. Finally, by constructing ITP models using Pf4 cre-Tgfb1 fl/fl and Pf4 cre+Tgfb1fl/fl mice, we found that TGF-β1 inhibition and platelet TGF-β1-deficence, both, could counteract the elevated platelet count and MDSCs percentage, as well as improved immunosuppressive capacity of MDSCs trigged by TPO-RA modulation. Conclusion: To sum up, it was demonstrated that platelet TGF-β1 induced the expansion and functional reprogramming of MDSCs via TGF-β/Smad pathway. These data indicate that platelet recovery not only serves as an endpoint of remission, but also paves the way for tolerance induction in immune-mediated thrombocytopenia. Our exploratory study deciphered immunological changes induced by TPO-RAs and helped to identify molecular mechanisms responsible for long-term remission after TPO-RA discontinuation.

CD138 promotes the accumulation and activation of autoreactive T cells in autoimmune MRL/lpr mice.

Autoreactive T cells, specifically CD138+ (syndecan-1) T cells produced in Fas-deficient systemic lupus erythematosus (SLE) mouse models, were shown to significantly promote the generation of autoantibodies. In the present study, Murphy Roths Large lymphoproliferative (MRL/lpr) lupus mice were used to investigate the role of CD138 protein expression in T cells in the progression of SLE. Measurement of flow cytometry, immunofluorescence and Luminex were performed to determine the effect of CD138 on T cells in MRL/lpr mice. The results demonstrate that CD138+ T cells induce apoptosis via a Fas-dependent pathway. CD138 protein expression in T cells of MRL/lpr mice significantly reduced T cell apoptosis and contributed to the accumulation of T cells and double negative (DN) T cells, whilst simultaneously promoting T cell activation in Fas-deficient lupus mice. CD138 protein expression in DN T cells also significantly increased the protein expression of Fas ligand to enhance the cytotoxicity of DN T cells. Furthermore, phorbol 12-myristate 13-acetate and ionomycin (PI) stimulation reduced CD138 protein expression in CD3+ T cells and prevented CD138+ T cell accumulation by inducing specific apoptosis. PI stimulation also activated T cells in MRL/lpr mice to increase CD69 protein expression. CD69 protein expression in CD138+ T cells significantly increased the frequency of apoptotic CD138+ T cells. In addition, results from the present study demonstrated that CD138- T cells of MRL/lpr lupus mice had an activation defect. CD138 protein expression in T cells significantly reversed the defective activation and activating T cells could significantly reduce CD138 protein expression in CD3+ T cells of MRL/lpr mice. This suggests that CD138 protein expression in CD3+CD138- T cells of MRL/lpr mice may be a consequence of the impaired activation in autoreactive T cells prior to exposure to self-antigens by the immune system. CD138 expression in autoreactive T cells has a central role in promoting the progression and development of autoimmune response in MRL/lpr mice.

Involvement of Innate Immune System in the Pathogenesis of Sepsis-Associated Acute Kidney Injury.

Although experimental models have shown that the innate immune system is a main contributor to acute kidney injury (AKI), its involvement in human sepsis-associated AKI (SA-AKI) remains unclear. We retrospectively evaluated 19 patients with SA-AKI who were treated with continuous renal replacement therapy (CRRT). Serum cytokine, complement components, and the proportion and functions of innate immune cells, such as CD56+ T cells, CD56+ natural killer (NK) cells, and monocytes, were analyzed. There were no differences in the proportions of CD56+ T and NK cells between patients with SA-AKI and healthy controls. In patients with SA-AKI, fas ligand (FasL) expression in CD56+ T cells was significantly upregulated, and the proportion of perforin-positive CD56+ T cells tended to be higher than that in healthy controls. The positive rate of both FasL and perforin of CD56+ T cells was significantly higher than that of CD56- T cells, which include cytotoxic T cells. Antigen-presenting capacity and phagocytic activity of monocytes in patients with SA-AKI were significantly decreased compared to those of healthy controls and did not recover soon after the initiation of CRRT. CD56+ T cells are involved in the disease processes of human SA-AKI through effector molecules such as FasL or perforin.

Caspase activation in tumour-infiltrating lymphocytes is associated with lymph node metastasis in oral squamous cell carcinoma.

Oral squamous cell carcinomas (OSCCs) are genetically heterogeneous and exhibit diverse stromal and immune microenvironments. Acquired resistance to standard chemo-, radio-, and targeted therapies remains a major hurdle in planning effective treatment modalities for OSCC patients. Since Caspase 8 (CASP8) is frequently mutated in OSCCs, we were interested to explore a potential interaction between tumour-infiltrating lymphocytes (TILs) and CASP8 activation using high-content image analysis of human tumour (n = 32) sections. Despite the lymphocyte-rich tumour microenvironment, we observed lower activation of CASP8 (0-10% of tumour area) and its downstream effector CASP3 (0-6%) in tumours than in normal oral epithelium. Conversely, we found apoptosis was high for all the lymphocyte subtypes examined (38-52% of lymphocytes within tumour islands). Tumours with higher Fas ligand (FasL) expression had a significantly higher proportion of cleaved CASP3/8 positive cytotoxic T cells within the tumour islands (p = 0.05), and this was associated with the presence of lymph node metastatic disease [odds ratio: 1.046, 95% confidence interval (1.002-1.091), p = 0.039]. Our finding of extensive activation of the extrinsic pathway of apoptosis in TILs, together with evidence of higher FasL in CASP8 mutated tumours, may be useful in predicting the course of disease in individual patients. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Abstract 3228: Photodynamic priming enhances immunotherapy responses overcoming oncogenic drivers in pancreatic patient derived tumor organoids

Abstract Photodynamic therapy (PDT) is an anti-cancer therapy that utilizes light, a photoresponsive photosensitizer (PS), and oxygen to confer direct cytotoxicity or vascular damage. PDT alters the tumor microenvironment (TME) transiently in a process termed photodynamic priming (PDP), making it more receptive to subsequent therapies. Previous studies reported that PDP exerts immune stimulatory effects via the release of damage associated molecular patterns (DAMPs) as a potent inducer of immunogenic cell death. Pancreatic cancer (PC) is a lethal human malignancy with no curable treatments available thus far. The desmoplastic stroma in PC limits the delivery of therapeutic agents contributing to treatment resistance. More, the stroma promotes an immunosuppressive TME that leads to immune exclusion, which is largely responsible for the immunotherapy failure in PC. Therefore, there is an unmet need to find more effective combination therapies against PC. In this work, we evaluated whether PDP enhances anti-PD1 therapy response by modulating immunogenicity in PC patient-derived organoids (PDOs). Visudyne (PS) and varied light doses of 25-100 J/cm2 were used for PDP treatments. Our data showed light dose dependent cytotoxicity and the expression of DAMPs including HSP60, calreticulin and HMGB1 in PDOs. Gene expression analysis of PDP treated PDOs showed increases in IFNγ, TNFα and CXCL12 while decreases in PDL1, TGFβ1 and FOXP1. Coculture of healthy donor (HD) monocyte-derived dendritic cells (mDCs) for 24 h with PDOs treated with a sublethal dose of PDP (25 J/cm2) showed increased CD40, CD86 and MHC11 activation markers on mDCs. Addition of matched HD naïve T cells 24 h post mDC-PDO coculture led to the excessive activation of CD4+ and CD8+ T cells highlighted by the PD1 expression on T cells from 0 h of initial T cell coculture to 48 h. This led us to treat PDO-mDC-T cell cocultures at 0 h to 48 h of initial T cell coculture with the anti-PD1 monoclonal antibody, Pembrolizumab. The treatment response was measured by live imaging of PDO growth followed by the analysis of changes in PDO diameter/area from day 0 of PDP to day 7 post-PDP. There were significant decreases in PDO growth in cultures treated with PDP followed by Pembrolizumab at different time points compared to untreated PDOs, untreated PDO-mDC-T cell cocultures, PDOs treated with PDP alone, PDO-mDC-T cell cocultures treated with PDP only or PDO-mDC-T cell cocultures treated with anti-PD1 alone. Also, we sorted T cells from the same cocultures at day 7 of PDP for gene expression analysis of T cell activation markers. There was enhanced expression of IFNγ, TNFα, FAS ligand, Granzyme B, IL2 and IL21 in T cells collected from combination therapy treated cocultures compared to others. Lastly, our data show that PDP can enhance anti-PD1 immunotherapy responses providing insights into a combination therapy option to treat PDAC. Citation Format: Pushpamali De Silva, Demi Wekking, Joanna Joeun Choe, Dario Missael Castellanos, Piotr Zelga, Russell Jenkins, Andrew Scott Liss, Tayyaba Hasan. Photodynamic priming enhances immunotherapy responses overcoming oncogenic drivers in pancreatic patient derived tumor organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3228.

Zonisamide improves Fas/FasL-mediated apoptosis and inflammation in a degenerative cervical myelopathy rat model

Degenerative cervical myelopathy (DCM) is a severe condition of the spinal cord caused by chronic compression. However, no studies to date have examined the effects of zonisamide (ZNS) on DCM via the Fas/FasL-mediated pathway. The aim of this study was to investigate the effects of ZNS on a DCM rat model and to explore the potential mechanisms. First, 40 adult Sprague–Dawley rats were used to establish the DCM rat model and were individually divided into four groups: the Sham group, DCM model group (DCM), ZNS group (DCM model rats treated with ZNS, 30 mg/kg/day), and ZNS + CD95 group (DCM model rats treated with ZNS and CD95). Histopathology injury and cell apoptosis, Fas and Fas ligand (FasL) expression and Fas/FasL relative protein levels were detected by hematoxylin and eosin staining, TUNEL assay, and immunofluorescence and western blotting, respectively. The results of our study demonstrated that ZNS could promote motor recovery while reversing histopathological injury and cell apoptosis in DCM rats. Moreover, Iba-1, Fas and FasL expression in DCM rats was decreased, accompanied by a decrease in cleaved caspase-3/caspase-3, cleaved caspase-8/caspase-8, cleaved caspase-9/caspase-9, cleaved caspase-10/caspase-10 and B-cell lymphoma-2 (Bcl-2)/Bcl-2 associated X (Bax) levels. All these results revealed that ZNS attenuates DCM injury in a rat model via the regulation of Fas and FasL signaling. Our study indicated that ZNS had beneficial effects on DCM and thus provided a novel theoretical approach for subsequent academic and clinical research on DCM injury.

Platelets Bind Senescent Red Blood Cells and Mediate Their Clearance from the Circulation

The bloodstream of mice and humans contain consistent proportions of red blood cells (RBC) which are bound to platelets (1-2% and 0.1-0.3%, respectively). This study identifies senescent RBC as the main constituent of platelet-RBC complexes (Plt-RBC) and their importance in RBC clearance. RBC have a defined circulation lifespan and upon reaching senescence are eliminated by phagocytes, thereby ensuring homeostasis and preventing accumulation of dead cells and toxic hemolytic byproducts. Senescent RBC are characterized by cellular and molecular changes, including reduced deformability, externalization of membrane phosphatidylserine (PS), and altered expression of membrane proteins such Fas ligand (CD95; upregulated) and integrin associated protein (CD47; downregulated), which act as pro-phagocytic signals. Similar changes to deformability and membrane composition occur in RBC compromised by sickle cell disease or Plasmodium infection. These pathologic cells also display a 10x higher platelet-binding capacity which is related to disease pathogenesis. Thus, we investigated how platelets may interact with senescent RBC and the purpose of Plt-RBC. Platelet binding to senescent RBC was studied using mice pulse-labelled with biotin-NHS, which enabled tracking of RBC age and clearance over time. The biotin+ RBC remaining for longer times represented older cells; the oldest detected were aged ≥58 days consistent with the reported maximal RBC lifespan in mice. During the first 45 days of observation Plt-RBC were similar in biotin+ versus newly replenished biotin- RBC (1.6-2.2%), but were significantly higher in biotin+ cells detected at 52 and 58 days (average ± SD: 6.7 ± 5.1% and 15 ± 8.1%, respectively; p < 0.001). These observations were replicated when biotin-NHS was replaced with CFSE. The aged RBC and Plt-RBC also contained significantly increased PS and CD95 compared to the replenished and non-complexed cells, although CD47 levels were not different. Flow cytometry imaging revealed Plt-RBC were composed of single RBC bound by 1-2 platelets. Collectively this indicated that platelets preferentially bind senescent RBC. To investigate if platelet binding affected senescent RBC clearance, platelet-depleting antibodies (anti-GPIbα or anti-GPIIb/IIIa) were given to temporarily reduce Plt-RBC when biotin+ Plt-RBC levels first began to diverge (Day 46-49 in multiple experiments). The treatment caused a maximal 10-fold reduction in Plt-RBC levels sustained for 2-3 days, and a marked retention of biotin+ RBC which coincided with Plt-RBC reductions. Consistent with these results, the clearance of transfused RBC in platelet-depleted mice was also significantly inhibited compared to control mice. Erythrophagocytes in the spleen, but not the liver, from the platelet-depleted mice also contained fewer transfused cells, and erythrophagocytes containing platelets were observed using flow cytometry and microscopy. Thus, platelets are important for the clearance and/or phagocytosis of both senescent and transfused RBC. We also studied the Plt-RBC clearance mechanism by transfusing ex vivo generated and labelled Plt-RBC and RBC. The Plt-RBC were cleared at least 5x faster than RBC (p=0.0014) suggesting that once formed, their lifespan in the circulation is transient. Similar transfusions in mice depleted of phagocytes (using clodronate liposomes) impeded Plt-RBC clearance, indicating a phagocyte-dependent pathway for their removal. An additional mechanistic insight, Plt-RBC displayed an increased retention in a microsphiltration assay system, indicating platelet binding may hinder the passage of cells through the microcirculation. Our studies have thus revealed an unexpected contribution by platelets in senescent RBC clearance. In our proposed model (Figure 1) senescent RBC have an enhanced ability to bind platelets, possibly due to upregulated PS and CD95, which accelerates their removal via splenic retention and phagocytosis. Platelets may also present additional pro-phagocytic signals to stimulate erythrophagocytosis. A normal platelet count provides surplus platelets to maintain RBC clearance, but in conditions of acute platelet loss, reduction of plt-RBC may cause accumulation of senescent RBC to pathological levels. Conversely, conditions where more RBC bind platelets such as in malarial infection will increase plt-RBC and result in thrombocytopenia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Amiodarone-drove XBP1s aggravates endoplasmic reticulum stress and apoptosis in Hashimoto’s thyroiditis through regulating LINC00842/miR-214/FASL axis

Hashimoto’s thyroiditis (HT) is an organ-specific autoimmune disease, that eventually lead to hypothyroidism. XBP1s is an endoplasmic reticulum stress related protein and participates in the pathogenesis of several diseases. Nevertheless, the potential role of XBP1s in amiodarone (AMIO)-treated HT patients remains unknown. In this study, AMIO aggravated the endoplasmic reticulum stress responses in HT patients and thyroid epithelial follicular cells. Moreover, MTT assay and flow cytometry analysis revealed that knockdown of XBP1s suppressed AMIO-induced thyroid epithelial follicular cells apoptosis. Mechanically, the Chromatin Immunoprecipitation (ChIP) and luciferase activity assay proved that XBP1s enhanced LINC00842 expression in HT patients and thyroid epithelial follicular cells via binding to LINC00842 promoter. LINC00842 functioned as a miR-214 sponge in HT patients and thyroid epithelial follicular cells. Besides, LINC00842 up-regulated Fas ligand (FASL) expression via inhibition of miR-214. In rescue experiments, overexpression of FASL reversed shXBP1s-induced suppression of cell apoptosis in AMIO-treated thyroid epithelial follicular cells. These findings concluded that AMIO-drove XBP1s aggravated endoplasmic reticulum stress and apoptosis in HT via modulating LINC00842/miR-214/FASL axis, providing a new sight for the therapeutic strategy of AMIO-induced HT.

Interleukin-18 serum level before and after intralesional immunotherapy with tuberculin purified protein derivative in patients with cutaneous and genital warts.

Several studies demonstrated the efficacy of intralesional purified protein derivative (PPD) immunotherapy in warts eradication. Nevertheless, the precise induced immune mechanisms are undetermined. Injected PPD is hypothesized to induce a delayed hypersensitivity reaction associated with cytokines release. Interleukin (IL)-18 has a major role in defense against viral infection via inducing interferon-γ release from T-helper 1 and natural killer (NK) cells. Moreover, IL-18 triggers Fas ligand expression on cytotoxic T cells and NK cells enhancing their cytotoxicity against virally infected cells. The aim of this study was to assess the role of IL-18 in the response to intralesional PPD injection in patients with warts. The study included 25 patients with warts and 25 HCs. Patients underwent PPD skin test, and only patients with positive tests were included and received intralesional PPD injections starting 72 h after the test then every 2 weeks until wart clearance or a maximum of 3 sessions. Serum IL-18 level was measured via enzyme-linked immune-sorbent assay in patients (pre-treatment and 2 weeks after the last injection) and HCs. After 3 sessions of injection, six (24%) patients were designated responders, nine (36%) patients showed partial response, and 10 (40%) patients were designated non-responders. Serum IL-18 level, post-treatment, was significantly higher than pre-treatment level (p =0.025) and level in HCs (p =0.036). Furthermore, the post-treatment level was significantly higher in responders than non-responders (p= 0.025). IL-18 is probably implicated in the immune mechanisms induced by PPD injection that cause eradication of warts.

Potential cardioprotective effects of Amentoflavone in doxorubicin-induced cardiotoxicity in mice

Doxorubicin (DOX) is an available chemotherapeutic drug for treating various tumors. However, its effectiveness is limited by cardiotoxicity. Amentoflavone (AMF), a natural biflavonoid separated from Cycas thouarsii ethyl acetate fraction, displays promising anticancer, anti-inflammatory, and antioxidant effects. Thus, our research aims to explore whether AMF could boost cardioprotective effects against DOX cardiotoxicity and reveal the potential underlying mechanisms of cardioprotection. Mice were classified into four groups; Normal control, Untreated DOX group, and DOX groups treated with AMF (40 and 80 mg/kg, respectively) intraperitoneal injection daily for four days before doxorubicin administration and for additional three days following DOX administration to assess cardiotoxicity. Echocardiography showed that AMF 80 treated group was protected from DOX cardiotoxicity. Additionally, it alleviated histopathological structural alterations and effectively restored heart weight and body weight ratio. These effects were confirmed biochemically by a substantially reduced serum creatine kinase-MB (CK-MB) and aspartate aminotransferase (AST) levels. AMF effectively restored nuclear respiratory factor-1(NRF-1), mitochondrial transcription factor A (TFAM), and normalized heat shock protein − 27(HSP-27) expression levels compared to the DOX group. Moreover, AMF mitigated oxidative stress conditions and significantly suppressed NADPH oxidase (NOX) expression levels. It also showed significant anti-inflammatory effects via suppressing interleukin-6 (IL-6) expression and decreasing nuclear factor Kabba B (NF-κb) immune-staining. In addition, AMF markedly reduced FAS ligand (FASL) expression and p53 immune staining in cardiac tissue. This study is the first for the in vivo potential beneficial effects of AMF against acute DOX cardiotoxicity, possibly via exerting antioxidant, anti-inflammatory, and anti-apoptotic effects and restoring mitochondrial function.