Neutrophils are conspicuous components of gastric cancer (GC) tumors, increasing with tumor progression and poor patient survival. However, the phenotype, regulation and clinical relevance of neutrophils in human GC are presently unknown. Most intratumoral neutrophils showed an activated CD54⁺ phenotype and expressed high level B7-H3. Tumor tissue culture supernatants from GC patients induced the expression of CD54 and B7-H3 on neutrophils in time-dependent and dose-dependent manners. Locally enriched CD54⁺ neutrophils and B7-H3⁺ neutrophils positively correlated with increased granulocyte-macrophage colony stimulating factor (GM-CSF) detection ex vivo; and in vitro GM-CSF induced the expression of CD54 and B7-H3 on neutrophils in both time-dependent and dose-dependent manners. Furthermore, GC tumor-derived GM-CSF activated neutrophils and induced neutrophil B7-H3 expression via JAK-STAT3 signaling pathway activation. Finally, intratumoral B7-H3⁺ neutrophils increased with tumor progression and independently predicted reduced overall survival. Collectively, these results suggest B7-H3⁺ neutrophils to be potential biomarkers in GC.

Keywords: B7-H3; Gastric cancer; Granulocyte-macrophage colony stimulating factor; Neutrophils.

MiR-29a belongs to one of the subtypes of miRNAs known as non-coding single-stranded RNAs and is preferentially expressed in normal tissues. B7-H3, a member of the B7/CD28 immunoglobulin superfamily, was shown to be overexpressed in several solid malignant tumors, including colon cancer. In addition, it is associated with tumor progression and poor prognosis. We used immunohistochemical and Western blotting to assess B7-H3 protein expression levels in colon cancer and adjacent normal tissues and then compared their relationships with clinicopathological factors. Quantitative real-time reverse-transcription PCR was used to assess B7-H3 and miRNA-29a mRNA expression levels, and then their relationship and clinical significance were evaluated. In addition, colon cancer Caco-2 cells, which constitutively overexpress B7-H3, were transfected with lentivirus particles for miR-29a upregulation. Invasion and migration assays were carried out in vitro along with the establishment of a subcutaneous xenograft model in vivo to determine the role of miRNA-29a in colon cancer progression. The B7-H3 protein showed elevated expression in colon carcinoma and was relevant to TNM staging, lymph node metastasis, and reduced survival. Meanwhile, miR-29a was preferentially expressed in normal colon tissues, while B7-H3 transcript levels had no marked differences between tumor and normal tissue specimens. In vitro, miR-29a upregulation resulted in reduced B7-H3 expression. Furthermore, miR-29a upregulation reduced the invasive and migratory abilities of colon carcinoma cells. In animal models, upregulation of miR-29a slowed down the growth of subcutaneous xenotransplanted tumors and resulted in prolonged survival time. MiR-29a downregulates B7-H3 expression and accordingly inhibits colon cancer progression, invasion, and migration, indicating miR-29a and B7-H3 might represent novel molecular targets for advanced immunotherapy in colon cancer.

Keywords: B7-H3; Colon cancer; Immunotherapy; MiR-29a.

Targeting B7-H3 chimeric antigen receptor (CAR) T cells has antitumor potential for therapy of non-small cell lung cancer (NSCLC) in preclinical studies. However, CAR T cell therapy remains a formidable challenge for the treatment of solid tumors due to the heterogeneous and immunosuppressive tumor microenvironment (TME). Nanozymes exhibit merits modulating the immunosuppression of the tumor milieu. Here, a synergetic strategy by combination of nanozymes and CAR T cells in solid tumors is described. This nanozyme with dual photothermal-nanocatalytic properties is endowed to remodel TME by destroying its compact structure. It is found that the B7-H3 CAR T cells infused in mice engrafted with the NSCLC cells have superior antitumor activity after nanozyme ablation of the tumor. Importantly, it is found that the changes altered immune-hostile cancer environment, resulting in enhanced activation and infiltration of B7-H3 CAR T cells. The first evidence that the process of combination nanozyme therapy effectively improves the therapeutic index of CAR T cells is presented. Thus, this study clearly supports that the TME-immunomodulated nanozyme is a promising tool to improve the therapeutic obstacles of CAR T cells against solid tumors.

Keywords: B7-H3 immune checkpoint; chimeric antigen receptor T cell; immunotherapy; nanozyme; photothermal-nanocatalytic effect.

Purpose: The purpose of this study was to investigate B7 homolog 3 (B7-H3) expression patterns and define its associations with programmed cell-death ligand 1 (PD-L1), pathological features, and survival in patients with cervical cancer.

Patients and methods: Immunohistochemical staining was used to investigate B7-H3 and PD-L1 expression in tissue microarrays from 552 patients with stage IB1 and IIA1 cervical cancer, including 406 with squamous cell carcinoma and 146 with endocervical adenocarcinoma.

Results: B7-H3 was expressed in the tumor cells (TCs) of 32.1% of the samples as well as in the stromal cells of 92.9% of the specimens. B7-H3 was co-expressed with PD-L1 in 21.0% of the samples, while only one or the other was expressed in 41.7% of the samples. B7-H3 expression in TCs was more frequent in squamous cell carcinoma, PD-L1-positive samples, and tissues from patients with lymph node metastasis; moreover, its expression was an independent predictor of shorter survival.

Conclusion: B7-H3 positivity in TCs is a promising prognostic biomarker, and targeting B7-H3 alone or in combination with PD-1/PD-L1 may be a potential immunotherapeutic strategy for patients with cervical cancer.

Keywords: B7 homolog 3; carcinoma of the cervix; immunotherapy; programmed cell-death ligand 1.

Tumor-infiltrating T cells are highly expressive of inhibitory receptor/immune checkpoint molecules that bind to ligand expressed by tumor cells and antigen-presenting cells, and eventually lead to T cell dysfunction. It is a hot topic to restore T cell function by targeting immune checkpoint. In recent years, immunotherapy of blocking immune checkpoint and its receptor, such as PD-L1/PD-1 targeted therapy, has made effective progress, which brings hope for patients with advanced malignant tumor. However, only a few patients benefit from directly targeting these checkpoints or their receptors by small compounds or antibodies. Since the complexity of the regulation of immune checkpoints in tumor cells, further research is needed to identify the novel endogenous regulators of immune checkpoints which can help for developing effective drug target to improve the effect of immunotherapy. Here, we verified that microRNA-326 (miR-326) repressed the gene expression of immune checkpoint molecules PD-L1 and B7-H3 in lung adenocarcinoma (LUAD). We detected that the expression of miR-326 in LUAD tissue was negatively correlated with PD-L1/B7-H3. The repression of PD-L1 and B7-H3 expression through miR-326 overexpression leads to the modification the cytokine profile of CD8⁺ T cells and decreased migration capability of tumor cells. Meanwhile, the downregulation of miR-326 promoted tumor cell migration. Moreover, blocking PD-L1 and B7-H3 attenuated the tumor-promoting effect induced by miR-326 inhibitor. In tumor-bearing mice, the infiltration of CD8⁺ T cells was significantly increased and the expression of TNF-α, and IFN-γ was significantly enhanced which contributed to tumor progression after miR-326 overexpression. Collectively, miR-326 restrained tumor progression by downregulating PD-L1 and B7-H3 expression and increasing T cell cytotoxic function in LUAD. Our findings revealed a novel perspective on the complex regulation of immune checkpoint molecules. A new strategy of using miR-326 in tumor immunotherapy is proposed.

The cosignal molecule B7-H3 is gaining attention due to its abnormal expression and abundant signal transduction in many types of malignancies. B7-H3-induced signaling includes at least three cascades: PI3K/AKT, JAK2/STAT3, and Raf/MEK/ERK1/2, which are also involved in epidermal growth factor receptor- (EGFR-) triggered signaling in lung adenocarcinoma cells. However, the correlation between B7-H3-induced signaling and EGFR signaling, and between B7-H3-targeted immunotherapy and EGFR-targeted therapy in lung adenocarcinoma, remains to be elucidated. Herein we find that knockout of B7-H3 gene decreased cell survival and increased EGFR-tyrosine kinase inhibitor gefitinib susceptibility of both H3B7-H3 deletion resulted in dramatic reduction of phosphorylation level of AKT and STAT3 in H3B7-H3 deletion both in H3B7-H3 ablation had significant synergistic effects with gefitinib in HCC827 cells. Collectively, our study reveals B7-H3-induced signaling in lung adenocarcinoma cell lines with divergent EGFR mutations, and a translational potential of combined targeted therapy of B7-H3 and EGFR in lung adenocarcinoma with EGFR Del E746-A750 mutation.

Receptors expressed on the plasma membrane and their interacting partners critically regulate cellular communication during homeostasis and disease, and as such represent main therapeutic targets. Despite its importance for drug development, receptor-ligand proteomics has remained a daunting field, in part because of the challenges associated to the study of membrane-expressed proteins. Here, to enable sensitive detection of receptor-ligand interactions in high throughput, we implement a new platform, the Conditioned Media AlphaScreen, for interrogation of a library consisting of most single transmembrane human proteins. Using this method to study key immune receptors, we identify and further validate the interleukin receptor IL20RA as the first binding partner for the checkpoint inhibitor B7-H3. Further, KIR2DL5, a natural killer cell protein that had remained orphan, is uncovered as a functional binding partner for the poliovirus receptor (PVR). This interaction is characterized using orthogonal assays, which demonstrate that PVR specifically engages KIR2DL5 on natural killer cells leading to inhibition of cytotoxicity. Altogether, these results reveal unappreciated links between protein families that may importantly influence receptor-driven functions during disease. Applicable to any target of interest, this technology represents a versatile and powerful approach for elucidation of receptor-ligand interactomes, which is essential to understand basic aspects of the biology of the plasma membrane proteins and ultimately inform the development of novel therapeutic strategies.

Keywords: assay development; automation; cancer therapeutics; cell-cell interactions; protein-protein interactions; secretome.

Coinhibitory pathways in the B7-CD28 family provide critical inhibitory signals that regulate immune homeostasis, defense and protect tissue integrity. CD276 (B7-H3) is an important immune checkpoint member of this family, which is induced on antigen-presenting cells (APCs), and plays an important role in the inhibition of T-cell function. We have characterized the CD276 gene of olive flounder, Paralichthys olivaceus. OfCD276 has an ORF of 912 bp that codes for 303 amino acids with a predicted molecular mass of 33 kDa. It is a type I transmembrane protein with a single extracellular V- and C-like Ig domains, a transmembrane region, and a highly diverse cytoplasmic tail. This gene was distinctly expressed in gill, spleen, and skin, and sparsely expressed in other tissues. Pathogen stimulation by VHSV revealed that transcription of OfCD276 was induced on early hours in liver and expressed late in head kidney, spleen, intestine and gill tissues. Flow cytometry analysis of leukocytes revealed the percentage of granulocytes and lymphocytes that expressed OfCD276 molecules on their cell surface was 85.1% and 3.1%, respectively. Our study shows a significant role played by this coinhibitory molecule that participate in the regulation of the cell mediated immune response.

Seeking for the novel biomarkers for Mycoplasma pneumoniae pneumonia (MPP) could be not only helpful for disease diagnosis but also useful for treatment efficacy monitoring. The aim of present study was to evaluate the role of plasma soluble B7-H3 (sB7-H3) in MPP diagnosis and treatment efficacy prediction, and involvement of B7-H3 in MPP disease course. A total of 108 MPP patients and 40 control subjects were recruited into this study for changes of sB7-H3 levels in MPP. In addition, a mouse model of MPP was also established for confirmation of the involvement of sB7-H3 in MPP in vivo. Significantly increased levels of sB7-H3 were found in both mild and severe MPP patients compared to control patients. Moreover, significantly increased level of sB7-H3 was also found in severe MPP patients compared to mild subjects. The ROC curve showed sB7-H3 had severity prediction capacity in mild and severe MPP. Plasma sB7-H3 correlated positively with IFN-r and GM-CSF in mild or severe MPP patients. Moreover, significantly increased level of plasma sB7-H3 level were found in acute phase MPP patients compared to control subjects, whereas significantly decreased level of plasma sB7-H3 was found in recovery phase MPP patients compared to acute phase patients. In addition, decreased levels of sB7-H3 were found in mice from Dexamethasone group compared to LAMP group. Plasma sB7-H3 level might serve as biomarker for severity MPP prediction and treatment efficacy evaluation. Furthermore, direct involvement of B7-H3 was confirmed in vivo during the MPP disease course.

OBJECTIVE

To investigate whether the plasma level of platelet auto- antibodies in ITP patients is related to that of co-stimulatory molecules sB7-H2 and sB7-H3.

METHODS

A total of 61 ITP patients and 25 healthy controls from the First Affiliated Hospital of Soochow University from June 2012 to August 2013 were enrolled in this study. The expression levels of platelet auto-antibodies against 5 glycoproteins (GPIX, GP Ib, GP IIIa, GPIIb and P-selectin) in plasma were detected by flow cytometric immuno-beads array, and the expression of soluable co-stimulatory molecules sB7-H2 and sB7-H3 was measured by ELISA.

RESULTS

The plasma levels of 5 auto-antibodies against platelet membrance glycoproteins significantly increased in ITP patiens (P < 0.01). Compared with healthy controls, sB7-H2 levels increased (P < 0.05), while the sB7-H3 level did not significantly change (r = 0.13, P>> 0.05). However, the correlation analysis showed that sB7-H3 negatively correlated with platelet P-selectin auto-antibody (r = -0.46, P < 0.05), and sB7-H2 and sB7-H3 significantly reduced in ITP patients with positive P-selectin auto-antibody (P < 0.01). In ITP patients, platelet counts negatively correlated with sB7-H2 (r = -0.3907, P < 0.01), but did not correlate with sB7-H3.

CONCLUSIONS

Soluble costimulatory molecule sB7-H2 elevates in ITP patients, and the level of sB7-H3 is associated with auto-antibodies against P-selectin, suggesting that costimulatory molecules B7-H2 and B7-H3 may be involved in the pathogenesis of immune regulation abnormality in ITP.

[This corrects the article DOI: 10.1182/blood-2014-09-603357.].

The immune costimulatory molecule B7-H3 has been shown to be involved in the regulation of murine bone formation. However, the role of B7-H3 in bone metabolic diseases remains unknown. In our study, matrix metalloproteinase 2 (MMP-2) and soluble B7-H3 (sB7-H3) were found to be correlatively up-regulated in the sera of osteoporosis patients. Furthermore, our results showed that MG63 cells treated with MMP-2 inhibitors produced lower amounts of sB7-H3 while cells with recombinant MMP-2 had an increased membrane B7-H3 (mB7-H3) shedding. Therefore, elevated MMP-2 levels resulted in an elevation of serum sB7-H3 and reduction of osteoblastic mB7-H3. B7-H3 knockdown in MG63 cells significantly decreased osteoblastic markers and substantially decreased the number of mineralized nodules after 21days. Thus, B7-H3-deficient MG63 cells exhibited impaired bone formation. These results suggest that mB7-H3 is required for the later phases of osteoblast differentiation and that MMP-2/B7-H3 plays a negative regulatory role in osteoporosis.

OBJECTIVE

To construct a recombinant eukaryotic expression vector containing the human B7-H3Ig gene and investigate the effects of B7-H3 signal on T cells.

METHODS

Being constructed by overlap extension PCR, the recombinant gene fragment of B7-H3Ig was inserted into pGEZ-Term vector. The recombinant pGEZ-Term/B7-H3Ig vector was used to transfect L929 cells to purify B7-H3Ig from conditioned medium harvested from cultured L929/B7-H3Ig cells. T cells were then stimulated with agonistic anti-CD3 mAb in the presence or absence of purified B7-H3Ig, and cell proliferation and the secretion of IL-10 and IFN-γ were analyzed 72 h later.

RESULTS

The concentration of B7-H3Ig was 0.559 mg/mL with a purity of more than 90% after protein G affinity chromatography. B7-H3Ig binding test showed that the putative B7-H3 receptor was presented in activated T cells and the maximum binding capacity was observed 48 h later. B7-H3Ig enhanced, in a dose-dependent manner, the proliferation and IL-10 and IFN-γ secretion of T cells stimulated with agonistic anti-CD3 mAb.

CONCLUSIONS

L929/B7-H3Ig transfectants have been successfully constructed. B7-H3 is a co-stimulator for T cell proliferation and IL-10 and IFN-γ expressions.

OBJECTIVE

To investigate the expression and subcellular distribution of costimulatory molecules B7-H1, B7-H3 and B7-H4 in human hematologic malignancy cell lines.

METHODS

The expression and subcellular distribution of B7-H1, B7-H3 and B7-H4 in 13 human hematologic malignancy cell lines were determined by RT-PCR, qPCR, Western blot and flow cytometry, the peripheral blood mononuclear cells (PB MNC) of 12 volunteers were used as control.

RESULTS

The mRNA of B7-H1, B7-H3 and B7-H4 was widely expressed in PB MNC and hematologic malignancy cell lines, with a lower level of B7-H4. The mRNA expression of 3 molecules was highest in Maver, Z138, and HL-60, respectively, while among them the B7-H3 and B7-H4 had no expression in CZ1. The nuclear and cytoplasmic protein of 3 costimulatory molecules abnormally overexpressed only in hematologic malignancy cell lines, with the highest level in U937, Z138, and Raji, respectively, while the B7-H3 and B7-H4 had no expression in CZ1. There were differences among mRNA expression, nuclear and cytoplasmic protein expression of 3 molecules in cell lines derived from the same type of tumor, but the differences of expression in mRNA and protein levels were not exactly the same. The B7-H3 expression abundance in membrane localization was higher in U937, Maver and Z138, while the membrane protein of B7-H1 and B7-H4 had no or low expression in 13 cell lines.

CONCLUSIONS

The mRNA expression of costimulatory molecules B7-H1, B7-H3 and B7-H4 can be widely detected. The protein level of 3 costimulatory molecules abnormally overexpressed only in hematologic malignancy cell lines, moreover the subcellular localizations mostly was found in nucleus and cytoplasm, while the membrane protein expresses in low level or had no expression. There are differences among the expression of 3 molecules in cell lines derived from the same type of tumor.

B7-H3, a new member of the B7 superfamily, acts as both a T cell costimulator and coinhibitor. Recent studies identified B7-H3 plays a critical role in the development of asthma. But the definitive mechanism is not clear. In this study, we further report that B7-H3 participates in the development of OVA-induced asthma in a murine model. And study its mechanism through the vitro and vivo experiment. Exogenous administration of B7-H3 strongly amplified the inflammatory response and augmented proinflammatory cytokines in vitro and vivo. These B7-H3-associated proinflammatory effects were not dependent on TLR2 signaling, as airway inflammation, eosinophils infiltration and cytokins (IL-4, IL-5, IL-13 and IFN-gamma) augment were still amplified in TLR2-deficient mice after administrated recombinant mouse B7-H3. These results indicated an important role for B7-H3 in the development of Th1 and Th2 cells in a murine model of asthma and its proinflammatory effects are not dependent on TLR2 signaling.

Objective: To investigate the expression and clinical significance of soluble B7-H3 (sB7-H3) in patients with secondary hemophagocytic lymphohistiocytosis (sHLH).

Methods: The plasma samples of 85 newly diagnosed sHLH patients from December 2012 to April 2018 were collected. The patients were divided into lymphoma-related HLH（LHLH）group and infection-related HLH（IHLH）group. The expression of sB7-H3 in plasma was detected by ELISA, and the clinical data were collected for analysis. Fifteen healthy people were chosen as control group.

Results: The expression level of sB7-H3 in lymphoma-related HLH and infection-related HLH group significant increased as compared with the control group, (P＜0.05), and the expression level of sB7-H3 in lymphoma-related HLH group was significant higher than that in infection-related HLH group [(35.75± 9.90) vs (28.70±8.95) ng/ml)] (P＜0.001). There were no significant statistical difference in the expression of some clinical factors (including age, fever, splenomegaly, ANC, Plt, FIB, calcium ion, serum albumin, LDH, serum ferritin, sCD25) in lymphoma-related HLH and infection-related HLH group (P＞0.05). The evaluation of expression and significance of sB7-H3 in sHLH by using ROC curve, showed that the area under ROC curve comparison of patients in lymphoma-related HLH group and infection-related HLH group was 0.718 (95% CI 0.610-0.810) (P=0.0002), and predicting the sensitivity and specificity of the lymphoma-related HLH patients were 77.36% and 59.38%, respectively. The best cut-off value of patients in sB7-H3 was 29.81 ng/ml, the overall survival time of sB7-H3 high-expression group (≥29.81 ng/ml) was significant shorter than that in low-expression group (＜29.81 ng/ml) (24 vs 440 d) (P＜0.001). The clinical factors affecting the survival status of sHLH patients were neutrophils, albumin, serum ferritin, serum calcium ions and sB7-H3 levels.

Conclusion: sB7-H3 associates with poor prognosis of sHLH patients, and may be involved in disease progression.

题目: 可溶性B7-H3在继发性噬血细胞性淋巴组织细胞增多症患者中的表达及临床意义.

目的: 探讨可溶性B7-H3（sB7-H3）在继发性噬血细胞性淋巴组织细胞增多症（sHLH）患者中的诊断及预后意义.

方法: 收集2012年12月至2018年4月收治的85例初诊sHLH患者初次就诊时的血浆标本，并按照病因将患者分为淋巴瘤相关HLH（LHLH）组和感染相关HLH（IHLH）组。应用ELISA实验检测患者血浆中sB7-H3的表达，同时收集患者的临床资料进行分析并以15例健康体检者为对照组.

结果: 与对照组相比较，淋巴瘤相关HLH组和感染相关HLH组sB7-H3的表达均明显升高（P＜0.05），淋巴瘤相关HLH组sB7-H3的表达明显高于感染相关HLH组（(35.75±9.90) vs (28.70±8.95) ng/ml）（P＜0.001）。淋巴瘤相关HLH组与感染相关HLH组患者部分临床指标(包括年龄、发热、脾肿大、ANC、Plt、FIB、钙离子、血清白蛋白、LDH、血清铁蛋白、sCD25)的表达均无明显统计学差异（P＞0.05）。应用ROC曲线评价sB7-H3在sHLH的表达及意义结果显示，在与感染相关HLH患者的对比中，ROC（AUC）曲线下面积为0.718（95% CI 0.610-0.810）（P=0.0002)，预测淋巴瘤相关HLH患者的敏感性和特异性分别为77.36%和59.38%。ROC曲线分析sB7-H3的最佳cut-off值为29.81 ng/ml，sB7-H3高表达组（≥29.81 ng/ml）总生存中位时间较低表达组（＜29.81 ng/ml）明显缩短（24 vs 440 d）（P＜0.001）。影响sHLH患者生存状态的临床指标分别是中性粒细胞、白蛋白、血清铁蛋白、血清钙离子和sB7-H3水平.

结论: sB7-H3与sHLH影响不良预后的相关因素并可能参与疾病进展.

OBJECTIVE

To explore the single nucleotide polymorphisms (SNPs) of co-stimulatory molecule B7-H3 gene in blood cells of patients with rheumatoid arthritis (RA) through the in vitro sequencing method and analyze the correlation between the SNPs and the susceptibility of RA.

METHODS

We sequenced directly B7-H3 gene in 86 cases of RA patients and 73 cases of health controls, and then sequence specific primer-PCR (SSP-PCR) genotyping method was carried out in 377 cases of RA patients and 321 health controls for 6 sites of SNPs.

RESULTS

Six sites of SNPs 19 589[chr.73 992 036, 19 630(chr.73 992 077), 19 948(chr. 73 992 394), 19 956(chr. 73 992 602), 20 214(chr. 73 992 660), 28 969(chr. 73 993 889)] were found in the CDS region and some introns of B7-H3 gene. Apart from 28 969 site (chr. 73 993 889), the others had significant differences in the genotypic frequency and allelic frequency between RA patients and health controls (P<0.05).

CONCLUSIONS

Six sites of B7-H3 gene have SNP variations and five sites are related to the pathogenesis of RA in Han populations.

Cancer immunotherapies have revolutionized how we can treat adult malignancies and are being translated to pediatric oncology. Chimeric antigen receptor T-cell therapy and bispecific antibodies targeting CD19 have shown success for the treatment of pediatric patients with B-cell acute lymphoblastic leukemia. Anti-GD2 monoclonal antibody has demonstrated efficacy in neuroblastoma. In this review, we summarize the immunotherapeutic agents that have been approved for treating childhood cancers and provide an updated review of molecules expressed by pediatric cancers that are under study or are emerging candidates for future immunotherapies. Advances in our knowledge of tumor immunology and in genome profiling of cancers has led to the identification of new tumor-specific/associated antigens. While cell surface antigens are normally targeted in a major histocompatibility complex (MHC)-independent manner using antibody-based therapies, intracellular antigens are normally targeted with MHC-dependent T cell therapies. Glypican 2 (GPC2) and B7-H3 (CD276) are two cell surface antigens that are expressed by a variety of pediatric tumors such as neuroblastoma and potentially can have a positive impact on the treatment of pediatric cancers in the clinic.

Keywords: B7-H3; chimeric antigen receptor (CAR); glypican 2 (GPC2); monoclonal antibody; pediatric cancer.

B7x is an immune checkpoint molecule which belongs to the B7 family of ligands which includes PD-L1, PD-L2, B7-H3 and HHLA2. B7x belongs to the Immunoglobulin superfamily and its protein structure is similar to other members with a N terminus peptide, IgV and IgC like extracellular domain with four cysteine residues. Its receptor is yet to be identified. B7x inhibits T cell proliferation and expansion by IL-2 dependent and non-IL-2 dependent pathways. Even though high levels of B7x mRNA can be detected in most tissues its protein expression is highly limited suggesting significant post translational control. In vivo data, show that B7x plays an important role in limiting autoimmunity in the peripheral tissues and fine-tuning autoimmune responses. B7x is highly expressed in various cancers and in prostate cancer its expression is corelated with poorer outcomes. Local production of IL-6 and IL-10 in various cancers promotes B7x expression and tumor immune evasion. B7x is especially expressed in PD-L1 negative tumors suggesting that this may be an important method of immune evasion in these tumors. Currently drug development, targeting B7x through various mechanisms including monoclonal antibodies and antibody drug conjugates are in development in cancers and increasing B7x expression with fusion proteins in autoimmune diseases is underway.

The purpose of this study is to explore the correlations of interleukin 36 (IL-36) and Soluble B7-H3 (sB7-H3) levels in bronchoalveolar lavage fluid (BALF) with clinical characteristics and laboratory findings.

A total of 35 children with M. pneumnoiae pneumonia (MPP) and 15 control subjects were enrolled. BALF concentrations of sB7-H3 and IL-36 were detected using enzyme-linked immunosorbent assays and clinical profiles of children with MPP were obtained.

Children with MPP had significantly higher levels of sB7-H3 and IL-36 compared to control subjects (both P < 0.05). Meanwhile, children with pleural effusion had significantly higher levels of sB7-H3 and IL-36 compared to children without pleural effusion (both P < 0.05). BALF concentration of sB7-H3 was strongly associated with concentration of IL-36 (r = 0.796, P < 0.0001) and sB7-H3 was correlated with duration of fever (r = 0.427, P = 0.11) and length of stay (r = 0.345, P = 0.043). Both concentrations of sB7-H3 and IL-36 were significantly decreased in convalescent phase after treatment (both P < 0.05).

Both soluble B7-H3 and IL-36 may play an important role in pathogenesis of M. pneumoniae infection and sB7-H3 could be useful as a prognostic predictor or biomarker of MPP.

Immune checkpoints balance initial antigen-driven T cell stimulation by enhancing or dampening activation, allowing co-existence of efficient immune responses and maintenance of self-tolerance. In oncology, checkpoints currently targeted by inhibitors to amplify activity of T cell, NK cells or myeloid cells responses comprise CTLA-4 (cytolytic T-lymphocyte-associated antigen 4 or CD152), PD-1 (programmed cell death 1, or CD279), PD-L1 ( programmed cell death-ligand 1, or CD274), LAG-3 (Lymphocyte-activation gene 3, or CD223), TIM3 (T-cell immunoglobulin and mucin-domain containing-3), TIGIT (T cell immunoreceptor with Ig and ITIM domains ), VISTA (V-domain Ig suppressor of T cell activation), B7/H3 (or CD276), KIR (killer-cell immunoglobulin-like receptor), NKG2A, CD39, CD73, CSF1R (colony-stimulating factor 1 receptor), CD47 or CD172a. Other "checkpoints" are being pharmacologically triggered in order to directly amplify T cell co-stimulation. Among these molecules, CD28, CD137 (also called 4-1BB), OX40 [also called tumor necrosis factor receptor superfamily, member 4 (TNFRSF4)], GITR (Glucocorticoid-induced tumor necrosis factor receptor family-related protein) or CD40 are also tested in oncology, most often in combination with an inhibitory checkpoint inhibitor. In autoimmune and inflammatory diseases, checkpoint inhibitors or activators (LAG-3, CD28, CD40L) are also being tested. In this review, we focus on some modulators of immune checkpoints for which the mechanism of action has been particularly studied. As this description cannot be exhaustive, we have grouped in Table I all monoclonal antibodies (MAbs) or recombinant proteins in clinical use (to our knowledge), modulating the action of a control point of the immune system.

B7-H3 is a type I membrane protein that has contradictory co-stimulatory and co-inhibitory effects in adaptive and anti-tumour immunity. B7-H3 is up-regulated in many malignant tumours, including breast cancer. Therefore, we hypothesise that B7-H3, which has an immunosuppressive role, suppresses anti-tumour immunity. The aim of this study was to clarify the role of B7-H3 in the tumor microenvironment in breast cancer, explore the possibility of B7-H3 as a target for clinical immunotherapy, and provide reference for clinical work. We knocked down B7-H3 with siRNA in MCF7 breast cancer cells, which we termed MCF7-B7-H3-KD cells, and the expression of B7-H3 was assessed by flow cytometry. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) knockdown was used as a control (MCF7-Gapdh). MCF7-B7-H3-KD and MCF7-Gapdh cells were co-cultured with peripheral blood mononuclear cells (PBMCs) and CD3⁺ T cells from healthy donors to assess the effect of B7-H3 loss. PBMCs cultured with MCF7-Gapdh cells showed decreased activation, proliferation, and function of CD8⁺ T cells, but there was no effect on the proliferation of CD4⁺ T cells. However, when MCF7-B7-H3-KD cells were co-cultured with PBMCs, the proliferation ability of CD4⁺ T cells and CD8⁺ T cells was significantly higher than that observed in MCF7-Gapdh cell co-culture. Additionally, co-culture with MCF7-Gapdh cells decreased the expression of IFN-γ (Interferon-γ). However, after co-culture with MCF7-B7-H3-KD cells, there was an increase in IFN-γ. We further found that this inhibitory effect on IFN-γ was because of decreased mTOR (the mammalian target of rapamycin) phosphorylation in T cells. Treatment of T cells co-cultured with MCF7-B7-H3-KD cells with an mTOR inhibitor blocked the secretion of IFN-γ. B7-H3 on tumour cells inhibits the proliferation of CD4⁺ and CD8⁺ T cells and inhibits the release of IFN-γ by decreasing mTOR signalling. A better understanding of these complex immune regulatory mechanisms should facilitate the generation of more powerful and selective tools to manipulate cancer therapy.

Keywords: B7-H3; Breast cancer; MCF7; mTOR.