Interleukin-2 is required for NKp30-dependent NK cell cytotoxicity by preferentially regulating NKp30 expression.

Natural killer (NK) cells are key effectors in cancer immunosurveillance, eliminating a broad spectrum of cancer cells without major histocompatibility complex (MHC) specificity and graft-versus-host diseases (GvHD) risk. The use of allogeneic NK cell therapies from healthy donors has demonstrated favorable clinical efficacies in treating diverse cancers, particularly hematologic malignancies, but it requires cytokines such as IL-2 to primarily support NK cell persistence and expansion. However, the role of IL-2 in the regulation of activating receptors and the function of NK cells expanded for clinical trials is poorly understood and needs clarification for the full engagement of NK cells in cancer immunotherapy. Here, we demonstrated that IL-2 deprivation significantly impaired the cytotoxicity of primary expanded NK cells by preferentially downregulating NKp30 but not NKp46 despite their common adaptor requirement for expression and function. Using NK92 and IL-2-producing NK92MI cells, we observed that NKp30-mediated cytotoxicity against myeloid leukemia cells such as K562 and THP-1 cells expressing B7-H6, a ligand for NKp30, was severely impaired by IL-2 deprivation. Furthermore, IL-2 deficiency-mediated NK cell dysfunction was overcome by the ectopic overexpression of an immunostimulatory NKp30 isoform such as NKp30a or NKp30b. In particular, NKp30a overexpression in NK92 cells improved the clearance of THP-1 cells in vivo without IL-2 supplementation. Collectively, our results highlight the distinct role of IL-2 in the regulation of NKp30 compared to that of NKp46 and suggest NKp30 upregulation, as shown here by ectopic overexpression, as a viable modality to harness NK cells in cancer immunotherapy, possibly in combination with IL-2 immunocytokines.

B7-H3 Expression in Breast Cancer and Brain Metastasis.

Brain metastasis is a significant challenge for some breast cancer patients, marked by its aggressive nature, limited treatment options, and poor clinical outcomes. Immunotherapies have emerged as a promising avenue for brain metastasis treatment. B7-H3 (CD276) is an immune checkpoint molecule involved in T cell suppression, which is associated with poor survival in cancer patients. Given the increasing number of clinical trials using B7-H3 targeting CAR T cell therapies, we examined B7-H3 expression across breast cancer subtypes and in breast cancer brain metastases to assess its potential as an interventional target. B7-H3 expression was investigated using immunohistochemistry on tissue microarrays of three clinical cohorts: (i) unselected primary breast cancers (n = 347); (ii) brain metastatic breast cancers (n = 61) and breast cancer brain metastases (n = 80, including a subset of 53 patient-matched breast and brain metastasis cases); and (iii) mixed brain metastases from a range of primary tumours (n = 137). In primary breast cancers, B7-H3 expression significantly correlated with higher tumour grades and aggressive breast cancer subtypes, as well as poorer 5-year survival outcomes. Subcellular localisation of B7-H3 impacted breast cancer-specific survival, with cytoplasmic staining also correlating with a poorer outcome. Its expression was frequently detected in brain metastases from breast cancers, with up to 90% expressing B7-H3. However, not all brain metastases showed high levels of expression, with those from colorectal and renal tumours showing a low frequency of B7-H3 expression (0/14 and 2/16, respectively). The prevalence of B7-H3 expression in breast cancers and breast cancer brain metastases indicates potential opportunities for B7-H3 targeted therapies in breast cancer management.

A high-content screen of FDA approved drugs to enhance CAR T cell function: ingenol-3-angelate improves B7-H3-CAR T cell activity by upregulating B7-H3 on the target cell surface via PKCα activation.

BACKGROUND: CAR T cell therapy is a promising approach to improve outcomes and decrease toxicities for patients with cancer. While extraordinary success has been achieved using CAR T cells to treat patients with CD19-positive malignancies, multiple obstacles have so far limited the benefit of CAR T cell therapy for patients with solid tumors. Novel manufacturing and engineering approaches show great promise to enhance CAR T cell function against solid tumors. However, similar to single agent chemotherapy approaches, CAR T cell monotherapy may be unable to achieve high cure rates for patients with difficult to treat solid tumors. Thus, combinatorial drug plus CAR T cell approaches are likely required to achieve widespread clinical success. METHODS: We developed a novel, confocal microscopy based, high-content screen to evaluate 1114 FDA approved drugs for the potential to increase expression of the solid tumor antigen B7-H3 on the surface of osteosarcoma cells. Western blot, RT-qPCR, siRNA knockdown and flow cytometry assays were used to validate screening results and identify mechanisms of drug-induced B7-H3 upregulation. Cytokine and cytotoxicity assays were used to determine if drug pre-treatment enhanced B7-H3-CAR T cell effector function. RESULTS: Fifty-five drugs were identified to increase B7-H3 expression on the surface of LM7 osteosarcoma cells using a novel high-content, high-throughput screen. One drug, ingenol-3-angelate (I3A), increased B7-H3 expression by up to 100%, and was evaluated in downstream experiments. Validation assays confirmed I3A increased B7-H3 expression in a biphasic dose response and cell dependent fashion. Mechanistic studies demonstrated that I3A increased B7-H3 (CD276) mRNA, total protein, and cell surface expression via protein kinase C alpha activation. Functionally, I3A induced B7-H3 expression enhanced B7-H3-CAR T cell function in cytokine production and cytotoxicity assays. CONCLUSIONS: This study demonstrates a novel high-content and high-throughput screen can identify drugs to enhance CAR T cell activity. This and other high-content technologies will pave the way to develop clinical trials implementing rational drug plus CAR T cell combinatorial therapies. Importantly, the technique could also be repurposed for an array of basic and translational research applications where drugs are needed to modulate cell surface protein expression.

Bioinformatic Analysis and Clinical Case Studies Identify CD276 as a Promising Diagnostic Biomarker for Clear Cell Renal Cell Carcinoma.

OBJECTIVE: This study aimed to explore the relationship between CD276 and clear cell renal carcinoma (ccRCC) and assess the diagnostic value of CD276 in ccRCC. METHODS: Expression levels of CD276 in ccRCC and para-cancer tissues were compared and analyzed retrospectively using data obtained from TCGA and GEO databases. The clinical data was analyzed prospectively. Immunohistochemistry and RT-PCR analyses were used to analyze the expression of CD276 at the mRNA and protein levels. These analyses compared the expression between ccRCC tissues and para-cancer tissues obtained from 70 patients with ccRCC. Next, ELISA was used to analyze peripheral blood samples from 70 patients with ccRCC and 72 healthy individuals, facilitating the differentiation of ccRCC patients from normal controls. Finally, we utilized the Kaplan-Meier method to generate ROC curves for assessing the diagnostic value of CD276 for ccRCC. RESULTS: Analysis of TCGA and GEO data revealed that the mRNA expression of CD276 was higher in ccRCC tissues than in para-cancer tissues (P < .05). Clinical validation using IHC and RT-PCR confirmed that the expression of CD276 was higher in ccRCC tissues than in para-cancer tissues, both at the mRNA and protein levels (P < .05). ELISA demonstrated that the expression of CD276 was higher in ccRCC patients than in normal individuals, and patients with a higher pathological grade showed higher expression of CD276 in the peripheral blood than those with a lower pathological grade (P < .05). ROC curves drawn from the above three datasets demonstrated that CD276 had a high diagnostic value for ccRCC (AUC = .894, .795, .938, respectively). CONCLUSION: The expression of CD276 was higher in ccRCC tissues and positively associated with the pathological grade. Therefore, CD276 may serve as a molecular biomarker for ccRCC prediction.

Expression and function of VISTA on myeloid cells.

V-domain containing Ig Suppressor of T cell Activation (VISTA) is predominantly expressed on myeloid cells and functions as a ligand/receptor/soluble molecule. In inflammatory responses and immune responses, VISTA regulates multiple functions of myeloid cells, such as chemotaxis, phagocytosis, T cell activation. Since inflammation and immune responses are critical in many diseases, VISTA is a promising therapeutic target. In this review, we will describe the expression and function of VISTA on different myeloid cells, including neutrophils, monocytes, macrophages, dendritic cells (DCs), myeloid-derived suppressor cells (MDSCs). In addition, we will discuss whether the functions of VISTA on these cells impact the disease processing.

Fully human anti-B7-H3 recombinant antibodies inhibited tumor growth by increasing T cell infiltration.

Mortality due to malignant tumors is one of the major factors affecting the life expectancy of the global population. Therapeutic antibodies are a cutting-edge treatment method for restricting tumor growth. B7-H3 is highly expressed in tumor tissues, but rarely in normal tissues. B7-H3 is closely associated with poor prognosis in patients with tumors. B7-H3 is an important target for antitumor therapy. In this study, the fully human anti-B7H3 single-chain antibodies (scFvs) were isolated and screened from the fully human phage immune library with B7H3 as the target. The antibodies screened from a fully human phage library had low immunogenicity and high affinity, which was more beneficial for clinical application. Leveraging B7-H3 scFvs as a foundation, we constructed two distinct recombinant antibody formats, scFv-Fc and IgG1, characterized by elevated affinity and a prolonged half-life. The results demonstrated that the recombinant antibodies had high specificity and affinity for the B7-H3 antigen and inhibited tumor cell growth by enhancing the ADCC. After treatment with anti-B7H3 recombinant antibody, the number of infiltrating T cells in the tumor increased and the secretion of IFN- γ by infiltrating T cells increased in vivo. Additionally, the use of pleural fluid samples obtained from tumor-afflicted patients revealed the ability of anti-B7-H3 recombinant antibodies to reverse CD8+ T cell exhaustion. In summary, we screened the fully human anti-B7H3 recombinant antibodies with specificity and high affinity that increase immune cell infiltration and IFN-γ secretion, thereby inhibiting tumor cell growth to a certain extent. This finding provides a theoretical basis for the development of therapeutic tumor antibodies and could help promote further development of antibody-based drugs.

Interplay between B7-H3 and HLA class I in the clinical course of pancreatic ductal adenocarcinoma.

Human leukocyte antigen (HLA) class I defects are associated with cancer progression. However, their prognostic significance is controversial and may be modulated by immune checkpoints. Here, we investigated whether the checkpoint B7-H3 modulates the relationship between HLA class I and pancreatic ductal adenocarcinoma (PDAC) prognosis. PDAC tumors were analyzed for the expression of B7-H3, HLA class I, HLA class II molecules, and for the presence of tumor-infiltrating immune cells. We observed defective HLA class I and HLA class II expressions in 75% and 59% of PDAC samples, respectively. HLA class I and B7-H3 expression were positively related at mRNA and protein level, potentially because of shared regulation by RELA, a sub-unit of NF-kB. High B7-H3 expression and low CD8+ T cell density were indicators of poor survival, while HLA class I was not. Defective HLA class I expression was associated with unfavorable survival only in patients with low B7-H3 expression. Favorable survival was observed only when HLA class I expression was high and B7-H3 expression low. Our results provide the rationale for targeting B7-H3 in patients with PDAC tumors displaying high HLA class I levels.

Dimerization of the 4Ig isoform of B7-H3 in tumor cells mediates enhanced proliferation and tumorigenic signaling.

B7-H3 (CD276) has two isoforms (2Ig and 4Ig), no confirmed cognate receptor, and physiological functions that remain elusive. While differentially expressed on many solid tumors correlating with poor survival, mechanisms of how B7-H3 signals in cis (tumor cell) versus in trans (immune cell co-regulator) to elicit pro-tumorigenic phenotypes remain poorly defined. Herein, we characterized a tumorigenic and signaling role for tumor cell-expressed 4Ig-B7-H3, the dominant human isoform, in gynecological cancers that could be abrogated upon CRISPR/Cas9 knockout of B7-H3; tumorigenesis was rescued upon re-expression of 4Ig-B7-H3. Size exclusion chromatography revealed dimerization states for the extracellular domains of both human 4Ig- and murine 2Ig-B7-H3. mEGFP lifetimes of expressed 4Ig-B7-H3-mEGFP fusions determined by FRET-FLIM assays confirmed close-proximity interactions of 4Ig-B7-H3 and identified two distinct homo-FRET lifetime populations, consistent with monomeric and homo-dimer interactions. In live cells, bioluminescence imaging of 4Ig-B7-H3-mediated split luciferase complementation showed dimerization of 4Ig-B7-H3. To separate basal from dimer state activities in the absence of a known receptor, C-terminus (cytosolic) chemically-induced dimerization of 4Ig-B7-H3 increased tumor cell proliferation and cell activation signaling pathways (AKT, Jak/STAT, HIF1α, NF-κß) significantly above basal expression of 4Ig-B7-H3 alone. These results revealed a new, dimerization-dependent intrinsic tumorigenic signaling role for 4Ig-B7-H3, likely acting in cis, and provide a therapeutically-actionable target for intervention of B7-H3-dependent tumorigenesis.

Dual silencing of tumor-intrinsic VISTA and CTLA-4 stimulates T-cell mediated immune responses and inhibits MCF7 breast cancer development.

BACKGROUND: As inhibitory immune checkpoint molecules, cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and V-domain Ig suppressor of T-cell activation (VISTA) can be expressed in tumoral cells and facilitate immune evasion of tumoral cells. Herein, we studied the significance of tumor-intrinsic CTLA-4 and VISTA silencing in tumor development and inflammatory factors expression in a co-culture system with MCF7 and T-cells. METHODS: MCF7 cells were transfected with 60 pmol of CTLA-siRNA, VISTA-siRNA, and dual VISTA-/CTLA-4-siRNA. The MTT assay was performed to study the effect of CTLA-4 and VISTA knockdown on the viability of MCF7 cells. Colony formation and wound-healing assays were performed to investigate the effect of CTLA-4 and VISTA silencing on the clonogenicity and migration of MCF7 cells. Flow cytometry was used to study the significance of CTLA-4 and VISTA knockdown on the apoptosis and cell cycle of MCF7 cells. Also, a co-culture system with MCF7 and T-cells was developed to study the expression levels of IL-2, IFN-γ, TNF-α, TGF-ß, and IL-10 following CTLA-4 and VISTA knockdown. The expression levels of caspase3, Bax, Bcl2, and MMP-9 were also investigated using quantitative real-time PCR. Finally, the TCGA Breast Cancer and GSE45827 datasets were analyzed to study the potential prognostic values of VISTA and CTLA-4, their expression difference in luminal A breast cancer and non-tumoral tissues, and their correlation in luminal A breast cancer tissues. RESULTS: Combined knockdown of tumor-intrinsic VISTA and CTLA-4 is superior in upregulating IL-2, IFN-γ, and TNF-α, downregulating TGF-ß and IL-10 in T lymphocytes. Also, the combined silencing arrests the cell cycle at the sub-G1 phase, decreases migration, inhibits clonogenicity, and reduces cell viability of MCF7 cells. This combined treatment upregulates caspase 9 and BAX and downregulates MMP-9 in MCF7 cells. Our in-silico results have demonstrated a significant positive correlation between CTLA-4 and VISTA in luminal A breast cancer. CONCLUSION: The additive effect of the combined knockdown of tumor-intrinsic VISTA and CTLA-4 can substantially upregulate pro-inflammatory factors, downregulate anti-inflammatory factors, and inhibit tumor development in MCF7 cells. The significant positive correlation between VISTA and CTLA-4 in luminal A breast cancer might support the idea that a network of inhibitory immune checkpoint molecules regulates anti-tumoral immune responses; thus, combinational immune checkpoint molecules blockade can be suggested.

Disruption of LPA-LPAR1 pathway results in lung tumor growth inhibition by downregulating B7-H3 expression in fibroblasts.

BACKGROUND: Lysophosphatidic acids (LPAs) belong to a class of bioactive lysophospholipids with multiple functions including immunomodulatory roles in tumor microenvironment (TME). LPA exerts its biological effects via its receptors that are highly expressed in fibroblasts among other cell types. As cancer-associated fibroblasts (CAFs) are a key component of the TME, it is important to understand LPA signaling and regulation of receptors in fibroblasts or CAFs and associated regulatory roles on immunomodulation-related molecules. METHODS: Cluster analysis, immunoblotting, real-time quantitative-PCR, CRISPR-Cas9 gene editing system, immunohistochemical staining, coculture model, and in vivo xenograft model were used to investigate the effects of LPA-LPAR1 on B7-H3 in tumor promotion of CAFs. RESULTS: In this study, we found that LPAR1 and CD276 (B7-H3) were generally highly expressed in fibroblasts with good expression correlation. LPA induced B7-H3 up-expression through LPAR1, and stimulated fibroblasts proliferation that could be inhibited by silencing LPAR1 or B7-H3 as well as small molecule LPAR1 antagonist (Ki16425). Using engineered fibroblasts and non-small cell lung carcinoma (NSCLC) cell lines, subsequent investigations demonstrated that CAFs promoted the proliferation of NSCLC in vitro and in vivo, and such effect could be inhibited by knocking out LPAR1 or B7-H3. CONCLUSION: The present study provided new insights for roles of LPA in CAFs, which could lead to the development of innovative therapies targeting CAFs in the TME. It is also reasonable to postulate a combinatory approach to treat malignant fibrous tumors (such as NSCLC) with LPAR1 antagonists and B7-H3 targeting therapies.

Soluble B7-H3 in Colorectal Cancer.

We present the results of comparative ELISA of the concentration of soluble form of immunity checkpoint B7-H3 (sB7-H3) in the serum of patients with colorectal cancer (CRC) at different stages before treatment and healthy control donors. The analysis revealed a statistically significant difference between the median levels of sB7-H3 in the blood serum of CRC patients (19.66 ng/ml) and healthy donors (16.76 ng/ml) (p=0.0025). ROC analysis showed 62.9% sensitivity and 56.7% specificity for CRC patients (cut-off 17.62 ng/ml; p=0.0028). An association of sB7-H3 levels with tumor progression was revealed. We demonstrated that sB7-H3 levels were significantly lower in patients with regional metastases than in patients without metastases (p=0.039) and that sB7-H3 concentration tends to decrease at the late stages of the disease. Thus, high serum level of sB7-H3 in CRC patients can be a favorable prognostic factor in future.

Determination of PDK1, SLC2A1, EGFR, PTEN, CD276 Gene Expression Levels and IDH1 Gene R132H Polymorphism in Brain Tumor Tissues.

AIM: To determine IDH1 R132H codon and the mRNA levels of PDK1, SLC2A1, EGFR, PTEN, and CD276 genes in brain tumors. MATERIAL AND METHODS: This study included 15 brain tumor tissues [pituitary adenoma (1), pilocytic astrocytoma (1), mixed meningioma (2), mesothelial meningioma (2), atypical meningioma (1), immature teratoma (1), glioblastoma (4), meningioma (2), and bladder cancer metastasis (1)]. The expression levels of genes in brain tumor tissues were analyzed using real-time PCR. Sanger sequencing was performed to identify the IDH1 gene R132H codon. RESULTS: All cases were wild-type in terms of IDH1 R132H: nucleotide 395 G > A; codon CGT > CAT. The mRNA level of PDK1 was lower in grade I tumor tissues (0.675-fold) and increased in grades II-III-IV (7.135, 16.912, and 7.081-fold, respectively) (p < 0.001). The mRNA level of SLC2A1 decreased in all grades I-II-III-IV [(0.424-, 0.093-, 0.234 (p < 0.001), and 0.141-fold (p < 0.005), respectively)]. The mRNA level of EGFR increased in all grades I-II-III-IV [1.388, 5.452 (p < 0.017), 4.624-, and 4.137-fold, respectively]. The mRNA level of PTEN increased in grades I-II-III [1.802-, 1.702-, and 1.5-fold, respectively] and decreased in grade IV (0.176-fold). The mRNA level of CD276 increased in all grades I-II-III-IV [1.8-, 5.756-(p < 0.001), 10.303 (p < 0.001), and 2.5-fold, respectively]. CONCLUSION: We obtained similar findings for previously reported PDK1, EGFR, PTEN, and CD276 gene expression levels. In contrast, SLC2A1 expression was markedly downregulated, as reported in other tumor studies. These findings may be due to the unique nature of brain tumor tissues. Additionally, a decrease in PTEN gene expression has been observed in grade IV brain tumors, including glioblastoma and meningioma. Although the size of the analyzed study group was limited, the gene expression results showed similarities in the behavior of genes during cancer staging.

Hub gene of disulfidptosis-related immune checkpoints in breast cancer.

Disulfidptosis and immune checkpoint genes play an important role in tumor treatment. But there has been less research on the relationship between disulfidptosis and immune checkpoint of breast cancer. The objective of this study was to identify the hub genes of disulfidptosis- related immune checkpoints in breast cancer. We downloaded breast cancer expression data from The Cancer Genome Atlas database. The expression matrix of disulfidptosis-related immune checkpoints genes was established by mathematical method. A protein-protein interaction networks was established based on this expression matrix, and differential expression analysis was performed between normal and tumor samples. Additionally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to functionally annotate putative diferentially expressed genes. Two hub genes CD80 and CD276 were obtained by mathematical statistics and machine learning. Differential expression of these two genes, prognostic survival analysis, combined diagnostic ROC curve and immune results all showed that they were closely related to the occurrence, development and death of breast tumors. The results of this study open up a new way to explore immunotherapy for breast cancer.

Influence of ADT on B7-H3 expression during CRPC progression from hormone-naïve prostate cancer.

Androgen deprivation therapy (ADT) is the standard care for advanced prostate cancer (PCa) patients. Unfortunately, although tumors respond well initially, they enter dormancy and eventually progress to fatal/incurable castration-resistant prostate cancer (CRPC). B7-H3 is a promising new target for PCa immunotherapy. CD276 (B7-H3) gene has a presumptive androgen receptor (AR) binding site, suggesting potential AR regulation. However, the relationship between B7-H3 and AR is controversial. Meanwhile, the expression pattern of B7-H3 following ADT and during CRPC progression is largely unknown, but critically important for identifying patients and determining the optimal timing of B7-H3 targeting immunotherapy. In this study, we performed a longitudinal study using our unique PCa patient-derived xenograft (PDX) models and assessed B7-H3 expression during post-ADT disease progression. We further validated our findings at the clinical level in PCa patient samples. We found that B7-H3 expression was negatively regulated by AR during the early phase of ADT treatment, but positively associated with PCa proliferation during the remainder of disease progression. Our findings suggest its use as a biomarker for diagnosis, prognosis, and ADT treatment response, and the potential of combining ADT and B7-H3 targeting immunotherapy for hormone-naïve PCa treatment to prevent fatal CRPC relapse.

B7-H3 immunoregulatory roles in cancer.

B7 homolog 3 (B7-H3, also called CD276) is a checkpoint of B7 family that is aberrantly and consistently expressed in several human cancers, and its overexpression correlates with weak prognosis. B7-H3 is expressed on a number of cells, and it acts as a driver of immune evasion. This is mediated through hampering T cell infiltration and promoting exhaustion of CD8+ T cells. Increased B7-H3 activity also promotes macrophage polarity toward pro-tumor type 2 (M2) phenotype. In addition, high B7-H3 activity induces aberrant angiogenesis to promote hypoxia, a result of which is resistance to common immune checkpoint inhibitor (ICI) therapy. This is mediated through the impact of hypoxia on dampening CD8+ T cell recruitment into tumor area. The immunosuppressive property of B7-H3 offers insights into targeting this checkpoint as a desired approach in cancer immunotherapy. B7-H3 can be a target in blocking monoclonal antibodies (mAbs), combination therapies, chimeric antigen receptor-modified T (CAR-T) cells and bispecific antibodies.

Optimal combination of immune checkpoint and senescence molecule predicts adverse outcomes in patients with acute myeloid leukemia.

BACKGROUND: High expression of immune checkpoints (ICs) and senescence molecules (SMs) contributes to T cell dysfunction, tumor escape, and progression, but systematic evaluation of them in co-expression patterns and prognosis in acute myeloid leukemia (AML) was lacking. METHODS: Three publicly available datasets (TCGA, Beat-AML, and GSE71014) were first used to explore the effect of IC and SM combinations on prognosis and the immune microenvironment in AML, and bone marrow samples from 68 AML patients from our clinical center (GZFPH) was further used to validate the findings. RESULTS: High expression of CD276, Bcl2-associated athanogene 3 (BAG3), and SRC was associated with poor overall survival (OS) of AML patients. CD276/BAG3/SRC combination, standard European Leukemia Net (ELN) risk stratification, age, and French-American-British (FAB) subtype were used to construct a nomogram model. Interestingly, the new risk stratification derived from the nomogram was better than the standard ELN risk stratification in predicting the prognosis for AML. A weighted combination of CD276 and BAG3/SRC positively corrected with TP53 mutation, p53 pathway, CD8+ T cells, activated memory CD4+ T cells, T-cell senescence score, and Tumor Immune Dysfunction and Exclusion (TIDE) score estimated by T-cell dysfunction. CONCLUSION: High expression of ICs and SMs was associated with poor OS of AML patients. The co-expression patterns of CD276 and BAG3/SRC might be potential biomarkers for risk stratification and designing combinational immuno-targeted therapy in AML.Key MessagesHigh expression of CD276, BAG3, and SRC was associated with poor overall survival of AML patients.The co-expression patterns of CD276 and BAG3/SRC might be potential biomarkers for risk stratification and designing combinational immuno-targeted therapy in AML.

The Concordant Disruption of B7/CD28 Immune Regulators Predicts the Prognosis of Oral Carcinomas.

Immune modulation is a critical factor in determining the survival of patients with malignancies, including those with oral squamous cell carcinoma (OSCC) and head and neck SCC (HNSCC). Immune escape or stimulation may be driven by the B7/CD28 family and other checkpoint molecules, forming ligand-receptor complexes with immune cells in the tumor microenvironment. Since the members of B7/CD28 can functionally compensate for or counteract each other, the concomitant disruption of multiple members of B7/CD28 in OSCC or HNSCC pathogenesis remains elusive. Transcriptome analysis was performed on 54 OSCC tumors and 28 paired normal oral tissue samples. Upregulation of CD80, CD86, PD-L1, PD-L2, CD276, VTCN1, and CTLA4 and downregulation of L-ICOS in OSCC relative to the control were noted. Concordance in the expression of CD80, CD86, PD-L1, PD-L2, and L-ICOS with CD28 members was observed across tumors. Lower ICOS expression indicated a worse prognosis in late-stage tumors. Moreover, tumors harboring higher PD-L1/ICOS, PD-L2/ICOS, or CD276/ICOS expression ratios had a worse prognosis. The survival of node-positive patients was further worsened in tumors exhibiting higher ratios between PD-L1, PD-L2, or CD276 and ICOS. Alterations in T cell, macrophage, myeloid dendritic cell, and mast cell populations in tumors relative to controls were found. Decreased memory B cells, CD8+ T cells, and Tregs, together with increased resting NK cells and M0 macrophages, occurred in tumors with a worse prognosis. This study confirmed frequent upregulation and eminent co-disruption of B7/CD28 members in OSCC tumors. The ratio between PD-L2 and ICOS is a promising survival predictor in node-positive HNSCC patients.

Analysis of genetically determined gene expression suggests role of inflammatory processes in exfoliation syndrome.

BACKGROUND: Exfoliation syndrome (XFS) is an age-related systemic disorder characterized by excessive production and progressive accumulation of abnormal extracellular material, with pathognomonic ocular manifestations. It is the most common cause of secondary glaucoma, resulting in widespread global blindness. The largest global meta-analysis of XFS in 123,457 multi-ethnic individuals from 24 countries identified seven loci with the strongest association signal in chr15q22-25 region near LOXL1. Expression analysis have so far correlated coding and a few non-coding variants in the region with LOXL1 expression levels, but functional effects of these variants is unclear. We hypothesize that analysis of the contribution of the genetically determined component of gene expression to XFS risk can provide a powerful method to elucidate potential roles of additional genes and clarify biology that underlie XFS. RESULTS: Transcriptomic Wide Association Studies (TWAS) using PrediXcan models trained in 48 GTEx tissues leveraging on results from the multi-ethnic and European ancestry GWAS were performed. To eliminate the possibility of false-positive results due to Linkage Disequilibrium (LD) contamination, we i) performed PrediXcan analysis in reduced models removing variants in LD with LOXL1 missense variants associated with XFS, and variants in LOXL1 models in both multiethnic and European ancestry individuals, ii) conducted conditional analysis of the significant signals in European ancestry individuals, and iii) filtered signals based on correlated gene expression, LD and shared eQTLs, iv) conducted expression validation analysis in human iris tissues. We observed twenty-eight genes in chr15q22-25 region that showed statistically significant associations, which were whittled down to ten genes after statistical validations. In experimental analysis, mRNA transcript levels for ARID3B, CD276, LOXL1, NEO1, SCAMP2, and UBL7 were significantly decreased in iris tissues from XFS patients compared to control samples. TWAS genes for XFS were significantly enriched for genes associated with inflammatory conditions. We also observed a higher incidence of XFS comorbidity with inflammatory and connective tissue diseases. CONCLUSION: Our results implicate a role for connective tissues and inflammation pathways in the etiology of XFS. Targeting the inflammatory pathway may be a potential therapeutic option to reduce progression in XFS.

Multi-omics analyses of CD276 in pan-cancer reveals its clinical prognostic value in glioblastoma and other major cancer types.

BACKGROUND: CD276 (also known as B7-H3) is one of the most important immune checkpoints of the CD28 and B7 superfamily, and its abnormal expression is closely associated with various types of cancer. It has been shown that CD276 is able to inhibit the function of T cells, and that this gene may potentially be a promising immunotherapy target for different types of cancer. METHODS: Since few systematic studies have been published on the role of CD276 in cancer to date, the present study has employed single-cell sequencing and bioinformatics methods to analyze the expression patterns, clinical significance, prognostic value, epigenetic alterations, DNA methylation level, tumor immune cell infiltration and immune functions of CD276 in different types of cancer. In order to analyze the potential underlying mechanism of CD276 in glioblastoma (GBM) to assess its prognostic value, the LinkedOmics database was used to explore the biological function and co-expression pattern of CD276 in GBM, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. In addition, a simple validation of the above analyses was performed using reverse transcription-quantitative (RT-q)PCR assay. RESULTS: The results revealed that CD276 was highly expressed, and was often associated with poorer survival and prognosis, in the majority of different types of cancer. In addition, CD276 expression was found to be closely associated with T cell infiltration, immune checkpoint genes and immunoregulatory interactions between lymphoid and a non-lymphoid cell. It was also shown that the CD276 expression network exerts a wide influence on the immune activation of GBM. The expression of CD276 was found to be positively correlated with neutrophil-mediated immunity, although it was negatively correlated with the level of neurotransmitters, neurotransmitter transport and the regulation of neuropeptide signaling pathways in GBM. It is noteworthy that CD276 expression was found to be significantly higher in GBM compared with normal controls according to the RT-qPCR analysis, and the co-expression network, biological function and chemotherapeutic drug sensitivity of CD276 in GBM were further explored. In conclusion, the findings of the present study have revealed that CD276 is strongly expressed and associated with poor prognosis in most types of cancer, including GBM, and its expression is strongly associated with T-cell infiltration, immune checkpoint genes, and immunomodulatory interactions between lymphocytes and non-lymphoid cells. CONCLUSIONS: Taken together, based on our systematic analysis, our findings have revealed important roles for CD276 in different types of cancers, especially GBM, and CD276 may potentially serve as a biomarker for cancer.

Wilms Tumor 1-Associated Protein Expression Is Linked to a T-Cell-Inflamed Phenotype in Pancreatic Cancer.

BACKGROUND: The molecular driving forces of anti-tumor immunity in pancreatic ductal adenocarcinoma (PDAC) remain unclear, which causing great difficulty in identifying an appropriate treatment strategy. AIMS: This study aims to explore the associations between expression of Wilms tumor 1-associated protein (WTAP) and effector T-cell infiltration in PDAC. METHODS: In this study, we explored the association between WTAP expression and infiltration level of CD8+ T cells in PDAC. 178 PDAC samples were selected from The Cancer Genome Atlas (TCGA) database. The associations between diverse immune-cell infiltration, Tumor Mutation Burden (TMB), immune checkpoints, and WTAP expression were performed via R software. Transcriptional hallmarks of anti-tumor immunity and known T-cell-inflamed signature of PDAC were both selected to explore the relevance to WTAP expression. Potential immune checkpoint blockade (ICB) response to different WTAP expression was predicted with tumor immune dysfunction and exclusion (TIDE) algorithm. RESULTS: WTAP was closely linked to CD8+ T-cell infiltration (r ≥ 0.5, P value < 0.05) and did not show notable association with TMB in PDAC. WTAP positively linked to T-cell-inflamed gene expression profiles (GEP) (IL2RB, IL2RA, ZAP70, ITK, CD3E, CD38, CD27, CD276, CD8A, CMKLR1, CXCR6, HLA-DQA1, HLA-DRB1, HLA-E, NKG7, and STAT1), cytolytic activity (GZMA and PRF1), various immune checkpoints (IDO1, CD274, HAVCR2, PDCD1, CTLA4, LAG3, and PDCD1LG2) and 4-chemokine signature (CCL4, CCL5, CXCL9, and CXCL10). Besides, increased expression of WTAP was related to a higher TIDE score. CONCLUSIONS: WTAP marks PDAC tumors with an active anti-tumor phenotype and might help the identification of PDAC patients who might benefit from immunotherapies.