Immune checkpoint-targeted immunotherapy has achieved unprecedented success, yet its limitations remain evident. Human leukocyte antigen-G (HLA-G), a novel immune checkpoint, exhibits restricted physiologic expression but is broadly expressed in various tumors, conferring systemic immune suppressive functions via different types of immune inhibitory receptors, and is associated with a poor prognosis for patients with cancer, making it an attractive tumor-site-agnostic candidate target for cancer immunotherapy. Since 2020, clinical trials employing different strategies of HLA-G-targeted immunotherapy for various advanced solid cancers have been conducted. Herein, the molecular characteristics of HLA-G, HLA-G-receptor binding interactions, and HLA-G-targeted preclinical investigations and clinical trials for solid cancer immunotherapy are highlighted, and the challenges associated with translating these findings into clinical settings are also discussed.

IntroductionThe concept that human leukocyte antigen-G (HLA-G) expression is specifically in various malignant lesions but absent in adjacent normal tissues has been well documented, and HLA-G is acknowledged as a novel immune checkpoint.MethodsIn this study, HLA-G expression in 523 gastric cancer (GC) lesions and 283 case-matched peritumoral tissues (PTTs) was analyzed by immunohistochemistry. The clinical significance of HLA-G expression in GC lesions and case-matched PTTs was evaluated, and a specific HLA-G-positive subpopulation in PTTs was identified.ResultsData showed that HLA-G expression was significantly different between GC lesions (370/523, 70.7%) and PTTs (238/283, 84.1%; p<0.001). HLA-G in GC lesions was more frequently observed in older patients (76.2% vs. 65.3%, p=0.006), and HLA-G in PTTs was more commonly observed in female patients (92.0% vs. 80.5%, p=0.014). Survival analysis revealed that HLA-G expression in PTTs (overall survival: 42.0 months vs. 56.7 months, p=0.023), but not in GC lesions (52.1 months vs. 50.9 months, p=0.623), is significantly associated with poor prognosis. The fundic gland mucous neck cells were the only subpopulation that expressed HLA-G.DiscussionOur findings, for the first time, suggest that fundic gland mucous neck cell HLA-G expression in PTTs is associated with GC progression.

Background:Human leukocyte antigen (HLA)-G has multiple isoforms with unique molecular structures and receptor-binding specificities. Different HLA-G isoform(s) may have distinct clinical relevance. Because of the lack of isoform-specific monoclonal antibodies (mAbs), the clinical significance of HLA-G isoforms (HLA-G1 to HLA-G7), except HLA-G1 and HLA-G5, remains largely unknown. Methods:In this study, mAbs against HLA-G2/6 and HLA-G1/4/5 isoforms were generated and characterized. Expression of HLA-G2/6 and HLA-G1/4/5 isoforms was analyzed by immunohistochemistry, and clinical significance was evaluated retrospectively in 345 patients with colorectal cancer (CRC). Results:The expression rate of HLA-G2/6 (90/345, 26.1%) was significantly lower than that of HLA-G1/4/5 (275/345, 79.7%; p < 0.001). Patients with HLA-G2/6 expression had significantly poorer overall survival (OS) (median OS: 6.3 years [95% CI: 4.1-8.5] vs. 10.0 years [95% CI: 7.6-12.4]; p = 0.008). Multivariate Cox proportional-hazard model results indicated that HLA-G2/6 was an independent prognostic factor for CRC (hazard ratio [HR] = 1.530, 95% CI: 1.125-2.081; p = 0.007). Moreover, HLA-G2/6 expression showed stratified prognostic significance among several CRC patient subgroups, specifically in female patients (p = 0.003), younger patients (<66 years p < 0.001), patients with colon cancer (p = 0.045), those at stage pT3 (p = 0.008), pN1 (p = 0.020), pM0 (p = 0.009), and AJCC stage III (p = 0.005). However, no statistical significance was found between HLA-G1/4/5 isoform expression and patient prognosis in CRC. Conclusions:This is the first study to generate mAbs for the HLA-G2/6 and HLA-G1/4/5 isoforms. Our findings reveal that HLA-G2/6-but not HLA-G1/4/5-expression is an independent prognostic indicator for patients with CRC. In the context of precision medicine, our study also suggests that HLA-G isoform typing may be necessary for HLA-G-targeted cancer immunotherapy.

BACKGROUND:Primary spontaneous pneumothorax is a rare disease commonly found in young adults, with unknown aetiology. We aimed to investigate the susceptibility genes and the downstream signalling involved in the development of primary spontaneous pneumothorax. METHODS:We conducted the first large-scale genome-wide association study (GWAS) composed of 2223 patients and 3838 controls. The functional role of the novel susceptibility loci was investigated by both in vivo and in vitro assays. Gene expression profiling in lung epithelial cell lines was performed, and conditional gene knockout mice were generated. RESULTS:We identified four novel susceptibility loci at 14q32.2 near C14orf177, at 15q26.3 near CHSY1, at 16q23.1 near CFDP1 and at 22q13.1 near CBX7. The fine-mapping of 22q13.1 revealed a functional variant which regulated CBX7 expression by disrupting the binding activity of transcription factor CREB1. Conditional knockout of Cbx7 in mouse lung epithelial cells resulted in lung cyst formation. Meanwhile, downregulation of the CBX7 elevated the expression of MMP9 and MMP16, which are part of extracellular matrix regulators and may lead to lung injury. CONCLUSIONS:Our GWAS discovered four novel susceptibility loci of primary spontaneous pneumothorax and presented a mechanistic basis for the genetic association with primary spontaneous pneumothorax. The novel susceptibility gene CBX7 and downstream MMP signalling give a new clue to the pathogenesis of primary spontaneous pneumothorax.

The prognostic significance of metastasis-associated in colon cancer-1 (MACC1) has been explored in a variety of malignancies. However, its clinical relevance in patients with gastric cancer (GC) is limited, also remains controversial. In this study, we retrospectively evaluated the prognostic value of lesion MACC1 expression in 347 GC patients. Lesion MACC1 expression was analyzed with immunohistochemistry and grouped as MACC1low (n = 172) and MACC1high (n = 175) cases. Data revealed that the degree of MACC1 expression is not related to patient sex, age and disease stage (all p > 0.05). Survival analysis showed that only post-operation advanced pT (p = 0.018), pN (p < 0.001), pM (p = 0.001) and AJCC stages (p < 0.001) are significantly associated with shorter survival, while no obvious difference was observed between MACC1low and MACC1high cases (p = 0.158). However, we found that survival for female (p = 0.032), older (p = 0.028), and early disease stage (pT stage I + II, p = 0.033) patients with MACC1high are remarkably worse than those with MACC1low. In summary, our findings revealed that, though MACC1 expression is not associated with the survival of the whole cohort, the prognostic risk stratification value of lesion MACC1 expression in subgroups of patients with gastric cancer should be noted.

Background: A biobank is a central resource that supports basic and clinical research. RNA quality of fresh-frozen tissue specimens in the biobank is highly associated with the success of downstream applications. Therefore, it is very important to evaluate the impact of tissue processing and storage conditions on RNA quality. Methods: A total of 238 surgically removed tissue specimens, including esophagus, lung, liver, stomach, colon, and rectal cancer, were used to evaluate RNA quality. Two tissue homogenization methods, manual and TissueLyser, were compared and the impacts of temperature fluctuation, tissue types, storage period, and clinicopathological parameters on RNA quality were analyzed. Results: RNA integrity was not influenced by tissue homogenization methods and tissue types. However, RNA integrity number (RIN) values were significantly correlated with temperature fluctuation. When the power of a -80°C freezer was cut off, RNA integrity of frozen tissues was not significantly affected until the temperature increased to 0°C. When the temperature rose to room temperature and remained for 4 hours, RNA integrity was almost completely destroyed. In addition, various cancer tissues with short-term storage at -80°C (<5 years) or high tumor differentiation had higher RINs. Conclusions: Tissue processing and storage conditions affected RNA quality of fresh-frozen cancer tissues. It is necessary to keep storage temperature stable and keep specimens at ultralow temperatures during homogenization. Also, for a biobank containing multiple types of cancer tissue samples, it is better to store them in liquid nitrogen if the storage duration is more than 5 years.

Although detailed underlying mechanisms for the pathogenesis of autoimmune diseases remain largely unknown, multiple factors such genetic and epigenetic background, environmental inducers, and dysregulation of immune tolerance for self-antigens have been considered to be associated with a variety of autoimmune diseases. Human leukocyte antigen (HLA) system, the most polymorphic complex in human genome, functions fundamental roles in immune homeostasis by interaction with both immune activating and inhibitory receptors expressed on different types of immune cells. In line with this, an increasing evidence of genetic predisposition and molecular functions of the non-classical HLA class I antigen HLA-G has been revealed in the clinical significance of autoimmune diseases. Tissue HLA-G expression was firstly observed on the extravillous cytotrophoblasts, and later its immune suppressive features have become well documented. In contrast to its high polymorphic classical HLA class I (HLA-A, HLA-B, and HLA-C) counterparts, HLA-G is with very limited genetic variations, while at least four membrane-bound (HLA-G1–HLA-G4) and three soluble (HLA-G5–HLA-G7) isoforms have been identified, which differs either in extracellular domains or in intracellular cytoplasmic tails generated by HLA-G primary transcript alternative splicing. Functionally, HLA-G can induce profound immune inhibition by interacting with a panel of HLA-G-binding immune receptors such as immunoglobulin-like transcripts (ILT)-2, ILT-4, and killer inhibitory receptor (KIR) 2DL4 differentially expressed on autoimmune diseases related T cells, natural killer cells (NK), B cells, dendritic cells (DCs), or macrophages. In this context, induction of HLA-G expression could have a protective effect to diminish auto-reactive immune responses among autoimmunity. In this chapter, we focus on the genetic association, molecular function, and mechanisms of HLA-G-mediated immune tolerance in autoimmune diseases.

ObjectiveTo evaluate the prognostic significance of peripheral lymphocyte count and its derived inflammatory markers, such as neutrophil-to-lymphocyte ratio (NLR), in a cohort of patients with gastric cancer (GC).MethodsIn this retrospective study, the clinical characteristics and follow-up information, both pre- and post-operative within one week of laboratory findings, of 338 patients with GC who underwent radical gastrectomy were retrieved, and their prognostic significance was evaluated.ResultsBoth lower pre- and post-operative lymphocyte counts and higher NLR and SSI were significantly related to advanced tumour (pT) and disease stages (American Joint Committee on Cancer [AJCC]) in patients with GC. Log-rank survival analysis showed that, in addition to traditional pT, pN, pM, and AJCC stages, both lower pre- (p = 0.041) and post-operative (p = 0.002) lymphocyte counts and higher NLR (ppre < 0.001 and ppost = 0.008) and SSI (ppre = 0.014 and ppost = 0.145) were associated with worse survival. Cox proportional hazards analysis revealed that pre-operative NLR (p = 0.018; hazard ratio = 1.778) was an independent predictor of prognosis in patients with GC. Moreover, when the pre-operative NLR was divided into NLRlow and NLRhigh, NLRhigh showed stratified prognostic value for patient sex (male, p = 0.001; female, p = 0.044), age (younger, p = 0.005; older, p = 0.005), and AJCC stage III (p = 0.007).ConclusionPre-operative NLR is an independent prognostic factor for patients with GC and has stratified prognostic value for patients with AJCC stage III GC.

ObjectiveHuman leukocyte antigen-G (HLA-G) and its receptors, including immunoglobulin-like transcripts (ILT)-2 and ILT-4, are closely associated with cancer development and clinical outcomes of patients. However, the clinical significance of HLA-G and ILT-2/-4 in gastric cancer (GC) is limited.MethodsIn this study, the percentage of HLA-G-, ILT-2 and ILT-4 positive tumor cells in 127 GC lesion suspensions of tumor cells gated for epithelialcelladhesionmolecule(EpCAM) was determined using multicolor flow cytometry and their clinical significance was evaluated.ResultsOur data showed that the median percentages of HLA-G-, ILT-2, and ILT-4 expressing GC cells were 18.0%, 67.80%, and 1.42%, respectively, and co-expression of HLA-G/ILT-2, HLA-G/ILT-4, and ILT-2/ILT-4 was 16.9%, 1.42%, and 1.70%, respectively. Kaplan-Meier survival results revealed that besides post-operation N status (p = 0.006), M status (p = 0.001), and AJCC clinical stage (p < 0.001), only high percentage of ILT-4+ GC cells was a significant factor for worse survival of patients with GC (overall survival [OS]: 42.9 months vs. 84.5 months; p = 0.031). However, among female patients with GC (n = 31), high percentage of either HLA-G+ (OS: 18.5 months vs. 89.3 months; p = 0.001) or ILT-4+ (OS: 17.9 months vs. 85.8 months; p = 0.002) GC cells was markedly associated with a poor prognosis.ConclusionOur findings revealed that among HLA-G, ILT-2, and ILT-4, only a high percentage of ILT-4+ GC cells was significantly related to poor prognosis in the entire cohort of patients with GC. However, high percentage of HLA-G+ and ILT-4+ GC cells is associated with poor clinical outcome among female patients with GC.

Immune checkpoint inhibitors (ICIs) have become a promising immunotherapy for cancers. Human leukocyte antigen-G (HLA-G), a neoantigen, its biological functions and clinical relevance have been extensively investigated in malignancies, and early clinical trials with “anti-HLA-G strategy” are being launched for advance solid cancer immunotherapy. The mechanism of HLA-G as a new ICI is that HLA-G can bind immune cell bearing inhibitory receptors, the immunoglobulin-like transcript (ILT)-2 and ILT-4. HLA-G/ILT-2/-4 (HLA-G/ILTs) signaling can drive comprehensive immune suppression, promote tumor growth and disease progression. Though clinical benefits could be expected with application of HLA-G antibodies to blockade the HLA-G/ILTs signaling in solid cancer immunotherapy, major challenges with the diversity of HLA-G isoforms, HLA-G/ILTs binding specificity, intra- and inter-tumor heterogeneity of HLA-G, lack of isoform-specific antibodies and validated assay protocols, which could dramatically affect the clinical efficacy. Clinical benefits of HLA-G-targeted solid cancer immunotherapy may be fluctuated or even premature unless major challenges are addressed.

Immune checkpoint inhibitors (ICIs) have become a promising area of research for cancer treatment. In addition to the well-known ICIs targeting PD-1/PD-L1, HLA-G/ILT-2/-4 is relatively new immune checkpoint that has been evaluated in early clinical trials in patients with advanced solid tumors. In this study, the expression of HLA-G (n=157), ILT-2/4 (n=82), and PD-L1 (n=70) in epithelial cell adhesion molecule (EpCAM)-positive colorectal cancer (CRC) cells was analyzed by multicolor flow cytometry, and the prognostic significance of these molecules was evaluated. In EpCAM+ CRC cells, the median percentages of HLA-G, ILT-2, ILT-4, and PD-L1 were 14.90%, 67.70%, 8.55% and 80.30%, respectively. In addition, a positive correlation was observed between them (all p<0.001). Higher levels of these immune checkpoint proteins are associated with lymph node metastasis. In addition to the AJCC stage (p=0.001), Kaplan-Meier survival analysis showed that higher levels of HLA-G (p=0.041), ILT-2 (p=0.060), ILT-4 (p<0.001), PD-L1 (p=0.012), HLA-GILT4 (p<0.001) and ILT-2ILT-4 (p<0.001) were significantly associated with shorter survival of CRC patients. When CRC patients were stratified by early and advanced AJCC stages, HLA-G levels were only related to the survival among CRC patients with early disease stage (p=0.024), while ILT-4 levels were significant for both CRC patients with early (p=0.001) and advanced (p=0.020) disease stages. Multivariate cox regression analysis revealed that advanced AJCC stage (HR=2.435; p=0.005) and higher ILT-4 levels (HR=2.198; p=0.063) were independent risk factors for poor outcomes in patients with CRC. In summary, among the immune checkpoints, HLA-G/ILT-2/4 and PD-L1, ILT-4 is the most significant prognostic indicator of CRC. This finding indicated that a combination of immunotherapy strategies, such as ILT-4 blockade, could improve the clinical outcomes in patients with cancer. Moreover, multicolor flow cytometry can be employed as a reliable and efficient, alternative to immunohistochemistry, for evaluating the immune checkpoint proteins expressed in tumor lesions.

COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has threatened public health worldwide. Host antiviral immune responses are essential for viral clearance and disease control, however, remarkably decreased immune cell numbers and exhaustion of host cellular immune responses are commonly observed in patients with COVID-19. This is of concern as it is closely associated with disease severity and poor outcomes. Human leukocyte antigen-G (HLA-G) is a ligand for multiple immune inhibitory receptors, whose expression can be upregulated by viral infections. HLA-G/receptor signalling, such as engagement with immunoglobulin-like transcript 2 (ILT-2) or ILT-4, not only inhibit T and natural killer (NK) cell immune responses, dendritic cell (DC) maturation, and B cell antibody production. It also induces regulatory cells such as myeloid-derived suppressive cells (MDSCs), or M2 type macrophages. Moreover, HLA-G interaction with CD8 and killer inhibitory receptor (KIR) 2DL4 can provoke T cell apoptosis and NK cell senescence. In this context, HLA-G can induce profound immune suppression, which favours the escape of SARS-CoV-2 from immune attack. Although detailed knowledge on the clinical relevance of HLA-G in SARS-CoV-2 infection is limited, we herein review the immunopathological aspects of HLA-G/receptor signalling in SARS-CoV-2 infection, which could provide a better understanding of COVID-19 disease progression and identify potential immunointerventions to counteract SARS-CoV-2 infection.

Objective: Re-positivity of SARS-CoV-2 in discharged COVID-19 patients have been reported; however, early risk factors for SARS-CoV-2 re-positivity evaluation are limited. Methods: This is a prospective study, a total of 145 COVID-19 patients were treated and all discharged according to the guideline criteria by Mar 11th 2020. After discharge, clinical visits and viral RT-PCR tests by the second and fourth week follow-up were carried-out. Patient demographic and clinical characteristics and laboratory data on admission and discharge were retrieved, and predictive factors for SARS-CoV-2 re-positivity were analyzed. Results: 13 out of 145 (9.0%) COVID-19 patients were confirmed re-positivity of SARS-CoV-2 by RT-PCR test. The median interval between disease onset to recurrence was 38 days. SARS-CoV-2 re-positive cases were of significantly longer virus shedding duration, notably higher body temperature, heart rate and lower TNF-alpha and IgG levels on admission. Covariate logistic regression analysis revealed virus shedding duration and IgG levels are independent risk factors for SARS-CoV-2 return positive after discharge. Conclusion: Longer viral shedding duration and lower IgG levels are risk factors for re-positivity of SARS-CoV-2 for discharged COVID-19 patients.

Signaling pathway between human leukocyte antigen (HLA)-G and immune inhibitory receptors immunoglobulin-like transcript (ILT)-2/4 has been acknowledged as one of immune checkpoints, and as a potential target for cancer immunotherapy. Like other immune checkpoints, inter- and even intra-tumor heterogeneity of HLA-G could render a rather complexity for HLA-G-target immunotherapy. However, little information for intra-tumor heterogeneity of HLA-G is available. In this study, HLA-G expression in a serial section of colorectal cancer (CRC) lesions from three CRC patients (each sample with serial section of 50 slides, 10 randomized slides for each antibody), three different locations within a same sample (five CRC), and three case-matched blocks that each includes 36 esophageal cancer samples, were evaluated with immunohistochemistry using anti-HLA-G antibodies (mAbs 4H84, MEM-G/1 and MEM-G/2 probing for all denatured HLA-G isoforms, 5A6G7 and 2A12 probing for denatured HLA-G5 and HLA-G6 isoforms). Our results revealed that, in addition to the frequently observed inter-tumor heterogeneity, intra-tumor heterogeneous expression of HLA-G is common in different areas within a tumor in CRC and esophageal cancer samples included in this study. Moreover, percentage of HLA-G expression probed with different anti-HLA-G antibodies also varies dramatically within a tumor. Given HLA-G has been considered as an important immune checkpoint, intra-tumor heterogeneity of HLA-G expression, and different specificity of anti-HLA-G antibodies being used among studies, interpretation and clinical significance of HLA-G expression in cancers should be with caution.

Objectives: Pandemic COVID-19 has become a seriously public health priority worldwide. Comprehensive strategies including travel restrictions and mask-wearing have been implemented to mitigate the virus circulation. However, detail information on community transmission is unavailable yet. Methods: From January 23 to March 1, 2020, 127 patients (median age: 46 years; range: 11-80) with 71 male and 56 female, were confirmed to be infected with the SARS-CoV-2 in Taizhou, Zhejiang, China. Epidemiological trajectory and clinical features of these COVID-19 cases were retrospectively retrieved from electronic medical records and valid individual questionnaire. Results: The disease onset was between January 9 to February 14, 2020. Among them, 64 patients are local residents, and 63 patients were back home from Wuhan from January 10 to 24, 2020 before travel restriction. 197 local residents had definite close-contact with 41 pre-symptomatic patients back from Wuhan. 123 and 74 of them contact with mask-wearing or with no mask-wearing pre-symptomatic patients back from Wuhan, respectively. Data showed that incidence of COVID-19 was significantly higher for local residents close-contact with no mask-wearing Wuhan returned pre-symptomatic patients (19.0% vs. 8.1%, p < 0.001). Among 57 close contact individuals, 21 sequential local COVID-19 patients originated from a pre-symptomatic Wuhan returned couple, indicated dense gathering in congested spaces is a high risk for SARS-CoV-2 transmission. Conclusions: Our findings provided valuable details of pre-symptomatic patient mask-wearing and restriction of mass gathering in congested spaces particularly, are important interventions to mitigate the SARS-CoV-2 transmission.

本指导文件更新于 2020 年 2 月 10 日,修订内容摘要如下: 1.增加"认证"一词,以说明任何有可能产生气溶胶或飞沫的实验均应在经认证的二级生物安全柜(BSC)中进行.

Background: Abnormal peripheral immunological features are associated with the progression of coronavirus disease 2019 (COVID-19). Methods: Clinical and laboratory data were retrieved in a cohort of 146 laboratory-confirmed COVID-19 patients. Potential risk factors for the development of severe COVID-19 were evaluated. Results: On admission, lymphocytes, CD3+, CD4+ and CD8+ T cells, eosinophils, and albumin and pre-albumin were dramatically lower, whereas neutrophils, and interleukin (IL)-10, C-reactive protein (CRP), aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT) were significantly higher in severe cases. By the second week after discharge, all variables improved to normal levels. Covariate logistic regression results showed that the CD8+ cell count and CRP level were independent risk factors for severe COVID-19. Conclusion: Lower peripheral immune cell subsets in patients with severe disease recovered to normal levels as early as the second week after discharge. CD8+ T cell counts and CRP levels on admission are independent predictive factors for severe COVID-19.

Aim: To identify methylation-associated genes in the carcinogenesis of colorectal cancer (CRC). Materials & methods: Genome-wide patterns of DNA methylation and gene expression in CRC tissues and adjacent normal tissues were determined and further validated in The Cancer Genome Atlas data and Chinese CRC patients, respectively. Gene overexpression and knockdown cells were constructed to investigate their biological roles in CRC. Results: After validations, hypermethylation of eight genes were found to be correlated with their reduced transcription, and hypomethyaltion of three genes were associated with their upregulation. CADM3, CNRIP1, GRHL2, GRIA4, GSTM2 and NRXN1 were associated with the overall survival of CRC patients. CNRIP1 and GSTM2 were mainly responsible for the proliferation in CRC cells. Conclusion: A total of 11 genes may be promising biomarkers for CRC.