Gene Expression In CD4 Research Articles

Evaluation of CD24 and CD44 as cancer stem cell markers in squamous cell carcinoma and epithelial dysplasia of the oral cavity by q- RT-PCR

Squamous cell carcinoma (SCC) is the most common cancer of the oral cavity and may be preceded by dysplastic epithelial lesion. Oral SCC (OSCC) contains a rare subset of cancer cells with self-renewal ability, termed as cancer stem cells (CSCs). CD24 and CD44 as CSC markers are cell surface glycoproteins. These markers contribute to the onset, maintenance, and extension of tumor growth, as well as angiogenesis. In the present study, these two markers were simultaneously evaluated to provide a specific phenotype for carcinogenesis process in oral cavity. In this analytical-cross-sectional study, the expression of CD24 and CD44 genes was evaluated in 45 OSCCs (20 low-grade and 25 high-grade) and 15 oral epithelial dysplasia specimens by real-time quantitative reverse transcription polymerase chain reaction. Kruskal-Wallis and Mann-Whitney U-test, Kendall, and Spearman tests were used for statistical analysis. The significance level was considered <0.05. High expression of both markers genes was reported in two-thirds of samples. There was no significant difference between studied groups in gene expression of CD24 and CD44 whereas statistically significant association between CD24 and CD44 was observed in all three groups. This correlation was more significant in OSCC groups (P < 0.001). High expression of CSC markers in OSCC and oral epithelial dysplasia revealed the importance of accurate examination of dysplastic lesions with high expression of these markers and the possibility of malignant transformation. Regarding a significant association of two markers, further studies are necessary to provide a specific phenotype (CD44 high CD24 high) for these lesions.

Differential gene expression of tumor-infiltrating CD4+ T cells in advanced versus early stage colorectal cancer and identification of a gene signature of poor prognosis

ABSTRACT Tumor-infiltrating lymphocytes (TILs) play indispensable roles in the progression and response to treatment of solid tumors. However, the prognostic significance of CD4+ TILs is not fully disclosed in cancers generally and in CRC in particular, mainly due to the existence of different functional subsets of CD4+ T cells. We performed transcriptomic profiling of CD4+ TILs isolated from CRC patients in order to identify differentially expressed genes and their functional pathways in early versus advanced disease stages. We found that in advanced stages, genes related to immune and inflammatory responses, in particular Th1-mediated immune response and cytotoxicity-mediated genes, were downregulated; while epigenetic-mediated silencing genes were upregulated. Interestingly, we identified genes, which were steadily upregulated or downregulated in CD4+ TILs with CRC progression from stage I to IV. Additionally, of the top 200 deregulated genes, 43 upregulated and 64 downregulated genes showed similar deregulation trends in the cancer genome atlas CRC dataset. From these 97 deregulated genes, we identified a “poor prognosis CD4 gene signature (ppCD4sig)”. Patients with high ppCD4sig score showed shorter disease-specific survival (DSS) and progression-free interval (PFI). The ppCD4sig was an independent prognostic indicator for DSS (HR = 1.73, 95% CI 1.32–2.27, P = 0.0001) and PFI (HR = 1.75, 95% CI 1.3–2.35, P = 0.0016). Additionally, patients at advanced stages and at a younger age (<55 years) were more likely to have a high ppCD4sig score. Altogether, our data provide novel insights and a unique prognostic gene signature of CD4+ TILs in the CRC microenvironment.

Deciphering the CXCL9-CXCL10-CXCL11/CXCR3 Axis in CLL-like Monoclonal B-Cell Lymphocytosis and Chronic Lymphocytic Leukemia: A New Target for Immune Activation?

Deciphering the CXCL9-CXCL10-CXCL11/CXCR3 Axis in CLL-like Monoclonal B-Cell Lymphocytosis and Chronic Lymphocytic Leukemia: A New Target for Immune Activation?

The JAK1/2 Inhibitor Ruxolitinib Downregulates CXCL12 Secretion from Bone Marrow Stromal Cells and M2 Macrophage Polarization in Multiple Myeloma

Introduction: The JAKSTAT pathway plays a critical role in the regulation of hematopoietic pathways and immunological cytokine signaling. The JAK pathway is also involved in tumor cell proliferation and drug resistance in multiple myeloma (MM). Thus, inhibition of the JAK pathway should be a potentially effective strategy for treating MM patients. B7-H3 is an immune checkpoint protein in the B7 superfamily and has been shown overexpressed in several tumors. Immune checkpoint blockade may suppress tumor progression or enhance anti-tumor immune responses. In this study, we investigated the effects of the JAK1/2 inhibitor ruxolitinib (Rux) on B7-H3 in MM. Materials and Methods: Bone marrow mononuclear cells (BMMCs) were collected from MM patients after obtaining IRB approval. Single-cell suspensions were prepared from human MM LAGλ-1A xenografts which had been grown in severe combined immunodeficient mice. HS-5 stromal and SUP-T1 T cells were purchased from ATCC. The cells were cultured and treated with or without RUX and then subjected to qRT-PCR, flow cytometric analysis, and western blot analysis. For qRT-PCR, total RNA was extracted and applied to cDNA synthesis, followed by qPCR. Gene expression was analyzed in MM BMMCs alone or co-cultured with stromal cells or T cells with or without Rux treatment (1μM) in vitro. Results: We identified increased B7-H3 expression in MMBMMCs from patients with progressive disease (PD) patients compared to those in complete remission (CR). Rux significantly reduced B7-H3 expression in MMBMMCs in patients with PD, MM cells (U266), and BM from patients in PD when co-cultured with stromal cells (HS-5) after 48-72 hours. Rux decreased B7-H3 expression in the human MM xenograft model LAGλ-1A when cultured ex vivo. In addition, Rux suppressed B7-H3 at protein levels as shown with flow cytometric analysis and western blotting, consistent with the gene expression results. Next, we tested whether B7-H3 blockade by Rux could potentially restore exhausted T cell activity against myeloma cells in MMBM. We found that Rux can increase IL-2 and CD8 gene expression in MMBM with lower plasma percentages (&lt; 30%) but not among those with higher plasma cell percentages (&gt;70%). Rux also elevated IL-2 and CD8 gene expression in BM when it was cocultured with T cells (SUP-T1), suggesting Rux may mediate immunological cytokine signaling. B7-H3-neutralizing antibody increased CD8 gene expression in MMBM in vitro, suggesting that one of the mechanisms through which Rux upregulates CD8 T cells in MMBM may be via downregulation of B7-H3. Conclusion: The immune checkpoint protein B7-H3 is overexpressed in MMBM in PD compared to CR patients. The JAK1/2 inhibitor Rux can decrease B7-H3 expression and increase IL-2 and CD8 expression in BM in vitro. Our results provide evidence for Rux inhibiting the immune checkpoint protein B7-H3 which may potentially restore exhausted T-cell activity in the MMBM tumoral microenvironment. Disclosures Chen: Oncotraker Inc: Equity Ownership. Berenson:OncoTracker: Equity Ownership, Other: Officer; OncoTracker: Equity Ownership, Other: Officer; Bristol-Myers Squibb: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Incyte Corporation.: Consultancy, Research Funding; Incyte Corporation.: Consultancy, Research Funding; Takeda: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Amag: Consultancy, Speakers Bureau; Amag: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Sanofi: Consultancy; Sanofi: Consultancy.

The JAK1/2 Inhibitor Ruxolitinib Downregulates the Immune Checkpoint Protein B7-H3 in Multiple Myeloma

Introduction: The JAKSTAT pathway plays a critical role in the regulation of hematopoietic pathways and immunological cytokine signaling. The JAK pathway is also involved in tumor cell proliferation and drug resistance in multiple myeloma (MM). Thus, inhibition of the JAK pathway should be a potentially effective strategy for treating MM patients. B7-H3 is an immune checkpoint protein in the B7 superfamily and has been shown overexpressed in several tumors. Immune checkpoint blockade may suppress tumor progression or enhance anti-tumor immune responses. In this study, we investigated the effects of the JAK1/2 inhibitor ruxolitinib (Rux) on B7-H3 in MM. Materials and Methods: Bone marrow mononuclear cells (BMMCs) were collected from MM patients after obtaining IRB approval. Single-cell suspensions were prepared from human MM LAGλ-1A xenografts which had been grown in severe combined immunodeficient mice. HS-5 stromal and SUP-T1 T cells were purchased from ATCC. The cells were cultured and treated with or without RUX and then subjected to qRT-PCR, flow cytometric analysis, and western blot analysis. For qRT-PCR, total RNA was extracted and applied to cDNA synthesis, followed by qPCR. Gene expression was analyzed in MM BMMCs alone or co-cultured with stromal cells or T cells with or without Rux treatment (1μM) in vitro. Results: We identified increased B7-H3 expression in MMBMMCs from patients with progressive disease (PD) patients compared to those in complete remission (CR). Rux significantly reduced B7-H3 expression in MMBMMCs in patients with PD, MM cells (U266), and BM from patients in PD when co-cultured with stromal cells (HS-5) after 48-72 hours. Rux decreased B7H3 expression in the human MM xenograft model LAGλ-1A when cultured ex vivo. In addition, Rux suppressed B7-H3 at protein levels as shown with flow cytometric analysis and western blotting, consistent with the gene expression results. Next, we tested whether B7-H3 blockade by Rux could potentially restore exhausted T cell activity against myeloma cells in MMBM. We found that Rux can increase IL-2 and CD8 gene expression in MMBM with lower plasma percentages (&lt; 30%) but not among those with higher plasma cell percentages (&gt;70%). Rux also elevated IL-2 and CD8 gene expression in BM when it was cocultured with T cells (SUP-T1), suggesting Rux may mediate immunological cytokine signaling. B7-H3-neutralizing antibody increased CD8 gene expression in MMBM in vitro, suggesting that one of the mechanisms through which Rux upregulates CD8 T cells in MMBM may be via downregulation of B7-H3. Conclusion: The immune checkpoint protein B7-H3 is overexpressed in MMBM in PD compared to CR patients. The JAK1/2 inhibitor Rux can decrease B7-H3 expression and increase IL-2 and CD8 expression in BM in vitro. Our results provide evidence for Rux inhibiting the immune checkpoint protein B7-H3 which may potentially restore exhausted T-cell activity in the MMBM tumoral microenvironment. Disclosures Chen: Oncotraker Inc: Equity Ownership. Berenson:Amgen: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Sanofi: Consultancy; Sanofi: Consultancy; Amag: Consultancy, Speakers Bureau; Amag: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; OncoTracker: Equity Ownership, Other: Officer; OncoTracker: Equity Ownership, Other: Officer; Bristol-Myers Squibb: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Incyte Corporation.: Consultancy, Research Funding; Incyte Corporation.: Consultancy, Research Funding; Takeda: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

B2M Gene Expression Reflects an Immunologically Active Tumor Microenvironment in DLBCL

Introduction Interactions between tumor cells and the immune system play a critical role in regulating tumor development. Immune-based therapies have shown variable responses in diffuse large B-cell lymphoma (DLBCL) which suggests that the immune cell composition of the tumor microenvironment (TME) influences response to these agents. However, the key determinants of the immune TME are poorly understood. We have recently shown that indolent lymphomas can be stratified as immunologically 'hot' or 'cold' (Tobin et al J. Clin Oncol, in press) and sought to test for this in DLBCL. Also, given that beta-2-microglobulin (B2M) has a key role in antigen presentation and its loss is a frequent immune evasion mechanism in DLBCL, we determined the effect of B2M expression on the nature and magnitude of immune infiltration in the tumor microenvironment of DLBCL. Methods Ninety-seven de novo systemic DLBCL FFPE biopsies underwent targeted exon re-sequencing of B2M (Illumina) in addition to quantitative gene expression of Β2M, immune effector (CD4, CD8, CD56, CD137), immunosuppressive macrophage markers (CD68, CD163) and immune checkpoints (TIM3, LAG3, PD1 and PDL1) (Nanostring Technologies). B2M promoter methylation by mass array, immunohistochemistry for the above markers and high throughput T-cell receptor β sequencing (Adaptive Biotechnologies) were performed on a subset of cases. Genomic findings were validated in a whole exome and transcriptome cohort of approximately 1000 DLBCL samples (Reddy et al Cell 2017). Results Β2MMut were detected in 14/97 (14.4%) samples and had lower Β2M gene expression compared to Β2MWT (p = 0.0094) and all of these showed B2M protein loss. However, 29/40 (72%) of B2MWT samples tested also had B2M protein loss. There was no differential methylation of B2M promoter regions observed compared to lymph node controls. Results indicate that mechanisms other than mutation and methylation status contribute to loss of B2M surface expression and that a more comprehensive assessment of B2M expression within tumor tissues is achieved by B2M digital gene quantification. Consistent with this, there were no significant differences in expression of intra-tumoral immune markers between B2MMut and B2MWT tissues, whereas gene expression of B2M was significantly associated and positively correlated with the gene expression of CD4, CD8, CD56, CD137, CD68, CD163, PD1, PDL1, LAG3 and TIM3 (all p &lt;0.01) and with the protein expression of CD8, CD56, CD137, PDL1 and LAG3 (all p &lt;0.01), irrespective of their classification as an immune-effector, immune-checkpoint or macrophage markers. These observations are consistent with a co-ordinately regulated immune response, indicating an adaptive immune-checkpoint response to regulate immune-effector activation. In keeping with this, the housekeeper genes did not correlate with immune gene expression indicating that high or low co-ordinate expression of immune genes was not reflecting tissue RNA quality or quantity (Fig 1). Next, we tested the discovery cohort for relationships with the TCR repertoire. High B2M gene expression was significantly associated with reduced TCR diversity (p = 0.0101) compared to low B2M gene expression, suggesting that clonal T cell expansions are more likely with intact antigen presentation. The validation cohort also demonstrated that B2M gene expression correlated with immune cell infiltration and additionally showed that B2M positively correlated with the gene expression of HLA Class I//II molecules and a range of regulatory, transport and assembly molecules involved in the antigen presentation machinery pathway. However, no differential survival benefit was observed in patients with high versus low B2M. Conclusions In summary, digital gene expression is a robust measure of B2M quantification in the TME. Our data show that high B2M gene expression reflects an immunologically active or 'hot' tumor microenvironment in DLBCL characterised by higher levels of immune cell infiltration. These findings indicate that B2M gene expression level could be used as a biomarker of an active intra-tumoral immune response in DLBCL. Further studies are required to determine if B2M gene expression may have a role in stratifying the selection of patients in whom immune-based therapies are more likely to be effective. Disclosures Gould: NovoNordisk: Other: Travel funding - domestic flights to attend education, May 2018. Keane:MSD: Consultancy; BMS: Research Funding; Celgene: Consultancy; Gilead: Consultancy; Roche: Consultancy, Other: Travel Grant. Hertzberg:Takeda: Consultancy, Honoraria; MSD: Consultancy; Roche: Consultancy, Honoraria. Gandhi:Gilead: Honoraria, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Other: Travel Support; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria.

The effect of exosome derived from gastric cancer cell line MKN-28, MKN-45, SGC-7901 on CD8+ T cell

Objective To detect exosome from gastric cancer cell line MKN-28, MKN-45, SGC-7901 on CD8+ T cell. Methods CD8+ T cell was isolated from health person peripheral blood by density gradient centrifugation and immunomagnetic bread, exosome of SGC-7901, MKN-45 and MKN-28 cell lines was obtained by ultracentrifugation. After coculture exosome and CD8+ T cell with different concentration for 48 h, CD8+ T cell was collected, and analyzed cell cycle by flow cytometry, cell apoptosis by Annexin V-propidium iodide (PI) stain, expression of cytokine by enzyme linked immunosorbent assay (ELISA), gene expression by real-time quantitative polymerase chain reaction (Real-time PCR). Results Exosome was isolated successfully from three gastric cancer cell lines and expressed molecular surface markers TSG101 and Alix. CD8+ T cells were isolated in peripheral blood mononuclear cell (PBMC) with a purity of over 98%. The effect of MKN-28 exosome on apoptosis was dose dependent. After stimulation with 100 mg/L MKN-45 exosome for 48 h, CD8+ T cells secreted IL-2, IL-6, IL-10, IFN-γ [(305.44±42.07) ng/L vs. (129.89±11.07) ng/L, P<0.01; (344.39±25.34) ng/L vs. (149.67±15.23) ng/L, P<0.01; (413.31±38.76) ng/L vs. (35.19±24.95) ng/L, P<0.01; (303.78±44.98) ng/L vs. (277.67±40.66) ng/L, P<0.05], and the difference was statistically significant. After SGC-7901 exosome stimulated, CD8+ T cells secreted IL-4, IL-6, IL-10, IFN-γ [(318.61±41.56) ng/L vs. (238.89±35.84) ng/L, P<0.05; (501.06±55.05) ng/L vs. (149.67 ±15.23) ng/L, P<0.01; (486.75±71.20) ng/L vs. (35.19±24.95) ng/L, P<0.01; (621.00±55.14) ng/L vs. (277.67±40.66) ng/L, P<0.01], the difference was statistically significant. After MKN-28 exosome stimulated, CD8+ T cells secreted IL-2, IL-6, IL-10, IFN-γ [ (411.56±48.62) ng/L vs. (129.89±11.07) ng/L, P<0.01; (403.83±52.29) ng/L vs. (149.67±15.23) ng/L, P<0.01; (678.31±78.02) ng/L vs. (35.19±24.95) ng/L, P<0.01; (751.88± 63.16) ng/L vs. (277.67±40.66) ng/L, P<0.01], and the difference was statistically significant. The expression of IL-10 secreted by CD8+ T cells stimulated by 100 mg/L MKN-28 exosome was about 19.53 times compared to the control group. After MKN-45 exosome stimulated, CD8+ T cells expressed forkhead/winged helix transcription factor P3 (Foxp3), interleukin (IL)-10, interferon (IFN)-γ, phosphatase and tensin homologue deleted on chromosome ten (PTEN), signal transducer and activators of transcription 3 (STAT3) (3.46±0.39 vs. 1.00±0.09, P<0.01; 3.29±0.39 vs. 1.01±0.16, P<0.01; 2.24±0.30 vs. 1.00±0.06, P<0.01; 2.14±0.12 vs. 1.00±0.08, P<0.01; 1.24±0.12 vs. 1.00±0.07, P<0.05). After SGC-7901 exosome stimulated, CD8+ T cell expressed Eomes, Foxp3, IL-10, IFN-γ, 2B4, CD160, GATA3, STAT3, PTEN (2.35±0.17 vs. 1.01±0.13, P<0.01; 3.19±0.21 vs. 1.00±0.09, P<0.01; 2.38±0.31 vs. 1.01±0.16, P<0.01; 5.44±0.71 vs. 1.00±0.06, P<0.01; 3.20±0.31 vs. 1.00±0.12, P<0.01; 3.19±0.13 vs. 1.01±0.16, P<0.01; 1.35±0.17 vs. 1.00±0.07, P<0.05; 1.44±0.18 vs. 1.00±0.07, P<0.05; 1.31±0.15 vs. 1.00±0.08, P<0.05) mRNA; after MKN-28 exosome stimulated, CD8+ T cell expressed Eomes, Foxp3, IL-10, IFN-γ, STAT3, PTEN, CD160, 2B4 (3.36±0.18 vs. 1.01±0.13, P<0.01; 4.35±0.30 vs. 1.00±0.09, P<0.01; 5.40±0.63 vs. 1.10±0.16, P<0.01; 6.43±0.84 vs. 1.00±0.06, P<0.01; 1.54±0.28 vs. 1.00±0.07, P<0.05; 0.78±0.11 vs. 1.00±0.08, P<0.01; 1.68±0.42 vs. 1.01±0.16, P<0.05; 1.31±0.22 vs. 1.01±0.12, P<0.05) mRNA expression. The gene expression of CD8+ T cells stimulated by MKN-28 exosome was relatively more changes, such as Foxp3, IL-10 and IFN-γ. Conclusion Gastric cancer cell line exosome stimulated CD8+ T cells to induce cell apoptosis, and change gene expression and cytokine secretion, but the proportion of negative regulatory molecules was much higher. Cancer exosome interacted with CD8+ T cells, which inhibited its immune function, resulting in tumor immune escape, which may be an important factor to promote disease progression. Key words: Gastric cancer; Exosome; CD8+ T cell; Gene expression; Cytokine

Programmed Death Ligand 1 Indicates Pre-Existing Adaptive Immune Response by Tumor-Infiltrating CD8+ T Cells in Non-Small Cell Lung Cancer.

Aberrant expression of programmed death ligand 1 (PD-L1) on tumor cells impedes antitumor immunity and instigates immune evasion. The remarkable efficacy of immune checkpoint blockade has been confirmed in various solid tumors. However, the correlation between PD-L1 expression and host immunological landscape remains of considerable controversy in non-small cell lung cancer (NSCLC). In the present study, PD-L1 expression and CD8+ tumor-infiltrating lymphocyte (TIL) infiltration levels were determined by immunohistochemistry (IHC) in tumor sections of 138 NSCLC patients. The expression level of PD-L1 was positively correlated with the abundance of CD8 + TILs (p < 0.0001). Furthermore, no constitutive expression of PD-L1 was observed in the majority of six NSCLC cell lines detected by Western blot; but exposure to interferon-γ (IFN-γ), a primary cytokine secreted by activated CD8+ T cells, prominently increased PD-L1 expression. Notably, a significantly positive association was determined within PD-L1, CD8 and IFN-γ gene expression by qRT-PCR, which was corroborated by RNA-sequencing from TCGA lung cancer dataset. These findings demonstrate that PD-L1 expression indicates an adaptive immune resistance mechanism adopted by tumor cells in the aversion of immunogenic destruction by CD8+ TILs. Both higher expression of PD-L1 and infiltration of CD8+ TILs were correlated with superior prognosis (p = 0.044 for PD-L1; p = 0.002 for CD8). Moreover, Cox multivariate regression analysis showed that the combination of PD-L1 and CD8 were independent prognostic factors, which was more accurate in prediction of prognosis in NSCLC than individually. Finally, we found that IFN-γ induced the upregulation of PD-L1 in NSCLC cells, mainly through the JAK/STAT1 signaling pathway. In conclusion, PD-L1 expression is mainly induced by activated CD8+ TILs via IFN-γ in the immune milieu and indicates pre-existing adaptive immune response in NSCLC.

Interferon γ and α Have Differential Effects on SAMHD1, a Potent Antiviral Protein, in Feline Lymphocytes.

Sterile alpha motif and histidine/aspartic domain-containing protein 1 (SAMHD1) is a protein with anti-viral, anti-neoplastic, and anti-inflammatory properties. By degrading cellular dNTPs to constituent deoxynucleoside and free triphosphate, SAMHD1 limits viral DNA synthesis and prevents replication of HIV-1 and some DNA viruses such as HBV, vaccinia, and HSV-1. Recent findings suggest SAMHD1 is broadly active against retroviruses in addition to HIV-1, such as HIV-2, FIV, BIV, and EIAV. Interferons are cytokines produced by lymphocytes and other cells that induce a wide array of antiviral proteins, including some with activity again lentiviruses. Here we evaluated the role of IFNs on SAMHD1 gene expression, transcription, and post-translational modification in a feline CD4+ T cell line (FeTJ) and in primary feline CD4+ T lymphocytes. SAMHD1 mRNA in FetJ cells increased in a dose-related manner in response to IFNγ treatment concurrent with increased nuclear localization and phosphorylation. IFNα treatment induced SAMHD1 mRNA but did not significantly alter SAMHD1 protein detection, phosphorylation, or nuclear translocation. In purified primary feline CD4+ lymphocytes, IL2 supplementation increased SAMHD1 expression, but the addition of IFNγ did not further alter SAMHD1 protein expression or nuclear localization. Thus, the effect of IFNγ on SAMHD1 expression is cell-type dependent, with increased translocation to the nucleus and phosphorylation in FeTJ but not primary CD4+ lymphocytes. These findings imply that while SAMH1 is inducible by IFNγ, overall activity is cell type and compartment specific, which is likely relevant to the establishment of lentiviral reservoirs in quiescent lymphocyte populations.

CD44(+) tumor cells promote early angiogenesis in head and neck squamous cell carcinoma

The role of CD44 in progression of head and neck squamous cell carcinoma (HNSCC) has been controversial. The goal of this study was to study the effects of CD44(+) tumor cells on the initial stages of tumor angiogenesis and to evaluate CD44 as a potential marker of tumor angiogenesis. The CD44 gene expression was studied using the Cancer Genome Atlas (TCGA) Head and Neck Cancer data base. Expression levels of CD44 and of microvascular density (MVD) markers were assessed by immunohistochemistry performed with tissue microarrays in a cohort of 49 HNSCC patients, 11 patients with dysplasia and 12 control oral mucosa tissues. The 4-nitroquinoline-1-oxide oral carcinogenesis mouse model was used to study CD44 expression during carcinogenesis. Gelatin sponges seeded with CD44(+), CD44(-) and unsorted cancer cells suspended in Matrigel were implanted in NOD/SCID mice into a dorsal skinfold chamber and compared to non-seeded sponges as controls. Angiogenic response was assessed by intravital microscopy. In the TCGA analysis, CD44 gene expression correlated with various pro-angiogenic genes. In human HNSCC tissues, CD44 expression was upregulated and was associated with blood vessels, although no correlation between MVD and CD44 expression was found. During oral carcinogenesis CD44 expression was upregulated. In dorsal skinfold chambers, CD44(+) cells showed a significantly higher MVD than CD44(-) or unsorted cells (p < 0.001). The results indicate that CD44(+) cells contain pro-angiogenic factors and stimulate tumor angiogenesis in HNSCC. Thus, CD44 might emerge as a potential angiogenic biomarker and a therapeutic target for anti-angiogenic therapies.

Genome Wide DNA Methylation Landscape Reveals Glioblastoma Mediated Epigenetic Modification in Tumor Infiltrating CD4+ T-Cells

Abstract INTRODUCTION CD4+ helper T (Th) cells initiate and maintain adaptive immune responses and play a critical role in orchestrating effective antitumor immune response. Although recent immunotherapeutic strategies have shown promising results against glioblastoma, the full potential of this modality has yet to be achieved. One of the major limitations of immunotherapy is the poor efficacy of antiglioblastoma T-cell response in the tumor microenvironment. We hypothesized that glioblastoma modulates antitumor T-cell response by epigenetic modification of tumor infiltrating Th cells (TIThC). METHODS To investigate the influence of glioblastoma on TIThCs, we isolated CD4+ T-cells from the tumor and peripheral blood (PB) of 5 steroid naïve patients with newly diagnosed glioblastoma and performed whole-genome bisulfite sequencing (WGBS) as well as RNAseq and identified differentially methylated and expressed genes between the two cell populations. RESULTS Our results show that glioblastoma mediated epigenetic modifications define the molecular characteristics of glioblastoma infiltrating CD4+ T-cells. Tegmentation based WGBS revealed more than 25 000 regions that are methylated differentially in pairwise comparison of TIThC and PB CD4+ T-cells. Methylation status correlated with the gene expression profile with more than 20 000 differentially expressed genes in TIThCs compared to PB. Of the CD4 lineage specific genes, TBX21, GATA3, RORC, and FOXP3, TBX21, GATA3, and FOXP3 showed differential methylation and expression level in TIThC; whereas, RORC only showed difference in methylation status but not in gene expression level. There was a significant difference in overall and CD4 lineage specific methylation and gene expression profile between patients. Pathway analysis of differentially methylated regions and differentially expressed genes indicated several pathways of tumor induced deregulation, including those involved in T-cell activation, lymphocyte differentiation, regulation of immune effector process, and cytokine production. CONCLUSION Glioblastoma multiforme (GBM) regulates antitumor immune response by significant epigenetic reprogramming of TIThC; thus, influencing their lineage specific differentiation and function.

Altered immune parameters associated with Koala Retrovirus (KoRV) and Chlamydial infection in free ranging Victorian koalas (Phascolarctos cinereus)

Koala Retrovirus (KoRV) has been widely speculated to cause immune suppression in koalas (Phascolarctos cinereus) and to underlie the koala’s susceptibility to infectious disease, however evidence for immunomodulation is limited. The aim of this study is to determine whether immunophenotypic changes are associated with KoRV infection in free ranging Victorian koalas. qPCR was used to examine mRNA expression for Th1 (IFNγ), Th2-promoting (IL6, IL10) and Th17 (IL17A) cytokines, along with CD4 and CD8 in whole blood of koalas (n = 74) from Mt Eccles and Raymond Island in Victoria, Australia, with and without natural chlamydial infection. KoRV positive koalas had significantly lower levels of IL17A (p`0.023) and IFNγ (p = 0.044) gene expression along with a decreased CD4:CD8 gene expression ratio (p = 0.025) compared to negative koalas. No effect of chlamydial infection or combined effect of KoRV and chlamydial infection was detected in these populations. The decreased expression of IFNγ could make KoRV infected koalas more susceptible to persistent chlamydial infection, and a decrease in IL17A could make them more susceptible to gram negative bacterial, fungal and mycobacterial infection; but more tolerant of chlamydial infection.

CD38 Predicts Favorable Prognosis by Enhancing the Immune Infiltration and Antitumor Immunity in Epithelial Ovarian Cancer Microenvironment

Background: Identifying predictive biomarker and novel target to optimize the immunotherapy strategies for epithelial ovarian cancer (EOC) is urgent needed. CD38 is a multifunctional glycoprotein that acts as ectoenzyme and immune receptor. However, the underlying immunological mechanisms and prognostic value of CD38 in EOC is remain unclear. Methods: CD38 gene expression in EOC was evaluated in Gene Expression Profiling Interactive Analysis (GEPIA) and TISIDB database. The prognostic value was calculated using GEPIA and Kaplan-Meier plotter. And Gene Set Enrichment Analysis was conducted to study the roles of CD38 in EOC microenvironment. Furthermore, the relationship between CD38 expression level and immune cells infiltration were analyzed by Tumor Immune Estimation Resource (TIMER) and TISIDB. Results: The GEPIA and TISIDB database shown that CD38 expression in EOC was higher than normal tissue and expressed highest in immunoreactive subtype among four molecular types. 424 cases from GEPIA revealed that high levels of CD38 was associated with longer disease-free survival (HR=0.66, P=0.00089) and increased overall survival (HR=0.67, P=0.0016), Kaplan-Meier plotter also confirms the prognostic value of CD38 in EOC. Data from TCGA database demonstrated that gene signatures in many categories, such as immune response and adaptive immune response, were enriched in EOC samples with CD38 highly expression. Besides, CD38 was positively correlated with immune cells infiltrating, especially activated CD8+ T cells, CD4+ T cells and B cells. Conclusions: CD38 is positively correlated with prognosis and immune cells infiltrating in EOC microenvironment, and contributes to regulation of antitumor immunity. CD38 could be used as a prognostic biomarker and potentially immunotherapy target. Funding Statement: This study was supported in part through the Natural Science Foundation of Zhejiang Province (LY17H160028, ZGZ). Declaration of Interests: The authors stated: No potential conflicts of interest were disclosed. Ethics Approval Statement: Not required.

Abstract 3217: Preclinical evaluation of the recombinant dendritic cell growth factor CDX-301 (Flt3L), and AST-008, a TLR9 agonist SNA

Abstract Recent studies have highlighted the critical role of CD103+/CD141+ dendritic cells (DCs) in antitumor immunity. CDX-301 is a soluble, recombinant human FLT3 ligand (Flt3L). Flt3L is a hematopoietic cytokine which stimulates the proliferation and differentiation of various blood cell progenitors, including CD103+/CD141+ DCs. CDX-301 is in clinical development for multiple cancers and may hold significant opportunity for synergistic development in combination with other immunotherapies, in particular toll-like receptor 9 (TLR9) agonists that are known to promote maturation and activation of DCs. AST-008 is a TLR9 agonist oligonucleotide in a spherical nucleic acid (SNA) format and is in clinical development for multiple cancers in combination with pembrolizumab. SNAs are densely packed, radial arrangements of oligonucleotides around a nanoparticle core. SNAs have increased cellular uptake, nuclease stability, and affinity to targets compared with linear oligonucleotides. AST-008 induces potent TH1-type immune responses in vitro, in mice and non-human primates, and has shown potent antitumor activity as a monotherapy and enhanced checkpoint inhibitor activity in several murine tumor models. AST-008 increases tumor-infiltrating lymphocytes, interferon-inducible gene expression, and activation and expansion of CD8+ T cells with reduced T-regulatory cells in the tumor microenvironment. We examined the effects of the combination of CDX-301 and a murine version of AST-008, muAST-008, in mice on DCs and on antitumor efficacy in a tumor model. Mice were implanted with MC38 murine colon adenocarcinoma tumor cells (day 0) and animals were treated with either CDX-301 (5 μg, days 2-8 i.p.) only, muAST-008 only (1 mg/kg or 3 mg/kg, days 9, 16, 23 and 30 p.t.), or the combination of CDX-301 (5 μg, days 2-8) with muAST-008 (1 mg/kg or 3 mg/kg, days 9, 16, 23 and 30). Treatment with CDX-301 and muAST-008 showed an additive effect in retarding tumor growth and prolonging survival. To help understand the mechanisms involved in the antitumor effects, animals were treated with a similar course of CDX-301 and muAST-008 and spleens were harvested for flow cytometry evaluation 2 days after the last CDX-301 dose. For these studies, we also included a second DC activating agent, an agonist anti-CD40 mAb FGK45.5 (50 μg i.p. together with the last dose of CDX-301) among the combinations. We observed a significant increase in the percentage of CD103+CD8+ cDCs by the addition of muAST-008 to CDX-301 treatment. In addition, muAST-008 led to the up-regulation of activation markers on dendritic cells, which was markedly enhanced when combined with CD40 activation. These data demonstrate that muAST-008 leads to systemic activation of CDX-301 expanded DCs, leading to more potent anti-tumor immunity and support the potential of combining CDX-301 and AST-008 in augmenting the immunotherapy of cancers. Citation Format: Lawrence J. Thomas, Li-Zhen He, Lauren E. Gergel, Eric M. Forsberg, Elizabeth Q. Do, James M. Boyer, April R. Baronas, Mallary Rocheleau, Kathleen M. Borrelli, Anna Wasiuk, Jeffrey Weidlick, Henry C. Marsh, Bart R. Anderson, SubbaRao Nallagatla, Richard Kang, Ekambar R. Kandimalla, Tibor Keler. Preclinical evaluation of the recombinant dendritic cell growth factor CDX-301 (Flt3L), and AST-008, a TLR9 agonist SNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3217.

The Role of Interferon Regulatory Factor 5 in Macrophage Inflammation During Osteoarthritis.

Mounting evidence suggests that aberrant immune responses are involved in the pathogenesis of osteoarthritis (OA). Synovial macrophages are likely involved. In this study, we sought to investigate the role of interferon regulatory factor 5 (IRF5). In vitro M1-polarized macrophages presented significantly higher IRF5 expression than M2-polarized macrophages. Interestingly, IRF5 expression was observed in macrophages from the synovial fluid of OA patients, and the level of IRF expression was positively correlated with disease severity, such that stage 4 OA synovial macrophages presented significantly higher levels of IRF5 than stage 2 and stage 3 OA synovial macrophages. Circulating monocytes from OA patients, on the other hand, expressed little IRF5. However, synovial fluid from OA patients could significantly upregulate IRF5 expression in circulating monocytes. Synovial macrophages also expressed significantly higher IL-12 than circulating monocytes, and circulating monocytes conditioned in OA synovial fluid demonstrated significantly higher IL-12 expression. Direct IRF5 transfection could increase IL-12 expression in circulating monocytes. Interestingly, IRF5-transfected monocytes promoted the expression of Th1-associated genes in naive CD4 T cells via an IL-12-dependent mechanism. Overall, our study demonstrated that IRF5 expression was associated with OA severity and could contribute to the activation of the M1-Th1 axis.

Loss of MafA and MafB expression promotes islet inflammation

Maf transcription factors are critical regulators of beta-cell function. We have previously shown that reduced MafA expression in human and mouse islets is associated with a pro-inflammatory gene signature. Here, we investigate if the loss of Maf transcription factors induced autoimmune processes in the pancreas. Transcriptomics analysis showed expression of pro-inflammatory as well as immune cell marker genes. However, clusters of CD4+ T and B220+ B cells were associated primarily with adult MafA−/−MafB+/−, but not MafA−/− islets. MafA expression was detected in the thymus, lymph nodes and bone marrow suggesting a novel role of MafA in regulating immune-cell function. Analysis of pancreatic lymph node cells showed activation of CD4+ T cells, but lack of CD8+ T cell activation which also coincided with an enrichment of naïve CD8+ T cells. Further analysis of T cell marker genes revealed a reduction of T cell receptor signaling gene expression in CD8, but not in CD4+ T cells, which was accompanied with a defect in early T cell receptor signaling in mutant CD8+ T cells. These results suggest that loss of MafA impairs both beta- and T cell function affecting the balance of peripheral immune responses against islet autoantigens, resulting in local inflammation in pancreatic islets.

Cyclooxygenase and CD147 expression in oral squamous cell carcinoma patient samples and cell lines

In oral squamous cell carcinoma (OSCC), cyclooxygenases (COX-1 and COX-2) contribute to inflammation, and cluster of differentiation factor 147 (CD147) contributes to invasiveness, but their relationship has not been previously examined within a cohort of patients with OSCC or OSCC cell lines. COX-2 and CD147 expression was determined by using immunohistochemistry on 39 surgical biopsy specimens of OSCC. Expression in tumor cells, stroma, and adjacent oral epithelium was characterized by using a visual grading system. COX-1, COX-2, and CD147 expression was determined in vitro by using OSCC cell lines (SCC25, BHY, and HN) and reverse transcriptase-quantitative polymerase chain reaction. Secretion of prostagladin E2 (PGE2) from OSCC cell lines was determined by using PGE2 enzyme-linked immunosorbent assay. Biopsy specimens showed higher COX-2 expression in tumor cells compared with stroma and adjacent epithelium (P < .05). There was no difference in CD147 expression among the tumor cells, stroma, and adjacent epithelium. In OSCC cell lines, there was a trend for COX-2 and CD147 gene expression to be coordinated. Interestingly, PGE2 secretion was more closely related to COX-1 expression than to COX-2 expression. COX-1, COX-2, and CD147 appear to be independently regulated in OSCC, potentially representing 2 therapeutic targets for future investigation. COX-1 expression in OSCC deserves further study because it may be an important determinant of PGE2 secretion from OSCC cells.

Effects of prophylactic administration of glutamine on CD4+ T cell polarisation and kidney injury in mice with polymicrobial sepsis.

The present study investigated the effects of glutamine (GLN) pretreatment on CD4+ T cell polarisation and remote kidney injury in mice with gut-derived polymicrobial sepsis. Mice were randomly assigned to three groups: normal control fed with American Institute of Nutrition (AIN)-93G diet and two sepsis groups provided with either AIN-93G-based diet or identical components, except part of casein was replaced by GLN. Mice were given their respective diets for 2 weeks. Then, mice in the sepsis groups were performed with caecal ligation and puncture and were killed 72 h after the surgery. Blood, spleens and kidneys were collected for further examination. The results showed that sepsis resulted in decreased circulating and splenic total Tlymphocyte and CD4+ T cell percentages, whereas IL-4-, and forkhead box p3 (Foxp3)-expressing CD4+ T cells percentages were up-regulated. Compared with the sepsis control group, pretreatment with GLN maintained blood T and CD4+ T cells and reduced percentages of IL-4- and Foxp3-expressing CD4+ T cells. Also, a more pronounced activation and increased anti-apoptotic Bcl-2 gene expression of splenic CD4+ T cells were observed. Concomitant with the decreased plasma IL-6, keratinocyte-derived chemokine (KC) levels, the gene expression of KC, macrophage inflammatory protein-2 and renal injury biomarker kidney injury molecule-1 (Kim-1) were down-regulated when GLN was administered. These findings suggest that antecedent of GLN administration elicit a more balanced blood T helper cell polarisation, sustained T cell populations, prevented splenic CD4+ T cell apoptosis and attenuated kidney injury at late phase of polymicrobial sepsis. GLN may have benefits in subjects at risk of abdominal infection.

Biologicals targeting T helper cell subset differentiating cytokines are effective in the treatment of murine anti-myeloperoxidase glomerulonephritis

Anti-myeloperoxidase nephritogenic autoimmunity induces severe glomerulonephritis. To assess the therapeutic potential of monoclonal antibodies targeting T helper (Th) subset differentiation determining cytokines, we studied a murine model of anti-myeloperoxidase glomerulonephritis. The temporal participation of T helper subsets was determined by quantitating gene expression of CD4+ T-cells isolated from nephritic kidneys and cytokine production by lymphocytes from nodes draining myeloperoxidase immunization sites. Th17 cytokines (IL-17A and IL-6) rose rapidly but declined as autoimmunity matured when Th1 cytokines (IL-12 and TNF) predominated. Therefore, T helper subset participation in anti-myeloperoxidase autoimmunity is biphasic, with Th17 early and Th1 late. To confirm the functional relevance of this biphasic pattern, we compared systemic anti-myeloperoxidase autoimmunity in wild type, Th17 deficient and Th1 deficient mice. Early, Th1 deficient mice developed similar autoimmunity and glomerulonephritis to wild type mice. However, Th17 deficient mice had significantly reduced anti-myeloperoxidase autoimmunity. In late autoimmunity, Th1 deficient mice developed reduced autoimmunity and were protected from anti-myeloperoxidase glomerulonephritis. The therapeutic potential of these findings were demonstrated by neutralizing monoclonal antibodies. Targeting IL-23p19 attenuated early Th17 dominated anti-myeloperoxidase autoimmunity and glomerulonephritis but not late phase disease. Targeting IL-12p35 attenuated late phase Th1 dominated anti-myeloperoxidase autoimmunity and glomerulonephritis but not early autoimmunity or glomerulonephritis. Targeting both T helper subsets with an anti-IL-12p40 monoclonal antibody was effective during both early and late phases of anti-myeloperoxidase glomerulonephritis. Thus, definition of dominant T helper differentiating subsets in anti-myeloperoxidase glomerulonephritis by renal CD4+ T-cell cytokine gene expression allows effective proper phase monoclonal antibody treatment of anti-myeloperoxidase glomerulonephritis.

MAPK pathway activity plays a key role in PD-L1 expression of lung adenocarcinoma cells.

Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD‐1) and programmed death‐ligand 1 (PD‐L1) have improved the survival of patients with non‐small cell lung cancer (NSCLC). Still, many patients do not respond to these inhibitors. PD‐L1 (CD274) expression, one of the factors that influences the efficacy of immune checkpoint inhibitors, is dynamic. Here, we studied the regulation of PD‐L1 expression in NSCLC without targetable genetic alterations in EGFR, ALK, BRAF, ROS1, MET, ERBB2 and RET. Analysis of RNA sequencing data from these NSCLCs revealed that inferred IFNγ, EGFR and MAPK signaling correlated with CD274 gene expression in lung adenocarcinoma. In a representative lung adenocarcinoma cell line panel, stimulation with EGF or IFNγ increased CD274 mRNA and PD‐L1 protein and membrane levels, which were further enhanced by combining EGF and IFNγ. Similarly, tumor cell PD‐L1 membrane levels increased after coculture with activated peripheral blood mononuclear cells. Inhibition of the MAPK pathway, using EGFR inhibitors cetuximab and erlotinib or the MEK 1 and 2 inhibitor selumetinib, prevented EGF‐ and IFNγ‐induced CD274 mRNA and PD‐L1 protein and membrane upregulation, but had no effect on IFNγ‐induced MHC‐I upregulation. Interestingly, although IFNγ increases transcriptional activity of CD274, MAPK signaling also increased stabilization of CD274 mRNA. In conclusion, MAPK pathway activity plays a key role in EGF‐ and IFNγ‐induced PD‐L1 expression in lung adenocarcinoma without targetable genetic alterations and may present a target to improve the efficacy of immunotherapy. © 2019 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.