Higher Expression Of CTLA-4 Research Articles

Identification of immunologic subtype and prognosis of GBM based on TNFSF14 and immune checkpoint gene expression profiling.

Immune-checkpoint therapy has failed to show significant benefit in glioblastoma (GBM) patients. Immunologic subtypes of GBM are necessary to identify patients who might benefit from immune-checkpoint therapy. This study reviewed 152 GBM samples from The Cancer Genome Atlas (TCGA) and 214 GBM samples from Chinese Glioma Genome Atlas (CGGA). Correlation analysis showed that immune checkpoint genes (ICGs) were mainly positively correlated. The prognostic analysis of the overall survival showed that there was a significant correlation between the overall survival (OS) and the prognosis of ICGs, in which the TNFSF14 gene was a significant adverse prognostic factor. Combined with TMB and neoantigens, we found that TNFSF9 and CD27 were significantly negatively correlated with TMB and neoantigens. The association between adaptive immune pathway genes and ICG expression showed that they were positively correlated with ICGs, indicating that adaptive immune pathway genes have a certain regulatory effect on the expression of ICGs. The analysis of clinical features of the samples showed that the higher the expression of ICGs, the more likely to be correlated with mutant isocitrate dehydrogenase (IDH), while the lower the expression level of IDH, the more likely to be significantly correlated with the primary GBM. Survival analysis showed that low expression of PD-L1, IDO1, or CTLA4 with TNFSF14 in the low expression group had the best prognosis, while high expression of IDO1 or CD274 with TNFSF14 in the high expression group and low expression of CTLA4 with TNFSF14 in the high expression group had the worst prognosis. We conclude that TNFSF14 is a biomarker to identify immunologic subtype and prognosis with other ICGs in GBM and may serve as a potential therapeutic target.

Abstract P5-04-26: Expression of cytotoxic T lymphocyte antigen-4 (CTLA-4) is associated with tumor-infiltrating lymphocytes (TILs) levelin HER2-positive breast cancers

Abstract Though CTLA4 expression was reported in various tumor types, expression of CTLA4 is not well known in breast cancers, especially HER2-positive breast cancers. TILs level has significant role in treatment response and prognosis in HER2-positive breast cancers. Total 167 patients with HER2-positive breast cancers were included. All patients were pre-operatively chemotherapy and radiotherapy naïve. All received adjuvant standard treatment. Histologic TILs evaluation was performed with full face hematoxylin and eosin sections. Immunohistochemistry for CTLA4 was performed with tissue microarray sections. CTLA4 expression was evaluated separately for tumor cells and TILs. Cytoplasmic and/or membranous stains were assessed and human appendix was used as positive control. The intensity and percentage of positivity were evaluated collectively. Immunostaining score was calculated by multiplying percentage of positive cells by staining intensity. Total immunostaining score was obtained by adding tumor cells score and TILs score. Expression level was divided into two categories (low and high) according to mean value. Only 11 patients (6.6%), 47 patients (28.1), and 10 patients (6.0) showed total negative staining for CTLA4 on tumor cells, TILs and both on tumor cells and TILs, respectively. According to mean score on tumor cells, 97 (58.1%) patients showed low level of CTLA4 expression and 70 (41.9%) showed high level of CTLA4 expression. One hundred and two (61.1%) patients had low expression and 65 (38.9%) patients had high expression of CTLA4 on TILs based on the mean value of score of TILs. According to mean value of total score both on tumor cells and TILs, 70 patients (41.9%) were categorized as high expression and 97 patients (58.1%) were categorized as low CTLA4 expression. TILs level, CTLA4 immunostaining score on tumor cells, CTLA4 immunostaining score on TILs, and total CTLA4 immunostaining score on tumor cells and TILs were significantly correlated each other. High expression level of CTLA4 on tumor cells was significantly associated with negative expression of hormone receptor and high TILs level. Also, high CTLA4 expression on TILs significantly correlated with high histologic grade, negative expression of hormone receptor and high TILs level. High level of total CTLA4 expression both on tumor cells and TILs was significantly correlated with negative expression of hormone receptor and high TILs level. However, expression level of CTLA4 was not associated with overall survival and disease-free survival. In conclusions, CTLA4 expression was common in HER2-positive breast cancers and the level of CTLA4 expression was associated with hormone receptor negativity and TILs level in HER2-positive breast cancers. Table 1. Correlation of TILs level, CTLA4 immunostaining score on tumor cells, TILs, and total immunostaining scoreimmunostaining score on tumor cellsimmunostaining score on TILsTotal immunostaining score on tumor cells and TILsTILs level0.254 (<0.001)0.394 (<0.001)0.312 (<0.001)Immunostaining score of CTLA-4 in tumor cells0.565 (<0.001)0.815 (<0.001)Immunostaining score of CTLA-4 in TILs0.783 (<0.001) Table 2. Correlation between CTLA-4 expression and clinicopathologic factorsLowHighLowHighLowHighHistologic grade1.0000.0390.754Low (grade 1, 2)51 (52.6)36 (51.4)60 (58.8)27 (41.5)52 (53.6)35 (50.0)High (grade 3)46 (47.4)34 (48.6)42 (41.2)38 (58.5)45 (46.4)35 (50.0)Necrosis0.3251.0000.325Absent30 (30.9)27 (38.6)35 (34.3)22 (33.8)30 (30.9)27 (38.6)Present67 (69.1)43 (61.4)67 (65.7)43 (66.2)67 (69.1)43 (61.4)Lymphovascular invasion0.7540.2650.754Absent51 (52.6)39 (55.7)51 (50.0)39 (60.0)51 (52.6)39 (55.7)Present46 (47.4)31 (44.3)51 (50.0)26 (40.0)46 (47.4)31 (44.3)Hormone receptor expression0.0120.0400.005Negative37 (38.1)41 (58.6)41 (40.2)37 (56.9)36 (37.1)42 (60.0)Positive60 (61.9)29 (41.4)61 (59.8)28 (43.1)61 (62.9)28 (40.0)TILs level0.001<0.001<0.001≤10%42 (43.3)19 (27.1)50 (49.0)11 (16.9)44 (45.4)17 (24.3)20-60%45 (46.4)29 (41.4)43 (42.2)31 (47.7)43 (44.3)31 (44.3)>60%10 (10.3)22 (31.4)9 (8.8)23 (35.4)10 (10.3)22 (31.4) Citation Format: Ahrong Kim, Jee Yeon Kim, So Jeong Lee. Expression of cytotoxic T lymphocyte antigen-4 (CTLA-4) is associated with tumor-infiltrating lymphocytes (TILs) levelin HER2-positive breast cancers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-26.

CD28-Dependent CTLA-4 Expression Fine-Tunes the Activation of Human Th17 Cells.

SummaryPrevious work has demonstrated that Th17 memory cells but not Th1 cells are resistant to CD28/CTLA-4 blockade with CTLA-4 Ig, leading us to investigate the individual roles of the CD28 and CTLA-4 cosignaling pathways on Th1 versus Th17 cells. We found that selective CD28 blockade with a domain antibody (dAb) inhibited Th1 cells but surprisingly augmented Th17 responses. CD28 agonism resulted in a profound increase in CTLA-4 expression in Th17 cells as compared with Th1 cells. Consistent with these findings, inhibition of the CD28 signaling protein AKT revealed that CTLA-4 expression on Th17 cells was more significantly reduced by AKT inhibition relative to CTLA-4 expression on Th17 cells. Finally, we found that FOXO1 and FOXO3 overexpression restrained high expression of CTLA-4 on Th17 cells but not Th1 cells. This study demonstrates that the heterogeneity of the CD4+ T cell compartment has implications for the immunomodulation of pathologic T cell responses.

CTLA-4 correlates with immune and clinical characteristics of glioma

BackgroundCTLA-4 is a well-studied immune checkpoint protein that negatively regulates T cell-mediated immune responses. However, the expression of CTLA-4 in glioma and the effects of CTLA-4 on prognosis in patients with glioma have not yet been examined.MethodsWe investigated the protein level of CTLA-4 in human glioma samples, extracted genetic and clinical data from 1024 glioma patients to characterize CTLA-4 expression and its relationship with immune functions in gliomas. R language was used for statistical analysis.ResultsHigher CTLA-4 expression was found in patients with higher grade, isocitrate dehydrogenase (IDH)-wild-type, and mesenchymal-molecular subtype gliomas than in patients with lower grade, IDH-mutant, and other molecular subtype gliomas. Further analysis showed that there was a strong positive correlation between CTLA-4 and the specific marker gene expression of immune cells, including CD8+ T cells, regulatory T cells, and macrophages in both databases, suggesting that higher CTLA-4 expression in the glioma microenvironment induced greater immune cell infiltration compared with that in gliomas with lower CTLA-4 expression. We further explored the associations between CTLA-4 and other immune-related molecules. Pearson correlation analysis showed that CTLA-4 was associated with PD-1, CD40, ICOS, CXCR3, CXCR6, CXCL12 and TIGIT. Patients with glioma with lower CTLA-4 expression exhibited significantly longer overall survival. Thus, these findings suggested that increased CTLA-4 expression conferred a worse outcome in glioma.ConclusionsIn summary, our findings revealed the expression patterns and clinical characteristics of CTLA-4 in glioma and may be helpful for expanding our understanding of antitumor immunotherapy in gliomas.

Identification of CTLA-4 associated with tumor microenvironment and competing interactions in triple negative breast cancer by co-expression network analysis.

Background: The study of CTLA-4 inhibitors has been one of the hot spots in the field of tumor immunotherapy. As the most immunogenic subtype of breast cancer, Triple negative breast cancer (TNBC) has a great potential in the treatment strategy. The aim of this study was to explore the relevant genes and pathways of CTLA-4 in TNBC and to explore the prognostic value, so as to provide a theoretical basis for clinical studies.Materials and methods: We used the data from The Cancer Genome Atlas (TCGA) to analyze the expression of CTLA-4 in different types of breast cancer, and analyzed the TNBC data of CTLA-4 related co-expression genes by WGCNA and enrichment analysis. LncRNA-miRNA-CTLA-4 network was constructed to explore the immune infiltration and immune checkpoint associated with CTLA-4. The effect of CTLA-4 on clinical outcomes in TNBC patients was also evaluated. Finally, we used data from GEO database to verify the differences of CTLA-4 in different molecular types of breast cancer and related prognostic results.Results: CTLA-4 was significantly higher in TNBC than in Luminal subtype and Her-2 + subtype (P=0.019 and P<0.001, separately), and was significantly higher in ER and PR negative samples than in ER and PR positive samples (P<0.001). CTLA-4 related genes mainly enriched in biological process of leukocyte differentiation, regulation of leukocyte activation and T cell activation. Hsa-mir-92a was found to be a survival significance marker associated with CTLA-4 and lncRNA-miRNA-CTLA-4 network was constructed. The results of immune infiltration analysis showed that CTLA-4 was mainly related with T cell (r=0.74). For immune checkpoints analysis, CTLA-4 was mainly related to PDCD1(r=0.72) and CD28(r=0.64). In TNBC, high expression of CTLA-4 is related to good survival (P=0.0061). Results consistent with previous analysis were obtained in the GEO database, the expression of CTLA-4 in TNBC was significantly higher than that in non-TNBC (p<0.001), CTLA-4 was associated with favorable survival of TNBC (p<0.001).Conclusion: Among all types of breast cancer, the expression of CTLA-4 was the highest in TNBC.CTLA-4 in TNBC can be regulated by hsa-mir-92a to form ceRNA networks and influence the prognosis of TNBC patients through the leukocyte differentiation, regulation of leukocyte activation and T cell activation pathway.

ES17.02 Molecular Heterogeneity

ES17.02 Molecular Heterogeneity

Leishmania infantum induces expression of the negative regulatory checkpoint, CTLA-4, by human naïve CD8+ T cells.

CD8+ T cells are important in mediating protective responses to intracellular pathogens. However, an uncontrolled response may lead to pathology. The role of CD8+ T cells in different clinical manifestations of human leishmaniasis is controversial and poorly understood. We aim to study the response of CD8+ T cells to the first exposure to different strains of Leishmania, seeking to correlate these findings with clinical manifestations of disease. We have evaluated the expression of granzyme A, inflammatory and anti-inflammatory cytokines, as well as CTLA-4 by human naïve CD8+ T cells exposed to Leishmania braziliensis and two different strains of Leishmania infantum in vitro. We observed that while exposure to Lbraziliensis induced an inflammatory profile, as measured by the expression of granzyme A, IFN-gamma and IL-17, as well as a higher IFN/IL-10 ratio, exposure to Linfantum led to a regulatory profile, as measured by lower IFN/IL-10 ratio and higher expression of CTLA-4. These results may help explain why patients with the visceral clinical form present a weaker cellular response and, consequently, a worse outcome of the disease. The use of CTLA-4 checkpoint inhibitors may emerge as a potential immunotherapy to ameliorate the immune response in visceral leishmaniasis patients.

Abstract 905: Comprehensive immunogenomic profiling of anti-PD-1 treated melanoma patients reveals subject-specific tumor escape mechanisms

Abstract Background: Despite the remarkable response of some melanoma patients to checkpoint inhibitor therapy, the majority of patients do not achieve complete response. It is of great interest to identify biomarkers and mechanisms that influence immunotherapy effectiveness. Here we apply our comprehensive tumor immunogenomics platform (ImmunoID NeXT) to identify potential biomarkers of response to checkpoint blockade therapy related to both the tumor and tumor microenvironment. Methods: We characterized the immunogenomics of 50 stage III/IV melanoma patients who have undergone anti-PD-1 therapy to assess potential factors influencing response. Tumor response to therapy was evaluated using RECIST criteria with a median follow-up of 12 months. Immuno-genomic profiling was performed using Personalis’ ImmunoID NeXT platform; an augmented exome/transcriptome platform and analysis pipeline which from a single paired tumor FFPE and normal blood sample yielded comprehensive tumor mutation information, gene expression quantification, neoantigen characterization, TCR repertoire profiling, HLA typing and tumor microenvironment profiling. The molecular information of the tumors was then analyzed together with their corresponding clinical response. Results: Through comprehensive immunogenomic profiling we demonstrated that higher TCR clonality in pre-treatment biopsy was predictive of response to PD-1 blockade, and significantly associated with improved progression free survival. We also observed increased response to anti-PD-1 treatment in patients with elevated pretreatment neoantigen burden. Further investigation of patients with high neoantigen burden and TCR clonality that failed to achieve complete response revealed potential resistance mechanisms to anti-PD-1 therapy. Specifically, we identified two patients with high expression of IDO1 or CTLA4, which may facilitate immune escape in a PD-1 independent manner. Additionally, we found two patients with mutations in their antigen presentation machinery (APM). The first patient had two independent HLA mutations in HLA-A and HLA-B, leading to the likely loss of surface expression of the proteins. In the second APM mutation patient we observed a high frequency (80% AF) frameshift variant in B2M, which potentially prevents proper HLA class I folding and antigen presentation. These APM mutations suggest reduced neoantigen presentation in these patients, which are probable mechanisms for tumor escape. Conclusions: In summary, our comprehensive cancer immunogenomic analysis shows that genomic and immune profiling of pretreatment patient samples can identify biomarkers and resistance mechanisms to immune checkpoint blockade, suggesting the potential efficacy of these as a combinatorial biomarker to optimize patient selection for anti-PD-1 therapy. Citation Format: Charles W. Abbott, Sean M. Boyle, Eric Levy, Rena McClory, Sekwon Jang, Richard Chen. Comprehensive immunogenomic profiling of anti-PD-1 treated melanoma patients reveals subject-specific tumor escape mechanisms [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 905.

Abstract 4047: Tumor-infiltrating lymphocytes in renal cancer patients demonstrate a diverse PD-1 expression and characteristic Treg classification

Abstract Immunotherapy has been shown to be effective for a proportion of chemoradiotherapy-refractory cancer patients. In particular, immune checkpoint inhibitors such as anti-CTLA-4 and anti-PD-1 antibodies are more effective for patients with high tumor mutation burden (TMB) or high expression of immune checkpoint molecules. In this study, we investigated immune responses in patients with renal cancer (RCC). Previous studies have demonstrated that the clinical effect of immune checkpoint inhibitors was 20% to 30%, and TMB was moderate (high; melanoma and lung cancer, low: astrocytoma and acute lymphocytic leukemia). We performed immune profiling of lymphocytes in the tumor tissue, normal tissue, and peripheral blood from 13 RCC patients by flow cytometry. Firstly, we examined the difference of CD4+ T cells, CD8+ T cells, and regulatory T (Treg) cells among 3 individual sampling sites. The frequency of CD8+ T cells in the tumor and normal tissues was higher than that of CD4+ T cells in the peripheral blood. In contrast, the frequency of CD4+ T cells in the peripheral blood was the highest, and that of effector-Treg (eTreg) cells (defined by CD3+CD4+FOXP3highCD45RAlow) in the tumor tissue was the highest. Additionally, we found that the RCC patients were arbitrarily classified into three groups based on the frequency of eTreg cells (i.e. high: &amp;gt; 10%, moderate: &amp;gt; 5%, and low: &amp;gt; 0%). Although this simple eTreg classification has not yet been shown to be associated with patient prognosis, this pattern is distinct from that of other cancer types. Secondly, we examined the expression of immune checkpoint molecules in the CD8+ T cells and eTreg cells. The CD8+ T cells in tumor tissue showed various degrees of PD-1 expression. An extremely high PD-1 expression in CD8+ cells was correlated with a moderate to high frequency of eTreg cells. The tumor-infiltrating CD8+ T cells showed a low expression of other checkpoint/accessory molecules such as LAG-3, CTLA-4, and TIGIT. In contrast, tumor-infiltrating eTreg cells showed high TIGIT and CTLA-4 expression, moderate PD-1 expression, and no LAG-3 expression. With regards to other immune-related molecules, the eTreg cells showed high CCR4 and low ICOS expression. These preliminary data implicate that the clinical benefit of immune checkpoint inhibitors for RCC patients could be associated with the high level of PD-1 expression in CD8+ T cells and the magnitude of eTreg cell infiltration into their tumor tissues. By analyzing the mechanism of high PD-1 expression in the CD8+ T cells and assessing the eTreg classification in RCC patients, we speculate that it is possible to develop a new treatment strategy by combining the anti-PD-1 antibody and Treg-depletion therapies. These data, along with additional measurements of samples from further patients, will be presented at the meeting. Citation Format: Daisuke Sugiyama, Tomoaki Muramatsu, Yoichi Kobayashi, Naoto Sassa, Shoichi Maruyama, Momokazu Goto, Yoshiki Akatsuka, Hiroyoshi Nishikawa. Tumor-infiltrating lymphocytes in renal cancer patients demonstrate a diverse PD-1 expression and characteristic Treg classification [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 4047.

Regulatory roles of IL-10-producing human follicular T cells.

Mucosal lymphoid tissues such as human tonsil are colonized by bacteria and exposed to ingested and inhaled antigens, requiring tight regulation of immune responses. Antibody responses are regulated by follicular helper T (TFH) cells and FOXP3+ follicular regulatory T (TFR) cells. Here we describe a subset of human tonsillar follicular T cells identified by expression of TFH markers and CD25 that are the main source of follicular T (TF) cell-derived IL-10. Despite lack of FOXP3 expression, CD25+ TF cells resemble T reg cells in high CTLA4 expression, low IL-2 production, and their ability to repress T cell proliferation. CD25+ TF cell-derived IL-10 dampens induction of B cell class-switching to IgE. In children, circulating total IgE titers were inversely correlated with the frequencies of tonsil CD25+ TF cells and IL-10-producing TF cells but not with total T reg cells, TFR, or IL-10-producing T cells. Thus, CD25+ TF cells emerge as a subset with unique T and B cell regulatory activities that may help prevent atopy.

Transcriptome Profile and Clinical Characteristics of CTLA-4 in Diffuse Gliomas

CTLA-4, identified as an immune checkpoint, has been widely reported to contribute to downregulation of immune responses to anti-tumor activity. Anti-CTLA-4 provides a novel strategy to improve the outcome of cancer immunotherapy, but the role of CTLA-4 needs to be addressed in glioma. To investigate the role of CTLA-4 in diffuse gliomas, we analyzed the transcriptome level data of CTLA-4 and evaluated its clinical prognostic value. 1022 glioma samples with transcriptome data were enrolled into this study, including 325 RNA-seq data from CGGA and 697 RNA-seq data from TCGA. Clinical and molecular data were also obtained. R project was the main tool for statistical analysis and graphical work. High-grade gliomas exhibited high expression of CTLA-4 while IDH wildtype gliomas showed CTLA-4 enrichment. According to TCGA transcriptional classification in gliomas, mesenchymal subtype displayed significantly higher CTLA-4 expression than other molecular subtypes. Gene ontology analysis demonstrated that CTLA-4 and correlated genes were involved in immune responses and inflammatory activities in diffuse gliomas. Through the analysis of transcriptomic data, CTLA-4 expression strongly correlated with immune cell populations and inflammatory activity. Survival analysis suggested that high expression of CTLA-4 was associated with poor prognosis in gliomas and may serve as an indicator of outcome in gliomas. High CTLA-4 expression is closely related to malignancy in diffuse gliomas and predicts poor outcome. Meanwhile, CTLA-4 plays an important role in regulating T cell activation and antitumor responses in gliomas. Therefore, blocking CTLA-4 is a potential target for developing the immunotherapy of gliomas. Funding: This work was supported by the capital health research and development of special (2014-2-1072); Beijing Municipal Natural Science Foundation (7142054) and National Natural Science Foundation of China (81471229). Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: This study was approved by Capital Medical University Institutional Review Board (IRB).

New Molecular and Therapeutic Insights into Canine Diffuse Large B Cell Lymphoma Elucidates the Role of the Dog As a Model for Human Disease

Background. Diffuse large B-cell lymphoma (DLBCL) is the commonest lymphoma in both humans and dogs. Canine DLBCL (cDLBCL) is considered an ideal comparative model for drug development, but a complete genomic characterization of this tumor is still lacking. In this study, we report an integrated analysis to comprehensively define the molecular mechanisms of cDLBCL and possible associations with clinical outcome.Methods. Fifty cDLBCLs were analyzed by RNA-Seq, methyl-CpG-binding sequencing and array comparative genomic hybridization. Normal B-cells derived from lymph nodes of 11 healthy dogs were used as controls.Additionally, immunohistochemistry, in vitroand in vivoexperiments were performed as validation analyses.Results.Compared to normal B-cells, cDLBCL showed a marked up-regulation of genes involved in the PI3K/mTOR and NF-κB pathways, including several TLRs in association with MYD88, indicating mechanisms similar to the human activated B cell-like subtype DLBCL.Both RNA-Seq and methylation sequencing led to the identification of two groups of cDLBCLs bearing different clinical outcome. The two groups did not overlap with the human germinal center B-cell (GCB) and the activated B-cell-like (ABC) DLBCL subtypes or the human DLBCL consensus clusters. The dogs with the poorest outcome presented a signature largely defined by markers of T-cell-mediated immune responses, with a high expression of PDL-1, PD-1 and CTLA-4, also validated in an independent cohort of cDLBCL by immunohistochemistry. These data provide a strong rationale for the use of cDLBCL to study immune checkpoint modulators.The observed high expression of PI3K/mTOR pathway genes was confirmed and validated achieving a clear anti-tumor activity with the use of the PI3K-delta inhibitor idelalisib and of the novel dual PI3K/mTOR inhibitor bimiralisib in the cDLBCL cell line CLBL-1.The cDLBCLs showed an up-regulation of MYC and of its targets, sustained by recurrent gains in the chromosome 13, where the oncogene is located, in approximately half of the cases. Thus, we have exposed the cDLBCL cell line CLBL-1 to the BET inhibitor birabresib (OTX015) and to the BRD4 degrader MZ1. Both compounds caused a significant reduction in the proliferation of tumor cells, and this effect was stronger especially with the second compound. Exposure to MZ1 determined an important downregulation of MYC and also of LIN28B, the most overexpressed transcript in cDLBCL when compared to controls. While LIN28B does not seem to be a relevant gene for human DLBCL, its overexpression causes murine T-cell lymphomas (Beachy et al, Blood 2011), and there is a direct association of MYC with LIN28B promoter resulting in transcriptional transactivation (Chang et al, PNAS 2009). Here, LIN28B genetic silencing in the CLBL-1 lead to a reduction in cell growth, opening new therapeutic target perspectives in canine lymphoma.Conclusions. We have reported the first large next generation sequencing study investigating the cDLBCL transcriptome, methylome and the genome-wide CNVs. We identified deregulated pathways and individual transcripts providing therapeutic targets, including an immune-related signature affecting the outcome of a subgroup of cDLBCL. Our data sustain the use of cDLBCL as comparative models for human DLBCL but also highlight differences that must be kept in consideration. DisclosuresHillmann:PIQUR Therapeutics AG: Employment. Wymann:PIQUR Therapeutics AG: Employment, Equity Ownership, Patents & Royalties.

243. Human myometrial T cells at the maternal-fetal interface are tissue-resident memory T cells, which show site-specific adaptation within one tissue

Introduction The uterine myometrium is a unique immune environment, capable of harboring a ‘foreign’ fetus without eliciting an immune response. Objective In this pilot study we aimed to investigate the local presence, adaptation and function of human myometrial CD4 + T cells at the maternal-fetal interface in uncomplicated pregnancy. Methods Myometrial biopsies were obtained at caesarean section from placental and incision site. Lymphocytes were isolated through dissection and digestion with collagenase IV. CD4 + T cells were analyzed by flow cytometry or FACS sorted for RNA sequencing with CEL-seq protocol. Suppression assays were performed with FACS sorted uterine Tregs added to 15,000 healthy donor PBMC in different ratios, in 0.1 μg/mL anti-CD3 coated plates. Results Flow cytometry revealed that 70–80% of myometrial CD4 + T cells were CD69 + memory cells, suggesting a tissue-resident memory (TRM) phenotype. RNA sequencing confirmed a TRM-like profile in CD69 + cells, with high expression of CD49a, CXCR6, DUSP6, PD-1 and low expression of CD62L, KLF2/3 and S1PR1 compared to blood memory CD4 + T cells. Especially at the placental site, we observed a high expression of negative costimulatory molecules PD-1, TIGIT, Lag3, TIM-3 and CTLA4 and a high percentage of Tregs. Expression was intermediate at the incision site and low in blood. Suppression assays confirmed the suppressive capacity of myometrial Tregs. Conclusion Myometrial CD4 + T cells at the maternal-fetal interface are TRM cells with a high expression of negative costimulatory molecules and a high abundance of functional Tregs. The distribution pattern of these immunoregulatory features indicates that T cells may not only adapt to a tissue, but also to specific sites within one tissue, possibly depending on the local micro-environment.

PO-509 Novel molecular classification of muscle-invasive bladder cancer opens new treatment opportunities

IntroductionMuscle-invasive bladder cancer (MIBC) is associated with a high risk of relapse and metastasis. The standard treatment consists of radical cystectomy followed by cisplatin-based chemotherapy. Therefore, the molecular characterisation of MIBC in order to find new therapeutic targets is still necessary.Material and methodsIn this study, using The Cancer Genome Atlas (TCGA) cohort, functional structure and layer analyses were done to characterise MIBC tumours at functional level. Functional structure was developed using probabilistic graphical models and gene ontology analyses. Layer analyses were based on sparse k-means and Consensus Cluster Algorithm (CCA). Sparse k-means assigns a weight to each variable, based on its relevance in the sample classification. Then, a CCA using variables that were selected by the sparse K-means method was applied to define the optimum number of groups for each case. This sparse k-means-CCA workflow was performed successive times to explore the existence of independent informative molecular layers. Once relevant genes for one molecular layer are identified, these genes were removed from the dataset and the sparse k-means-CCA workflow was performed again, allowing the identification of different layers of molecular information and establishing different classifications based on various molecular features. Gene ontology analyses were done for each layer.Results and discussionsA probabilistic graphical model with a functional structure was obtained. Sixteen layers were functionally characterised and the first three layers were analysed in depth. The first layer defined luminal and basal groups. Luminal tumours had a better prognosis than basal ones. Strikingly, luminal tumours showed higher expression of the androgen receptor, suggesting that this group is a good candidate for receiving treatment with androgen receptor inhibitors. The second layer established two groups based on genes related to extracellular matrix function. Lastly, the third layer defined two groups on the basis of immune information. The immune high group had a better prognosis and was characterised by higher expression of CTLA4 and PDL1, so this group may be a candidate for receiving immunotherapy.ConclusionUsing probabilistic graphical models and layer analyses it is possible to establish independently a molecular and an immune classification. Using these approaches, we also proposed a specific treatment for luminal tumours (androgen receptor inhibitors) and for immune high tumours (immunotherapy).

High CTLA-4 expression correlates with poor prognosis in thymoma patients.

Thymomas, tumors that arise from epithelial cells of the thymus gland, are the most common neoplasms of the anterior mediastinum, with an incidence rate of approximately 2.5 per million/year. Cytotoxic T Lymphocyte Antigen 4 (CTLA-4 or CD152) exerts inhibitory activity on T cells, and since its oncogenic role in the progression of different types of tumors, it has emerged as a potential therapeutic target in cancer patients.In this study, we assessed the expression of CTLA-4 both at mRNA and protein levels in paraffin embedded-tissues from patients with thymomas. Furthermore, we evaluated the relationship between CTLA-4 expression and the clinical-pathologic characteristics and prognosis in patients with thymomas. Sixty-eight patients with median age corresponding to 62 years were included in this analysis. Thymomas were classified accordingly to the WHO and Masaoka-Koga for histochemical analysis and for prognostic significance.A statistical difference was found between CTLA-4 mRNA levels in human normal thymus compared with thymoma specimens. CTLA-4 expression was statistically found to progressively increase in A, B1, B2, AB and it was maximal in B3 thymomas. According to Masaoka-Koga pathological classification, CTLA-4 expression was lower in I, IIA and IIB, and higher in invasive III and IV stages. By confocal microscopy analysis we identified the expression of CTLA-4 both in tumor cells and in CD45+ tumor-infiltrating leukocytes, mainly in B3 and AB thymomas.Finally, CTLA-4 overexpression significantly correlates with reduced overall survival in thymoma patients and in atypical thymoma subgroup, suggesting that it represents a negative prognostic factor.

Immune rebound associates with a favorable clinical response to autologous HSCT in systemic sclerosis patients

AbstractTo evaluate the immunological mechanisms associated with clinical outcomes after autologous hematopoietic stem cell transplantation (AHSCT), focusing on regulatory T- (Treg) and B- (Breg) cell immune reconstitution, 31 systemic sclerosis (SSc) patients underwent simultaneous clinical and immunological evaluations over 36-month posttransplantation follow-up. Patients were retrospectively grouped into responders (n = 25) and nonresponders (n = 6), according to clinical response after AHSCT. Thymic function and B-cell neogenesis were respectively assessed by quantification of DNA excision circles generated during T- and B-cell receptor rearrangements. At the 1-year post-AHSCT evaluation of the total set of transplanted SSc patients, thymic rebound led to renewal of the immune system, with higher T-cell receptor (TCR) diversity, positive correlation between recent thymic emigrant and Treg counts, and higher expression of CTLA-4 and GITR on Tregs, when compared with pretransplant levels. In parallel, increased bone marrow output of newly generated naive B-cells, starting at 6 months after AHSCT, renovated the B-cell populations in peripheral blood. At 6 and 12 months after AHSCT, Bregs increased and produced higher interleukin-10 levels than before transplant. When the nonresponder patients were evaluated separately, Treg and Breg counts did not increase after AHSCT, and high TCR repertoire overlap between pre- and posttransplant periods indicated maintenance of underlying disease mechanisms. These data suggest that clinical improvement of SSc patients is related to increased counts of newly generated Tregs and Bregs after AHSCT as a result of coordinated thymic and bone marrow rebound.

Mixed signature of activation and dysfunction allows human decidual CD8+ T cells to provide both tolerance and immunity

Understanding how decidual CD8+ T cell (CD8+ dT) cytotoxicity is regulated and how these cells integrate the competing needs for maternal-fetal tolerance and immunity to infection is an important research and clinical goal. Gene-expression analysis of effector-memory CD8+ dT demonstrated a mixed transcriptional signature of T cell dysfunction, activation, and effector function. High protein expression of coinhibitory molecules PD1, CTLA4, and LAG3, accompanied by low expression of cytolytic molecules suggests that the decidual microenvironment reduces CD8+ dT effector responses to maintain tolerance to fetal antigens. However, CD8+ dT degranulated, proliferated, and produced IFN-γ, TNF-α, perforin, and granzymes upon in vitro stimulation, demonstrating that CD8+ dT are not permanently suppressed and retain the capacity to respond to proinflammatory events, such as infections. The balance between transient dysfunction of CD8+ dT that are permissive of placental and fetal development, and reversal of this dysfunctional state, is crucial in understanding the etiology of pregnancy complications and prevention of congenital infections.

Stereotactic Radiotherapy Increases Functionally Suppressive Regulatory T Cells in the Tumor Microenvironment

Radiotherapy (RT) enhances innate and adaptive antitumor immunity; however, the effects of radiation on suppressive immune cells, such as regulatory T cells (Treg), in the tumor microenvironment (TME) are not fully elucidated. Although previous reports suggest an increased Treg infiltration after radiation, whether these Tregs are functionally suppressive remains undetermined. To test the hypothesis that RT enhances the suppressive function of Treg in the TME, we selectively irradiated implanted tumors using the small animal radiation research platform (SARRP), which models stereotactic radiotherapy in human patients. We then analyzed tumor-infiltrating lymphocytes (TIL) with flow-cytometry and functional assays. Our data showed that RT significantly increased tumor-infiltrating Tregs (TIL-Treg), which had higher expression of CTLA-4, 4-1BB, and Helios compared with Tregs in nonirradiated tumors. This observation held true across several tumor models (B16/F10, RENCA, and MC38). We found that TIL-Tregs from irradiated tumors had equal or improved suppressive capacity compared with nonirradiated tumors. Our data also indicated that after RT, Tregs proliferated more robustly than other T-cell subsets in the TME. In addition, after RT, expansion of Tregs occurred when T-cell migration was inhibited using Fingolimod, suggesting that the increased Treg frequency was likely due to preferential proliferation of intratumoral Treg after radiation. Our data also suggested that Treg expansion after irradiation was independent of TGFβ and IL33 signaling. These data demonstrate that RT increased phenotypically and functionally suppressive Tregs in the TME. Our results suggest that RT might be combined effectively with Treg-targeting agents to maximize antitumor efficacy. Cancer Immunol Res; 5(11); 992-1004. ©2017 AACR.

Single-sex infection with female Schistosoma mansoni cercariae mitigates hepatic fibrosis after secondary infection.

BackgroundInfection with Schistosoma spp. affects more than 258 million people worldwide. Current treatment strategies are mainly based on the anthelmintic Praziquantel, which is effective against adult worms but neither prevents re-infection nor cures severe liver damage. The best long-term strategy to control schistosomiasis may be to develop an immunization. Therefore, we designed a two-step Schistosoma mansoni infection model to study the immune-stimulating effect of a primary infection with either male or female cercariae, measured on the basis of TH1/TH2-response, granuloma size and hepatic fibrosis after a secondary bisexual S. mansoni challenge.Methodology/Principle findingsAs a first step, mice were infected with exclusively female, exclusively male, or a mixture of male and female S. mansoni cercariae. 11 weeks later they were secondarily infected with male and female S. mansoni cercariae. At week 19, infection burden, granuloma size, collagen deposition, serum cytokine profiles and the expression of inflammatory genes were analyzed. Mice initially infected with female S. mansoni cercariae displayed smaller hepatic granulomas, livers and spleens, less hepatic fibrosis and higher expression of Ctla4. In contrast, a prior infection with male or male and female S. mansoni did not mitigate disease progression after a bisexual challenge.Conclusions/SignificanceOur findings provide evidence that an immunization against S. mansoni is achievable by exploiting gender-specific differences between schistosomes.

Stereotactic radiotherapy increases functionally suppressive regulatory T cells in the tumor microenvironment

Abstract Radiotherapy (RT) enhances innate and adaptive anti-tumor immunity; however, the effects of RT on immune-suppressive regulatory T cells (Tregs), in the tumor microenvironment (TME) have not yet been fully elucidated. Although previous reports suggest a post-RT increase in Tregs, whether these Tregs are functionally suppressive has not been determined. To test the hypothesis that RT enhances the suppressive function of Tregs in the TME, we selectively irradiated implanted tumors using the Small Animal Radiation Research Platform (SARRP), which models stereotactic RT in human patients– followed by flow-cytometric and functional analyses of tumor-infiltrating lymphocytes (TIL). Our data showed that RT significantly increased tumor-infiltrating Tregs (TIL-Tegs), and that these cells have higher expression of CTLA-4, 4-1BB, and Helios, consistent with activated/suppressive phenotype. This observation held true across several tumor models (B16/F10, RENCA, MC38). Notably, we found that post-RT, TIL-Tregs had equal or improved suppressive capacity compared with non-irradiated tumors. Our data also indicated that Tregs proliferate post-RT, more robustly than the other T cell subsets in the TME. In addition, the post-RT Treg expansion occurred when T cell migration was inhibited by Fingolimod - suggesting that the post-RT Treg increase is likely due to preferential proliferation of intratumoral Tregs. Our data also suggest that post-RT Treg expansion is independent of TGF-β and IL-33. Collectively, these data demonstrate that RT increases the phenotypically and functionally suppressive Tregs in the TME, and provide a rationale for treatment regimens that combine RT with Treg-targeting agents to maximize anti-tumor efficacy.