Immunoregulatory Gene Expression Research Articles

High CTLA-4 gene expression is an independent good prognosis factor in breast cancer patients, especially in the HER2-enriched subtype

BackgroundBreast cancer (BC) is the most common cancer in women and the leading cause of cancer-related death worldwide. This heterogeneous disease has been historically considered a non-immunogenic type of cancer. However, recent advances in immunotherapy have increased the interest in knowing the role of the immune checkpoints (IC) and other immune regulation pathways in this neoplasia.MethodsIn this retrospective study, we evaluated the correlation of mRNA expression of CTLA-4, PDCD1 (PD1), CD274 (PD-L1), PDCD1LG2 (PD-L2), CD276 (B7-H3), JAK2, and FOXO1 with clinicopathological factors and BC patient’s outcome by real-time quantitative polymerase chain reaction (qPCR).ResultsOur results showed that immunoregulatory gene expression depends on BC immunophenotype being CTLA-4 and PDCD1 (PD1) overexpressed on triple-negative/basal-like (TN/BL) and luminal B/HER2-positive phenotypes, respectively, and CD276 (B7-H3), JAK2 and FOXO1 associated with both luminal A and luminal B/HER2-negative tumors. In addition, we found that these genes can also be related to aggressive and non-aggressive clinicopathological characteristics in BC. Finally, survival analysis showed that CTLA-4 expression levels emerge as a significant independent factor of good prognosis in BC patients, especially in the HER2-enriched subtype.ConclusionConsidering all these data, we can conclude that the expression of immunoregulatory genes depends on tumor phenotype and has potential clinical implications in BC patients.

Clinical significance and immune landscape of a novel immune cell infiltration-based prognostic model in lung adenocarcinoma

Tumor-infiltrating immune cells (TICs) play a central role in the tumor microenvironment, which can reflect the host anti-tumor immune response. However, few studies have explored TICs in predicting the prognosis of lung adenocarcinoma (LUAD). In our study, we enrolled 2470 LUAD patients from TCGA and GEO databases, and the normalized enrichment scores for 65 immune cell types were quantified for each patient. An immune-related risk score (IRRS) was built on the basis of 17 selected TICs using LASSO regression analysis, and the results showed that high-risk patients were correlated with shorter survival time for the LUAD cohorts. Correlation analyses between IRRS and clinical characteristics were also evaluated to validate the clinical use of IRRS. In addition, we analyzed the differences in the distribution of immune cell infiltration and immunoregulatory gene expression, which may facilitate individual immunotherapy. Based on the above result, we conclude that IRRS can act as a powerful predictor for risk stratification and prognosis prediction, and may facilitate the decision-making process for LUAD patients.

Differential expression of immunoregulatory cytokines in adipose tissue and liver in response to high fat and high sugar diets in female mice.

A comprehensive understanding of how dietary components impact immunoregulatory gene expression in adipose tissue (AT) and liver, and their respective contributions to metabolic health in mice, remains limited. The current study aimed to investigate the metabolic consequences of a high-sucrose diet (HSD) and a high-fat diet (HFD) in female mice with a focus on differential lipid- and sucrose-induced changes in immunoregulatory gene expression in AT and liver. Female C57BL/6 J mice were fed a purified and macronutrient matched high fat, high sugar, or control diets for 12 weeks. Mice were extensively phenotyped, including glucose and insulin tolerance tests, adipose and liver gene and protein expression analysis by qPCR and Western blot, tissue lipid analyses, as well as histological analyses. Compared to the control diet, HSD- and HFD-fed mice had significantly higher body weights, with pronounced obesity along with glucose intolerance and insulin resistance only in HFD-fed mice. HSD-fed mice exhibited an intermediate phenotype, with mild metabolic deterioration at the end of the study. AT lipid composition was significantly altered by both diets, and inflammatory gene expression was only significantly induced in HFD-fed mice. In the liver however, histological analysis revealed that both HSD- and HFD-fed mice had pronounced ectopic lipid deposition indicating hepatic steatosis, but more pronounced in HSD-fed mice. This was in line with significant induction of pro-inflammatory gene expression specifically in livers of HSD-fed mice. Overall, our findings suggest that HFD consumption in female mice induces more profound inflammation in AT with pronounced deterioration of metabolic health, whereas HSD induced more pronounced hepatic steatosis and inflammation without yet affecting glucose metabolism.

Canine peripheral blood mononuclear cell-derived B lymphocytes pretreated with lipopolysaccharide enhance the immunomodulatory effect through macrophage polarization.

BackgroundPreconditioning with lipopolysaccharide (LPS) is used to improve the secretion of anti-inflammatory agents in B cells. However, there are only a few studies on canine B cells.ObjectiveThis study aimed to evaluate the immune regulatory capacity of canine peripheral blood mononuclear cell-derived B cells pretreated with LPS.MethodsCanine B cells were isolated from canine peripheral blood mononuclear cells, which were obtained from three healthy canine donors. The B cells were preconditioned with LPS, and then cell viability and the expression of the regulatory B cell marker were assessed. Finally, RNA extraction and immunofluorescence analysis were performed.ResultsLPS primed B cells expressed the interleukin (IL)-10 surface marker and immunoregulatory gene expression, such as IL-10, programmed death-ligand 1, and transforming growth factor beta. Macrophages in the inflammatory condition cocultured with primed B cells were found to have significantly down-regulated pro-inflammatory cytokine, such as tumor necrosis factor-α, and up-regulated anti-inflammatory cytokines such as IL-10. Additionally, it was revealed that co-culture with primed B cells re-polarized M1 macrophages to M2 macrophages.ConclusionsThis study revealed that LPS-primed B cells have an anti-inflammatory effect and can re-polarize macrophages, suggesting the possibility of using LPS-primed B cells as a therapeutic agent for its anti-inflammatory effects and immune modulation.

FGL2 promotes tumor progression in the CNS by suppressing CD103+ dendritic cell differentiation

Few studies implicate immunoregulatory gene expression in tumor cells in arbitrating brain tumor progression. Here we show that fibrinogen-like protein 2 (FGL2) is highly expressed in glioma stem cells and primary glioblastoma (GBM) cells. FGL2 knockout in tumor cells did not affect tumor-cell proliferation in vitro or tumor progression in immunodeficient mice but completely impaired GBM progression in immune-competent mice. This impairment was reversed in mice with a defect in dendritic cells (DCs) or CD103+ DC differentiation in the brain and in tumor-draining lymph nodes. The presence of FGL2 in tumor cells inhibited granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced CD103+ DC differentiation by suppressing NF-κB, STAT1/5, and p38 activation. These findings are relevant to GBM patients because a low level of FGL2 expression with concurrent high GM-CSF expression is associated with higher CD8B expression and longer survival. These data provide a rationale for therapeutic inhibition of FGL2 in brain tumors.

Alloresponses of Human T-Cells from Adult Peripheral Blood and Umbilical Cord Blood Are Differentially Impacted By Lenalidomide

Immunomodulatory drugs (IMiDs), such as lenalidomide provide a tool to enhance both direct anti-tumor and graft-versus-tumor effects after allogeneic haematopoietic stem-cell transplantation (AHCT). However, early clinical experience with IMiDs after AHCT using adult peripheral blood (APB) as a stem cell source has been limited by induction of graft-versus-host disease. Characterization of the mechanisms by which IMIDs can modulate alloresponses of T-cells and identification of differential effects on T-cells from disparate cell sources could facilitate more effective use of these drugs in the setting of AHCT. We therefore used in vitro modelling, multi-parameter flow cytometry and gene expression analysis to delineate the impact of the widely used IMiD lenalidomide on quantitative and qualitative alloresponses mediated by T-cells derived from APB and umbilical cord blood (UCB).We co-cultured carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled peripheral blood mononuclear cells (PBMC) from healthy adult donors or donated UCB units with irradiated allogeneic PBMC in the presence of 1μM lenalidomide or vehicle control. In this model, cellular and supernatant concentrations of lenalidomide (determined by mass spectrometry) were similar to in vivo levels achieved at doses used as maintenance after AHSCT in published studies. Functional alloresponses were quantified after 7-9 days of allogeneic co-culture by flow cytometry. In addition, co-culture responders were flow-sorted into alloproliferative or non-proliferative fractions and extracted RNA used for gene expression profiling.We demonstrate that lenalidomide potentiates net alloproliferation of APB derived T-cells cells by selectively enhancing allospecific proliferation of CD8+ T-cells (median 58% (lenalidomide-treated) versus 43% (untreated), n=40, p<0.001). Although pre-treatment of allogeneic stimulators and responders had a modest potentiating effect, lenalidomide was required during allogeneic co-culture for maximal potentiation of CD8+ alloresponses of APB T-cells. CD8+ T-cells had enhanced effector memory differentiation, were enriched for polyfunctional effectors (capable of producing two or more pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-2) and/or expressing the lysosomal membrane-associated protein CD107a), and had a distinct gene expression profile with altered expression of key immunoregulatory genes and depletion of cellular ikaros levels.In common with our findings in APB T-cells, lenalidomide also potentiated proliferation of alloreactive CD8+ T-cells from UCB (median 43% (lenalidomide-treated) vs 24% (untreated), n=17, p=0.0005) with similar polyfunctional effector memory differentiation, immunoregulatory gene expression changes and cellular ikaros depletion. Importantly, lenalidomide reduced allospecific proliferation of UCB CD4+ T-cells (median 58% (untreated) versus 41% (lenalidomide-treated), n=17, p<0.01) whereas alloproliferation of APB CD4+ T-cells was unaffected. The reduction in UCB CD4+ alloproliferation was accompanied by selectively expansion of CD4+CD25+FOXP3+ regulatory T-cells (median 7.6% (lenalidomide-treated) vs 5.2% (untreated), n=12, p 0.02), resulting in an overall reduction in net UCB T-cell alloproliferation after lenalidomide treatment.Our findings show that lenalidomide has a qualitatively different impact on alloresponses of T-cells from different cell sources; alloresponses of APB T-cells are increased by lenalidomide via selective expansion of polyfunctional CD8+ effectors while lenalidomide limits alloresponses of UCB T-cells by reducing CD4+ effector expansion and increasing tolerogenic Treg. These findings have important implications for the future use of IMiDs in the setting of AHCT. DisclosuresNo relevant conflicts of interest to declare.

Immunologic heterogeneity of tumor-infiltrating lymphocyte composition in primary melanoma

Tumor-infiltrating lymphocytes (TILs) in primary melanomas are thought to represent the host antitumor immune response, but controversy exists over whether TILs offer independent prognostication of survival. We studied a cohort of 1241 patients with primary melanoma to assess the association of absent, nonbrisk, and brisk TIL grade with survival outcomes. We tested whether quantitative TIL counts using immunohistochemical lymphocyte markers CD3, CD45, and FOXP3 add prognostic value to TIL grading compared with histology alone in 15% of the cohort. To assess for intergroup immunologic heterogeneity among TIL grades, we investigated differential expression of 594 immunoregulatory genes in 67 primary melanomas. On histologic evaluation of 1241 primary melanomas, TILs were graded as absent (n=388, 31%), nonbrisk (n=330, 27%), and brisk (n=523, 42%). Patients with brisk TILs had improved recurrence-free survival (P=.025) and overall survival (P=.006) compared with patients with nonbrisk and absent TILs, for which there were no differences in recurrence-free survival (P=.40) or overall survival (P=.41). TIL quantitation by immunohistochemistry did not improve prognostication compared with TIL grading on hematoxylin and eosin-stained sections. Melanomas with nonbrisk and absent TILs share similar immunoregulatory gene expression profiles. In contrast, melanomas with brisk TILs demonstrate upregulation of T-cell activation pathways and inhibition of upstream immune checkpoint regulators. The presence of TILs in primary melanomas represents a heterogeneous group, and caution in prognostic interpretation is warranted. Melanomas with brisk TILs are defined by an immunostimulatory gene expression profile and improved prognosis compared with melanomas with nonbrisk or absent TILs.

Immunomodulation and liver protection of Yinchenhao decoction against concanavalin A-induced chronic liver injury in mice

ObjectiveThis study investigated the immunoregulatory and protective roles of Yinchenhao decoction, a compound of Chinese herbal medicine, in a mouse model of concanavalin A (ConA)-induced chronic liver injury. MethodsFemale BalB/c mice were randomly divided into 4 groups: normal control, ConA model, ConA model treated with Yinchenhao decoction (400 mg/kg, orally), and ConA model treated with dexamethasone (0.5 mg/kg, orally). All treatments were given once a day for 28 d. Except of the normal control, mice received tail vein injection of ConA (10 mg/kg) on days 7, 14, 21, and 28, at 1 h after treatment with Yinchenhao decoction or dexamethasone or saline to induce chronic liver injury. ResultsRepeated ConA injection induced chronic liver injury, which was evidenced by inflammatory cell infiltration and necrosis, increased serum alanine aminotranferease activities, decreased albumin levels, and an imbalanced expression of immunoregulatory genes in the liver tissues including significantly enhanced interferon-γ, interleukin-4, monocyte chemotactic protein-1, and cluster of differentiation 163 mRNA levels, and reduced tumor necrosis factor-α and interleukin-6 mRNA levels. Treatment with Yinchenhao decoction significantly reversed the ConA-induced changes in immunoregulatory gene expression in the liver tissues, reduced serum alanine aminotranferease activity, enhanced serum albumin level, and attenuated the extent of liver inflammation and necrosis. Furthermore, Yinchenhao decoction did not result in hepatocyte degeneration and spleen weight loss that were observed in mice received long-term treatment with dexamethasone. ConclusionYinchenhao decoction treatment protected liver against the ConA-induced chronic liver damage and improved liver function, which were associated with the modulation of gene expression related to immune/inflammatory response.

Autologous hematopoietic SCT normalizes miR-16, -155 and -142-3p expression in multiple sclerosis patients.

Autologous hematopoietic SCT (AHSCT) has been investigated in the past as a therapeutic alternative for multiple sclerosis (MS). Despite advances in clinical management, knowledge about mechanisms involved with clinical remission post transplantation is still limited. Abnormal microRNA and gene expression patterns were described in MS and have been suggested as disease biomarkers and potential therapeutic targets. Here we assessed T- and B-cell reconstitution, microRNAs and immunoregulatory gene expression after AHSCT. Early immune reconstitution was mainly driven by peripheral homeostatic proliferation. AHSCT increased CD4(+)CD25(hi)FoxP3(+) regulatory T-cell counts and expression of CTLA-4 and GITR (glucocorticoid-induced TNFR) on CD4(+)CD25(hi) T cells. We found transient increase in exhausted PD-1(+) T cells and of suppressive CD8(+)CD28(-)CD57(+) T cells. At baseline, CD4(+) and CD8(+) T cells from MS patients presented upregulated miR-16, miR-155 and miR-142-3p and downregulated FOXP3, FOXO1, PDCD1 and IRF2BP2. After transplantation, the expression of FOXP3, FOXO1, PDCD1 and IRF2BP2 increased, reaching control levels at 2 years. Expression of miR-16, miR-155 and miR-142-3p decreased towards normal levels at 6 months post therapy, remaining downregulated until the end of follow-up. These data strongly suggest that AHSCT normalizes microRNA and gene expression, thereby improving the immunoregulatory network. These mechanisms may be important for disease control in the early periods after AHSCT.

Immunoregulatory gene expression and response to bevacizumab in ovarian cancer — An analysis of the cancer genome atlas

Immunoregulatory gene expression and response to bevacizumab in ovarian cancer — An analysis of the cancer genome atlas

Abstract 4763: Resveratrol synergizes with recombinant death ligands for potent antitumor activity against human metastatic colon cancers

Abstract Despite recent advances in the treatment of metastatic colon cancer (CC), most patients are not curable. Furthermore, whenever patients have exhausted all available standard chemotherapy and biologic agent treatment, patients are left with a very poor performance status. Thus, additional therapy for CC needs to be both active against the most advanced forms of the disease and tolerable for patients. Resveratrol (RV) is a phytoalexin from the skin of grapes that has been shown to have inhibitory effects on the proliferation of human tumors. It can be administered to cancer patients as an oral naturopathic/nutritional supplement. The current study investigated the capacity of RV to modulate apoptotic and immunoregulatory gene expression in CC cells and explored the functional consequences of modulation on in vitro tumor cell proliferation. Gene expression was assessed with a real time PCR assay and in vitro proliferation with a standard MTS assay. The ATCC cell line, T84 as well as cell suspensions prepared from metastatic CC specimens involving the stomach (SM), ovary (OM), and extensive peritoneal carcinomatosis (PC) were tested. Inhibition of proliferation was observed with RV alone in concentrations up to 5 ug/mL and this was enhanced further when RV was combined with the immunologic death ligands, rTNF and rTRAIL. For the T84 cell line, levels of proliferation inhibition were 28.0%, 0.5%, 5.4%, 44.5% and 51.8% with RV, TNF, TRAIL, RV+TNF, and RV+TRAIL respectively. The corresponding values for surgical specimens were SM: 5.6%, 1.0%, 0.3%, 28.3%, and 52.4% respectively; OM: 26.2%, 4.6%, 6.1%, 57.4%, and 75.0% respectively; and PC: 46.9%, 17.1%, 59.6%, 61.8% and 96.7%, respectively. These effects of RV + immunologic death ligands were reflected by changes in expression of apoptotic and immunoregulatory genes. For example, in OM cells some of the genes whose expression was up-regulated by RV, TRAIL, and RV+TRAIL were FAS (4.33, 1.57, &amp; 6.22-fold, respectively), DAPK (3.07, 1.16, 3.68), CARD6 (1.96, -1.09, 3.01), and TRADD (2.05, 1.29, 2.31). Consistently, there was increased gene expression in response to the combination of RV +TNF/TRAIL to levels greater than those elicited by any single agent for the following genes: TNFRSF9; CASP10; BCL2L1; BAK1; and BAX. The results of this study demonstrate that RV, a well tolerated oral naturopathic/nutritional supplement can inhibit the in vitro proliferation of human CC cells and collaborate with the immunologic death ligands TNF and TRAIL to inhibit proliferation further. These effects are correlated with changes in expression of apoptotic and immunoregulatory genes in CC cells. Taken together, the results suggest that RV could be combined with cytokine-based therapy or other forms of immunotherapy which elicit immunologic death ligands for the treatment of metastatic CC in patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4763. doi:10.1158/1538-7445.AM2011-4763

In this issue

In this issue

P78 HLA DRB1*1503: a predominant allelic haplotype in SLE correlated to immunoregulatory gene expression affecting regulatory T cells

P78 HLA DRB1*1503: a predominant allelic haplotype in SLE correlated to immunoregulatory gene expression affecting regulatory T cells

Reduced Immunogenicity of First-Trimester Human Fetal Pancreas

The use of human fetal pancreatic tissue may provide a potential source of transplantable beta-cells as a therapy for type 1 diabetes. Human fetal pancreas has a remarkable capacity to grow and differentiate in vivo and has been shown to reverse diabetes in rodents. However, it is known that human fetal pancreas obtained from the second trimester of gestation is immunogenic and is rejected after transplantation. Tissue obtained from earlier stages might prove to be immune privileged, as has been shown for other tissues. In this study, we determined the immunogenicity of human fetal pancreatic tissue obtained from the first trimester of gestation in a humanized mouse model. A microarray study of immunoregulatory gene expression in first- and second-trimester human fetal pancreas was also undertaken. The analysis of transplanted human fetal pancreata revealed a significantly decreased immunogenicity of the first-trimester tissue. The first-trimester grafts showed only limited cellular infiltration and contained numerous insulin-positive cells, whereas second-trimester tissue was completely infiltrated and rejected. Furthermore an analysis of immunoregulatory genes expressed in first- and second-trimester human fetal pancreas by microarray demonstrated the upregulation of several key immunoregulatory genes in the second-trimester tissue. This might account for the reduced immunogenicity of the younger tissue. Our results provide the first indication that the use of first-trimester human fetal pancreas for transplantation might increase the survival of the grafts and might decrease the requirement for immunosuppressive drugs.

Posttranslational Regulation of IRF-4 Activity by the Immunophilin FKBP52

Interferon regulatory factor-4 (IRF-4) plays an important role in immunoregulatory gene expression in B and T lymphocytes and is also highly expressed in human T cell leukemia virus type 1 infected cells. In this study, we characterize a novel interaction between IRF-4 and the FK506-binding protein 52 (FKBP52), a 59 kDa member of the immunophilin family with peptidyl-prolyl isomerase activity (PPIase). IRF-4-FKBP52 association inhibited IRF4-PU.1 binding to the immunoglobulin light chain enhancer E λ2–4 as well as IRF-4-PU.1 transactivation, effects that were dependent on functional PPIase activity. FKBP52 association also resulted in a structural modification of IRF-4, detectable by immunoblot analysis and by IRF-4 partial proteolysis. These results demonstrate a novel posttranslational mechanism of transcriptional control, mediated through the interaction of an immunophilin with a transcriptional regulator.

Human Cytomegalovirus Binding to Human Monocytes Induces Immunoregulatory Gene Expression

To continue our investigation of the cellular events that occur following human CMV (HCMV) infection, we focused on the regulation of cellular activation following viral binding to human monocytes. First, we showed that viral binding induced a number of immunoregulatory genes (IL-1beta, A20, NF-kappaB-p105/p50, and IkappaBalpha) in unactivated monocytes and that neutralizing Abs to the major HCMV glycoproteins, gB (UL55) and gH (UL75), inhibited the induction of these genes. Next, we demonstrated that these viral ligands directly up-regulated monocyte gene expression upon their binding to their appropriate cellular receptors. We then investigated if HCMV binding also resulted in the translation and secretion of cytokines. Our results showed that HCMV binding to monocytes resulted in the production and release of IL-1beta protein. Because these induced gene products have NF-kappaB sites in their promoter regions, we next examined whether there was an up-regulation of nuclear NF-kappaB levels. These experiments showed that, in fact, NF-kappaB was translocated to the nucleus following viral binding or purified viral ligand binding. Changes in IkappaBalpha levels correlated with the changes in NF-kappaB translocation. Lastly, we demonstrated that p38 kinase activity played a central role in IL-1beta production and that it was rapidly up-regulated following infection. These results support our hypothesis that HCMV initiates a signal transduction pathway that leads to monocyte activation and pinpoints a potential mechanism whereby HCMV infection of monocytes can result in profound pathogenesis, especially in chronic inflammatory-type conditions.