Spleen Cells Research Articles

Tailoring ZnO nanoparticles for biomedical applications: Surface functionalization of ZnO nanoparticles with 2-oxo-2H-chromene-3-carboxylic acid coupled beta-cyclodextrin for enhanced antimicrobial and anticancer effect

Nanomedicine has newly emerged as a superior alternative for the treatment of numerous life-threatening illnesses. Zinc oxide nanomaterials have attracted a lot of attention in this decade because of their enormous potential in a wide range of biomedical applications. This work describes the surface functionalization of zinc oxide nanoparticles (ZnO NPs) with 2-oxo-2H-chromene-3-carboxylic acid (3-CCA) coupled beta-cyclodextrin (β-CD) to create a biocompatible system (ZnO-β-CD-3-CCA) and the evaluation of its antimicrobial and anticancer properties. Different characterization techniques, such as X-ray powder diffraction, UV–Vis spectroscopy, FTIR, transmission, and scanning electron microscopy, were used to characterize the biocompatible system. The antimicrobial properties of the synthesized ZnO NPs and the surface functionalized ZnO-β-CD-3-CCA system were evaluated using the disc diffusion method. Modification with functionalized β-CD systems increased the bacteriostatic effect of ZnO nanoparticles against various microbes. In vitro cytotoxicity of ZnO NPs and surface functionalized ZnO-β-CD-3-CCA system was evaluated against two distinct cancer cell lines: MCF-7, Hep G2, and normal rat spleen cells employing MTT assay, and the results highlight that the selective cytotoxicity of the surface functionalized ZnO-β-CD-3-CCA system against the human breast cancer (MCF-7) and human liver cancer (Hep G2) cell lines were superior to that of ZnO NPs. Also, the developed systems were less toxic to normal rat spleen cells. This work is significant because it could result in next-generation nanoparticle-based cancer treatments that are more successful.

The Acetic Acid Produced by Lactobacillus Species Regulates Immune Function to Alleviate PEDV Infection in Piglets.

Porcine epidemic diarrhea virus (PEDV) infection results in significant mortality among newborn piglets, leading to substantial economic setbacks in the pig industry. Short-chain fatty acids (SCFA), the metabolites of intestinal probiotics, play pivotal roles in modulating intestinal function, enhancing the intestinal barrier, and bolstering immune responses through diverse mechanisms. The protective potential of Lactobacillus delbrueckii, Lactobacillus johnsonii, and Lactococcus lactis was first noted when administered to PEDV-infected piglets. Histological evaluations, combined with immunofluorescence studies, indicated that piglets receiving L. lactis displayed less intestinal damage, with diminished epithelial cell necrosis and milder injury levels. Differences in immunofluorescence intensity revealed a significant disparity in antigen content between the L. lactis and PEDV groups, suggesting that L. lactis might suppress PEDV replication, the intestine. We then assessed short-chain fatty acid content through targeted metabolomics, finding that acetate levels markedly varied from other groups. This protective impact was confirmed by administering acetate to PEDV-infected piglets. Data suggested that piglets receiving acetate exhibited resistance to PEDV. Flow cytometry analyses were conducted to evaluate the expression of innate and adaptive immune cells in piglets. Sodium acetate appeared to bolster innate immune defenses against PEDV, marked by elevated NK cell and macrophage counts in mesenteric lymph nodes, along with increased NK cells in the spleen and macrophages in the bloodstream. Acetic acid was also found to enhance the populations of CD8+ IFN-γ T cells in the blood, spleen, and mesenteric lymph, CD4+ IFN-γ T cells in mesenteric lymph nodes and spleen, and CD4+ IL-4+T cells in the bloodstream. Transcriptome analyses were carried out on the jejunal mucosa from piglets with PEDV-induced intestinal damage and from healthy counterparts with intact barriers. Through bioinformatics analysis, we pinpointed 189 significantly upregulated genes and 333 downregulated ones, with the PI3K-AKT, ECM-receptor interaction, and pancreatic secretion pathways being notably enriched. This transcriptomic evidence was further corroborated by western blot and qPCR. Short-chain fatty acids (SCFA) were found to modulate G protein-coupled receptor 41 (GPR41) and 43 (GPR43) in porcine intestinal epithelial cells (IPEC-J2). Post-acetic acid exposure, there was a notable upsurge in the ZO-1 barrier protein expression in IPEC-J2 compared to the unexposed control group (WT), while GPR43 knockdown inversely affected ZO-1 expression. Acetic acid amplified the concentrations of phosphorylated PI3K and AKT pivotal components of the PI3K/AKT pathway. Concurrently, the co-administration of AKT agonist SC79 and PI3K inhibitor LY294002 revealed acetic acid's role in augmenting ZO-1 expression via the P13K/AKT signaling pathway. This study demonstrates that acetic acid produced by Lactobacillus strains regulates intestinal barrier and immune functions to alleviate PEDV infection. These findings provide valuable insights for mitigating the impact of PEDV in the pig industry.

MyD88 exacerbates inflammation-induced bone loss by modulating dynamic equilibrium between Th17/Treg cells and subgingival microbiota dysbiosis.

This study aimed to investigate the contribution of myeloid differentiation primary-response gene 88 (MyD88) on the differentiation of T helper type 17 (Th17) and regulatory T (Treg) cells and the emerging subgingival microbiota dysbiosis in Porphyromonas gingivalis-induced experimental periodontitis. Alveolar bone loss, infiltrated inflammatory cells, immunostained cells for tartrate-resistant acid phosphatase (TRAP), the receptor activator of nuclear factor-kB ligand (RANKL), and osteoprotegerin (OPG) were quantified by microcomputerized tomography and histological staining between age- and sex-matched homozygous littermates (wild-type [WT, Myd88+/+] and Myd88-/- on C57BL/6 background). The frequencies of Th17 and Treg cells in cervical lymph nodes (CLNs) and spleen were determined by flow cytometry. Cytokine expression in gingival tissues, CLNs, and spleens were studied by quantitative polymerase chain reaction (qPCR). Analysis of the composition of the subgingival microbiome and functional annotation of prokaryotic taxa (FAPROTAX) analysis were performed. P. gingivalis-infected Myd88-/- mice showed alleviated bone loss, TRAP+ osteoclasts, and RANKL/OPG ratio compared to WT mice. A significantly higher percentage of Foxp3+CD4+ T cells in infected Myd88-/- CLNs and a higher frequency of RORγt+CD4+ T cells in infected WT mice was noted. Increased IL-10 and IL-17a expressions in gingival tissue at D14-D28 then declined in WT mice, whereas an opposite pattern was observed in Myd88-/- mice. The Myd88-/- mice exhibited characteristic increases in gram-positive species and species having probiotic properties, while gram-negative, anaerobic species were noted in WT mice. FAPROTAX analysis revealed increased aerobic chemoheterotrophy in Myd88-/- mice, whereas anaerobic chemoheterotrophy was noted in WT mice after P. gingivalis infection. MyD88 plays an important role in inflammation-induced bone loss by modulating the dynamic equilibrium between Th17/Treg cells and dysbiosis in P. gingivalis-induced experimental periodontitis.

Ferroptosis and iron mineralization involved in the death and survival of orange‐spotted groupers challenged with Pseudomonas plecoglossicida

AbstractPseudomonas plecoglossicida (P. plecoglossicida) is a pathogen in aquaculture that causes considerable economic loss. According to artificial infection experiments, the fish were classified into control group, moribund group, and survival group. Compared to the control group, both the moribund group and the survival group of fish had fewer red blood cells (RBCs) and lower oxygen saturation (SaO2). Furthermore, the fish in the survival group has more RBCs and SaO2 compared to the moribund group. The concentrations of total iron, ferrous iron, ferric iron, and mineralized iron in the fish spleen of the moribund and survival groups were lower compared to those of the control group. Additionally, the concentrations of these iron components in the fish spleen of the survival group were higher than those of the moribund group. The results demonstrated that iron mineralization is involved in the survival of fish challenged with P. plecoglossicida. Compared to the control and survival groups, the fish spleen had several distinguishing features in the moribund group, including less reduced glutathione (GSH), higher mitochondrial complex V activity, more lipid peroxidation, and reactive oxygen species, as well as reduced glutathione peroxidase 4 (gpx4) expression. Moreover, there were intact cell membranes, a normal nucleus size, no chromatin concentration, and disappearance of cristae in the mitochondria of the spleens of the moribund group. The characteristics of spleen cells in the moribund group were consistent with ferroptosis, suggesting that ferroptosis was involved in the death of fish challenged with P. plecoglossicida.

Development of a colloidal gold immunochromatographic strip for rapid detection of avian coronavirus infectious bronchitis virus

Avian infectious bronchitis virus (IBV) still causes serious economic losses in the poultry industry. Currently, there are multiple prevalent genotypes and serotypes of IBVs. It is imperative to develop a new diagnosis method that is fast, sensitive, specific, simple, and broad-spectrum. A monoclonal hybridoma cell, N2D5, against the IBV N protein was obtained after fusion of myeloma SP2/0 cells with spleen cells isolated from the immunized Balb/c mice. The N2D5 monoclonal antibody (mAb) and the previously prepared mouse polyclonal antibody against the IBV N protein were used to target IBV as a colloidal gold–mAb conjugate and a captured antibody, respectively, in order to develop an immunochromatographic strip. The optimal pH and minimum antibody concentration in the reaction system for colloidal gold–mAb N2D5 conjugation were pH 6.5 and 30 μg/mL, respectively. Common avian pathogens were tested to evaluate the specificity of the strip and no cross-reaction was observed. The sensitivity of the strip for detecting IBV was 10−1.4522 EID50/mL. The strip showed a broad-spectrum cross-reactive capacity for detecting IBV antigens, including multiple IBV genotypes in China and all of the seven serotypes of IBV that are currently prevalent in southern China. Additionally, the result can be observed within 2 min without any equipment. The throat and cloacal swab samples of chickens that were artificially infected with three IBV strains were tested using the developed strip and the qPCR method; the strip test demonstrated a high consistency in detecting IBV via qPCR gene detection. In conclusion, the immunochromatographic strip that was established is rapid, sensitive, specific, simple, practical, and broad-spectrum; additionally, it has the potential to serve as an on-site rapid detection method of IBV and can facilitate the surveillance and control of the disease, especially in resource-limited areas.

Abstract 2604: Evaluating the potency of antibody-drug conjugate (ADC) free-drugs on T cells

Abstract Antibody-drug conjugates (ADCs) combine the targeting specificity of antibodies with powerful cytotoxic payloads to direct antigen-specific tumor cell killing. Multiple ADCs have shown single-agent clinical activity against solid and hematological cancers and are currently being evaluated in combination with immune checkpoint inhibitors (CPIs). Therapeutic benefit from CPIs relies on the presence and quality of responding antitumor T cells, both in the tumor and from new T cell priming and expansion outside the tumor. Select cytotoxic ADC payloads, like monomethyl auristatin E (MMAE), have the potential to amplify T cell responses through immunogenic cell death (ICD) and, as such, may be optimal combination partners with CPIs. However, ADC payloads released in the tumor (bystander drug) or released systemically also have the potential to negatively impact T cell viability, proliferation, and priming. The compatibility of different ADC payload chemotypes (Auristatins, camptothecins (CPTs), Pyrrolobenzodiazepine (PBDs), Tubulysins) with CPIs may depend on both the direct and indirect effects of payloads and their associated free-drugs on immune cells. Here, we determined the relative potencies of ADC payload free-drugs MMAE and DXd on activated human and murine T cells in vitro and in a model of xeno-graft versus host disease (xeno-GVHD) to evaluate the impact of ADC bystander effects on human immune cell expansion and function in vivo. In vitro, ADC payloads showed a range of potencies on activated T cells, with IC50 values skewing lower for CD4 compared to CD8 T cells. For each payload, murine T cells were significantly less sensitive than human T cells, which may have implications for modeling combinations of ADCs and CPIs. Mouse CD8 T cells were ~10-fold less sensitive to MMAE and 20-25-fold less sensitive to clinical CPT payloads compared to human T cells. In an in vivo model of human immune cell activation, xeno-GVHD, bystander effects from a non-targeted MMAE-ADC did not impair the expansion of human immune cells, however, a non-targeted DXd-ADC reduced CD4 T cells and robustly reduced populations of non-T cells in spleens. Consistent with reduced immune cell expansion, the DXd-ADC significantly slowed the xeno-GVHD disease course compared to the MMAE-ADC treated and control groups. These data suggest that the most commonly used ADC payload classes, MMAE and DXd, may have different direct effects on proliferating human immune cells as bystander free-drugs, which may have clinical relevance for combinations of ADCs with CPIs. Continued research exploring the balance between ADC potency on tumor cells, direct and indirect effects of payloads on immune cells, and dose timing and sequencing could help maximize the potential of ADCs in combination with immunotherapies. Citation Format: Reice D. James, Michelle L. Ulrich, J Hartsuyker, Alyson J. Smith, Ryan A. Heiser. Evaluating the potency of antibody-drug conjugate (ADC) free-drugs on T 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 2604.

Abstract 5629: MEF2D regulates T cell function via the CD70-CD27 signaling axis and promotes immune escape in hepatocellular carcinomas

Abstract Background: Immune escape remains a challenge in the treatment of hepatocellular carcinoma (HCC), one of the most common and deadly cancers in the world. The transcription factor, myocyte enhancer factor 2D (MEF2D), is important in the regulation of differentiation and adaptive responses in many cancers, such that we tested its role in HCC. Methods: We knocked down MEF2D in HCC cell lines and analyzed HCC tissues and cell lines by RNA sequencing, Western blot and immunohistochemistry. MEF2D protein acetylation and proteins that interact with MEF2D were identified by coimmunoprecipitation assay. Chromatin immunoprecipitation was used to analyze the regulation of CD70 transcription by MEF2D. H22 cells, with MEF2D knockout or without (controls), were injected into the livers of syngeneic BALB/c mice. Flow cytometry was used to analyze the function of T cells in tumors, spleens, and lymph nodes. Results: Through database searches, we noted that MEF2D and CD70 were upregulated in HCC and associated with shorter patient survival times. Compared to WT tumors, injection of MEF2D-knockdown HCC cells into immunocompetent mice led to smaller tumors with increased T cell infiltration, activation, and impaired T-regulatory (Treg) cell suppressive function. Mechanistically, we found that MEF2D binds to the promoter region of CD70 gene and activates its transcription. Moreover, acetylation of MEF2D enhances the binding of MEF2D to the CD70 promoter and further promotes its transcription. CD70 blocking antibody inhibited activation of the CD70-CD27 signaling axis in murine HCC tumors, leading to impaired immunosuppressive function of Tregs and enhanced T cell-mediated anti-tumor immune responses. Thus, regulation of the CD70-CD27 signaling axis by MEF2D affects the numbers and functions of Treg cells and thereby controls T cell infiltration. Conclusions: MEF2D in HCC cells increases the expression of CD70 and blocks T cell-mediated anti-tumor immunity via the CD70-CD27 signaling axis. Strategies to manipulate this pathway might increase the efficacy of immune therapies for HCC. Citation Format: Fanhua Kong, Liqing Wang, Qifa Ye, Yan Xiong, Wayne W. Hancock. MEF2D regulates T cell function via the CD70-CD27 signaling axis and promotes immune escape in hepatocellular carcinomas [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 5629.

Abstract 2630: Identifying most promising combination therapies for immune suppressive breast cancer using a combined in vitro/in vivo approach

Abstract Combining epigenetic therapy with immunotherapy is a promising strategy to address the complexities of cancer biology. However, it's important to note that the field is still evolving, and more research is needed to understand the optimal combinations, timing, and patient populations that will benefit the most from these approaches. We evaluated a combination of two different checkpoint inhibitor therapies (anti-CTLA4 and anti-PD1) with two different hypomethylating agents (5-azacytidine, AZA and Decitabine, DEC) in vitro and in vivo using a mouse breast cancer cell line 4T1 known to be resistant towards checkpoint inhibition (CPi). Beside tumor cell killing and tumor volume measurement, cytokine expression and immune cell phenotyping was determined. In a co-culture assay with autologous T cells and 4T1 cells, antitumor efficacy weas increased in all combination arms using AZA but not in combination with DEC. The combination of AZA with anti-CTLA4 was significantly better than the respective monotherapies (oneway ANOVA, p< 0.001). This effect was observed as well in the combination with anti-PD1 and AZA, however it was less pronounced (oneway ANOVA, p< 0.01). Of note, AZA restored the CTLA4 expression on CD8+ T cells in vitro, which was reduced by co-culture with tumor cells alone. In the follow-up in vivo experiment we focus on the combination of both CPi with AZA on different treatment schedules in the combination arms. The combination of anti-CTLA4 and AZA in a staggered treatment schedule displayed significant efficacy compared to control (oneway ANOVA, p< 0.05). The implantation of 4T1 reduced the number of T cells in bone marrow (BM), peripheral blood (PB) and spleen of the mice, confirming the immune-suppressive nature of the breast cancer line. This effect was restored under treatment with AZA in monotherapy and combination with CPi. Interestingly AZA reduced the number of immunosuppressive MDSC in BM, spleen and in tumor tissue. Vice versa NK cells were increased in BM, spleen and tumor tissue under treatment with AZA. AZA reduced the number of circulation tumor cells in PB and increased as already shown in vitro the percentage of CD8/CTLA4++ in PB. The PCA analysis of 36 mouse cytokines in vivo displayed two main clusters separated mainly by the presence of absence of AZA in the treatment arms. The combination arms which displayed the highest efficacy showed an increase in CXCL1, G-CSF, IL-6, LIF, M-CSF, MCP-1 (CCL2) and MCP-3 (CCL7) and a decrease of IFN-gamma, CXCL2. This is in line with the more inflammatory pattern of the tumor infiltrating lymphocytes in those groups. In summary, we have strong evidence that AZA influences the immune landscape of breast cancer towards a more inflammatory subtype. The combination with CPi was superior to the monotherapy. In addition, our data indicate that the treatment schedule of the combination might have a crucial influence on efficacy. Citation Format: Hannah Silberzahn, Mattias Bleisch, Anne Löhr, Volker Knauff, Kanstantsin Lashuk, Julia B. Schueler. Identifying most promising combination therapies for immune suppressive breast cancer using a combined in vitro/in vivo approach [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 2630.

Biodistribution and dosimetry of 177Lu-DOTA-IBA for therapy of bone metastases

BackgroundWe designed and synthesized a novel bisphosphonate radiopharmaceutical (68 Ga- or 177Lu-labeled DOTA-ibandronate [68 Ga/177Lu-DOTA-IBA]) for the targeted diagnosis and treatment of bone metastases. The biodistribution and internal dosimetry of a single therapeutic dose of 177Lu-DOTA-IBA were evaluated using a series of single-photon emission computerized tomography (SPECT) images and blood samples. Five patients with multiple bone metastases were included in this prospective study. After receiving 1110 MBq 177Lu-DOTA-IBA, patients underwent whole-body planar, SPECT/CT imaging and venous blood sampling over 7 days. Dosimetric evaluation was performed for the main organs and tumor lesions. Safety was assessed using blood biomarkers.Results177Lu-DOTA-IBA showed fast uptake, high retention in bone lesions, and rapid clearance from the bloodstream in all patients. In this cohort, the average absorbed doses (ADs) in the bone tumor lesions, kidneys, liver, spleen, red marrow, bladder-wall, and osteogenic cells were 5.740, 0.114, 0.095, 0.121, 0.095, and 0.333 Gy/GBq, respectively. Although no patient reached the predetermined dose thresholds, the red marrow will be the dose-limiting organ. There were no adverse reactions recorded after the administration of 1110 MBq 177Lu-DOTA-IBA.ConclusionDosimetric results show that the ADs for critical organs and total body are within the safety limit and with high bone retention. It is a promising radiopharmaceutical alternative for the targeted treatment of bone metastases, controlling its progression, and improving the survival and quality of life of patients with advanced bone metastasis.

Abstract 1425: Epigenetic modulation synergizes with a novel TCR β chain directed antibody-fusion molecule to suppress checkpoint-refractory tumors irrespective of MHC I status

Abstract Background: Epigenetic silencing of MHC-I, and poor T cell infiltration and function in the tumor microenvironment (TME) are prevalent in solid malignancies and associate with lack of response to immune checkpoint blockade (ICB). Novel combination therapies may overcome these barriers, thereby providing clinical benefit across solid tumors. STAR0602 is a first-in-class bifunctional therapeutic molecule, that simultaneously engages a nonclonal mode of T cell receptor (TCR) activation with costimulation to promote activation and expansion of αβ T cell subsets expressing distinct variable β (Vβ) TCR chains. Here, we examined the tumor-suppressive ability and mode of action of a murine surrogate of STAR0602 (mSTAR1302) in combination with the class I HDAC inhibitor Entinostat, an epigenetic modulator that enhances tumor immune cell recognition, in multiple tumor models, including ICB-refractory. Methods: mSTAR1302 and/or Entinostat were administered to mice bearing ICB-responsive breast EMT6 wild-type (wt) and ICB-refractory EMT6 β2-microglobulin (B2m)-deleted and CT26 (Kras G12Dmut colon) tumors. Anti-tumor efficacy, survival, and protective memory were monitored. Immune cell depletion studies were conducted. Comprehensive proteomic and immune profiling of tumor, tumor-draining lymph node (tdLN), and spleen cell populations was conducted. Antigen-specific IFNg T cell responses were evaluated by ELISpot. Results: Combination therapy demonstrated significant enhancement in tumor suppression and prolonged survival in CT26 (MHC-I+), EMT6 wt (MHC-I+) and EMT6 β2m-deleted (MHC-Inull) tumors relative to single agent therapy, as well as enhanced tumor-specific protective memory responses. Tumor suppression was independent of CD4+ T cell immunity and associated with sustained activation and expansion of Vβ13 CD8+ T cells. Conclusions: Ongoing mechanistic studies indicate combination therapy to elicit complimentary mechanisms of tumor suppression beyond CD8+ T cells. Ongoing proteomic and single-cell transcriptomic investigation of MHC-I competent and null tumors will provide a deeper insight into the mode of action of this combination therapy. Collectively, these data support the use of STAR0602 in combination with Entinostat for the treatment of solid malignancies, including for patients harboring ICB-refractory tumors devoid of MHC class I. Citation Format: Katherine E. Lothstein, Asma S. Khelifa, Masaya Miyamoto, Christine M. Minnar, Lisa Poppe, Nicholas Roller, Andrew Bayliffe, Ke Liu, Jacques Moisan, Madan Katragadda, Zhen Su, Jeffrey Schlom, Sofia R. Gameiro. Epigenetic modulation synergizes with a novel TCR β chain directed antibody-fusion molecule to suppress checkpoint-refractory tumors irrespective of MHC I status [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 1425.

Abstract 4065: Entinostat synergizes with the IL-15 superagonist N-803 and anti-PD-1 to engage cDC1 and stem-like TCF-1+ CD8+ T and NK cells to overcome colorectal tumors refractory to anti-PD-1 therapy

Abstract Background: Immune checkpoint blockade (ICB) has limited efficacy in colorectal carcinoma (CRC), where ~85% of patients harbor microsatellite stability (MSS). A minor subset of CRC patients (~15%) harbor microsatellite instability (MSI) and demonstrate improved responses to ICB ranging from 30-50%. Combination therapy has the potential to increase the response rate. Entinostat, a class I HDAC inhibitor, has demonstrated potential to enhance ICB efficacy, such as αPD-1 and immunostimulatory cytokines. N-803, a heterodimeric IL-15 superagonist that enhances proliferation and activation of CD8+ and NK cells has demonstrated to enhance the clinical benefit of Nivolumab in non-small cell lung cancer patients that were relapsed or refractory to αPD-1 monotherapy. Here, we investigated the antitumor efficacy and mode of action of combination therapy comprising αPD1, Entinostat, and N-803 in αPD1-refractory CT26 and Jak1-deleted MC38 CRC tumor models. Methods: Entinostat, N-803, and αPD1 alone or in combination were administered to mice bearing established CT26 or Jak1-deleted MC38 tumors. Antitumor efficacy, survival, and protective memory upon tumor rechallenge were monitored. Comprehensive flow cytometric profiling of the tumor microenvironment (TME), tumor-draining lymph node (tdLN), and spleen cell populations was conducted. CT26 tumor and tdLN immunomes were examined by single-cell transcriptomics. Cytokines and chemokines were examined in the TME and sera. Antigen-specific IFNg T cell responses were evaluated by ELISpot. Results: We demonstrated that combination therapy with Entinostat, N-803 and αPD1 elicits potent suppression of CT26 and Jak1-deleted MC38 tumors, with median overall survival increasing 33% to not reached. These effects were associated with increased activation, infiltration, cytokine production, and cytolytic function of tumor CD8+ T and NK cells, and increased sera IFNg and TNFα. Tumor resolution and protective memory were associated with development of p15e-specific T cell responses. Increased migration of CD8+ T cells and cross-presenting dendritic cells (cDC1) to the tdLN, as well as significant enrichment of cross-presentation genes in tumor cDC1s was observed. Transcriptomic and phenotypic analyses demonstrated increased stem-like TCF-1+ CD8+ T cell accumulation in the tdLNs and TME of mice treated with triple combination. Conclusions: Combination therapy with Entinostat plus N-803 and αPD1 synergized to promote a sustained antitumor response and prolonged survival. We hypothesize the antitumor effects were due to collaborative efforts of CD8+ T and NK cells, and cDC1 cells in tdLN and TME. These results provide a rationale for the clinical testing of αPD1, Entinostat and N-803 combination for patients with CRC, including those with acquired resistance to ICB. Citation Format: Christine M. Minnar, Masaya Miyamoto, Asma S. Khelifa, Grace Lui, Katherine L. Lothstein, Paul L. Chariou, Thomas J. Meyer, Alexei Lobanov, Margaret Cam, Jeffrey Schlom, Sofia R. Gameiro. Entinostat synergizes with the IL-15 superagonist N-803 and anti-PD-1 to engage cDC1 and stem-like TCF-1+ CD8+ T and NK cells to overcome colorectal tumors refractory to anti-PD-1 therapy [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 4065.

Lack of ATP2B1 in CD4+ T Cells Causes Colitis.

The ATP2B1 gene encodes for a calcium pump, which plays a role in removing Ca2+ from cells and maintaining intracellular Ca2+ homeostasis. Reduction of the intracellular Ca2+ concentration in CD4+ T cells is thought to reduce the severity of colitis, while elevation of Ca2+ in CD4+ T cells induces T cell hyperactivity. Our aim was to clarify the role of ATP2B1 in CD4+ T cells and in inflammatory bowel disease development. A murine CD4+ T cell-specific knockout (KO) of ATP2B1 was created using a Cre-loxP system. CD4+ T cells were isolated from thymus, spleen, and blood using fluorescence-activated cell sorting. To quantify messenger RNA levels, quantitative real-time polymerase chain reaction was performed. Although the percentages of CD4+ T cells in both KO mouse spleen and blood decreased compared with those of the control samples, both T-bet (a T helper 1 [Th1] activity marker) and GATA3 (a Th2 activity marker) expression levels were further increased in KO mouse blood CD4+ T cells (vs control blood). Diarrhea and colonic wall thickening (with mucosal changes, including crypt distortion) were seen in KO mice but not in control mice. Prior to diarrhea onset, the KO mouse colon length was already noted to be shorter, and the KO mouse stool water and lipid content were higher than that of the control mice. Tumor necrosis factor α and gp91 expressions were increased in KO mouse colon. Lack of ATP2B1 in CD4+ T cells leads to Th1 and Th2 activation, which contributes to colitis via elevation of tumor necrosis factor α and oxidative stress.

Expression analysis and antiviral activity of koi carp (Cyprinus carpio) viperin against carp edema virus (CEV)

Viperin, also known as radical S-Adenosyl methionine domain containing 2 (RSAD2), is an IFN stimulated protein that plays crucial roles in innate immunity. Here, we identified a viperin gene from the koi carp (Cyprinus carpio) (kVip). The ORF of kVip is 1047 bp in length, encoding a polypeptide of 348 amino acids with neither signal peptide nor transmembrane protein. The predicted molecular weight is 40.37 kDa and the isoelectric point is 7.7. Multiple sequence alignment indicated that putative kVip contains a radical SAM superfamily domain and a conserved C-terminal region. kVip was highly expressed in the skin and spleen of healthy koi carps, and significantly stimulated in both natural and artificial CEV-infected koi carps. In vitro immune stimulation analysis showed that both extracellular and intracellular poly (I: C) or poly (dA: dT) caused a significant increase in kVip expression of spleen cells. Furthermore, intraperitoneal injection of recombinant kVip (rkVip) not only reduced the CEV load in the gills, but also improved the survival of koi carps following CEV challenge. Additionally, rkVip administration effectively regulated inflammatory and anti-inflammatory cytokines (IL-6, IL-1β, TNF-α, IL-10) and interferon-related molecules (cGAS, STING, MyD88, IFN-γ, IFN-α, IRF3 and IRF9). Collectively, kVip effectively responded to CEV infection and exerted antiviral function against CEV partially by regulation of inflammatory and interferon responses.

Generation and characterization of mAb 61H9 against junctional adhesion molecule-a with potent antitumor activity.

Junctional adhesion molecule-A (JAM-A) is an adhesion molecule that exists on the surface of certain types of cells, including white blood cells, endothelial cells, and dendritic cells. In this study, the cDNA sequences of JAM-A-Fc were chemically synthesized with optimization for mammalian expression. Afterward, we analyzed JAM-A protein expression through transient transfection in HEK293 cell lines. Mice were immunized with JAM-A-Fc protein, and hybridoma was prepared by fusing myeloma cells and mouse spleen cells. Antibodies were purified from the hybridoma supernatant and four monoclonal strains were obtained and numbered 61H9, 70E5, 71A8, and 74H3 via enzyme-linked immunosorbent assay screening. Immunofluorescence staining assay showed 61H9 was the most suitable cell line for mAb production due to its fluorescence signal being the strongest. Flow cytometric analysis proved that 61H9 possessed high affinity. Moreover, antagonism of JAM-A mAb could attenuate the proliferative, migrative, and invasive abilities of ESCC cells and significantly inhibit tumor growth in mice. By examining hematoxylin-eosin staining mice tumor tissues, we found inflammatory cells infiltrated lightly in the anti-JAM-A group. The expression of BCL-2 and IκBα in the anti-JAM-A group were decreased in mice tumor tissues compared to the control group. Ultimately, a method for preparing high-yield JAM-A-Fc protein was created and a high affinity mAb against JAM-A with an antitumor effect was prepared.

Achyranthes bidentata polysaccharides improve cyclophosphamide-induced adverse reactions by regulating the balance of cytokines in helper T cells

The manuscript aimed to study the immune function maintenance effect of Achyranthes bidentata polysaccharides (ABPs). The mice were divided into the control group, cyclophosphamide-induced (CTX) group, and ABPs-treated (ABP) group. The results showed that, compared with the CTX group, ABPs could significantly improve the spleen index and alleviate the pathological changes in immune organs. Ex vivo study of whole spleen cells, the levels of interleukin-2 (IL-2), interleukin-6 (IL-6), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) were increased. The proliferation of lymphocytes and the proportion of CD3+CD4+ Th cells in peripheral blood mononuclear cells were increased. The transcription of GATA-3, Foxp3, and ROR γ t were decreased, while the transcription of T-bet was increased. The transcriptome sequencing analysis showed that the differentially expressed genes (DEGs) caused by ABPs-treated were mostly downregulated in CTX-induced mice. The Th2-related genes were significantly enriched in DEGs, with representative genes, including Il4, II13, Il9, etc., while increasing the expression of immune effector genes simultaneously, including Ccl3, Ccr5, and Il12rb2. It was suggested that ABPs possibly regulated the balance of cytokines in helper T cells to ameliorate the immune function of CTX-induced mice.

Rho-kinase inhibitor alleviates CD4+T cell mediated corneal graft rejection by modulating its STAT3 and STAT5 activation

Penetrating keratoplasty remains the most common treatment to restore vision for corneal diseases. Immune rejection after corneal transplantation is one of the major causes of graft failure. In recent years, Rho-associated protein kinase (ROCK) inhibitors have been found to be associated with the activation of the STATs pathway and are widely studied in autoimmune diseases. Therefore, it may be possible that the ROCK inhibitors also participate in the local and systemic immune regulation in corneal transplantation through activation of the STATs pathway and affect the CD4+ T cell differentiation. This study aimed to explore the role of ROCK-STATs pathway in the occurrence of immune rejection in corneal transplantation by applying Y27632, a ROCK inhibitor, to the recipient mice and peripheral CD4+ T cells. We found that Y27632 significantly up-regulated the phosphorylation level of STAT5 in both spleen and lymph nodes, down-regulated the phosphorylation level of STAT3 in the CD4+ T cells in the spleen. It also increased the proportion of CD4+CD25+Foxp3+Helios+ Tregs while decreased CD4+IL17A+ -Th17 cells. Moreover, Y27632 also reduced the proportion of dendritic cells in both spleen and lymph nodes, as well as the expression level of CD86 on their surfaces in the spleen, while the proportion of macrophages was not affected. The expression levels of ROCK1, ROCK2, CD11c and IL-17A mRNA were also found to be low in the graft tissue while the expression of Helios was upregulated. Rho-kinase inhibitor can modulate the balance of Tregs/Th17 by regulating the phosphorylation levels of both STAT3 and STAT5, thereby inhibiting the occurrence of immune rejection in allogeneic corneal transplantation.

Adjuvant effects of β-defensin on DNA vaccine OmpC against edwardsiellosis in flounder (Paralichthys olivaceus)

β-defensin of flounder plays an important role in immunomodulation by recruiting immune cells and has a potential vaccine adjuvant effect in addition to its bactericidal activity. In this study, adjuvant effects of β-defensin on DNA vaccine OmpC against edwardsiellosis in flounder (Paralichthys olivaceus) were investigated. The bicistronic eukaryotic expression plasmid pBudCE4.1 plasmid vector with two independent coding regions was selected to construct DNA vaccine of p-OmpC which express only the gene for the outer membrane protein of Edwardsiella tarda and the vaccine of p-OmpC-βdefensin which express both the outer membrane protein of the bacterium and β-defensin of flounder. In vitro and in vivo studies have shown that the constructed plasmids can be expressed in flounder embryonic cell lines and injection sites of muscles. After vaccination by intramuscular injection, both p-OmpC and p-OmpC-βdefensin groups showed significant upregulation of immune-response. Compared to the pBbudCE4.1 and the p-OmpC vaccinated groups, the p-OmpC-βdefensin vaccinated group showed significantly more cell aggregation at the injection site and intense immune response. The proportion of sIgM+ cells, as well as the CD4-1+ and CD4-2+ cells in both spleen and kidney was significantly higher in the p-OmpC-βdefensin vaccinated group at peak time point than in the control groups. The relative survival rate of the p-OmpC-βdefensin vaccine was 74.17%, which was significantly higher than that of the p-OmpC vaccinated group 48.33%. The results in this study determined that β-defensin enhances the responses in cellular and humoral immunity and evokes a high degree of protection against E. tarda, which is a promising candidate for vaccine adjuvant.

Imbalance of Tfollicular helper cell subsets trigger the differentiation of pathogenic B cells in idiopathic membranous nephropathy.

This study aims to elucidate the role of T follicular helper (Tfh) cells and their subsets in idiopathic membranous nephropathy (IMN). The frequencies of Tfh cell subsets and B cell subsets in peripheral blood (PB) were detected in both IMN patients and healthy controls (HCs). The involvement of Tfh cells in the disease pathogenesis was examined by coculturing human Tfh cells with B cells. The dynamic changes of Tfh cells in PB or spleen were monitored in passive Heymann nephritis (PHN) rats. The frequencies of circulating Tfh (cTfh) cells, cTfh2 cells, and plasmablasts were enriched in the PB of patients with IMN. cTfh cells expressed higher ICOS, and lower BTLA than healthy counterparts. The frequency of ICOS + cTfh2 was associated with the severity of IMN, including 24h urine protein, IgG4 concentration and the IgG4: IgG ratio. Positive correlations were also observed between the frequency of cTfh2 cells with plasmablasts, serum IL-21 and IL-4 levels. Importantly, cTfh cells isolated from IMN patients were able to induce the differentiation of B cells to memory B cells (MBC) and plasmablasts, this process could be substantially attenuated by blocking the IL-21. Similar increases of ICOS + cTfh cells were also detected in spleen of PHN rats, concomitant with elevated urine protein levels. Collectively, our results demonstrate that the imbalance of cTfh cell subsets play a crucial pathogenic role in IMN by inducing the differentiation of B cells through IL-21, and cTfh2 cells might serve as useful markers to evaluate the progression of IMN.

Immunomodulatory Effects of Sodium Hyaluronate Health Drink on Immunosuppressed Mice.

This study aimed to explore the immunomodulatory effects and mechanism of a sodium hyaluronate health drink in immunosuppressed mice. The results showed that the sodium hyaluronate health drink could improve thymus atrophy, repair spleen cell damage, promote the release of IL-2, IL-6 and TNF-α in serum, restore immune deficiency, and enhance immune function. In addition, 16s rRNA sequencing results of intestinal flora showed that different doses of health drink had different effects on the intestinal flora of mice. The low-dose group of mice showed a significant up-regulation of the abundance of Lactobacillus and promoted the formation of a new genus Akkermansia, while the medium- and high-dose group up-regulated the abundance of Lactobacillus and norank-f-Muribaculaceae, and stimulated the production of the new genus Alistipes. Sodium hyaluronate health drink may enhance the immune function of mice by changing the composition and abundance of intestinal flora, which provided a theoretical basis for the subsequent product development.

Nifuroxazide suppresses PD-L1 expression and enhances the efficacy of radiotherapy in hepatocellular carcinoma

Radiation therapy is a primary treatment for hepatocellular carcinoma (HCC), but its effectiveness can be diminished by various factors. The over-expression of PD-L1 has been identified as a critical reason for radiotherapy resistance. Previous studies have demonstrated that nifuroxazide exerts antitumor activity by damaging the Stat3 pathway, but its efficacy against PD-L1 has remained unclear. In this study, we investigated whether nifuroxazide could enhance the efficacy of radiotherapy in HCC by reducing PD-L1 expression. Our results showed that nifuroxazide significantly increased the sensitivity of tumor cells to radiation therapy by inhibiting cell proliferation and migration while increasing apoptosis in vitro. Additionally, nifuroxazide attenuated the up-regulation of PD-L1 expression induced by irradiation, which may be associated with increased degradation of PD-L1 through the ubiquitination-proteasome pathway. Furthermore, nifuroxazide greatly enhanced the efficacy of radiation therapy in H22-bearing mice by inhibiting tumor growth, improving survival, boosting the activation of T lymphocytes, and decelerating the ratios of Treg cells in spleens. Importantly, nifuroxazide limited the increased expression of PD-L1 in tumor tissues induced by radiation therapy. This study confirms, for the first time, that nifuroxazide can augment PD-L1 degradation to improve the efficacy of radiation therapy in HCC-bearing mice.