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Background Brucellosis, as a serious zoonotic infectious disease, has been recognized as a re-emerging disease in the developing countries worldwide. In china, the incidence of brucellosis is increasing each year, seriously threatening the health of humans as well as animal populations. Despite a quite number of diagnostic methods currently being used for brucellosis, innovative technologies are still needed for its rapid and accurate diagnosis, especially in area where traditional diagnostic is unavailable. Methodology/Principal findings In this study, a total of 22 B cell linear epitopes were predicted from five Brucella outer membrane proteins (OMPs) using an immunoinformatic approach. These epitopes were then chemically synthesized, and with the method of indirect ELISA (iELISA), each of them displayed a certain degree of capability in identifying human brucellosis positive sera. Subsequently, a fusion protein consisting of the 22 predicted epitopes was prokaryotically expressed and used as diagnostic antigen in a newly established brucellosis testing method, nano-ZnO modified paper-based ELISA (nano-p-ELISA). According to the verifying test using a collection of sera collected from brucellosis and non-brucellosis patients, the sensitivity and specificity of multiepitope based nano-p-ELISA were 92.38% and 98.35% respectively. The positive predictive value was 98.26% and the negative predictive value was 91.67%. The multiepitope based fusion protein also displayed significantly higher specificity than Brucella lipopolysaccharide (LPS) antigen. Conclusions B cell epitopes are important candidates for serologically testing brucellosis. Multiepitope fusion protein based nano-p-ELISA displayed significantly sensitivity and specificity compared to Brucella LPS antigen. The strategy applied in this study will be helpful to develop rapid and accurate diagnostic method for brucellosis in human as well as animal populations., Author summary Brucellosis is one of the most important zoonosis in the world and has caused tremendous economic losses in agriculture and animal husbandry in many countries. Developing rapid, sensitive and specific diagnostic methods is very important for early detection and treatment of brucellosis patients. In this study, a novel diagnostic technique, nano-ZnO modified paper ELISA, was established. The antigen used in this technique was a fusion protein containing multiple B cell epitopes, which were predicted from Brucella major outer membrane proteins such as Bp26, Omp31, Omp16, Omp2b and Omp25. Comparing to traditional LPS antigen, this multiepitope based antigen displayed considerably higher sensitivity and higher specificity in laboratory. With the strategy described in this paper, more efficient epitopes and protein antigen can be identified in the future. Currently, LPS antigen is only prepared from live Brucella, while protein antigen can be produced in large quantities in prokaryotic expression system. In addition to nano-p-ELISA, this protein antigen can also be used for development other methods such as fluorescent polarization assay (FPA) and immunochromatographic assay (ICA) to meet the varied demand for brucellosis testing.

Summary Haemato‐oncological patients are at risk in case of severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infection. Currently, vaccination is the best‐evaluated preventive strategy. In the present study, we aimed to assess serological response, predictive markers, and safety of BNT162b2 in haemato‐oncological patients. A total of 259 haemato‐oncological patients were vaccinated with two 30 µg doses of BNT162b2 administered 21 days apart. Serological response was assessed by ELECSYS® Anti‐SARS‐CoV‐2‐S immunoassay before vaccination, and at 3 and 7 weeks after the first dose (T1, T2). Safety assessment was performed. At T2 spike protein receptor binding domain (S/RBD) antibodies were detected in 71·4% of haematological and in 94·5% of oncological patients (P

The immune cells and the repertoire of T cells and B cells play an important role in the pathogenesis of systemic lupus erythematosus (SLE). Exploring their expression and distribution in SLE can help us better understand this lethal autoimmune disease. In this study, we used a single-cell 5' RNA sequence and single-cell T cell receptor (TCR)/B cell receptor (BCR) to study the immune cells and the repertoire from ten SLE patients and the paired normal controls (NC). The results showed that 9732 cells correspondence to 12 cluster immune cell types were identified in NC, whereas 11042 cells correspondence to 16 cluster immune cell types were identified in SLE. The results demonstrated that neutrophil, macrophage, and dendritic cells were accumulated in SLE by annotating the immune cell types. Besides, the bioinformatics analysis of differentially expressed genes (DEGs) in these cell types indicates their role in inflammation response. In addition, patients with SLE showed increased TCR and BCR clonotypes compared with the healthy controls. Furthermore, patients with SLE showed biased usage of TCR and BCR V(D)J genes. Taken together, we characterized the transcriptome and TCR/BCR immune repertoire profiles of SLE patients, which may provide a new avenue for the diagnosis and treatment of SLE.

FAM83D has been demonstrated to contribute to tumorigenesis. However, its immune effects in hepatocellular carcinoma (HCC) have not been reported. This study aimed to identify the immune role of FAM83D in HCC. FAM83D was over-expressed in HCC and contributed to poor prognosis according to the results of data analysis based on The Cancer Genome Atlas (TCGA). Afterward, the levels of immune cells infiltration were found to be correlated with the expression level of FAM83D in HCC. Through TISIDB and cBioPortal network tools, a total of 82 FAM83D-associated genes were screened out, including 12 immunoinhibitors, 20 immunostimulators and 50 tightly co-expressed genes. TCGA cohort was divided into train set and test set on the basis of the proportion of 7:3. According to FAM83D-associated immunomodulators, a four gene predicted model was established using train set via the Cox regression analysis. Survival analysis demonstrated that the overall survival (OS) of high-risk HCC patients was poor compared with the patients in low-risk group. The reliability and predicted power of the risk-score model were identified by a receiver operating characteristic (ROC) curve. A risk-score based nomogram as well as a calibration curve, which were created could be used to anticipate patient’s 1-year, 3-year and 5-year survival probabilities. The test set was used to validate these results. Our findings showed that the FAM83D gene was related with HCC immunity. The immune marker chosen could be a promising biomarker for HCC prognosis.

This study aims to explore biomarkers associated with vitiligo and analyze the pathological role of immune cell infiltration in the disease. We used the robust rank aggregation (RRA) method to integrate three vitiligo data sets downloaded from gene expression omnibus database, identify the differentially expressed genes (DEGs) and analyze the functional correlation. Then, the comprehensive strategy of combined weighted gene coexpression network analysis (WGCNA) and logical regression of the selection operator (LASSO), support vector machine recursive feature elimination (SVM-RFE), and random forest (RF) machine learning algorithm are employed to screen and biomarkers associated with vitiligo. Finally, the immune cell infiltration of vitiligo was evaluated by CIBERSORT, and the correlation between biomarkers and infiltrating immune cells was analyzed. Herein, we identified 131 robust DEGs, and enrichment analysis results showed that robust DEGs and melanogenesis were closely associated with vitiligo development and progression. TYR, TYRP1, DCT and LARP7 were identified as vitiligo-related biomarkers. Immune infiltration analysis demonstrated that CD4 T Cell, CD8 T Cell, Tregs, NK cells, dendritic cells, and macrophages were involved in vitiligo’s pathogenesis. In summary, we adopted a comprehensive strategy to screen biomarkers related to vitiligo and explore the critical role of immune cell infiltration in vitiligo. Abbreviations: TYR, Tyrosinase; TYRP1, Tyrosinase-related protein-1; DCT, dopachrome tautomerase; LARP7, La ribonucleoprotein domain family, member-7; RRA, robust rank aggregation; DEGs, differentially expressed genes; WGCNA, weighted gene coexpression network analysis; LASSO, logical regression of the selection operator; SVM-RFE, support vector machine recursive feature elimination; RF, random forest; GWAS, Genome-wide association study; FasL, Fas-Fas ligand; Tregs, T-regulatory cells; NK, natural killer; GEPCs, gene expression profiling chips; GO, gene ontology; GSEA, gene set enrichment analysis; FDR, false discovery rate; AUC, area under the curve; ROC, receiver-operating characteristic; BP, biological process; CC, cellular component; MF, molecular function., GraphicalAbstract

The present in vitro study showed that IL-2/IL-2R antibody complex facilitates Treg-induced neuroprotection in the oxygen glucose deprivation/reoxygenation (OGD/R) model of stroke. First, we examined the role of IL-2/IL-2R-treated Tregs in OGD/R-exposed rat primary cortical cells (PCCs), which represent the cell type of the ischemic gray matter in the stroke brain. Here, OGD/R induced cell death, which was attenuated by Tregs and more robustly by IL-2/IL-2R-treated Tregs, but not by IL-2/IL-2R treatment alone. Second, we next assessed IL-2/IL-2R effects in OGD/R-exposed human oligodendrocyte progenitor cells (OPCs), which correspond to the white matter injury after stroke. Results revealed that a similar pattern neuroprotection as seen in the gray matter, in that OGD/R triggered cell death, which was ameliorated by Tregs and more effectively by IL-2/IL-2R-treated Tregs, but IL-2/IL-2R treatment alone was not therapeutic. Third, as we begin to understand the mechanism underlying IL-2/IL-2R engagement of Tregs, we investigated the inflammatory response in OGD/R-exposed human neural progenitor cells (NPCs), which recapitulate both ischemic gray and white matter damage in stroke. Similar to PCCs and OPCs, OGD/R produced cell death and was blocked by Tregs and more efficiently by IL-2/IL-2R-treated Tregs, whereas IL-2/IL-2R treatment alone did not alter the ischemic insult. Moreover, the inflammatory marker, TNF-α, was upregulated after OGD/R, dampened by both Tregs and more efficiently by IL-2/IL-2R-treated Tregs but more pronounced in the latter, and not affected by IL-2/IL-2R treatment alone, suggesting IL-2/IL-2R-Treg-mediated modulation of inflammatory response in stroke. Altogether, these observations support the use of IL-2/IL-2R treatment in enhancing the anti-inflammatory effects of Tregs in stroke.

Pancreatic cancer ranks one of the worst in overall survival outcome with a 5 year survival rate being less than 10%. Pancreatic cancer faces unique challenges in its diagnosis and treatment, such as the lack of clinically validated biomarkers and the immensely immunosuppressive tumor microenvironment. Recently, the LY6 gene family has received increasing attention for its multi-faceted roles in cancer development, stem cell maintenance, immunomodulation, and association with more aggressive and hard-to-treat cancers. A detailed study of mRNA expression of LY6 gene family and its association with overall survival (OS) outcome in pancreatic cancers is lacking. We used publicly available clinical datasets to analyze the mRNA expression of a set of LY6 genes and its effect on OS outcome in the context of the tumor microenvironment and immunomodulation. We used web-based tools Kaplan-Meier Plotter, cBioPortal, Oncomine and R-programming to analyze copy number alterations, mRNA expression and its association with OS outcome in pancreatic cancer. These analyses demonstrated that high expression of LY6 genes is associated with OS and disease free survival (DFS) outcome. High expression of LY6 genes and their association with OS outcome is dependent on the composition of tumor microenvironment. Considering that LY6 proteins are anchored to the outer cell membrane or secreted, making them readily accessible, these findings highlight the potential of LY6 family members in the future of pancreatic cancer diagnosis and treatment.

Circulating Klotho peptide hormone has anti-aging activity and affects tissue maintenance. Hypomorphic mutant Klotho [kl/kl] mice on C57BL/6xC3H, BALB/c and 129 genetic backgrounds, show decreased Klotho expression that correlate with accelerated aging including pre-mature death due to abnormally high levels of serum vitamin D. These mice also show multiple impairments in the immune system. However, it remains unresolved if the defects in the immune system stem from decreased Klotho expression or high vitamin D levels in the serum. Transfer of the kl/kl allele to pure C57BL/6 genetic background [B6-kl/kl] significantly reduced expression of Klotho at all ages. Surprisingly, B6-kl/kl mice showed normalized serum vitamin D levels, amelioration of severe aging-related phenotypes and normal lifespan. This paper reports a detailed analysis of the immune system in B6-kl/kl mice in the absence of detrimental levels of serum vitamin D. Remarkably, the data reveal that in the absence of overt systemic stress, such as abnormally high vitamin D levels, reduced expression of Klotho does not have a major effect on the generation and maintenance of the immune system.

Background: Focused ultrasound (FUS) activation of microbubbles (MBs) for blood-brain (BBB) and blood-tumor barrier (BTB) opening permits targeted therapeutic delivery. While the effects of FUS+MBs mediated BBB opening have been investigated for normal brain tissue, no such studies exist for intracranial tumors. As this technology advances into clinical immunotherapy trials, it will be crucial to understand how FUS+MBs modulates the tumor immune microenvironment. Methods and Results: Bulk RNA sequencing revealed that FUS+MBs BTB/BBB opening (1 MHz, 0.5 MPa peak-negative pressure) of intracranial B16F1cOVA tumors increases the expression of genes related to proinflammatory cytokine and chemokine signaling, pattern recognition receptor signaling, and antigen processing and presentation. Flow cytometry revealed increased maturation (i.e. CD86) of dendritic cells (DCs) in the meninges and altered antigen loading of DCs in both the tumor and meninges. For DCs in tumor draining lymph nodes, FUS+MBs had no effect on maturation and elicited only a trend towards increased presentation of tumor-derived peptide by MHC. Neither tumor endothelial cell adhesion molecule expression nor homing of activated T cells was affected by FUS+MBs. Conclusion: FUS+MBs-mediated BTB/BBB opening elicits signatures of inflammation; however, the response is mild, transient, and unlikely to elicit a systemic response independent of administration of immune adjuvants.

The application of cancer chronotherapy is to treat cancers based on at specific times during circadian rhythms. Previous studies have characterized the impact of circadian clock on tumorigenesis and specific immune cells. Here, by using multi-omics computation techniques, we systematically characterized the distinct roles of core circadian clock genes in thoracic cancers including lung adenocarcinoma, lung squamous cell carcinoma, and esophageal carcinoma. Strikingly, a wide range of core clock genes are epigenetically altered in lung adenocarcinomas and lung squamous cell carcinomas but not esophageal carcinomas. Further cancer hallmark analysis reveals that several core clock genes highly correlate with apoptosis and cell cycle such as RORA and PER2. Interestingly, our results reveal that CD4 and CD8 T cells are correlated with core clock molecules especially in lung adenocarcinomas and lung squamous cell carcinomas, indicating that chrono-immunotherapy may serve as a candidate option for future cancer management.

The distribution and abundance of immune cells, particularly T‐cell subsets, play pivotal roles in cancer immunology and therapy. T cells have many subsets with specific function and current methods are limited in estimating them, thus, a method for predicting comprehensive T‐cell subsets is urgently needed in cancer immunology research. Here, Immune Cell Abundance Identifier (ImmuCellAI), a gene set signature‐based method, is introduced for precisely estimating the abundance of 24 immune cell types including 18 T‐cell subsets, from gene expression data. Performance evaluation on both the sequencing data with flow cytometry results and public expression data indicate that ImmuCellAI can estimate the abundance of immune cells with superior accuracy to other methods especially on many T‐cell subsets. Application of ImmuCellAI to immunotherapy datasets reveals that the abundance of dendritic cells, cytotoxic T, and gamma delta T cells is significantly higher both in comparisons of on‐treatment versus pre‐treatment and responders versus non‐responders. Meanwhile, an ImmuCellAI result‐based model is built for predicting the immunotherapy response with high accuracy (area under curve 0.80–0.91). These results demonstrate the powerful and unique function of ImmuCellAI in tumor immune infiltration estimation and immunotherapy response prediction., Immune Cell Abundance Identifier (ImmuCellAI) is a gene set signature‐based method for precisely estimating the abundance of 24 immune cell types including 18 T‐cell subsets. Application of ImmuCellAI to immunotherapy datasets reveals the dynamic change of immune cell abundance. An ImmuCellAI result‐based model for predicting the immunotherapy response achieves high accuracy with area under curve 0.80–0.91.

Background: Gastric cancer is associated with high mortality, and effective methods for predicting prognosis are lacking. We aimed to identify potential prognostic markers associated with the development of gastric cancer through bioinformatic analyses. Methods: Gastric cancer-associated gene expression profiles were obtained from The Cancer Genome Atlas and Gene Expression Omnibus databases. The key genes involved in the development of gastric cancer were obtained by differential expression analysis, coexpression analysis, and short time-series expression miner (STEM) analysis. The potential prognostic value of differentially expressed genes was further evaluated using a Cox regression model and risk scores. Hierarchical clustering was applied to validate the impact of key genes on the overall survival of gastric cancer patients. Results: A total of 1381 genes were consistently dysregulated in the development of gastric cancer. Among them, 186 genes affected the overall survival of gastric cancer patients. The following genes had areas under the receiver operating characteristic curve greater than 0.9 in both datasets and were therefore considered key genes: ADAM12, CEP55, LRFN4, INHBA, ADH1B, DPT, FAM107A, and LOC100506388. LRFN4, DPT, and LOC100506388 were identified as potential prognostic genes for gastric cancer through a nomogram. Overexpression of LRFN4 and LOC100506388 was associated with a higher risk of gastric cancer. Finally, we found that tumors were infiltrated with high levels of Th2 cells and mast cells, and the infiltration levels were associated with overall survival in gastric cancer patients. Conclusions: We found that key dysregulated genes may have a prognostic value for the development of gastric cancer.

Several abnormalities have been reported in the peripheral blood mononuclear cells of keloid-forming patients and particularly in the monocyte cell fraction. The goal of this in vitro study was to determine whether monocytes from keloid-prone patients contribute to the keloid phenotype in early developing keloids, and whether monocyte differentiation is affected by the keloid microenvironment. Therefore, keloid-derived keratinocytes and fibroblasts were used to reconstruct a full thickness, human, in vitro keloid scar model. The reconstructed keloid was co-cultured with monocytes from keloid-forming patients and compared to reconstructed normal skin co-cultured with monocytes from non-keloid-formers. The reconstructed keloid showed increased contraction, dermal thickness (trend) and α-SMA+ staining, but co-culture with monocytes did not further enhance the keloid phenotype. After 2-week culture, all monocytes switched from a CD11chigh/CD14high/CD68low to a CD11chigh/CD14low/CD68high phenotype. However, only monocytes co-cultured with either reconstructed keloid scar or normal skin models skewed towards the more fibrotic M2-macrophage phenotype. There was negligible fibroblast and fibrocyte differentiation in mono- and co-cultured monocytes. These results indicate that monocytes differentiate into M2 macrophages when in the vicinity of early regenerating and repairing tissue, independent of whether the individual is prone to normal or keloid scar formation. Electronic supplementary material The online version of this article (10.1007/s00403-019-01942-9) contains supplementary material, which is available to authorized users.

Barrett's esophagus (BE) is metaplasia of the squamous epithelium to a specialized columnar epithelium. BE progresses through low- and high-grade dysplasia before developing into esophageal adenocarcinoma. The BE microenvironment is not well defined. We compare 12 human clinical BE and adjacent normal squamous epithelium biopsies using single cell immunophenotyping by flow cytometry. A cassette of 19 epithelial and immune cell markers was used to detect differences between cellular compartments in normal and BE tissues. We found that the BE microenvironment has an immunological landscape distinct from adjacent normal epithelium. BE has an increased percentage of epithelial cells with a concomitant decrease in the percentage of immune cells, accompanied by a shift in the immune landscape from a predominantly T cell rich microenvironment in normal tissue to a B cell rich landscape in BE tissue. Hierarchical clustering separates BE and normal samples into two discrete groups based upon our 19-marker panel, but also reveals unexpected, shared phenotypes for three patients. Our results suggest that flow based single cell analysis may have the potential for revealing clinically relevant differences between BE and normal adjacent tissue, and that surface immunophenotypes could identify specific subpopulations from dysplastic tissue for further investigation.

As a means of obtaining reproducible and accurate numbers of larvae for laboratory rearing, we tested a large-particle flow-cytometer type device called the ‘Automated Particle Counter’ (APC). The APC is a gravity-fed, self-contained unit that detects changes in light intensity caused by larvae passing the detector in a water stream and controls dispensing by stopping the flow when the desired number has been reached. We determined the accuracy (number dispensed compared to the target value) and precision (distribution of number dispensed) of dispensing at a variety of counting sensitivity thresholds and larva throughput rates (larvae per second) using < 1-day old Anopheles gambiae and Aedes aegypti larvae. All measures were made using an APC algorithm called the ‘Smoothed Z-Score’ which allows the user to define how many standard deviations (Z scores) from the baseline light intensity a particle’s absorbance must exceed to register a count. We dispensed a target number of 100 An. gambiae larvae using Z scores from 2.5–8 and observed no difference among them in the numbers dispensed for scores from 2.5–6, however, scores of 7 and 8 under-counted (over-dispensed) larvae. Using a Z score ≤ 6, we determined the effect of throughput rate on the accuracy of the device to dispense An. gambiae larvae. For rates ≤ 98 larvae per second, the accuracy of dispensing a target of 100 larvae was - 2.29% ± 0.72 (95% CI of the mean) with a mode of 99 (49 of 348 samples). When using a Z score of 3.5 and rates ≤ 100 larvae per second, the accuracy of dispensing a target of 100 Ae. aegypti was - 2.43% ± 1.26 (95% CI of the mean) with a mode of 100 (6 of 42 samples). No effect on survival was observed on the number of An. gambiae first stage larvae that reached adulthood as a function of dispensing.

Despite the close association between Kaposi’s sarcoma (KS) and immune dysfunction, it remains unclear whether tumor infiltrating immune cells (TIIC), by their absence, presence, or dysfunction, are mechanistically correlated with KS pathogenesis. Therefore, their potential capacity to serve as prognostic biomarkers of KS disease progression or control is unclear. Because epidemic-KS (EpKS) occurs with HIV-1 co-infection, it is particularly important to compare TIIC between EpKS and HIV-negative African endemic-KS (EnKS) to dissect the roles of HIV-1 and Kaposi Sarcoma-associated herpesvirus (KSHV) in KS pathogenesis. This cross-sectional study of 13 advanced KS (4 EnKS, 9 EpKS) patients and 3 healthy controls utilized single-color immunohistochemistry and dual-color immunofluorescence assays to characterize and quantify KSHV infected cells in relation to various TIIC in KS biopsies. Analysis of variance (ANOVA) and Mann-Whitney tests were used to assess differences between groups where P-values < 0.05 were considered significant. The abundance of KSHV infected cells was heterogeneous in KS biopsies. Despite the presence of T-cell chemoattractant chemokine CxCL-9 in biopsies, CD8+ T-cells were sparsely distributed in regions with evident KSHV infected cells but were readily detectable in regions devoid of KSHV infected cells (P < 0.0001). CD68+ (M1) macrophages were evenly and diffusely distributed in KS biopsies, whereas, the majority of CD163+ (M2) macrophages were localized in regions devoid of KSHV infected cells (P < 0.0001). Overall, the poor immune cell infiltration or co-localization in KS biopsies independent of HIV-1 co-infection suggests a fundamental tumor immune evasion mechanism that warrants further investigation.

Colon cancer (CC) is the third most common cancer diagnosed in the United States and the incidence has been rising among young adults. We and others have shown a relationship between the immune infiltrate and prognosis, with improved disease-free survival (DFS) being associated with a higher expression of CD8+ T cells. We hypothesized that a microbial signature might be associated with intratumoral immune cells as well as DFS. We found that the relative abundance of one Operational Taxonomic Unit (OTU), OTU_104, was significantly associated with recurrence even after applying false discovery correction (HR 1.21, CI 1.08 to 1.36). The final multivariable model showed that DFS was influenced by three parameters: N-stage, CD8+ labeling, as well as this OTU_104 belonging to the order Clostridiales. Not only were CD8+ labeling and OTU_104 significant contributors in the final DFS model, but they were also inversely correlated to each other (p=0.022). Interestingly, CD8+ was also significantly associated with the microbiota composition in the tumor: CD8+ T cells was inversely correlated with alpha diversity (p=0.027) and significantly associated with the beta diversity. This study is the first to demonstrate an association among the intratumoral microbiome, CD8+ T cells, and recurrence in CC. An increased relative abundance of a specific OTU_104 was inversely associated with CD8+ T cells and directly associated with CC recurrence. The link between this microbe, CD8+ T cells, and DFS has not been previously shown.

Conventional combinatorial anticancer therapy has shown promising outcomes; still, a significant interest in developing new methods to reinforce and possibly merge chemotherapy and immunotherapy persists. Here, a new one‐step method that immediately modifies immune cells into a targeted form of chemoimmunotherapy through spontaneous and rapid incorporation of hydrophobized antibody–drug conjugates (ADCs) on the surface of immune cells is presented. Therapeutic objectives of this approach include targeted delivery of a potent chemotherapeutic agent to avoid adverse effects, enhancing the mobilization of infused immune cells toward tumor sites, and preserving the intense cytotoxic activities of immune cells against tumor cells. The embedding of hydrophobized ADCs on the immune cell membrane using the strategy in this study provides noninvasive, nontoxic, and homogenous modifications that transiently arm immune cells with highly potent cytotoxic drugs targeted toward cancer cells. The resulting surface‐engineered immune cells with ADCs significantly suppress the tumor growth and drive the eradication of target cancer cells through combinatorial anticancer effects. This novel strategy allows convenient and timely preparation of advanced chemoimmunotherapy on a single immune cell to treat various types of cancer.

// Niklas K. Finnberg 1 , Prashanth Gokare 1 , Avital Lev 1 , Sergei I. Grivennikov 2 , Alexander W. MacFarlane IV 3 , Kerry S. Campbell 3 , Ryan M. Winters 4 , Karen Kaputa 4 , Jeffrey M. Farma 5 , Abbas El-Sayed Abbas 6 , Luigi Grasso 7 , Nicholas C. Nicolaides 7 and Wafik S. El-Deiry 1 1 Department of Hematology/Oncology and Molecular Therapeutics Program, Laboratory of Translational Oncology and Experimental Cancer Therapeutics, Fox Chase Cancer Center, Philadelphia, PA, USA 2 Cancer Prevention and Control Program, Fox Chase Cancer Center, Philadelphia, PA, USA 3 Division of Basic Science, Fox Chase Cancer Center, Philadelphia, PA, USA 4 Biosample Repository Facility, Fox Chase Cancer Center, Philadelphia, PA, USA 5 Division of General Surgery, Department of Surgical Oncology, Fox Chase Cancer Center, Philadelphia, PA, USA 6 Division of Thoracic Surgery, Department of Surgical Oncology, Fox Chase Cancer Center, Philadelphia, PA, USA 7 Morphotek Inc., Philadelphia, PA, USA Correspondence to: Wafik S. El-Deiry, email: // Keywords : 3D, organoid, colorectal cancer, immune cells, biomarkers Received : June 24, 2017 Accepted : July 20, 2017 Published : August 05, 2017 Abstract We have developed 3D-tumoroids and tumor slice in vitro culture systems from surgical tumor specimens derived from patients with colorectal cancer (CRC) or lung cancer to evaluate immune cell populations infiltrating cultured tissues. The system incorporates patient’s peripherally and tumor-derived immune cells into tumoroid in vitro cultures to evaluate the ability of the culture to mimic an immunosuppressive tumor microenvironment (ITM). This system enables analysis of tumor response to standard therapy within weeks of surgical resection. Here we show that tumoroid cultures from a CRC patient are highly sensitive to the thymidylate synthase inhibitor 5-fluorouracil (adrucil) but less sensitive to the combination of nucleoside analog trifluridine and thymidine phosphorylase inhibitor tipiracil (Lonsurf). Moreover, re-introduction of isolated immune cells derived from surrounding and infiltrating tumor tissue as well as CD45+ tumor infiltrating hematopoietic cells displayed prolonged (>10 days) survival in co-culture. Established tumor slice cultures were found to contain both an outer epithelial and inner stromal cell compartment mimicking tumor structure in vivo . Collectively, these data suggest that, 3D-tumoroid and slice culture assays may provide a feasible in vitro approach to assess efficacy of novel therapeutics in the context of heterogeneous tumor-associated cell types including immune and non-transformed stromal cells. In addition, delineating the impact of therapeutics on immune cells, and cell types involved in therapeutic resistance mechanisms may be possible in general or for patient-specific responses.

Corticosteroids, such as dexamethasone (DEX), are the mainstays for the treatment of moderate to severe inflammatory bowel disease (IBD). However, their relatively poor bioavailability and lack of specificity is often the origin of short and long-term adverse effects. Here, spherical polymeric nanoconstructs (SPNs) encapsulating dexamethasone are proposed for the systemic treatment of IBD. In a mouse model of colitis, the accumulation of SPNs within the inflamed intestine is firstly assessed using near infra-red fluorescent (NIRF) imaging at different stages of the disease - 5, 7 and 10 days of Dextran Sulfate Sodium (DSS) administration. Then, the efficacy of DEX-SPNs is tested in vitro over macrophages and in vivo by monitoring the animal weight, food and water intake; expression of inflammatory cytokines (TNF-α, IL-1β, IL-6); intestinal density of macrophages; rectal bleeding and histological scoring. 150 nm DEX-SPNs are shown to deposit within the hyper-permeable inflamed intestine in a disease severity-dependent fashion. DEX-SPNs exposed to LPS-stimulated RAW 264.7 cells reduce the expression of inflammatory cytokines as rapidly as free DEX. In DSS-administered mice, DEX-SPNs treatments improve weight loss, reduce the macrophage infiltration, expression of inflammatory cytokines, rectal bleeding and histological scoring, as compared to free DEX. Moreover, DEX-SPNs exert a strong systemic anti-inflammatory effect and facilitate animal recovery. This work confirms the benefits of using sufficiently small nanoconstructs for targeting inflamed, hyper-permeable tissues and efficiently delivering high doses of corticosteroids for the treatment of intestinal and systemic inflammation.

Anti-retroviral therapy (ART) improves life expectancy in people living with HIV (PWH), but it remains unclear how chronic HIV infection affects normal aging of the immune system. Plasma cell-free protein expression and immune phenotypes were assessed in blood from ART treated PWH (19–77yrs, n=106) and age-matched, HIV-negative controls (HC, n=103). Age-associated parameters were identified in HC and PWH independently by univariate Spearman correlation (p Plasma biomarker analysis resulted in 8-parameter inflammatory index that was higher in PWH compared to HC and correlated with age. Combining flow and plasma data generated 277 and 491 age-associated parameters (out of total 1,357) in HC and PWH, respectively. PWH exhibited different immunological profiles associated with age compared to HC. However, CD38 expression on T cells declined with age in both HC and PWH. Predictive modeling using age-associated parameters in HC generated a 14-parameter signature with 76.9% accuracy to predict chronological age in HC, however this model failed to predict age in PWH (18.6% accuracy) highlighting the effect of HIV infection on immunological aging. A 25-parameter model (IMmunological Age Prediction, IMAP-25) however showed 70% and 53% accuracy in HC and PWH and was used to show an accelerated aging rate in PWH by 5.6 yrs compared to HC. We applied a statistical approach to generate immune signatures, easily monitored in human blood samples, that could be used as an indicator of one’s ‘immunological age’ during ART-treated HIV infection.

Hepatocellular carcinoma (HCC) is an immune-related tumor, that the type and number of tumor-infiltrated immune cells can serve as biomarkers for the clinical application. In this study, we constructed the immune model for diagnostic and prognostic prediction of HCC based on the systematic bioinformatics analyses on the component of immune cells from large samples transcriptome. CIBERSORT analysis found that the component of immune cells between 513 HCC and 473 adjacent normal tissues was different. M0 macrophages and regulatory T cells were mainly enriched in tumor tissues, whereas the CD8+ T cell and activated CD4+ memory T cells were the most in normal tissues. Using random forest and LASSO analyses, eleven immune cell types were mined out to construct the immune diagnostic model (IDG), which showed high efficiency in distinguishing cancer from normal tissues both in testing and validation groups. In addition, the immune prognostic model (IPG) consisting of five types of immune cells was constructed using the LASSO-Cox algorithm. It showed that HCC patients of the high-risk group had a significantly shorter survival time than those of low-risk group in testing, validation, and entire cohorts. Besides, Nomogram plots and decision curve analyses revealed that the IPG was positively associated with the HCC clinical classification of the Barcelona Clinic Liver Cancer (BCLC) stage, and showing more accuracy of prediction than independent BCLC stage. Related analyses found that IDG positively correlated with epithelial-mesenchymal transition (EMT) and cytotoxic factor-related genes and negatively correlated with immune checkpoint regulators related genes. From the GSEA analysis of the biological function of genes related to IPG, it was found that the genes of the high-risk group were enriched in some tumorigenesis related pathways, such as DNA replication, cell cycle, and PPARA. Therefore, this study identified IDG and IPG as efficient biomarkers for the diagnosis and prognosis of HCC., Highlights • We comprehensive analyzed the infiltrated immune cells between HCC and adjacent tissues in big data samples. • The IDG and IPG models were potential biomarkers for the diagnosis and prognosis of HCC, respectively. • The IPG model was associated with the HCC clinical characteristics.

International audience; Sarcomatoid mesothelioma (SM) is a devastating cancer associated with one of the poorest outcome. Therefore, representative preclinical models reproducing different tumor microenvironments (TME) observed in patients would open up new prospects for the identification of markers and evaluation of innovative therapies. Histological analyses of four original models of rat SM revealed their increasing infiltrative and metastatic potential were associated with differences in Ki67 index, blood-vessel density, and T-lymphocyte and macrophage infiltration. In comparison with the noninvasive tumor M5-T2, proteomic analysis demonstrated the three invasive tumors F4-T2, F5-T1 and M5-T1 shared in common a very significant increase in the abundance of the multifunctional proteins galectin-3, prohibitin and annexin A5, and a decrease in proteins involved in cell adhesion, tumor suppression, or epithelial differentiation. The increased metastatic potential of the F5-T1 tumor, relative to F4-T2, was associated with an increased macrophage vs T-cell infiltrate, changes in the levels of expression of a panel of cytokine genes, an increased content of proteins involved in chromatin organization, ribosome structure, splicing, or presenting anti-adhesive properties, and a decreased content of proteins involved in protection against oxidative stress, normoxia and intracellular trafficking. The most invasive tumor, M5-T1, was characterized by a pattern of specific phenotypic and molecular features affecting the presentation of MHC class I-mediated antigens and immune cell infiltration, or involved in the reorganization of the cytoskeleton and composition of the extracellular matrix. These four preclinical models and data represent a new resource available to the cancer research community to catalyze further investigations on invasiveness.

The neurotransmitter γ-aminobutyric acid (GABA) is an extracellular signaling molecule in the brain and in pancreatic islets. Here, we demonstrate that GABA regulates cytokine secretion from human peripheral blood mononuclear cells (PBMCs) and CD4+ T cells. In anti-CD3 stimulated PBMCs, GABA (100 nM) inhibited release of 47 cytokines in cells from patients with type 1 diabetes (T1D), but only 16 cytokines in cells from nondiabetic (ND) individuals. CD4+ T cells from ND individuals were grouped into responder or non-responder T cells according to effects of GABA (100 nM, 500 nM) on the cell proliferation. In the responder T cells, GABA decreased proliferation, and inhibited secretion of 37 cytokines in a concentration-dependent manner. In the non-responder T cells, GABA modulated release of 8 cytokines. GABA concentrations in plasma from T1D patients and ND individuals were correlated with 10 cytokines where 7 were increased in plasma of T1D patients. GABA inhibited secretion of 5 of these cytokines from both T1D PBMCs and ND responder T cells. The results identify GABA as a potent regulator of both Th1- and Th2-type cytokine secretion from human PBMCs and CD4+ T cells where GABA generally decreases the secretion., Highlights • GABA regulates cytokine secretion in anti-CD3-stimulated peripheral blood mononuclear cells (PBMCs) and CD4+ T cells. • GABA inhibits secretion of 47 cytokines in PBMCs from type 1 diabetes patients. • GABA regulates secretion of pro- and anti-inflammatory cytokines in a concentration-dependent manner. GABA is a signal molecule in the brain, blood and pancreatic islets where it is secreted by the insulin-producing β cells. GABA has many roles in human islets including optimizing function and survival of β cells. Bhandage et al. now show that GABA is a potent regulator of secretion of both pro- and anti-inflammatory cytokines in stimulated immune cells. In type 1 diabetes the β-cell mass is diminished and thus the protective effect of GABA in the islets although not in blood. Targeting GABA signaling in diabetes mellitus is likely to be a part of the solution when curing diabetes.

Background: TFF1 deficiency induces a mucosal pro-inflammatory phenotype that contributes to gastric tumorigenesis in mouse and human. Methods: We utilized the Tff1-KO mouse model to assess the impact of TFF1 loss on immune cells infiltration in the stomach. We used single cell suspension, flow cytometry, immunohistochemistry, and quantitative PCR (qPCR) assays. Results: The Tff1-KO gastric mucosa demonstrated high chronic inflammatory scores (score: 3-4) at age 2 months, which exacerbated at age 8 months (score: 4-6). We next used single-cell suspensions for flow cytometry analysis of total leukocytes (CD45+ cells), total T lymphocytes (CD45+CD3+cells), T cell subsets (CD4+, CD8+, and CD3+CD4-CD8-cells), and monocytes/macrophages (CD45+F4/80+cells). The results demonstrated an age-dependent (2 → 8 month age) significant increase of leukocytes (p

Background: Central obesity (CO) is an inflammatory disease. Because immune cells and adipocytes are catecholamines(CA)-producing cells, we studied the expression of adrenoceptors (AR) in peripheral blood mononuclear cells (PBMCs) hypothesizing a distinct adrenergic pattern in inflammatory obesity. Methods: AR expression was assessed in blood donors categorized by waist circumference (WC) (CO: WC≥0.80 m in women and ≥0.94 m in men). Following a pilot study for all AR subtypes, we measured β2AR expression in fifty-seven individuals and correlated this result with anthropometric, metabolic and inflammatory parameters. A ratio (R) between AR mRNA of CO and non-CO2.0 over expression. Results: The pilot study revealed no differences between groups, except for β2AR mRNA. CO individuals showed underexpression of β2AR relatively to those without CO (R=0.08; p=0.009). β2AR expression inversely correlated with triacylglycerol (r=-0.271; p=0.041), very low-density lipoprotein-cholesterol (r=-0.313; p=0.018) and leptin (r=-0.392; p=0.012) and positively with high-density lipoprotein-cholesterol (r=0.310: p=0.045) plasma levels. Multiple logistic regression analysis showed a protective effect of β2AR expression (≥2x10-6) [odds ratio (OR) 0.177 with respective confidence interval of 95% (95% CI) (0.040- 0.796)] for the occurrence of CO. A higher association was found for women as compared to men (Ξ9:1) [OR 8.972 (95% CI) (1.679-47.949)]. Conclusion: PBMCs β2AR, underexpressed in centrally obese, are associated with a better metabolic profile and showed a protective role for the development of CO. The discovery of β2AR as a new molecular marker of obesity subphenotypes in PBMCs might contribute to clarify the adrenergic immunomodulation of inflammatory obesity.

The crosstalk between immune cells, cancer cells, and extracellular vesicles (EVs) secreted by cancer cells remains poorly understood. We created three-dimensional (3D) cell culture models using human leiomyoma discs and Myogel to study the effects of immune cells on highly (HSC-3) and less (SCC-25) invasive oral tongue squamous cell carcinoma (OTSCC) cell lines. Additionally, we studied the effects of EVs isolated from these cell lines on the cytotoxicity of CD8+ T and NK cells isolated from three healthy donors. Our analysis included the effects of these EVs on innate immunity in zebrafish larvae. Activated immune cells significantly decreased the proliferation of both OTSCC cell lines and associated with a diminished invasion area of HSC-3 cells. In general, EVs from SCC-25 increased the cytotoxic activity of CD8+ T and NK cells more than those from HSC-3 cells. However, this effect varied depending on the source and the immune and cancer cell subgroups. In zebrafish, the amount of IL-13 mRNA was decreased by SCC-25 EVs. This study describes promising in vitro and in vivo models to investigate interactions between immune cells, cancer cells, and EVs.

// Maria del Mar Valenzuela-Membrives 1 , Francisco Perea-Garcia 3 , Abel Sanchez- Palencia 2 , Francisco Ruiz-Cabello 3 , Mercedes Gomez-Morales 4 , Maria Teresa Miranda-Leon 5 , Inmaculada Galindo-Angel 4 , Maria Esther Farez-Vidal 6 1 Department of Pneumology, San Cecilio University Hospital, Granada, Spain 2 Department of Thoracic Surgery, Virgen de las Nieves University Hospital, Granada, Spain 3 Institute for Biomedical Research, Virgen de las Nieves University Hospital, Granada, Spain 4 Department of Pathology, School of Medicine, University of Granada, Granada, Spain 5 Department of Statistics and Operative Research, School of Medicine, University of Granada, Granada, Spain 6 Department of Biochemistry and Molecular Biology, School of Medicine, University of Granada, Granada, Spain Correspondence to: Maria Esther Farez-Vidal, email: efarez@ugr.es Keywords: immunological response, lung cancer, immunohistochemistry, flow cytometry, lymphocyte subsets Received: May 18, 2016 Accepted: September 19, 2016 Published: September 26, 2016 ABSTRACT Immune cell infiltration is a common feature of many human solid tumors. Innate and adaptative immune systems contribute to tumor immunosurveillance. We investigated whether tumors evade immune surveillance by inducing states of tolerance and/or through the inability of some immune subpopulations to effectively penetrate tumor nests. Immunohistochemistry and flow cytometry analysis were used to study the composition and distribution of immune subpopulations in samples of peripheral blood, tumor tissue (TT), adjacent tumor tissue (ATT), distant non-tumor tissue (DNTT), cancer nests, cancer stroma, and invasive margin in 61 non-small-cell lung cancer (NSCLC) patients. A significantly higher percentage of T and B cells and significantly lower percentage of NK cells were detected in TT than in DNTT. Memory T cells (CD4 + CD45RO + , CD8 + CD45RO + ) and activated T cells (CD8 + DR + ) were more prevalent in TT. Alongside this immune activation, the percentage of T cells with immunosuppressive activity was higher in TT than in DNTT. B- cells were practically non-existent in tumor nests and were preferentially located in the invasive margin. The dominant NK cell phenotype in peripheral blood and DNTT was the cytotoxic phenotype (CD56 + CD16 + ), while the presence of these cells was significantly decreased in ATT and further decreased in TT. Finally, the immunologic response differed between adenocarcinoma and squamous cell carcinoma and according to the tumor differentiation grade. These findings on the infiltration of innate and adaptative immune cells into tumors contribute to a more complete picture of the immune reaction in NSCLC.

Contains fulltext : 171567.pdf (Publisher’s version ) (Open Access) Sunitinib improves the outcomes of patients with solitary fibrous tumours (SFTs). The aim of this study was to investigate and contextualise sunitinib-induced morpho-functional changes in order to gain insights into the drug's mechanism of action.To this end, four surgical specimens obtained from two sunitinib-responsive patients with malignant SFT, and one primary cell culture obtained from fresh tumoral tissue and its stabilised cell line, were studied by means of immunohistochemistry, bright field in situ hybridisation, immunofluorescence/confocal microscopy, and biochemistry.The post-sunitinib surgical samples were characterised by two biologically relevant morpho-functional changes: clear areas and necrotic foci. The first were associated with the attenuation/loss of PDGFRB expression and decreased mTOR signalling, and corresponded to a pathological response. The second were associated with the over-expression of PDGFRB and VEGFA, strong mTOR signalling activation, and the appearance of HIF1alpha expression, hallmarks of pathological progression. The analysis clearly showed that sunitinib reduces the vascular supply network and inhibits tumoral cells. It also either induces autophagy, thus favouring drug response, or impairs autophagy as a result of lysosome sequestration, thus favouring disease progression. These distinct autophagic events were associated with different myeloid immune contextures. Finally, we also found that PDGFRB is one of the components of a complex that includes Beclin 1 and VPS34.The results of these tissue-based analyses provide new insights into sunitinib's mechanism of action in SFT patients.