Expression Levels Of CD8 Research Articles

Altered Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1/T-Cell Immunoglobulin Mucin-3 Signaling Causes the Dysregulation of Decidual CD8+T Cells in the Third Trimester during Preeclamptic Pregnancies

Abstract Objective: To investigate the frequency and function of Tim-3+CD8+T cells in the third trimester of normal pregnancies (NPs) and preeclamptic (PE) pregnancies. Methods: T-cell immunoglobulin mucin-3 (Tim-3) expression levels of CD8+T cells in the decidua, peripheral blood, and umbilical cord blood obtained from women showing NPs and PE pregnancies were analyzed using flow cytometry. Decidual CD8+T cells were cultured in the presence of recombinant human carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) protein and/or Tim-3-specific neutralizing antibodies for analyzing CD107a and intracellular cytokine expression. The placental CEACAM1 protein expression was analyzed using immunohistochemistry. Results: Tim-3+CD8+T cells were more abundant in the decidua than in the peripheral blood. Tim-3 expression in the decidual CD8+T cells was significantly lower in PE patients. Decidual Tim-3+CD8+T cells from PE patients expressed higher levels of CD107a and the Th1-type cytokine IFN-γ, but lower levels of the Th2-type cytokine IL-4. CEACAM1 altered the CD107a, IFN-γ, and IL-4 levels; this was reversed by anti-Tim-3 antibodies. The CEACAM1 protein levels were lower in the placental tissues of women with PE pregnancies than in those of women with NPs. Conclusions: Abnormal CEACAM1/Tim-3 regulation may participate in the development of PE, accompanied by disturbed Th2 cell predominance and higher cytotoxicity of decidual CD8+T cells.

Application of Immunoprofiling Using Multiplexed Immunofluorescence Staining Identifies the Prognosis of Patients with High-Grade Serous Ovarian Cancer.

Immunoprofiling has an established impact on the prognosis of several cancers; however, its role and definition in high-grade serous ovarian cancer (HGSOC) are mostly unknown. This study is to investigate immunoprofiling which could be a prognostic factor in HGSOC. We produced tumor microarrays of 187 patients diagnosed with HGSOC. We performed a multiplexed immunofluorescence staining using Opal Multiplex IHC kit and quantitative analysis with Vectra-Inform system. The expression intensities of programmed death-ligand 1 (PD-L1), CD4, CD8, CD20, FoxP3, and CK in whole tumor tissues were evaluated. The enrolled patients showed general characteristics, mostly FIGO stage III/IV and responsive to chemotherapy. Each immune marker showed diverse positive densities, and each tumor sample represented its immune characteristics as an inflamed tumor or noninflamed tumor. No marker was associated with survival as a single one. Interestingly, high ratios of CD8 to FoxP3 and CD8 to PD-L1 were related to the favorable overall survival (77 vs. 39 months, 84 vs. 47 months, respectively), and CD8 to PD-L1 ratio was also a significant prognostic factor (HR 0.621, 95% CI 0.420–0.917, p = 0.017) along with well-known clinical prognostic factors. Additionally, CD8 to PD-L1 ratio was found to be higher in the chemosensitive group (p = 0.034). In conclusion, the relative expression levels of CD8, FoxP3, and PD-L1 were significantly related to the clinical outcome of patients with HGSOC, which could be a kind of significant immunoprofiling of ovarian cancer patients to apply for treatment.

Deficiency of miR-15a/16 upregulates NKG2D in CD8+ T cells to exacerbate dextran sulfate sodium-induced colitis

The miR-15a/16 gene cluster is located in human chromosome 13 (13q14.3) and mouse chromosome 14 (14qC3). These genes are involved in cancer development and immune regulation. Our group has previously verified the binding of the 3′-untranslated region of NKG2D gene by miR-16 through dual-luciferase reporter assay. Herein, we found that miR-16 overexpression inhibited the NKG2D expression of CD8+ T cells, and that CD8+ NKG2D+ T cell frequency increased in miR-15/16−/− mice. CD8+ NKG2D+ T cells derived of miR-15/16−/− mice displayed activatory phenotype with enhanced IFN-γ production and cytotoxicity. The transfection of lentivirus containing antago-miR-16 sequences enhanced the NKG2D expression level of CD8+ T cells. However, no significant differences in CD8+ NKG2D+ T cell frequencies existed between wild-type and miR-15/16-transgenic mice because NKG2D was not expressed on the rest CD8+ T cells. When CD8+ T cells of miR-15/16-transgenic mice were treated with IL-2 in vitro, the magnitude of NKG2D expression and activation of CD8+ T cells was lower than that of wild-type mice. miR-15/16−/− mice showed that the exacerbation of colitis induced by dextran sulfate sodium (DSS) with more CD8+ T cells accumulated in inflamed colons, whereas miR-15/16-transgenic mice ameliorated DSS-induced colitis with less infiltration of CD8+ T cells. When NKG2D+ cells were depleted with NKG2D antibody in miR-15/16−/− mice, the aggravated colitis disappeared. All these results demonstrated that NKG2D could be upregulated by decreased miR-16 in CD8+ T cells to mediate inflammation. Thus, gene therapy based on the overexpression of miR-16 in CD8+ T cells can be used for patients with inflammatory diseases.

Immune-related gene expression in the kidneys and spleens of goslings infected with goose nephritic astrovirus

Goose nephritic astrovirus (GNAstV) was first isolated in 2018, causing great economic losses to the goose industry. However, little is known about host immune response to GNAstV infection. In this study, forty 2-day-old goslings were randomly divided into 2 groups: infection and negative control groups. Each gosling in the infection group was challenged with 0.5 mL GNAstV-JSHA intramuscularly, whereas the gosling in the negative control group was inoculated with the same amount of PBS. Histopathological changes and virus location in the spleen and kidney were examined, and the expression of immune-related genes was determined by qPCR at 7 and 14 d after infection. Our results showed that GNAstV infection induced degeneration and necrosis of splenic lymphocytes and renal epithelial cells, and these cells were positive for the virus. In addition, GNAstV infection induced the activation of pattern recognition receptors (RIG-I, MDA-5, and TLR3) and key adaptor molecules (MyD88, MAVS, and IRF7) in the spleen and kidney, and upregulated the gene expression of interferon-α in the spleen and antiviral proteins (MX1, OASL, and IFITM3) in the spleen and kidney. Moreover, high expression levels of interleukin (IL)-1β and IL-8 in the spleen and iNOS in the spleen and kidney were found. These results indicated that GNAstV infection activated host innate immune response. Furthermore, GNAstV infection increased the expression levels of CD8+, MHCI, and MHCII, indicating that adaptive immune response was activated. Besides, TGF-β was highly expressed in the spleen and kidney, which may be an immune evasion strategy of GNAstV to cause infection. Interestingly, both IL-1β and IL-6 mRNA levels were decreased in the kidney, which may help reduce kidney lesions. This is the first study to report changes in immune-related gene expression in response to GNAstV infection, and our results provide insights into viral pathogenesis.

An immune-based risk-stratification system for predicting prognosis in pulmonary sarcomatoid carcinoma (PSC)

ABSTRACT Pulmonary sarcomatoid carcinoma (PSC) is an uncommon subtype of lung cancer, and immune checkpoint blockade promises in clinical benefit. However, virtually nothing is known about the expression of common immune checkpoints in PSC. Here, we performed immunohistochemistry (IHC) to detect nine immune-related proteins in 97 PSC patients. Based on the univariable Cox regression, random forests were used to establish risk models for OS and DFS. Moreover, we used the GSEA, CIBERSORT, and ImmuCellAI to analyze the enriched pathways and microenvironment. Univariable analysis revealed that CD4 (P = 0.008), programmed cell death protein 1 (PD-1; P = 0.003), galectin-9 (Gal-9) on tumor cells (TCs; P = 0.021) were independent for DFS, while CD4 (P = 0.020), PD-1 (P = 0.004), Gal-9 (P = 0.033), and HLA on TILs (P = 0.031) were significant for OS. Meanwhile, the expression level of CD8 played a marginable role in DFS (P = 0.061), limited by the number of patients. The combination of Gal-9 on TC with CD4 and PD-1 on TILs demonstrated the most accurate prediction for DFS (AUC: 0.636-0.791, F1-score: 0.635–0.799), and a dramatic improvement to TNM-stage (P < 0.001 for F1-score of 1-y, 3-y, and 5-yDFS). A similar finding was also observed in the predictive ability of CD4 for OS (AUC: 0.602-0.678, F1-score: 0.635–0.679). CD4 was negatively associated with the infiltration of neutrophils (P = 0.015). PDCD1 (coding gene of PD-1) was positively correlated to the number of exhausted T cells (Texs; P = 0.020) and induced regulatory T cells (iTregs; P = 0.021), and LGALS9 (coding gene of Gal-9) was positively related to the level of dendritic cells (DCs; P = 0.021). Further, a higher combinational level of CD4, PDCD1 on TILs, and LAGLS9 on TCs were proved to be infiltrated with more M1-type macrophages (P < 0.05). We confirmed the expression status of nine immune-related proteins and established a TNM-Immune system for OS and DFS in PSC to assist clinical risk-stratification.

Abstract 4973: Comparison of tumor immune microenvironment in triple negative breast cancer (TNBC) of African American versus that of white patients

Abstract Introduction: Although there are many possible causes, it has been observed that Triple Negative Breast Cancer (TNBC) has a worse overall outcome in African American (AA) than white patients. Recently, there is also evidence that the patients response to the tumor, or the biological response expressed as the tumor immune microenvironment, impacts outcome. The goal of this study was to compare the Tumor Microenvironment (TME) of TNBC in the two mentioned groups in order to seek molecular parameters that are different in TNBC in AA patients as compared to whites. Methods: We selected N=50 AA and N=50 white TNBC samples from the Yale Pathology archives that were matched by diagnosis date. We measured CD45, CD3, CD8, CD20, CD14, CD68, PD-L1, THY1, aSMA and Fibroblast Activation Protein (FAP) expression in both the tumor and stromal compartments in whole slides from formalin fixed paraffin embedded (FFPE) tissues using multiplexed quantitative immunofluorescence (QIF). We also assessed the activation status of CD3-positive cells using Ki67 and Granzyme B markers. Median of each marker expression was calculated for each case by using quantitation from all Fields of View (FOV) that contained at least 10% tumor area. Results: AA tumors contained a significantly higher CD45-positive cells in overall, within tumor and stromal compartments (p = 0.0102, 0.0007 and 0.0280 respectively). Being more specific about CD45 marker, AA also showed to have a significantly higher level of overall lymphocyte population, measured by quantitating CD45+ CD14- cells (p =0.0081). AA tumors also had a significantly higher level of CD8 expression (p &amp;lt;0.0001). Even though we could not find any differences in CD3 expressions between the two groups, AA tumors contained a significantly higher level of activated T-cells (p =0.0432), where activation is defined as CD3 cells with high Ki67 or Granzyme B. AA tumors contained a significantly higher level of CD68 positive cells (p &amp;lt; 0.0001) and PD-L1 expression in CD68+ cells (p =0.0101). All other markers showed no difference between the two groups. Conclusion: Significant differences are seen in the TME of AA vs. white TNBCs. Further characterization of macrophages, fibroblasts, regulatory T-cells and Extracellular Matrix are underway to more specifically define TME and determine the relationship between TME features and outcome. Citation Format: Vesal Yaghoobi, Vasiliki Pelekanou, Tess O'Meara, Andrea Silber, Lajos Pusztai, David Rimm, Kim Blenman. Comparison of tumor immune microenvironment in triple negative breast cancer (TNBC) of African American versus that of white patients [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 4973.

Expression and Significance of PD-1, TIM-3 and VISTA on T Cell of Acute Myeloid Leukemia Patients

To study the expression of multiple negative costimulatory molecules on peripheral blood T cells in patients with acute myeloid leukemia (AML) and its affection on prognosis. The peripheral blood samples from patients with newly diagnosed AML, complete remission (CR), and no-remission (NR) were collected, the expression levels PD-1、VISTA and TIM-3 in CD4+ and CD8+ T cells were detected by flow cytometry , and the clinical data of patients were analyzed. The expression levels of PD-1、VISTA and TIM-3 of CD4+ and CD8+ T cells in the newly diagnosed AML patients were significantly higher than those in control group (P＜0.05). The expression levels of PD-1、TIM-3 and VISTA of CD4+ and CD8+ T cells in the CR group were significantly lower than those in newly diagnosed and the NR group (P＜0.05). The TIM-3 expression level positively correlated with VISTA expression level of CD4+ and CD8+ T cells in newly diagnosed AML patients (r=0.85 and 0.73). The VISTA and PD-1 expression level of CD4+ T cells in newly diagnosed AML, NR after first induction chemotherapy and high risk patients significantly increased (P＜0.05), the TIM-3 expression level of CD8+ T cells in high risk group significantly increased (P＜0.05), and the VISTA expression level of CD8+ T cells in CBFβ-MYH11 mutation-positive group significantly decreased (P＜0.05). The expression of PD-1、TIM-3 and VISTA in AML peripheral blood T cells may be involved in the immune escape of AML and can be the targets of treatment for acute myeloid leukemia patients.

Increased expression of CD8 marker on T-cells in COVID-19 patients

BackgroundCell-mediated immunity including T-cells (T helper and cytotoxic) plays an essential role in efficient antiviral responses against coronavirus disease-2019 (COVID-19). Therefore, in this study, we evaluated the ratio and expression of CD4 and CD8 markers in COVID-19 patients to clarify the immune characterizations of CD4 and CD8 T-cells in COVID-19 patients. MethodsPeripheral blood samples of 25 COVID-19 patients and 25 normal individuals with similar age and sex as the control group were collected. White blood cells, platelets, and lymphocytes were counted and CD4 and CD8 T lymphocytes were evaluated by flow cytometry. ResultsThe number of white blood cells, lymphocytes, and platelets were reduced significantly in COVID-19 patients (P < 0.05). The difference in CD4:CD8 ratio, CD4 T-cell frequency, CD8 T-cell frequency, and CD4 mean fluorescence intensity (MFI) was not significant between COVID-19 patients and healthy individuals (P > 0.05); however, the CD8 MFI increased significantly in COVID-19 infected patients (P < 0.05). ConclusionAlthough, there is no significant difference in the ratio of CD4 to CD8 between two groups, the expression level of CD8 in COVID-19 patients was significantly higher than the normal individuals. This result suggested that the cellular immune responses triggered by COVID-19 infection were developed through overexpression of CD8 and hyperactivation of cytotoxic T lymphocytes.

Relationship between prognosis of Lumina B breast cancer and tumor infiltration of forxhead box protein 3+ regulatory T lymphocytes and CD8+ cytotoxic T lymphocytes

Objective To detect the expression level of forxhead box Protein 3 (FoxP3) and CD8 in invasive ductal carcinoma of breast Lumina B, investigate the relevance between FoxP3+ regulatory T lymphocyte (Treg) and CD8+ cytotoxic T lymphocyte (CTL) as well as prognosis of breast cancer patients. Methods Immunohistochemistry was used to detect the expression level of FoxP3 and CD8 in the paraffin-embedded breast cancer samples. One-sample t test was applied to analyze the relationship among the expression levels of FoxP3 and CD8 of patient’s age, tumor size, nodal status, and histophathologic grade. Kaplan meier Log-rank was used to analyze the correlation between the expression level of CD8 and FoxP3 and the overall survival and disease-free survival of breast cancer patients. Results The expression of CD8 is associated with tumor diameter ≤2 cm, lymph node negative metastasis, and with lower histopathologic grade (gradeⅠ/Ⅱ) (P 2 cm, lymph node metastasis (P<0.01). High expression level of FoxP3 infiltrating in tumor was could reduce the desease-free survival of breast cancer patients (P<0.05), while low expression of CD8 prolonged desease-free survival (P<0.01). Conclusion Tumor-infiltrating CD8+ CTLs are associated with good prognosis, whereas tumor-infiltrating FoxP3+ Tregs are associated with adverse outcomes. Key words: Invasive ductal carcinoma of breast; Tumor-infiltrating T lymphocyte; Regulatory T lymphocyte; Cytotoxic T lymphocytes; Forxhead box protein 3; CD8

Role of Immune Response, Inflammation, and Tumor Immune Response–Related Cytokines/Chemokines in Melanoma Progression

To investigate the role of tumor cytokines/chemokines in melanoma immune response, we estimated the proportions of immune cell subsets in melanoma tumors from The Cancer Genome Atlas, followed by evaluation of the association between cytokine/chemokine expression and these subsets. We then investigated the association of immune cell subsets, chemokines, and cytokines with patient survival. Finally, we evaluated the immune cell tumor-infiltrating lymphocyte (TIL) score for correlation with melanoma patient outcome in a separate cohort. There was good agreement between RNA sequencing estimation of T-cell subset and pathologist-determined TIL score. Expression levels of cytokines IL-12A, IFNG, and IL-10, and chemokines CXCL9 and CXCL10 were positively correlated with PDCD1, CTLA-4, and CD8+ T-cell subset, but negatively correlated with tumor purity (Bonferroni-corrected P < 0.05). In multivariable analysis, higher expression levels of cytokines IFN-γ and TGFB1, but not chemokines, were associated with improved overall survival. A higher expression level of CD8+ T-cell subset was also associated with improved overall survival (hazard ratio [HR]= 0.06, 95% confidence interval [CI]= 0.01-0.35, P= 0.002). Finally, multivariable analysis showed that patients with a brisk TIL score had improved melanoma-specific survival than those with a nonbrisk score (HR= 0.51, 95% CI= 0.27-0.98, P= 0.0423). These results suggest that the expression of specific tumor cytokines represents important biomarkers of melanoma immune response.

Changes in plasma EBV-DNA and immune status in patients with nasopharyngeal carcinoma after treatment with intensity-modulated radiotherapy

BackgroundPrevious studies reported the early diagnostic values of plasma Epstein-Barr virus (EBV)-DNA. The present study aimed to assess the relationship between the concentration of plasma EBV-DNA and the number of CD8+PD-1+(programmed cell death-1,PD-1) and regulatory T (Treg) cells in patients with nasopharyngeal carcinoma (NPC) who were treated with intensity-modulated radiotherapy (IMRT).MethodsThis study included 37 patients treated with IMRT. Peripheral blood samples were collected two times for each patient, before radiation therapy and 1 week after the treatment. Further, the numbers of CD4+, Treg, CD8+, and CD8+PD1+ cells were determined by flow cytometry.ResultsThe changes after IMRT were determined by comparing the numbers of neutrophils, lymphocytes, CD4+, Treg, CD8+, CD8+PD1+ cells, and the concentration of plasma EBV-DNA between pretreatment and post-treatment groups. IMRT could reduce the expression level of PD-1 and the number of Treg cells. The concentration of plasma EBV-DNA and the expression level of CD8+PD-1+ were closely associated with the occurrence and development of NPC. Thus, EBV-DNA can be used as an important marker for early diagnosis, and IMRT can strongly reduce the copies of EBV-DNA.ConclusionsThis study showed that IMRT could reverse T-cell exhaustion and reduce the copies of EBV-DNA. In clinical practice, plasma EBV-DNA is a sensitive biomarker for diagnosis, prognosis, and evaluation of clinical efficacy.

Prognostic impact of CD8 and programmed death-ligand 1 expression in patients with resectable non-small cell lung cancer

BackgroundThe prognostic impact of the expression of CD8 and programmed death-ligand 1 (PD-L1) has not been established in patients with resectable non-small cell lung cancer (NSCLC).MethodsSurgical tissue specimens were obtained from 136 patients with NSCLC who underwent surgical resection. The expression levels of CD8 and PD-L1 were assessed using tissue microarrays and immunohistochemistry.ResultsThe CD8-positive group showed significant increases in overall survival (OS) (median, not reached [NR] vs. 28.452 months) and relapse-free survival (RFS) (median, NR vs. 14.916 months) compared with the CD8-negative group. In contrast to CD8, the PD-L1-negative group demonstrated significant increases in OS (median, NR vs. 29.405 months) and RFS (median, 63.573 vs. 17.577 months) compared with the PD-L1-positive group. Two prognostic groups were stratified according to CD8/PD-L1 expression: group 1 (CD8-positive/PD-L1-negative) vs. group 2 (CD8/PD-L1: positive/positive, negative/negative, negative/positive). Group 1 had better OS (median, NR vs. 29.405 months) and RFS (median, NR vs. 17.577 months) than group 2. Multivariate analysis indicated that group 1 constituted an independent favourable prognostic factor for OS (hazard ratio [HR], 0.329, p = 0.001) and RFS (HR, 0.293; p < 0.001).ConclusionsPositive CD8 and negative PD-L1 expression together may be favourable prognostic markers in resectable NSCLC.

Correlation between tumor microenvironment-associated factors and the efficacy and prognosis of neoadjuvant therapy for rectal cancer.

The tumor microenvironment contributes to the survival and development of tumor cells and is therefore a key target for cancer therapy. The tumor microenvironment has unique physical and chemical properties and is associated with inflammation and immunity. To examine the correlation between tumor microenvironment-associated factors and the efficacy and prognosis of neoadjuvant therapy for rectal cancer, and to compare the differences between two treatments [neoadjuvant chemotherapy (NAC) vs. neoadjuvant chemoradiotherapy (NACR)], an immunohistochemical method was used to measure the expression levels of CD4+ tumor-infiltrating lymphocytes (TILs), cluster of differentiation (CD)8+TILs, forkhead box P3 (FOXP3)+TILs, cytotoxic T lymphocyte-associated antigen-4+TILs and programmed death ligand-1 (PD-L1)+TILs in 109 patients with rectal cancer, pre- and post-neoadjuvant therapy. The significance of these protein expression patterns was also analyzed using tissue microarrays, and the prognostic significance of these findings evaluated. The results indicated that high levels of CD4+TILs, CD8+TILs and PD-L1+TILs may be associated with favorable responses to neoadjuvant therapy, whereas high levels of FOXP3+TILs were associated with poor therapeutic responses. Expression levels of CD8+TILs and FOXP3+TILs following neoadjuvant therapy were independent prognostic factors and affected the total survival of patients subjected to neoadjuvant therapy for the treatment of rectal cancer. Moreover, the effects of NAC and NACR on the tumor microenvironment may be different.

Au-CGKRK Nanoconjugates for Combating Cancer through T-Cell-Driven Therapeutic RNA Interference.

Numerous prior studies on fighting cancer have been based on using inhibitors of JAK-STAT pathway (signal transducer and activator of transcription 3 (STAT3) inhibitor in particular), a signaling pathway responsible for progression of many types of cancer cells. However, recent studies have shown that STAT3 activation leads to upregulation of program death receptor-ligand 1 (PD-L1, an immune checkpoint protein that plays a major role behind evasion of immune systems by growing tumors) expression levels in tumor cells, leading to enhanced immune suppression. This is why global efforts are being witnessed in combating cancer through use of immune checkpoint inhibitors. Herein, we report on the design, synthesis, physicochemical characterizations, and bioactivity evaluation of novel tumor- and tumor-vasculature-targeting noncytotoxic Au-CGKRK nanoconjugates (17–80 nm) for combating tumor. Using a syngeneic mouse tumor model, we show that intraperitoneal (i.p.) administration of the Au-CGKRK nanoparticles (NPs) complexed with both PD-L1siRNA (the immune checkpoint inhibitor) and STAT3siRNA (the JAK-STAT pathway inhibitor) results in significant (>70%) enhancement in overall survivability (OS) in melanoma-bearing mice (n = 5) when compared to the OS in the untreated mice group. The expression levels of CD8 and CD4 proteins in the tumor lysates of differently treated mice groups (by Western blotting) are consistent with the observed OS enhancement being a T-cell-driven process. Biodistribution study using near-infrared dye-loaded Au-CGKRK nanoconjugates revealed selective accumulation of the dye in mouse tumor. Notably, the overall survival benefits were significantly less (∼35%) when melanoma-bearing mice were treated (i.p.) with Au-CGKRK NPs complexed with only PD-L1siRNA or with STAT3siRNA alone. The presently described Au-CGKRK nanoconjugates are expected to find future use in therapeutic RNA-interference-based cancer immunotherapy.

Evaluation of CD30/CD4/CD8 in triple-negative invasive ductal carcinoma of breast in association with clinicopathological prognostic factors

Triple-negative breast cancer (TNBC) lacks the benefits of receptor-targeted therapeutic strategies. The limitations in treatment options along with poor patients' outcome heighten the need for novel approaches. Due to recent concentration on the role of biomarkers in prognosis, treatment, and survival of various cancer subtypes, this study involves an investigation of CD4, CD8, and CD30 markers detected by immunohistochemistry in TNBCs and their association with clinicopathological and prognostic factors. Tissue samples of 85 hormone receptor- and human epidermal growth factor receptor-2-negative ductal breast carcinomas extracted from the archive of pathology department. Regarding CD4/CD8 ratio, the infiltrated T-lymphocytes were investigated. The tumoral tissue regions were also identified to be immunohistochemically assessed for the CD30 expression levels. With an elevated CD4/CD8 ratio, a significant increase in lymph node involvement was observed (P < 0.05); in contrast, increased expression levels of CD8 were related to significant reduction of lymph node involvement. CD30 overexpression was found to be significantly associated with shortened overall survival (OS) and highly involvement of lymph nodes. Following the progression in stage and grade of tumor, CD4/CD8 ratio and CD30 expression levels are increased and are accompanied by adverse prognosis and poor OS, while CD8-enhanced expression carries a favorable prognostic impact as it improves OS status. Therefore, all these findings could be of interest in the field of target therapy.

Abstract 4687: Characterization of the cryoablation-induced immune response in kidney cancer patients

Abstract Cryoablation is one of promising treatment modalities for kidney cancer and expected to induce strong local immune response as well as systemic T cell-mediated immune reaction that may lead to regression of distant metastatic lesions. In this study, we collected tumor tissues from 14 kidney cancer patients, at the time points of pre-cryoablation and 3 months after cryoablation. In addition, blood samples were collected from 8 kidney cancer patients at the same time points. We applied a next generation sequencing approach to characterize T cell receptor beta (TCRB) repertoires using mRNA isolated from tumor tissues and peripheral blood mononuclear cells. To assess clonality of T cells, the diversity index of TCRB was calculated according to complementarity-determining region 3 sequences. Through TCRB repertoire analysis, we demonstrated expansion of certain T cell clones in tumor tissues by cryoablation. In addition, we found that proportions of abundant TCRB clonotypes (≥ 1% frequency among total TCRB reads) were significantly increased in the post-cryoablation tissue samples than those of pre-cryoablation tumor samples, suggesting that cryoablation could induce strong immune reactions in tumors with oligoclonal expansion of anti-tumor T cells. Interestingly, some of these TCRB clonotypes were also increased in peripheral blood, indicating that certain numbers of T cell clones may also circulate systemically and then attack tumor cells in distant regions. We also measured mRNA expression levels of immune-related genes in the tissue samples of pre- and post- cryoablation and found significantly elevated expression levels of CD8, CD4, Granzyme A, and CD11c along with high CD8/FOXP3 ratio in the post-cryoablation tissue samples. Furthermore, immunohistochemical analysis confirmed infiltration of a large number of CD11c-positive cells, probably representing macrophages and dendritic cells into the post-cryoablation tissues. Collectively, our findings revealed that cryoablation could induce both local and systemic immune responses associated with CD11c-positive cells infiltration and oligoclonal expansion of anti-tumor T cells. For the translation into clinics, unique TCR sequences identified in post-cryoablation tissues in individual patients can be applied to personalized TCR-engineered T cell therapy. Citation Format: Taigo Kato, Tomoyuki Iwasaki, Motohide Uemura, Akira Nagahara, Hiroki Higashihara, Keigo Osuga, Yuji Ikeda, Kazuma Kiyotani, Jae-Hyun Park, Norio Nonomura, Yusuke Nakamura. Characterization of the cryoablation-induced immune response in kidney cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4687. doi:10.1158/1538-7445.AM2017-4687

Clinical significance of T cell clonality and expression levels of immune-related genes in endometrial cancer.

Immune microenvironment characterized by T cell clonality as well as expression signatures of immune-related genes in endometrial cancer tissues may play significant roles in clinical outcome of patients. We aimed to investigate the clinical significance of immune-related gene expression and TCR repertoire in endometrial cancer. Using total RNAs extracted from 32 endometrioid endometrial cancer cases, we performed quantitative real-time PCR to measure mRNA expression levels of immune-related genes including TRB, CD8, GZMA, HLA-A, CD11c and PD-L1. Higher mRNA expression levels of CD8 (P=0.039) and CD11c (P=0.046) in the 32 tissue samples were significantly associated with longer progression-free survival (PFS). Expression levels of CD8 (P<0.001) and CD11c (P=0.048) were also significantly associated with longer PFS in 540 cases in TCGA database. We also performed T cell receptor β (TCRβ) sequencing of tumor-infiltrating lymphocytes (TILs) on an Illumina MiSeq platform. To evaluate clonal expansion of TCRβ clonotypes, we adjusted the number of abundant TCRβ clonotypes by TRB mRNA expression levels and examined TCR clonality with the expression levels of immune-related genes and clinicopathological factors. The cases with high clonal T cell expansion along with high PD-L1 expression in cancer tissues was related to higher mRNA expression levels of CD8 (P<0.001), GZMA (P<0.001) and HLA-A (P=0.027), showed a significantly longer PFS (P=0.015), indicating a possibility that these parameters may serve as faborable prognostic factors. Considering clinical stage, mRNA expression of CD8 (P=0.037), GZMA (P=0.027) and HLA-A (P=0.022) was significantly higher in tumors at an early stage. Thus, we identified clinical and prognostic significance of immune microenvironment including the T cell clonality of TILs as well as PD-L1 and CD11c mRNA expression levels in endometrial cancer tissues.

The Role of Indoleamine 2,3-Dioxygenase in Diethylnitrosamine-Induced Liver Carcinogenesis.

Indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing intracellular enzyme of the L-kynurenine pathway, causes preneoplastic cells and tumor cells to escape the immune system by inducing immune tolerance; this mechanism might be associated with the development and progression of human malignancies. In the present study, we investigated the role of IDO in diethylnitrosamine (DEN)-induced hepatocarcinogenesis by using IDO-knockout (KO) mice. To induce hepatocellular carcinoma (HCC), hepatic adenoma, and preneoplastic hepatocellular lesions termed foci of cellular alteration (FCA), male IDO-wild-type (WT) and IDO-KO mice with a C57BL/6J background received a single intraperitoneal injection of DEN at 2 weeks of age. The mice were sacrificed to evaluate the development of FCA and hepatocellular neoplasms. HCC overexpressed IDO and L-kynurenine compared to surrounding normal tissue in the DEN-treated IDO-WT mice. The number and cell proliferative activity of FCAs, and the incidence and multiplicity of HCC were significantly greater in the IDO-WT than in the IDO-KO mice. The expression levels of the IDO protein, of L-kynurenine, and of IFN-γ, COX-2, TNF-α, and Foxp3 mRNA were also significantly increased in the DEN-induced hepatic tumors that developed in the IDO-WT mice. The mRNA expression levels of CD8, perforin and granzyme B were markedly increased in hepatic tumors developed in IDO-KO mice. Moreover, Foxp3-positive inflammatory cells had infiltrated into the livers of DEN-treated IDO-WT mice, whereas fewer cells had infiltrated into the livers of IDO-KO mice. Induction of IDO and elevation of L-kynurenine might play a critical role in both the early and late phase of liver carcinogenesis. Our findings suggest that inhibition of IDO might offer a promising strategy for the prevention of liver cancer.

IFN-Stimulated Genes Upregulation Pattern in Chronic Hepatitis C

Background: The development of effective tools for the large-scale analysis of gene expression has provided new insights into the involvement of gene networks and regular pathways in various disease processes. The chemokine receptor CXCR3 is a G protein-coupled receptor found predominantly on T cells that is activated by three ligands as follow: CXCL9 (Mig), CXCL10 (IP-10) and CXCL11 (I-TAC), and play a key role in immune and inflammatory responses by promoting recruitment and activation of different subpopulations of leukocytes. Aim of the work: The study is a logical functional approach for the development of serum markers chemokines that bind to CXC chemokine receptor 3 to determine whether they play a role in the future of immune system to clear HCV, these chemokines: CXCL9, CXCL10 and CXCL11. Patients and methods: 131 male and female patients with chronic hepatitis C virus (CHCV) infection, their age ranges between 22 and 55 years, selected from the National Hepatology and Tropical Medicine Research Institute. The included patients were divided to two groups, the first group: 80 patients were investigated for the predictive values of CXCL9,10,11 and CXCR3 chemokines in peripheral blood mononuclear cells (PBMCs), the second group were fifty one patients analyzed for the expression of surface markers on CD8+T cells. Twenty healthy individuals were included to serve as controls for each group. All the patients and controls were subjected to the following: history, clinical examination, abdominal ultrasonography and collection of blood samples for routine laboratory investigation and serological assay. Results: Chemokine CXCL9, CXCL10, CXCL11 and their receptor CXCR3 expression levels are induced in PBMCs during CHCV infection, associated with increased the expression levels of CD8+T cells in CHCV patients. Conclusion: The interaction between chemokines and their receptors is essential in recruiting HCV-specific T cells to control the infection. Recommendations: The regulationof chemokines and their receptors could be a future potential therapeutic target to decrease liver inflammation and to increase specific T cell migration to the infected liver, the blocking of chemokines and chemokine receptor engagement is a therapeutic strategy that should be explored in the near future for non-responders to current anti-HCV therapy.

Prognostic Impact of Immune Microenvironment Markers in Tumor and Stroma in Resectable Nsclc

ABSTRACT Aim: Tumor-infiltrating immune cells have been proved to play an important role in tumor progression. In this study we have analyzed the expression of 11 immune related genes in both tumor and stroma samples of resectable NSCLC patients. Methods: In this retrospective study FFPE samples from 117 early-stage NSCLC patients of primary tumor tissue were used. The most representative areas of tumor and tumor stroma of each sample were carefully micro-dissected. RT-PCR using hydrolysis probes (TaqMan, Applied Biosystems) was performed to assess the expression of Treg markers such as: CD127, CD25, FOXP3, CTLA-4, IL-10, TGFB-1, LAG-3, GITR and TNFA as well as CD4 and CD8. Relative gene expression was assessed using GAPDH and CDKN1B as endogenous controls and results were normalized against a human cDNA (Clontech) as a reference. All statistical analysis were considered significant at p Results: In both tumor and stroma, we found over-expression of CD25 (19.46X and 11.59X, respectively), FOXP3 (4.96X and 4.08X, respectively), CTLA-4 (2.89X and 3.02, respectively) and TGBF-1 (3.24X and 2.11X, respectively) and down-expression of TNFA (0.39X and 0.27X, respectively). Survival analyses revealed that patients with lower expression levels of CD8 in the tumor have worse overall survival (OS) (median 37.20 vs 81.23 months, p = 0.001) and progression free survival (PFS) (median 19.43 vs 81.23 months, p = 0.002). Lower expression levels were also associated with worse OS for CD4 (42.90 vs 81.23 months, p= 0.026) and LAG-3 (36.20 vs 69 months, p = 0.017) in tumor. Furthermore, patients with a “Treg profile” (↑CD25 and ↓CD127) in stroma had worse PFS (median 12.7 vs 35.36 months, p = 0.004). We also found that those patients with higher levels of the ratio FOXP3 stroma/FOXP3 tumor had worse OS (median 42.9 vs NR, p = 0.002) and PFS (19.43 vs NR, p = 0.001), and the same was observed for the ratio FOXP3 stroma/CD4 tumor (for OS, median 46.66 vs 81.23 months, p = 0.025 and for PFS, median 22.13 vs 37.8 months, p= 0.027) and for ratio FOXP3 stroma/ CD8 tumor (for OS, median 46.43 vs 74.33 months, p = 0.017 and for PFS, median 22.13 vs 37.8 months, p= 0.022). Conclusions: Immune biomarkers in tumor microenvironment seem to play an important prognostic role in early-stage NSCLC patients. Supported by grants PS09-01149 and RD06/0020/1024 from ISCIII. Disclosure: All authors have declared no conflicts of interest.