Immunosuppressive Protein Research Articles

Association of circulating protein biomarkers with clinical outcomes of durvalumab in head and neck squamous cell carcinoma

ABSTRACT The potential for durvalumab, a programmed cell death ligand-1 (PD-L1)-blocking monoclonal antibody, to treat head and neck squamous cell carcinoma (HNSCC) is being evaluated in multiple clinical trials. We assessed circulating proteins at baseline to identify potential biomarkers and to understand pathways related to clinical outcomes for durvalumab. Prior to treatment, 66 serum proteins were measured using multiplex immunoassays for 158 durvalumab-treated HNSCC patients in the phase II HAWK and CONDOR trials as a discovery dataset and 209 durvalumab-treated HNSCC patients in the phase III EAGLE trial as a validation dataset. Multivariate Cox modeling of HAWK and CONDOR datasets established that higher baseline concentrations of interleukin-6 (IL-6), C-reactive protein, S100 calcium-binding protein A12, and angiopoietin-2 (ANGPT2) were associated with shorter overall survival (OS), while higher concentrations of osteocalcin correlated with longer OS after durvalumab treatment (p < .05). All five proteins remained significantly correlated with OS after adjusting for baseline clinical factors, with consistent results across clinical efficacy endpoints based on univariate correlation analyses. The validation dataset from the EAGLE trial confirmed the independent association of IL-6 and osteocalcin with OS, and preserved directional trends for the other biomarkers identified in the discovery dataset. Our results demonstrate the important role of immunosuppressive proteins in the resistance of HNSCC to durvalumab treatment. Osteocalcin showed a positive correlation with clinical outcomes, which remains to be further investigated.

Pregnancy Zone Protein (PZP) is significantly upregulated in the decidua of recurrent and spontaneous miscarriage and negatively correlated to Glycodelin A (GdA)

BackgroundPregnancy Zone Protein (PZP) is an immunosuppressive protein that is expressed by the placenta and has also been identified in immune cells. When PZP and Glycodelin A (GdA) are combined, they act synergistically to inhibit Th-1 immune response. Little is known about its combined expression and role in normal and disturbed first trimester pregnancy. Patients and methodsWe investigated the expression of PZP and GdA in placental tissue obtained from spontaneous miscarriage (SM) (n = 19) and recurrent miscarriage (RM) (n = 17) at gestational weeks 6–13 by immunohistochemistry and on mRNA-level by either TaqMan PCR or in situ hybridization. Placental tissue from legal terminations of healthy pregnancies (n = 15) served as control group. Immunofluorescence double staining was used to analyse the combined expression of PZP and GdA in decidual tissue. ResultsThe protein level of PZP was significantly increased in decidual stroma of SM samples compared to the decidua of control specimens and also significantly upregulated in the decidual stroma cells in the RM group. Concerning GdA, the decidual stroma revealed a significantly decreased protein level in the group with spontaneous abortions than in the group with healthy pregnancies. There was also a significant downregulation of GdA in the decidual stroma of RM samples compared to the control group. We observed a significant negative correlation of PZP and GdA in decidual stromal tissue of recurrent abortion. We could confirm the staining results for PZP as well as for GdA on mRNA level. Both proteins are co-localized in decidual stroma as analysed by immunofluorescence double staining. ConclusionA balanced expression of GdA and its carrier protein PZP in the decidua seems crucial for a successful ongoing pregnancy. According to our data, these immunosuppressive proteins are co-localized in the decidual tissue and show a negative correlation only in patients suffering from recurrent abortion.

Expression of a phenoloxidase cascade inhibitor enhances the virulence of the fungus Beauveria bassiana against the insect Helicoverpa armigera

Entomopathogenic fungi have high potential for controlling insect pests, although the slow killing speed has blocked their widespread application. To increase the virulence of entomopathogenic fungi, genetic modification can be employed. Egf1.0 is an immunosuppressive protein encoded by polydnavirus, carried by parasitoid wasp Microplitis demolitor, which blocks the prophenoloxidase (PPO) activation response of host insects. In this study, we explored the feasibility of genetically modifying entomopathogenic fungi with increased virulence by expressing Egf1.0. In comparison with the wild-type parents, the median lethal concentration (LC50) of Beauveria bassiana expressing Egf1.0 against Helicoverpa armigera was reduced by 2.7-fold, and the median lethal time (LT50) was reduced by 22.8%. In vitro assay showed that recombinant Egf1.0 was able to inhibit the PPO activation response of H. armigera. In vivo assay revealed that the expression of Egf1.0 in B. bassiana caused a higher degree of suppression to PPO activation response of H. armigera. These assays suggested that the increased virulence of the transgenic fungi is due to the increased ability to suppress the host insect's immune response. Moreover, colony growth, conidia yield, and germination assays revealed that the expression of Egf1.0 in B. bassiana had no effect on its growth and development. In conclusion, the expression of Egf1.0 can significantly enhance the pathogenicity of B. bassiana against host insects.

Transforming growth factor beta type 1 (TGF-β) and hypoxia-inducible factor 1 (HIF-1) transcription complex as master regulators of the immunosuppressive protein galectin-9 expression in human cancer and embryonic cells.

Galectin-9 is one of the key proteins employed by a variety of human malignancies to suppress anti-cancer activities of cytotoxic lymphoid cells and thus escape immune surveillance. Human cancer cells in most cases express higher levels of galectin-9 compared to non-transformed cells. However, the biochemical mechanisms underlying this phenomenon remain unclear.Here we report for the first time that in human cancer as well as embryonic cells, the transcription factors hypoxia-inducible factor 1 (HIF-1) and activator protein 1 (AP-1) are involved in upregulation of transforming growth factor beta 1 (TGF-β1) expression, leading to activation of the transcription factor Smad3 through autocrine action. This process triggers upregulation of galectin-9 expression in both malignant (mainly in breast and colorectal cancer as well as acute myeloid leukaemia (AML)) and embryonic cells. The effect, however, was not observed in mature non-transformed human cells. TGF-β1-activated Smad3 therefore displays differential behaviour in human cancer and embryonic vs non-malignant cells. This study uncovered a self-supporting biochemical mechanism underlying high levels of galectin-9 expression operated by the human cancer and embryonic cells employed in our investigations. Our results suggest the possibility of using the TGF-β1 signalling pathway as a potential highly efficient target for cancer immunotherapy.

Abstract PO-128: Lack of Aurora kinase signaling leads cancer cells to adopt an endocycle and form polyploid multinucleated giant cells that resist antimitotic drugs

Abstract Polyploid multinucleated (PP/MN) giant cancer cells are common in tumors and have been tightly linked with tumor heterogeneity, resistance to cancer therapy, tumor relapse, metastasis, malignancy, immunosuppression, modulation of the tumor microenvironment and cancer stem cells. The abundance of PP/MN cancer cells is markedly higher in high-grade malignant tumors than in low-grade tumors, in the metastatic foci than in the primary tumor, and in relapsing tumors post-chemotherapy than in tumors before therapy. They can remain viable and metabolically active for extended periods of time. They can secrete an array of growth factors, cytokines and chemokines. Immunosuppressive proteins including programmed death-ligand 1 (PD-L1) were also found to be overexpressed in these cells. Such cells are known to escape from cytotoxicity induced by major antimitotic agents including taxanes and vinca alkaloids. Therefore, they are responsible of contributing to a microenvironment advantageous for tumor growth and survival. However, the molecular mechanisms that cause these cells to form are yet known. PP/MN cancer cells can repopulate, and form macroscopic colonies and spheroids in vitro as they generate tumors when inoculated into mice. In comparison to diploid cancer cells, these daughter cells lower the expression of epithelial markers and acquire a mesenchymal phenotype, which is a key transformation for cancer development, progression and metastasis. Emerging evidence has demonstrated that PP/MN cancer cells arise in lung cancer, cervical carcinoma, ovarian cancer, prostate cancer, glioblastoma, colorectal cancer, and breast cancer. The role of PP/MN giant cancer cells in cancer is well documented. Therefore, revealing the molecular events that cause PP/MN giant cells to form can lead to clinically relevant approaches for treating recurrent and metastatic disease, which remains a major global health issue despite decades of substantial research. In this study, we discover that Aurora kinases are synergistic determinants of a switch from the proliferative cell cycle to polyploid growth and multinucleation in lung cancer cell lines. When Aurora kinases are inhibited together, lung cancer cells uniformly grow into PP/MN giant cells. These cells have adopted an endocycle in which the genome replicates, mitosis is omitted and cells grow in size. Consequently, such cells continue to safely grow in the presence of antimitotic agents. These polyploid multinucleated cancer cells can re-enter the proliferative cell cycle and grow in cell number when the treatment is terminated. We also demonstrate that chemical programming of the cells to endocycle with Aurora kinase inhibitors can serve as a methodology to identify antimitotic agents in a simple, high-throughput fashion. To the best of our knowledge, ours is the first study to describe the responsible genes involved in the formation of PP/MN giant cancer cells. Citation Format: Vural Tagal, Michael G. Roth. Lack of Aurora kinase signaling leads cancer cells to adopt an endocycle and form polyploid multinucleated giant cells that resist antimitotic drugs [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-128.

Coding transcriptome analyses reveal altered functions underlying immunotolerance of PEG-fused rat sciatic nerve allografts

BackgroundCurrent methods to repair ablation-type peripheral nerve injuries (PNIs) using peripheral nerve allografts (PNAs) often result in poor functional recovery due to immunological rejection as well as to slow and inaccurate outgrowth of regenerating axonal sprouts. In contrast, ablation-type PNIs repaired by PNAs, using a multistep protocol in which one step employs the membrane fusogen polyethylene glycol (PEG), permanently restore sciatic-mediated behaviors within weeks. Axons and cells within PEG-fused PNAs remain viable, even though outbred host and donor tissues are neither immunosuppressed nor tissue matched. PEG-fused PNAs exhibit significantly reduced T cell and macrophage infiltration, expression of major histocompatibility complex I/II and consistently low apoptosis. In this study, we analyzed the coding transcriptome of PEG-fused PNAs to examine possible mechanisms underlying immunosuppression.MethodsAblation-type sciatic PNIs in adult Sprague-Dawley rats were repaired using PNAs and a PEG-fusion protocol combined with neurorrhaphy. Electrophysiological and behavioral tests confirmed successful PEG-fusion of PNAs. RNA sequencing analyzed differential expression profiles of protein-coding genes between PEG-fused PNAs and negative control PNAs (not treated with PEG) at 14 days PO, along with unoperated control nerves. Sequencing results were validated by quantitative reverse transcription PCR (RT-qPCR), and in some cases, immunohistochemistry.ResultsPEG-fused PNAs display significant downregulation of many gene transcripts associated with innate and adaptive allorejection responses. Schwann cell-associated transcripts are often upregulated, and cellular processes such as extracellular matrix remodeling and cell/tissue development are particularly enriched. Transcripts encoding several potentially immunosuppressive proteins (e.g., thrombospondins 1 and 2) also are upregulated in PEG-fused PNAs.ConclusionsThis study is the first to characterize the coding transcriptome of PEG-fused PNAs and to identify possible links between alterations of the extracellular matrix and suppression of the allorejection response. The results establish an initial molecular basis to understand mechanisms underlying PEG-mediated immunosuppression.

Abstract 695: Treatment with oral mifepristone enables a patient with end-stage pancreatic cancer in hospice, on a morphine drip, to restore a decent quality of life

Abstract The progesterone (P) receptor modulator, mifepristone, has been previously found to provide significant improvement in length and quality of life in people with a variety of advanced cancers. A 57 year old man with severe abdominal pain, dyspnea, nausea weakness and vomiting, in hospice with marked sedation from a morphine drip, was treated with oral mifepristone 200mg/day, as an option to extend his lifespan with reasonable quality of life. He had never received treatment for his disease and was given a prognosis of only 1-3 weeks to live, by his oncologist. He showed quick improvement in both his nausea (which was intractable), and his abdominal pain, within one month of treatment. His first visit he was wheelchair ridden, next visit he had progressed to a cane and after 2 months walked in without any assistance. He has now been on oral mifepristone 200mg/day for 5 months, as his only treatment. His only complaint is slight weakness and mild dyspnea; he is considered ECOG-2 when initially he was ECOG-IV. He has had no side effects from the mifepristone and rarely needs his previous analgesics, or anti-emetics. The hypothesized mechanism of action of this drug is to inhibit the activation of a membrane P receptor in the cancer cells that causes the production of a 757 amino acid unique protein measuring 90 kDa that is involved in cell-cell regulation, but is converted to smaller intracytoplasmic splice variants, which are immunosuppressive proteins. It is called the progesterone induced blocking factor (PIBF). When these smaller splice variants are released into the tumor micro-environment, they suppress cytolytic activity of the cellular immune system, e.g., by stabilizing perforin granules in natural killer cells. The fact that treatment with mifepristone has been shown to improve length and quality of life, not only in cancer associated with the classic nuclear P receptor, e.g., breast, ovarian, and endometrial cancer, but also those cancers devoid of the classic nuclear receptor, e.g., small cell and non-small cell lung cancer, leukemia, colon cancer, thymic epithelial cell cancer, renal cell carcinoma, transitional cell carcinoma of the renal-pelvis, and glioblastoma multiforme, suggests that the PIBF protein may be important for a large variety of cancers to escape immune surveillance. Pancreatic cancer can be added to this list of advanced cancers that respond to mifepristone therapy. The fact that it seems to work even when standard chemotherapy, immunotherapy, and targeted therapies have failed to work on patients with widespread cancer, including a large variety of cancers, could suggest that the PIBF protein may be a mechanism used by stem cells to escape immune surveillance. Since there are no other treatment options, we have elected not to repeat computerized tomography scans to evaluate whether the lesions have regressed but we believe clinically they must have. Citation Format: Jerome H. Check, Diane Check, Carrie Wilson, Maya Srivastava, Trina Poretta, James Aikins. Treatment with oral mifepristone enables a patient with end-stage pancreatic cancer in hospice, on a morphine drip, to restore a decent quality of life [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 695.

Abstract 4509: Targeting tumor-derived exosomes using a lectin affinity hemofiltration device

Abstract Background: Tumor-derived exosomes (TEX) are 30-150 nm vesicles that are released by tumor cells and have systemic disease-promoting effects. Despite abundant research showing that TEX are purveyors of tumor growth-promoting and immunosuppressive cargo, a clinical strategy for addressing exosomes in oncology is lacking. Aethlon Medical is initiating clinical trials in cancer patients with the Hemopurifier®, a hollow fiber hemofiltration device that is operated using dialysis infrastructure, for removing exosomes from circulating blood. The Hemopurifier incorporates size exclusion and a lectin (Galanthus nivalis agglutinin; GNA) affinity matrix for capturing nanoparticles having high mannose glycoprotein surface structures. Experimental Procedures: A benchtop version of the Hemopurifier was operated by recirculating samples of cancer patients' plasma through the hollow fiber filters ex vivo. The exosomes remaining in plasma were quantified using nanoparticle tracking analysis measurements and the ExoView instrument. To identify the Hemopurifier-bound material, an elution technique was developed to displace the GNA-bound material from the Hemopurifier. The recovered vesicles were assessed using ExoView and flow cytometry and compared to an established exosome isolation method, size exclusion chromatography, which utilizes small-size ion exchange columns (mini-SEC). In vitro assays were also performed by co-culturing the recovered exosomes with T cells to evaluate their immunosuppressive functions. Results: The results demonstrate that the GNA affinity capture mechanism of the Hemopurifier is effective for clearing 92-99% of exosomes from input concentrations of 109-1010 exosomes per mL of plasma. The device captures exosomes from diverse tumor types including head and neck cancer, melanoma, ovarian cancer, esophageal cancer and breast cancer. Analysis of plasma samples from Stages III and IV triple negative and human epidermal growth factor receptor 2 overexpressing breast cancer showed that Hemopurifier-bound material comprised the appropriately-sized vesicles expressing tumor susceptibility gene 101 and tetraspanins. In comparison to mini-SEC-isolated exosomes, tumor-specific epitopes and immunosuppressive proteins exhibited equivalent expression among Hemopurifier-isolated exosomes. The immunosuppressive functions of Hemopurifier- and mini-SEC-isolated exosomes were also equivalent. Conclusions: The Hemopurifier effectively clears exosomes present in plasma that originate from diverse types of cancer. The Hemopurifier-captured exosomes exhibit signatures of malignancy and immunosuppression and are therefore relevant therapeutic targets. This research was supported by the National Cancer Institute of the National Institutes of Health under award number 1R43CA232977-01. Citation Format: Annette M. Marleau, Michael T. Jacobs, Nadia Gruber, Timothy C. Rodell, Soldano Ferrone, Theresa L. Whiteside. Targeting tumor-derived exosomes using a lectin affinity hemofiltration device [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4509.

PD-L1 Expression Is a Favorable Prognostic Marker in Gastric Carcinoma.

Gastric cancer (GC) is the fifth most common cancer and the third leading cause of cancer-related mortality worldwide. Although multidisciplinary therapeutic strategies have improved treatment outcomes, the overall prognosis for patients with GC remains poor. Recently, immunotherapeutic agents targeting immunosuppressive proteins such as anti-programmed death-1 receptor and anti-programmed death ligand-1 (PD-L1) have emerged as effective treatment options for various cancers, including GC. In addition to their therapeutic role, the expression of PD-L1 has been used as a predictive biomarker for programmed death-1/PD-L1 treatment response and has been shown to have a prognostic role in certain cancers. This study aims to evaluate the expression of PD-L1 in GC samples from Jordanian patients and assess its prognostic role as well as its correlation with clinicopathologic variables. Gastrectomy samples from 96 patients diagnosed with gastric adenocarcinoma were included in the study. Immunohistochemistry assay was employed for PD-L1 testing, and the scoring was based on a combined positive score (CPS). It was found that 66.7% of the study samples were positive for PD-L1 (CPS≥1). The expression of PD-L1 was not significantly associated with any of the assessed clinicopathologic variables; however, it was found to be an independent favorable prognostic factor for overall survival (hazard ratio: 0.481; 95% confidence interval: 0.231-1.001; P=0.050).

Effect of nitric oxide inhibition in Bacillus Calmette-Guerin bladder cancer treatment

BackgroundBacillus Calmette-Guerin (BCG) is the standard treatment for patients with high-risk non-muscle invasive bladder cancer (BC). Despite its success, about 30–50% of patients are refractory. It was reported that inducible nitric oxide synthase (iNOS) tumor expression is presented in 50% of human BC, associated with bad prognosis and BCG failure. Objectiveto evaluate in human bladder tumors the association between iNOS expression and the tumor microenvironment focusing on the immunosuppressive protein S100A9. Also, investigate in a preclinical murine MB49-BC model the tumor immunoresponse induced by BCG in combination with the nitric oxide production inhibitor l-NAME. ResultsIn human bladder tumors, we detected a positive association between iNOS and S100A9 tumor expression, suggesting a relationship between both immunomodulatory proteins. We also found a positive correlation between iNOS tumor expression and the presence of S100A9+ tumor-infiltrating cells, suggesting an immunosuppressive tumor microenvironment induced by the nitric oxide production.Using the subcutaneous murine BC model, we show that similarly to the human pathology, MB49 tumors constitutively expressed iNOS and S100A9 protein. MB49 tumor-bearing mice presented an immunosuppressive systemic profile characterized by fewer cytotoxic cells (CD8+ and NK) and higher suppressor cells (Treg and myeloid-derived suppressor cells -MDSC-) compared to normal mice. BCG treatment reduced tumor growth, increasing local CD8+-infiltrating cells and induced a systemic increase in CD8+ and a reduction in Treg. BCG combined with l-NAME, significantly reduced tumor growth compared to BCG alone, diminishing iNOS and S100A9 tumor expression and increasing CD8+-infiltrating cells in tumor microenvironment. This local response was accompanied by the systemic increase in CD8+ and NK cells, and the reduction in Treg and MDSC, even more than BCG alone. Similar results were obtained using the orthotopic BC model, where an increase in specific cytotoxicity against MB49 tumor cells was detected. ConclusionThe present study provides preclinical information where NO inhibition in iNOS-expressing bladder tumors could contribute to improve BCG antitumor immune response. The association between iNOS and S100A9 in human BC supports the hypothesis that iNOS expression is a negative prognostic factor and a promising therapeutic target.

TNF-\u03b1 and INF-\u03b3 primed canine stem cell-derived extracellular vesicles alleviate experimental murine colitis

The inflammatory bowel diseases (IBD) are characterized by relapsing inflammation and immune activation diseases of the gastrointestinal tract. Extracellular vesicles, which elicit similar biological activity to the stem cell themselves, have been used experimentally to treat dextran sulfate sodium (DSS)-induced colitis in murine models though immunosuppressive potential. In this study, we investigated whether the Extracellular vesicles (EVs) obtained by stimulating inflammatory cytokine on canine adipose mesenchymal stem cells (cASC) improved anti-inflammatory and/or immunosuppressive potential of EVs, and/or their ability to alleviate inflammation in colitis. We also explored the correlation between immune cells and the inflammatory repressive effect of primed EVs. Pro-inflammatory cytokines such as TNF-α and IFN-γ increased immunosuppressive protein such as HGF, TSG-6, PGE2 and TGF-β in EVs. Moreover, the anti-inflammatory effect of EVs was improved through pretreatment with inflammatory cytokines. Importantly, EVs obtained from primed stem cells effectively induced macrophage polarization toward an anti-inflammatory M2 phenotype and suppressed activated immunity by enhancing regulatory T cells in inflamed colon in mice. Our results provide a new and effective therapy for the EVs obtained from ASC stimulated with TNF-α and IFN-γ against not only IBD, but also immune-mediated disease.

Protein kinase D3 regulates the expression of the immunosuppressive protein, PD‑L1, through STAT1/STAT3 signaling.

Oral squamous cell carcinoma (OSCC) is capable of constructing a favorable immune escape environment through interactions of cells with cells and of cells with the environment. Programmed death ligand-1 (PD-L1) is a well-recognized inhibitor of anti-tumor immunity that plays an important role in tumor immune escape. However, the molecular mechanisms regulating PD-L1 expression are not yet fully understood. In this study, to investigate the role of protein kinase D3 (PKD3) in the regulation of PD-L1 expression, the expression and correlation of PKD3 and PD-L1 were first analyzed by the immunostaining of human OSCC tissue sections, cell experiments and TCGA gene expression databases. The expression levels of PKD3 and PD-L1 were found to be significantly higher in OSCC cells than in normal tissues or cells. In addition, the expression levels of PKD3 and PD-L1 were found to be significantly positively correlated. Subsequently, it was found that the levsel of PD-L1 expression decreased following the silencing of PKD3 and that the ability of interferon (IFN)-γ to induce PD-L1 expression was also decreased in OSCC. The opposite phenomenon occurred following the overexpression of PKD3. It was also found that the phosphorylation of signal transducer and activator of transcription (STAT)1/STAT3 was reduced by the knockdown of PKD3 in OSCC. Moreover, the expression level of PD-L1 was decreased after the use of siRNA to knockdown STAT1 or STAT3. On the whole, the findings of this study confirm that PKD3 regulates the expression of PD-L1 induced by IFN-γ by regulating the phosphorylation of STAT1/STAT3. These findings broaden the understanding of the biological function of PKD3, suggesting that PKD is a potential therapeutic target for OSCC.

Melanoma cell-derived exosomes in plasma of melanoma patients suppress functions of immune effector cells

Melanoma patients’ plasma contains exosomes produced by malignant and normal cells. Plasma exosomes were isolated and separated by immunocapture into two fractions: melanoma cell-derived exosomes (MTEX) and normal cell-derived exosomes (non-MTEX). Immunosuppressive effects of MTEX on primary human immune cells were evaluated. Exosomes were isolated from plasma of 12 melanoma patients and six healthy donors (HDs). Expression levels of 19 immunoregulatory proteins in MTEX, non-MTEX and HDs exosomes were evaluated by on-bead flow cytometry. Functional/phenotypic changes induced in CD8+ T or natural killer (NK) cells by MTEX or non-MTEX were compared. Plasma protein levels were higher in patients than HDs (P < 0.0009). In patients, MTEX accounted for 23–66% of total exosomes. MTEX were enriched in immunosuppressive proteins (P = 0.03). MTEX, but not HDs exosomes, inhibited CD69 expression (P ≤ 0.0008), induced apoptosis (P ≤ 0.0009) and suppressed proliferation (P ≤ 0.002) in CD8+ T cells and downregulated NKG2D expression in NK cells (P = 0.001). Non-MTEX were enriched in immunostimulatory proteins (P = 0.002) and were only weakly immunosuppressive. Elevated MTEX/total exosome ratios and, surprisingly, non-MTEX ability to induce apoptosis of CD8+ T cells emerged as positive correlates of disease stage. MTEX emerge as the major mechanism of tumor-induced immune suppression and as an underestimated barrier to successful melanoma immunotherapy.

Molecular profiles and immunomodulatory activities of glioblastoma-derived exosomes.

BackgroundGlioblastoma is one of the most immunosuppressive human tumors. Emerging data suggest that glioblastoma-derived exosomes (GBex) reprogram the tumor microenvironment into a tumor-promoting milieu by mechanisms that not yet understood.MethodsExosomes were isolated from supernatants of glioblastoma cell lines by size exclusion chromatography. The GBex endosomal origin, size, protein cargos, and ex vivo effects on immune cell functions were determined. GBex were injected intravenously into mice to evaluate their ability to in vivo modulate normal immune cell subsets.ResultsGBex carried immunosuppressive proteins, including FasL, TRAIL, CTLA-4, CD39, and CD73, but contained few immunostimulatory proteins. GBex co-incubated with primary human immune cells induced simultaneous activation of multiple molecular pathways. In CD8+ T cells, GBex suppressed TNF-α and INF-γ release and mediated apoptosis. GBex suppressed natural killer (NK) and CD4+ T-cell activation. GBex activated the NF-κB pathway in macrophages and promoted their differentiation into M2 cells. Inhibition of the NF-κB pathway in macrophages reversed the GBex-mediated effects. GBex-driven reprogramming of macrophages involved the release of soluble factors that promoted tumor proliferation in vitro. In mice injected with GBex, the frequency of splenic CD8+ T cells, NK cells, and M1-like macrophages was reduced, while that of naïve and M2-like macrophages increased (P < .05).ConclusionsGBex reprogrammed functions of all types of immune cells in vitro and altered their frequency in vivo. By creating and sustaining a highly immunosuppressive environment, GBex play a key role in promoting tumor progression.

CD44v3 protein-carrying tumor-derived exosomes in HNSCC patients’ plasma as potential noninvasive biomarkers of disease activity

ABSTRACT The molecular cargo of tumor-cell-derived exosomes (TEX) mimics that of parental tumor cells. Thus, TEX could potentially serve as noninvasive biomarkers of cancer progression. However, separation of TEX from non-TEX in patients’ plasma requires tumor antigen-specific detection reagents. CD44v3 has been of interest as a potential biomarker of disease progression in HNSCC, because its overexpression in tumor cells associates with poor outcome. Here, CD44v3+ TEX immunocaptured from plasma of 44 HNSCC patients and 7 healthy donors (HDs) were evaluated as potential biomarkers of disease activity and stage. Exosomes were isolated from plasma of by size exclusion chromatography. Using anti-CD44v3 or anti-CD3 mAbs on beads, CD44v3+ TEX CD3(-)TEX-enriched exosomes were immunocaptured from supernatants of nonmalignant or HNSCC cell lines and from patients’ plasma. On-bead flow cytometry was used for the detection of FAS-L, PD-L1, TGFF-β. CSPG4 or EGFR on exosomes. The TEX expression profiles were correlated to clinicopathological parameters. Relative florescence intensity (RFI) values for CD44v3 were higher (p < .01) on TEX from HNSCC cell lines or on CD44v3+ CD3(-) plasma-derived exosomes. RFI values of CD44v3 on CD3(-) exosomes were higher (p < .005) in patients than in HDs and correlated (p < .05) with the UICC stage and lymph node metastasis. In HNSCC patients, CD44v3+ exosomes higher levels of immunosuppressive proteins compared to CD44v3(-) exosomes (p < .05-p < .005), and RFI values for these markers correlated with higher disease stages and lymph node metastasis. Isolation of CD44v3+ exosomes by immunocapture allowed for enrichment of TEX which are potentially promising liquid biomarkers of the tumor burden and disease stage in HNSCC.

Downregulation of Protein Inhibitor of Activated STAT (PIAS) 1 Is Possibly Involved in the Process of Allograft Rejection

BackgroundProtein inhibitors of activated STAT (PIAS) proteins are regarded as negative regulators of cytokine-signaling and potent immunosuppressive proteins. However, their role in the process of organ transplant rejection has not been elucidated. MethodsIn the current study, we compared transcript levels of PIAS1 to 4 in the peripheral blood of renal transplant recipients who experienced transplant rejection with those having normal transplant functions. Expression of PIAS1 was significantly higher in nonrejected group compared with the rejected group among male recipients; however, differences were insignificant among female recipients. Expressions of other PIAS genes were not different between study groups. Significant pairwise correlations were found between expression levels of PIAS genes in all study subgroups. The current investigation highlights the role of PIAS1 downregulation in the evolution of graft rejection and potentiates this gene as a predictive marker for transplant fate.

Breast cancer cells promote CD169+ macrophage-associated immunosuppression through JAK2-mediated PD-L1 upregulation on macrophages

Macrophages are recognized as one of the major cell types in tumor microenvironment, and macrophage infiltration has been predominantly associated with poor prognosis among patients with breast cancer. Using the murine models of triple-negative breast cancer in CD169-DTR mice, we found that CD169+ macrophages support tumor growth and metastasis. CD169+ macrophage depletion resulted in increased accumulation of CD8+ T cells within tumor, and produced significant expansion of CD8+ T cells in circulation and spleen. In addition, we observed that CD169+ macrophage depletion alleviated tumor-induced splenomegaly in mice, but had no improvement in bone loss and repression of bone marrow erythropoiesis in tumor-bearing mice. Cancer cells and tumor associated macrophages exploit the upregulation of the immunosuppressive protein PD-L1 to subvert T cell-mediated immune surveillance. Within the tumor microenvironment, our understanding of the regulation of PD-L1 protein expression is limited. We showed that there was a 5-fold higher relative expression of PD-L1 on macrophages as compared with 4T1 tumor cells; coculture of macrophages with 4T1 cells augmented PD-L1 levels on macrophages, but did not upregulate the expression of PD-L1 on 4T1 cells. JAK2/STAT3 signaling pathway was activated in macrophages after coculture, and we further identified the JAK2 as a critical regulator of PD-L1 expression in macrophages during coculture with 4T1 cells. Collectively, our data reveal that breast cancer cells and CD169+ macrophages exhibit bidirectional interactions that play a critical role in tumor progression, and inhibition of JAK2 signaling pathway in CD169+ macrophages may be potential strategy to block tumor microenvironment-derived immune escape.

Transovarial effect of the mixture of metal nanoparticles (Ag, Cu, Fe, MnO2) on biochemical indices of blood of one-day-old chickens compared to metal salts

Aim. Due to small sizes of metal nanoparticles (NPMe), after getting into the organism, they penetrate different internal organs, overcoming hematoencephalitic and placental barriers, and may cause different pathophysiological effects in the organism. Thus, the aim of our study was to study transovarial effect of NPMe mixture (Ag, Cu, Fe, MnO2 ) on biochemical indices of blood of one-day-old chickens compared to salts of relevant metals. Methods. The studies were conducted using Hisex White cross chickens (n = 28) with the sex ratio of roosters to hens of 1:6, which were fed for 37 days with the following additives: an experimental sample of NPМе mixture, containing nanoparticles of silver (31.5 ± 0.9 nm), iron (100.0 ± 10.0 nm), copper (70.0 ± 4.0 nm) and manganese peroxidase (50.0 ± 3.0 nm), in the doses of 0.3 and 4.0 mg/kg of bodyweight (experiments ІІ and ІІІ); solution of salt mixture – AgNO3 , (CuSO4 ∙ 5H2 O), (MnSO4 ∙ 5H2 O) and (FeSO4 ∙ 7H2 O) (experiment І); distilled water (control). Starting with Day 30 of the experiment, eggs were collected from poultry in each group for a week and placed for further incubation. Blood samples were taken from hatched one-day-old chickens (n = 93) for biochemical studies. The registration of biochemical indices was conducted using spectrophotometer SHIMADZU UV-1800 (Japan). Results. The experiments determined the mechanism of toxic effect of NPMe mixture after its administration to chickens in the dose of 4.0 mg/kg of bodyweight, which was manifested in decreasing the natural resistance (tendency to the increase in seromucoids – immunosuppressive proteins), excessive formation of toxic products of purine exchange (increase in the concentration of uric acid by 77.6 %; P &lt; 0.05) and straining of enzyme systems of natural detoxication (increase in the activity of alanine aminotransferase and aspartate aminotransferase by 23.8 and 31.6 %; P &lt; 0.05) with the development of oxidative stress (increase in the content of diene conjugates and malondialdehyde by 19.3 and 23.8 %; P &lt; 0.01) in the organism of one-day-old chickens. The mechanism of pharmacological effect of NPMe mixture at its administration to chickens in the dose of 0.3 mg/kg of bodyweight lies in enhancing immune reactions (increasing the amount of general proteins and globulins by 14.4 and 12.0 %; P &lt; 0.05) along with the antioxidant effect via the release of vitamins A and E from the endogenous depot (by 14.7 and 25.7 % above the control; P &lt; 0.05) in the organism of one-day-old chickens. Conclusions. In conditions of transovarial transmission, there is confi rmed advantage of the effect of NPMe mixture (Ag, Cu, Fe, MnO2 ) in the dose of 0.3 mg/kg of bodyweight of laying hens comparing to the effect of the analogue in the form of the mixture of metal salts, which may help in achieving a high percentage of liveability of young birds, forming defensive reactions in the organism of chickens and ensuring full realization of genetic potential in terms of meat or egg productivity of agricultural poultry. The mechanism of toxic effect of metal nanoparticles in the dose of 4.0 mg/kg of bodyweight was determined which will allow elaborating safe regulations for the application of non-organic nanomaterials in modern poultry breeding and timely management of the risks of their application.

Oxytocin Inhibition of Metastatic Colorectal Cancer by Suppressing the Expression of Fibroblast Activation Protein-α.

Oxytocin (OXT) and its receptor (OXTR) are present in the gastrointestinal system and are involved in gastrointestinal tumorigenesis. However, the effect of OXTR signaling on the development of colorectal cancer (CRC) and its underlying mechanisms remain unexplored. To address these issues, we first examined the expressions of OXT, OXTR, and several cancer-associated proteins using colon “tissue chips” from a spectrum of malignant progression of the colon, which included normal colon tissue, chronic colitis, colorectal adenoma, and colorectal adenocarcinoma (CAC). The results showed that the expressions of OXT and OXTR decreased gradually with the malignant progression of the disease. Stimulation of CAC tissues with OXT increased OXTR expression while down-regulated FAPα and CCL-2 protein expressions in a concentration- and time-dependent manner. Moreover, cell invasion experiment showed that OXT treatment reduced the invasion ability of colon cancer cells and blocking OXTR with atosiban blocked OXT-reduced invasion ability of human colon cancer cell lines Ls174T and SW480. The results indicate that OXT has the potential to inhibit CRC development via down-regulating the immunosuppressive proteins FAPα and CCL-2. When the OXTR signaling is weakened, colon tissues may transform to CRC. These findings also highlight the possibility of applying OXT to inhibit CRC development directly.

The let-7 family of microRNAs suppresses immune evasion in head and neck squamous cell carcinoma by promoting PD-L1 degradation

BackgroundAccumulation of immunosuppressive protein programmed death-ligand 1 (PD-L1) has been documented in several cancers and contributes to the evasion of the host immune system. However, cancer cell-intrinsic signaling-dependent control of PD-L1 expression remains to be elucidated. Herein, we aimed to identify the let-7 family of microRNAs as candidates that up-regulate tumor cell PD-L1 expression and mediates immune evasion of head and neck squamous cell carcinoma (HNSCC).MethodsThe expression of let-7 family and PD-L1 was quantified in HNSCC tissues and adjacent normal tissues. PD-L1 degradation was evaluated in HNSCC cells in response to elevated expressions of let-7a or let-7b. The regulation of let-7 family on PD-L1 degradation through a mechanism involving T-cell factor-4 (TCF-4) control of β-catenin/STT3 pathway was evaluated. Immune recognition of HNSCC in vivo was examined in subcutaneous tumor-bearing C3H mice in the presence of let-7a/b and/or CTLA-4 antibody.ResultsThe let-7 family were significantly down-regulated in the context of HNSCC, sharing a negative correlation with PD-L1 expression. Glycosylated PD-L1 was detected in HNSCC cells, which was reduced by let-7a/b over-expression. TCF-4, the target of let-7a/b, activated the β-catenin/STT3 pathway and promoted PD-L1 degradation. In vivo analysis demonstrated that let-7a/b over-expression potentiated anticancer immunotherapy by CTLA-4 blockade.ConclusionsTaken together, our findings highlight targeting let-7 family as a potential strategy to enhance immune checkpoint therapy for HNSCC.