Cell Costimulation Research Articles

Modulation of myeloid and T cells in vivo by Bruton’s tyrosine kinase inhibitor ibrutinib in patients with metastatic pancreatic ductal adenocarcinoma

BackgroundIn preclinical studies of pancreatic ductal adenocarcinoma (PDAC), ibrutinib improved the antitumor efficacy of the standard of care chemotherapy. This led to a phase 1b clinical trial to determine the...

Bioinformatics-based analysis of potential candidates chromatin regulators for immune infiltration in osteoarthritis

BackgroundThrough the bioinformatics analysis to screen out the potential chromatin regulators (CRs) under the immune infiltration of osteoarthritis (OA), thus providing some theoretical support for future studies of epigenetic mechanisms under OA immune infiltration.MethodsBy integrating CRs and the OA gene expression matrix, we performed weighted gene co-expression network analysis (WGCNA), differential analysis, and further screened Hub genes by protein-protein interaction (PPI) analysis. Using the OA gene expression matrix, immune infiltration extraction and quantification were performed to analyze the correlations and differences between immune infiltrating cells and their functions. By virtue of these Hub genes, Hub gene association analysis was completed and their upstream miRNAs were predicted by the FunRich software. Moreover, a risk model was established to analyze the risk probability of associated CRs in OA, and the confidence of the results was validated by the validation dataset.ResultsThis research acquired a total of 32 overlapping genes, and 10 Hub genes were further identified. The strongest positive correlation between dendritic cells and mast cells and the strongest negative correlation between parainflammation and Type I IFN reponse. In the OA group DCs, iDCs, macrophages, MCs, APC co-inhibition, and CCR were significantly increased, whereas B cells, NK cells, Th2 cells, TIL, and T cell co-stimulation were significantly decreased. The risk model results revealed that BRD1 might be an independent risk factor for OA, and the validation dataset results are consistent with it. 60 upstream miRNAs of OA-related CRs were predicted by the FunRich software.ConclusionThis study identified certain potential CRs and miRNAs that could regulate OA immunity, thus providing certain theoretical supports for future epigenetic mechanism studies on the immune infiltration of OA.

CHI3L1 enhances melanoma lung metastasis via regulation of T cell co-stimulators and CTLA-4/B7 axis.

ICOS/ICOSL and CD28/B7-1/B7-2 are T cell co-stimulators and CTLA-4 is an immune checkpoint inhibitor that play critical roles in the pathogenesis of neoplasia. Chitinase 3-like-1 (CHI3L1) is induced in many cancers where it portends a poor prognosis and contributes to tumor metastasis. Here we demonstrate that CHI3L1 inhibits the expression of ICOS, ICOSL and CD28 while stimulating CTLA-4 and the B7 moieties in melanoma lung metastasis. We also demonstrate that RIG-like helicase innate immune activation augments T cell co-stimulation, inhibits CTLA-4 and suppresses pulmonary metastasis. At least additive antitumor responses were seen in melanoma lung metastasis treated with anti-CTLA-4 and anti-CHI3L1 antibodies in combination. Synergistic cytotoxic T cell-induced tumor cell death and the heightened induction of the tumor suppressor PTEN were seen in co-cultures of T and tumor cells treated with bispecific antibodies that target both CHI3L1 and CTLA-4. Thus, CHI3L1 contributes to pulmonary metastasis by inhibiting T cell co-stimulation and stimulating CTLA-4. The simultaneous targeting of CHI3L1 and the CTLA-4 axis with individual and, more powerfully with bispecific antibodies, represent promising therapeutic strategies for pulmonary metastasis.

Discovery of Taxus chinensis fruit wine as potentially functional food against Alzheimer's disease by UHPLC-QE-MS/MS, network pharmacology and molecular docking.

Nowadays, there is no specific cure for Alzheimer's disease (AD), but the progression of AD can be improved by preventive interventions. The wine of Taxus chinensis fruit (TCFW) has the effect of improving human immunity and anti-aging as a long history of health care wine in folk, especially popular in the longevity villages in China, which may be potentially effective dietary products to improve AD. However, the chemical constituents and molecular mechanisms of TCFW still remain unknown. In this study, chemical profiling with UHPLC-QE-MS/MS, network pharmacology and molecular docking were integrated to fastly explore the potential chemicals and mechanisms of TCFW against AD. A total of 31 chemical components in TCFW were detected and identified compared with the solvent wine of TCFW by UHPLC-QE-MS/MS. Then, 27 potential key targets and 14 chemical compounds of TCFW were uncovered for the improvement of AD by network pharmacology and molecular docking. These 14 compounds were reported to have diverse bioactivities such as neuroprotective activity, antifibrotic activity, anticancer activity, antiviral activity and effectiveness in the treatment of neuronal injury, Alzheimer's disease, etc. Among these 27 targets affected by TCFW predicted by our approach, AKT1, PTGS2, NOS3, NOS2, INS, ESR1, ESR2, BDNF, IL6, IL1B, DRD2 and ACHE were significantly altered in AD. The GO and KEGG enrichment analyses revealed that TCFW mainly acted on oxidative response, inflammatory response, insulin secretion, amyloid fibril formation, neurodegenerative pathway-multiple diseases, Alzheimer's disease, longevity regulation pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, etc, which were the main pathogenesis of AD. PRACTICAL APPLICATIONS: Alzheimer's disease (AD) is a degenerative neurological disorder characterized by cognitive and behavioral dysfunction. Nowadays, there is no specific cure for AD, but the progression of AD can be improved by preventive interventions. The wine of Taxus chinensis fruit (TCFW) has the effect of improving human immunity and anti-aging as a long history of health care wine in folk, especially popular in the longevity villages in China, which may be potentially effective dietary products to improve AD. This study proposed a fastly integrated method to explore the potential chemicals and mechanisms of TCFW against AD by UHPLC-QE-MS/MS, network pharmacology and molecular docking. Here, we found that TCFW may ameliorate AD by reversing many biological events, including oxidative stress, inflammatory response, neuronal apoptosis, insulin secretion, amyloid fibril formation, and T cell co-stimulation, which may provide some insights for the development and research of anti-AD drugs.

Differential effects of periodontal microbiome on the rheumatoid factor induction during rheumatoid arthritis pathogenesis

Association between exposure to periodontal bacteria and development of autoantibodies related to rheumatoid arthritis (RA) has been widely accepted; however, direct causal relationship between periodontal bacteria and rheumatoid factor (RF) is currently not fully understood. We investigated whether periodontal bacteria could affect RF status. Patients with preclinical, new-onset, or chronic RA underwent periodontal examination, and investigation of subgingival microbiome via 16S rRNA sequencing. Degree of arthritis and RF induction was examined in collagen-induced arthritis (CIA) mice that were orally inoculated with different periodontal bacteria species. Subsequently, single-cell RNA sequencing analysis of the mouse spleen cells was performed. Patients with preclinical RA showed an increased abundance of the Porphyromonadacae family in the subgingival microbiome compared to those with new-onset or chronic RA, despite comparable periodontitis severity among them. Notably, a distinct subgingival microbial community was found between patients with high-positive RF and those with negative or low-positive RF (p=0.022). Oral infections with the periodontal pathogens P. gingivalis and Treponema denticola in CIA mice similarly enhanced arthritis score, but resulted in different levels of RF induction. Genes related to B cell receptor signaling, B cell proliferation, activation, and differentiation, and CD4+ T cell costimulation and cytokine production were involved in the differential induction of RF in mice exposed to different bacteria. In summary, periodontal microbiome might shape RF status by affecting the humoral immune response during RA pathogenesis.

Somatic Mutations in T Cells in Patients with Hematological Diseases

Somatic mutations accumulate in cells during aging, and the mutation rate and spectrum vary between tissues. Mutation accumulation can be associated with inflammation: for example, myeloid driver gene mutations in hematopoietic stem cells (clonal hematopoiesis, CH), has been linked with atherosclerosis. Somatic mutations can also accumulate in T cells, where they are connected with immune dysregulation in large granular lymphocytic leukemia, characterized by autoimmunity and STAT3 mutations in T cells. However, the mutation spectrum in T cells in other hematological disorders is not yet fully discovered. We hypothesized that T cell lineage specific somatic mutations are associated with T cell clonality in various diseases involving the hematopoietic system. We systematically characterized the spectrum of somatic mutations in CD4+ and CD8+ T cells in altogether 207 samples from 95 patients with chronic graft-versus-host disease (cGVHD, number of patients: 40), myelodysplastic syndrome (MDS, n = 15), immune thrombocytopenia (n = 2), aplastic anemia (AA, n = 21, previously published), and immunodeficiency (n = 17, previously published). Age at sampling was similar between disease cohorts. DNA was extracted from bead separated CD4+ and CD8+ cells from peripheral blood or bone marrow samples. We applied a custom-made targeted NGS panel, consisting of 2533 immune-related genes. Variant calling was performed using Mutect2. In variant filtering, we used 42 healthy T cell and skin samples as panel of normals. To explore the association between somatic mutations and clonal T cell expansions, we performed TCRb deep sequencing from 148 matched patient samples. With mean sequencing depth of 382x, we identified 888 non-synonymous somatic variants in total. 784 (86%) of the variants were only found in either CD4+ or CD8+ fraction. 52 variants were shared between individual patient's CD4+ and CD8+ T cells, suggesting that they were acquired prior to T cell lineage differentiation. On the average, we observed 3.8 fraction-specific variants per sample. Mean variant allele frequency (VAF) was 4.5% and 12.7% for fraction-specific and shared variants, respectively. Variant types included missense (91%), nonsense (5.1%), splice-site (1.7%), frameshift (1.3%), and non-stop (0.2%) mutations. The most common mutational signatures were age-associated Signature 1 and defective DNA mismatch repair associated Signature 15. Immunodeficiency patients had lower number of fraction-specific variants compared to AA, MDS and cGVHD patients (p = 0.027). In all cohorts, CD8+ T cells had acquired more variants than CD4+ T cells (p = 0.00012; mean [median] for CD8: 4.4 [4] and CD4: 3.2 [2]). The highest fraction-specific VAF in each sample was associated with the largest T cell clone size in CD8+ but not in CD4+ samples (R = 0.59, p = 3.1e-09 and p = ns, respectively). In CD8+ T cells, age was associated with number of fraction-specific variants (R = 0.38, p = 1.1e-4) but not with the highest CD8-specific VAF (p = ns). Among the variants shared between individual patients' CD4+ and CD8+ T cells, we found mutations in DNMT3A (n = 3) and TET2 (n = 2) and ASXL1 (n = 1), the most recurrently mutated CH genes. In addition to shared mutations, in 12% of patients had fraction-specific myeloid driver gene mutations (e.g. DNMT3A, TET2, KRAS, NRAS) in CD4+ or CD8+ T cells. To identify pathways enriched for putative driver mutations, we performed analyses of fraction-specific T cell variants with OncodriveFM. The most significantly mutated gene ontology pathways included T cell co-stimulation, leukocyte migration, Ras protein signal transduction, and positive regulation of MAP kinase activity. In conclusion, T cell fraction-specific somatic mutations are common in patients with various hematological diseases, and they are enriched in pathways important for T cell function. CD8+ T cells accumulate more mutations than CD4+ T cells. In CD8+ T cells, the number of somatic mutations was associated with age, reflecting stochastic accumulation of mutations. Based on mutational signatures, somatic mutations in T cells are mainly result of endogenous mutational processes. In addition, mutational clone size CD8+ T cells was significantly associated with the largest TCR clone, suggesting that mutations have accumulated in expanded CD8+ T cell clones. Further studies are warranted to determine the functional effects of somatic mutations in T cells.

Blocking T Cell Co-Stimulation Via CD70 As a Target for Gvhd in Allogeneic Stem Cell Transplantation (SCT)

Blocking T Cell Co-Stimulation Via CD70 As a Target for Gvhd in Allogeneic Stem Cell Transplantation (SCT)

Erythropoietin Levels in Patients with Polycythemia Vera and Secondary Erythrocytosis

BACKGROUND: Polycythemia Vera (PV) is a myeloproliferative neoplasm characterized by uncontrolled proliferation of erythrocytes with frequent co-stimulation of myeloid and megakaryocyte progenitor cells. Prior to the utilization of molecular testing, subnormal or low erythropoietin (EPO) levels were an important component in the diagnosis of PV. In 2005, the somatic JAK2 V617Fmutation was discovered to be present in greater than 95% of patients with PV. In turn, the 2008 WHO diagnostic criteria revision added the JAK2 mutation to the major criteria. The current WHO criteria for the diagnosis of PV designates the major criteria to be a hemoglobin greater than 16.5 g/dL in men and 16 g/dL in women (or a hematocrit greater than 49% in men and 48% in women), a bone marrow biopsy demonstrating hypercellularity and trilineage panmyelosis, and the presence of a JAK2 V617F or exon 12 mutation. A subnormal EPO level is a minor criterion. Based on these, PV can be diagnosed by the presence of all three major criteria, or the first two major criteria with a subnormal EPO level. Subnormal EPO levels have been reported to be found in only around 30% of patients otherwise meeting the diagnostic criteria for PV. The sensitivity and specificity of a subnormal EPO level in the diagnosis of PV has been shown to be 64% and 96%, respectively. The diagnostic value of the EPO level is confounded by the co-occurrence of comorbidities such as obstructive sleep apnea, chronic obstructive pulmonary disease, tobacco use, and obesity. As a result, controversies exist as to the role of EPO in the diagnosis of PV. In this study, we aimed to describe the EPO level in patients with primary erythrocytosis (or PV) and secondary erythrocytosis in a Los Angeles safety-net hospital where these comorbidities are prevalent. We also examined the prevalence of these comorbidities in both patient populations. METHODS: We performed a retrospective chart review of all patients aged greater than 18 years who presented to Harbor-UCLA Medical Center between 2015 and 2021 with the diagnosis of erythrocytosis using the International Classification of Disease-specific codes. We excluded patients who were erroneously given the diagnosis code for erythrocytosis when no erythrocytosis was documented or patients who had one documented hemoglobin level above the normal reference range with spontaneous resolution and no further work up. Patient demographics, baseline characteristics, laboratory values, bone marrow biopsy results, molecular testing, and comorbidities were recorded. RESULTS: There was a total of 141 patients who met the inclusion criteria (Table 1). Of these, 12 (8.5%) had the diagnosis of primary erythrocytosos or PV. Of those diagnosed with PV, all 12 (100%) had the JAK2 V617Fmutation. The average hemoglobin and hematocrit of patients with PV and secondary erythrocytosis was 17.8 g/dL and 54% and 18.1 g/dL and 57%, respectively. The average EPO for those with primary and secondary erythrocytosis was 3.4 mIU/mL and 23.7 mIU/mL, respectively (normal 2.6-18.5 mIU/mL). Of those with primary polycythemia, 6/11 (55%) had a subnormal EPO level at diagnosis. The average age of those with PV and secondary erythrocytosis was 69 years and 53 years, respectively. The average BMI for those with PV and erythrocytosis was 27.5 and 32.5, respectively. In those with secondary erythrocytosis versus PV, the most common comorbidities were pulmonary disease (58.3% versus 41.7%), smoking (36.7% versus 41.7%), heart disease (23.0% versus 25%), kidney disease (21.6% versus 25%), and testosterone use (5.0% versus 0%) (Table 2). CONCLUSIONS: In this study, we found that most patients with PV had a low EPO level at diagnosis with over half having a subnormal EPO level. In contrast, very few patients with secondary erythrocytosis had a subnormal EPO level. The prevalence of comorbidities was common in both primary and secondary erythrocytosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Deregulated Expression of Circular RNAs Is Associated with Immune Evasion and Leukemia Relapse after Allogeneic Hematopoietic Stem Cell Transplantation.

Circular RNAs (circRNAs) are a novel class of epigenetic regulators that participate in leukemogenesis. However, their roles in leukemia relapse after transplantation remain unclear. We defined the circRNAs profile of the bone-marrow-enriched CD34+ cells from ten acute myeloid leukemia (AML) patients after transplantation (five relapse [RE] and five continuous complete remission [CR]) and four healthy controls (HCs) by RNA-seq. Differentially expressed circRNAs were validated using real-time quantitative polymerase chain reaction (RT-qPCR) in an independent cohort of six AML patients with pairwise samples at diagnosis and at relapse and six controls. The bioinformatics analysis revealed a distinct circRNAs profile in relapse patients compared with controls (CR or HCs), while there was no significant difference between CR and HCs. Functional enrichment analysis demonstrated that mRNAs co-expressed with identified circRNAs were primarily involved in immune-related pathways, including the T cell receptor signaling pathway and lymphocyte differentiation. Moreover, we performed a protein-protein interaction network based on the immune-related genes and annotated 20 hub genes. The abnormal expression of hub genes was responsible for impairing T cell co-stimulation and activation, thus contributing to the immune escape of relapse blasts. We further constructed competing endogenous RNAs (ceRNA) regulatory networks based on immune-related genes and identified 10 key circRNAs that are associated with immune evasion. Six candidate circRNAs and their associated miRNA/mRNAs in the ceRNA network were randomly selected to be validated in another set by RT-qPCR. CircRNAs dysregulation may be involved in the immune evasion of relapse blasts and is associated with AML relapse. Our results identify several promising biomarkers and might provide novel insights into the biology of AML relapse post-transplantation.

A prognostic signature of cuproptosis and TCA-related genes for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common malignant tumor of the liver. Cuproptosis is a newly defined form of cell death. Copper ion induces cell death by binding to the tricarboxylic acid cycle (TCA). The effect of cuproptosis-related and TCA-related genes on the clinical prognosis of HCC is still unclear. In this study, we explores the genetic changes of cuproptosis-related genes that affect the TCA process and their potential therapeutic value in HCC patients. The cuproptosis and TCA-related genes were obtained from cuproptosis-related articles and the molecular signatures database. The prognosis signatures of eight related genes were constructed using the last absolute shrinkage and selection operator (LASSO), and Receiver Operating Characteristic (ROC) curves were used to evaluate the signature. In addition, we analyzed downstream functional enrichment and immune infiltration to explore cuproptosis-inducing drugs and immunotherapeutic responses. All these analyses were validated using multiple datasets of the International Cancer Genome Consortium (ICGC). TCA and copper malnutrition-related genes (CDKN2A, IDH1, OGDHL, IDH3G, IDH3B, GLS, DLAT, LIPT1) were finally included. According to the risk score, they were divided into high-risk and low-risk groups. Survival analysis showed that the overall survival (OS) of the high-risk group was significantly lower than that of the low-risk group. We established a risk prognostic feature to predict the OS of patients with HCC. Based on this feature and the clinical stage, we constructed a nomogram. Functional enrichment analysis revealed pathways related to organelle division and the cell cycle. Different risk scores had different immune abundances in immune cells (including macrophages and regulatory T-cells) and immune pathways (including antigen-presenting cells co-stimulation). Moreover, the drug sensitivity of eleschomol and PD-L1 in the high-risk group was better than that in the low-risk group. The status of TP53 somatic mutation was also closely related to the risk score. In this study, we established a new prediction signature of eight genes related to cuproptosis and the TCA process, which can effectively predict the prognosis of HCC patients.

Radiomics features for assessing tumor-infiltrating lymphocytes correlate with molecular traits of triple-negative breast cancer

BackgroundTumor-infiltrating lymphocytes (TILs) have become a promising biomarker for assessing tumor immune microenvironment and predicting immunotherapy response. However, the assessment of TILs relies on invasive pathological slides.MethodsWe retrospectively extracted radiomics features from magnetic resonance imaging (MRI) to develop a radiomic cohort of triple-negative breast cancer (TNBC) (n = 139), among which 116 patients underwent transcriptomic sequencing. This radiomic cohort was randomly divided into the training cohort (n = 98) and validation cohort (n = 41) to develop radiomic signatures to predict the level of TILs through a non-invasive method. Pathologically evaluated TILs in the H&E sections were set as the gold standard. Elastic net and logistic regression were utilized to perform radiomics feature selection and model training, respectively. Transcriptomics was utilized to infer the detailed composition of the tumor microenvironment and to validate the radiomic signatures.ResultsWe selected three radiomics features to develop a TILs-predicting radiomics model, which performed well in the validation cohort (AUC 0.790, 95% confidence interval (CI) 0.638–0.943). Further investigation with transcriptomics verified that tumors with high TILs predicted by radiomics (Rad-TILs) presented activated immune-related pathways, such as antigen processing and presentation, and immune checkpoints pathways. In addition, a hot immune microenvironment, including upregulated T cell infiltration gene signatures, cytokines, costimulators and major histocompatibility complexes (MHCs), as well as more CD8+ T cells, follicular helper T cells and memory B cells, was found in high Rad-TILs tumors.ConclusionsOur study demonstrated the feasibility of radiomics model in predicting TILs status and provided a method to make the features interpretable, which will pave the way toward precision medicine for TNBC.

Paeoniflorin improves autoimmune myocarditis in young rat by inhibiting CXCR5 to reduce follicular helper T cells

In the current study, we aimed to investigate the effect of paeoniflorin on autoimmune myocarditis. A total of 72 young Lewis rats were randomly divided into control, experimental autoimmune myocarditis (EAM), paeoniflorin low dose (Pae-L 20 mg/kg), paeoniflorin high dose (Pae-H, 40 mg/kg), EAM-NC, CXCR5 siRNA groups, respectively. The levels of TNF-α, IL-6, IFN-γ, and IL-21 were detected. H&E staining was used to investigate the histopathological changes of myocardial tissue. Flow cytometry and immunofluorescence double-labeling assay were used to detect the CD4 and CXCR5. Western blot was employed to investigate the expression of proteins. Pae enhanced the body weight and ameliorated the histopathology and inflammation score of myocardial tissue on day 21 and 35. In the peripheral blood, Pae diminished the proportion of CD4 + CXCR5 + Tfh cells on day 21 and day 35. Furthermore, Pae decreased the expression of CXCR5, CXCL13, programmed cell death-1 (PD-1), phosphorylated p38 (p-p38), phosphorylated ERK1/2 (p-ERK1/2), Bcl-6, and Inducible T cell CO-Stimulator (ICOS) in myocardial tissue on day 35. Our study indicated that paeoniflorin could effectively alleviate autoimmune myocarditis. The mechanism is possibly related to inhibit CXCR5 to reduce Tfh cells via p38 MAPK signaling.

Molecular Changes in Chronic Myeloid Leukemia During Tyrosine Kinase Inhibitors Treatment. Focus on Immunological Pathways.

IntroductionThe aim of our research was to investigate changes in the molecular background of the immune response in the chronic phase (CP) of chronic myeloid leukaemia (CML) during treatment with tyrosine kinase inhibitors (TKIs).MethodsGlobal gene and miRNA expression profiles were assessed using genome-wide RNA and miRNA microarray technology in bone marrow mononuclear cells. Fifty-one patients were recruited, and bone marrow samples were taken at diagnosis before treatment with TKIs and after 3, 6, and 12 months of treatment with TKIs. The largest number of upregulated genes was observed when the 0-month group (time of diagnosis) was compared to the 3-month group; 1774 genes were significantly upregulated, and 390 genes were significantly downregulated.DiscussionUpregulated biological processes according to gene ontology (GO) classification involved basic cellular processes such as cell division, cell cycle, cell–cell adhesion, protein transport, mitotic nuclear division, apoptosis, and DNA replication. Differentially expressed miRNAs were annotated using GO classification to several immunity-related processes, including the T cell receptor signalling pathway, T cell costimulation, immune response, and inflammatory response. TKI therapy exerts a significant impact on cellular cycle processes and T-cell activation, which was proven at the molecular level.

Pro-Apoptotic and Anti-Migration Properties of a Thiazoline-Containing Platinum(II) Complex in MDA-MB-231 Breast Cancer Cells: The Role of Melatonin as a Synergistic Agent.

Triple-negative breast cancer (TNBC) is an aggressive cancer insensitive to hormonal and human epidermal growth factor receptor 2 (HER2)-targeted therapies and has a poor prognosis. Therefore, there is a need for the development of convenient anticancer strategies for the management of TNBC. In this paper, we evaluate the antitumoral potential of a platinum(II) complex coordinated with the ligand 2-(3,5-diphenylpyrazol-1-yl)-2-thiazoline (DPhPzTn), hereafter PtDPhPzTn, against the TNBC cell line MDA-MB-231, and compared its effect with both cisplatin and its less lipophilic counterpart PtPzTn, the latter containing the ligand 2-(pyrazol-1-yl)-2-thiazoline (PzTn). Then, the putative potentiating actions of melatonin, a naturally occurring antioxidant with renowned antitumor properties, on the tumor-killing ability of PtDPhPzTn were also checked in TNBC cells. Our results show that PtDPhPzTn presented enhanced cytotoxicity compared to both the classical drug cisplatin and PtPzTn. In addition, PtDPhPzTn was able to induce apoptosis, being more selective for MDA-MB-231 cells when compared to non-tumor breast epithelial MCF10A cells. Likewise, PtDPhPzTn produced moderate S phase arrest and greatly impaired the migration ability of MDA-MB-231 cells. Most importantly, the co-stimulation of TNBC cells with PtDPhPzTn and melatonin substantially enhanced apoptosis and markedly improved the anti-migratory action compared to PtDPhPzTn alone. Altogether, our findings provide evidence that PtDPhPzTn and melatonin could be potentially applied to breast cancer treatment as powerful synergistic agents.

IL-2 Combined with IL-15 Enhanced the Expression of NKG2D Receptor on Patient Autologous NK Cells to Inhibit Wilms' Tumor via MAPK Signaling Pathway.

Objective The dysfunction of immune surveillance, a hot spot in cancer research, could lead to the occurrence and development in multicancers. However, the potential mechanisms of immunity in Wilms' tumor (WT) remain unclear on Wilms' tumor (WT). In this study, we aim to investigate the immune cell in WT and explore the underlying treatment strategy. Method We quantified stromal and immune scores by using ESTIMATE algorithm based on gene expression matrix of WT patients in TCGA and GEO databases. Different expression genes (DEGs) and functional enrichments were analyzed by R studio and DAVID tools. Flow cytometry, immunofluorescence staining, ELISA assay, and qRT-PCR were used for detecting the NK cells, cytotoxic cytokines (INF-γ, PRF, and GZMB), and NK cell receptor expression, respectively. WT patient autologous NK cells were stimulated by IL-2 and IL-15, and the cytotoxicity of NK cells against WT cell lines was detected by LDH assay. Western blot experiment was used for measuring the MAPK signaling pathway protein maker in NK cells. Results ESTIMATE indicated that WT tissue had a lower immune score than adjacent kidney tissue. Meanwhile, the low immune score group was associated with poorly outcomes. DEG functional enrichment analysis showed that NK cell-mediated cytotoxicity was significantly different in low and high immune score groups. Although few of proportion of NK cells in WT patients were increased, most of that were significantly lower than normal children. Moreover, the proportion of NK cells and the expression level of INF-γ, PRF, and GZMB in WT tissue were lower than adjacent kidney tissue. Importantly, the NKG2D expression level of NK cells was significantly lower in WT tissue. Furthermore, in vitro, compared with uncultured NK cells, IL-2 and IL-15 could effectively enhance the cytotoxicity of NK cells on killing the WT cell lines. The FACS and WB results showed that the NKG2D and p-PI3K ratio PI3K, MEK1/2, and p-ERK1/2 ratio ERK1/2 were significantly increased in IL-2 and IL15 group compared with uncultured groups. Conclusion The abnormal NK cell-mediated cytotoxicity may cause the occurrence of WT. Costimulation of WT patients autologous NK cells could effectively enhance the antitumor reaction which involved in activation of NKG2D-mediated MAPK signaling pathway.

Construction of ceRNA network and identification of hub genes in aniridia-associated keratopathy using bioinformatics analysis.

Aniridia-associated keratopathy (AAK) is characteristic at ocular surface of aniridia caused by haploinsufficiency of PAX6. Competing endogenous RNA (ceRNA) has been reported to play an important role in various diseases, whereas its function on AAK is unclear. The microarray data of 20 AAK patients and 20 healthy people were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed lncRNAs, miRNAs, and mRNAs were analyzed using “limma” packages and weighted gene co-expression network analysis (WGCNA). A ceRNA network was constructed by Cytoscape 3.9.1, and miR-224-5p, miR-30a-5p, and miR-204-5p were at the center of the network. CIBERSORTx algorithm and ssGSEA analyses revealed that AAK was associated with immune cell infiltration, showing that activated Mast cells increased while resting Mast cells decreased and NK cells decreased in AAK. Type II INF Response, CCR, parainflammation, T cell co-stimulation, and APC co-stimulation of AAK patients differed from healthy individuals. Additionally, the ROC curve of five genes, MITF(AUC = 0.988), RHOB(AUC = 0.973), JUN(AUC = 0.953), PLAUR (AUC = 0.925), and ARG2 (AUC = 0.915) with high confidence in predicting AAK were identified. Gene set enrichment analysis (GSEA) analysis of hub genes enriched in the IL-17 signaling pathway.

Co-Stimulatory Receptor Signaling in CAR-T Cells.

T cell engineering strategies have emerged as successful immunotherapeutic approaches for the treatment of human cancer. Chimeric Antigen Receptor T (CAR-T) cell therapy represents a prominent synthetic biology approach to re-direct the specificity of a patient’s autologous T cells toward a desired tumor antigen. CAR-T therapy is currently FDA approved for the treatment of hematological malignancies, including subsets of B cell lymphoma, acute lymphoblastic leukemia (ALL) and multiple myeloma. Mechanistically, CAR-mediated recognition of a tumor antigen results in propagation of T cell activation signals, including a co-stimulatory signal, resulting in CAR-T cell activation, proliferation, evasion of apoptosis, and acquisition of effector functions. The importance of including a co-stimulatory domain in CARs was recognized following limited success of early iteration CAR-T cell designs lacking co-stimulation. Today, all CAR-T cells in clinical use contain either a CD28 or 4-1BB co-stimulatory domain. Preclinical investigations are exploring utility of including additional co-stimulatory molecules such as ICOS, OX40 and CD27 or various combinations of multiple co-stimulatory domains. Clinical and preclinical evidence implicates the co-stimulatory signal in several aspects of CAR-T cell therapy including response kinetics, persistence and durability, and toxicity profiles each of which impact the safety and anti-tumor efficacy of this immunotherapy. Herein we provide an overview of CAR-T cell co-stimulation by the prototypical receptors and discuss current and emerging strategies to modulate co-stimulatory signals to enhance CAR-T cell function.

ICOS-Fc as innovative immunomodulatory approach to counteract inflammation and organ injury in sepsis.

Inducible T cell co-stimulator (ICOS), an immune checkpoint protein expressed on activated T cells and its unique ligand, ICOSL, which is expressed on antigen-presenting cells and non-hematopoietic cells, have been extensively investigated in the immune response. Recent findings showed that a soluble recombinant form of ICOS (ICOS-Fc) can act as an innovative immunomodulatory drug as both antagonist of ICOS and agonist of ICOSL, modulating cytokine release and cell migration to inflamed tissues. Although the ICOS-ICOSL pathway has been poorly investigated in the septic context, a few studies have reported that septic patients have reduced ICOS expression in whole blood and increased serum levels of osteopontin (OPN), that is another ligand of ICOSL. Thus, we investigated the pathological role of the ICOS-ICOSL axis in the context of sepsis and the potential protective effects of its immunomodulation by administering ICOS-Fc in a murine model of sepsis. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in five-month-old male wild-type (WT) C57BL/6, ICOS-/-, ICOSL-/- and OPN-/- mice. One hour after the surgical procedure, either CLP or Sham (control) mice were randomly assigned to receive once ICOS-Fc, F119SICOS-Fc, a mutated form uncapable to bind ICOSL, or vehicle intravenously. Organs and plasma were collected 24 h after surgery for analyses. When compared to Sham mice, WT mice that underwent CLP developed within 24 h a higher clinical severity score, a reduced body temperature, an increase in plasma cytokines (TNF-α, IL-1β, IL-6, IFN-γ and IL-10), liver injury (AST and ALT) and kidney (creatinine and urea) dysfunction. Administration of ICOS-Fc to WT CLP mice reduced all of these abnormalities caused by sepsis. Similar beneficial effects were not seen in CLP-mice treated with F119SICOS-Fc. Treatment of CLP-mice with ICOS-Fc also attenuated the sepsis-induced local activation of FAK, P38 MAPK and NLRP3 inflammasome. ICOS-Fc seemed to act at both sides of the ICOS-ICOSL interaction, as the protective effect was lost in septic knockout mice for the ICOS or ICOSL genes, whereas it was maintained in OPN knockout mice. Collectively, our data show the beneficial effects of pharmacological modulation of the ICOS-ICOSL pathway in counteracting the sepsis-induced inflammation and organ dysfunction.

ICOS is upregulated on T cells following radiation and agonism combined with radiation results in enhanced tumor control

Multiple preclinical studies have shown improved outcomes when radiation therapy is combined with immune modulating antibodies. However, to date, many of these promising results have failed to translate to successful clinical studies. This led us to explore additional checkpoint and co-stimulatory pathways that may be regulated by radiation therapy. Here, we demonstrate that radiation increases the expression of inducible T cell co-stimulator (ICOS) on both CD4 and CD8 T cells in the blood following treatment. Moreover, when we combined a novel ICOS agonist antibody with radiation we observed durable cures across multiple tumor models and mouse strains. Depletion studies revealed that CD8 T cells were ultimately required for treatment efficacy, but CD4 T cells and NK cells also partially contributed to tumor control. Phenotypic analysis showed that the combination therapy diminished the increased infiltration of regulatory T cells into the tumor that typically occurs following radiation alone. Finally, we demonstrate in a poorly immunogenic pancreatic tumor model which is resistant to combined radiation and anti-PD1 checkpoint blockade that the addition of this novel ICOS agonist antibody to the treatment regimen results in tumor control. These findings identify ICOS as part of a T cell pathway that is modulated by radiation and targeting this pathway with a novel ICOS antibody results in durable tumor control in preclinical models.

337.10: Monotherapy With TNX-1500, a Fc-Modified Anti-CD154mAb, Prolongs Cardiac Allograft Survival in Cynomolgus Monkeys

Purpose: Blockade of the CD40/CD154 T cell costimulation pathway is a promising approach to replace conventional clinical immunosuppressive therapy. TNX-1500 (TNX)* is a novel humanized anti-CD154 mAb that contains the hu5c8 Fab region and an Fc region engineered to modulate FcγR2 binding to reduce the risk of thromboembolic events seen with hu5c8 IgG1 in previous clinical trials. Its efficacy was evaluated in cynomolgus monkey heterotopic heart allograft recipients. Methods: Cynomolgus monkey heterotopic cardiac allograft recipients were treated with TNX 30mg/kg iv 2x weekly for 2 weeks, then either a ‘standard-dose’ regimen of 20mg/kg weekly from days 21-180 (sTNX, n=5) or a reduced-dose TNX maintenance regimen (10 mg/kg weekly x6, then 20mg/kg monthly; loTNX), with (loTNX+MMF, n=4) or without mycophenolate mofetil (MMF) at 200mg/d po (loTNX, n=4). In 3 sTNX animals therapy was discontinued on day 175. Results were compared to historical data with hu5c8 monotherapy dosed as for loTNX (n=5). Biopsies or explanted hearts were evaluated at three protocol-defined time-points (d45, d90, d180), at earlier explant. Results: sTNX (MST >232d, range 116-305) significantly prolonged cardiac allograft survival relative to hu5c8 alone (MST 133d; p=0.016), loTNX (MST 99d; p=0.014), or loTNX+MMF (MST 88d; p=0.011). All grafts treated with loTNX () or with loTNX+MMF () exhibited acute rejection histologically within 90 days, and 7 of 8 grafts failed before day 180. One sTNX graft rejected at 116d in association with bacterial infection; one graft was electively explanted at 181d; and three grafts survived for 57, 90, and 130 days after discontinuing TNX-1500. Neither sustained thrombocytopenia nor micro- or macro-vascular thrombosis were observed. At 90 days, loTNX+MMF had a significantly higher median ISHLT score (2.0) than sTNX grafts (0.2; p=0.016); loTNX+MMF also had a significantly higher median CAV score (1.8) at d90 than either loTNX (0.2; p=0.022) or sTNX (0.3; p=0.026). At subsequent graft explant or end-of-study biopsy (180d), loTNX+MMF had a higher median CAV score (2.4) than loTNX (0.4; p=0.002) or sTNX (0.8; p=0.002). sTNX suppressed the rise in Teff/Tregs ratio at the final time-point (180d or <180d if graft failure) more effectively than loTNX+MMF (3.5 ± 0.9 vs 9.5 ± 6.1, p=0.031). sTNX (5/5) but not loTNX (1/4) or loTNX+MMF (2/4) consistently prevented anti-donor antibody elaboration. Conclusions: Blockade of CD154 with sTNX monotherapy was not associated with thrombotic events, well tolerated, and consistently prevented pathogenic alloimmunity in this stringent preclinical model. Lower TNX maintenance dosing, with or without MMF, was less effective. *TNX-1500 is a pre-IND molecule and not approved for any clinical indication. NIH grant P01 HL158504. Tonix Pharmaceuticals. RC is supported by the Benjamin Research Fellowship from the German Research Foundation.