T-cell Immunoglobulin Mucin Research Articles

Transcriptomic Profiling of Tumor-Infiltrating CD4+TIM-3+ T Cells Reveals Their Suppressive, Exhausted, and Metastatic Characteristics in Colorectal Cancer Patients.

T cell immunoglobulin mucin-3 (TIM-3) is an immune checkpoint identified as one of the key players in regulating T-cell responses. Studies have shown that TIM-3 is upregulated in the tumor microenvironment (TME). However, the precise role of TIM-3 in colorectal cancer (CRC) TME is yet to be elucidated. We performed phenotypic and molecular characterization of TIM-3+ T cells in the TME and circulation of CRC patients by analyzing tumor tissues (TT, TILs), normal tissues (NT, NILs), and peripheral blood mononuclear cells (PBMC). TIM-3 was upregulated on both CD4+ and CD3+CD4− (CD8+) TILs. CD4+TIM-3+ TILs expressed higher levels of T regulatory cell (Tregs)-signature genes, including FoxP3 and Helios, compared with their TIM-3− counterparts. Transcriptomic and ingenuity pathway analyses showed that TIM-3 potentially activates inflammatory and tumor metastatic pathways. Moreover, NF-κB-mediated transcription factors were upregulated in CD4+TIM-3+ TILs, which could favor proliferation/invasion and induce inflammatory and T-cell exhaustion pathways. In addition, we found that CD4+TIM-3+ TILs potentially support tumor invasion and metastasis, compared with conventional CD4+CD25+ Tregs in the CRC TME. However, functional studies are warranted to support these findings. In conclusion, this study discloses some of the functional pathways of TIM-3+ TILs, which could improve their targeting in more specific therapeutic approaches in CRC patients.

IL-6 promotes metastasis of non-small-cell lung cancer by up-regulating TIM-4 via NF-κB.

ObjectivesInterleukin‐6 (IL‐6) is critical for the development of non‐small‐cell lung cancer (NSCLC). Recently, we identified T‐cell immunoglobulin domain and mucin domain 4 (TIM‐4) as a new pro‐growth player in NSCLC progression. However, the role of TIM‐4 in IL‐6‐promoted NSCLC migration, invasion and epithelial‐to‐mesenchymal transition (EMT) remains unclear.Materials and MethodsExpressions of TIM‐4 and IL‐6 were both evaluated by immunohistochemical staining in NSCLC tissues. Real‐time quantitative PCR (qPCR), Western blot, flow cytometry and RT‐PCR were performed to detect TIM‐4 expression in NSCLC cells with IL‐6 stimulation. The roles of TIM‐4 in IL‐6 promoting migration and invasion of NSCLC were detected by transwell assay. EMT‐related markers were analysed by qPCR and Western blot in vitro, and metastasis was evaluated in BALB/c nude mice using lung cancer metastasis mouse model in vivo.ResultsHigh IL‐6 expression was identified as an independent predictive factor for TIM‐4 expression in NSCLC tissues. NSCLC patients with TIM‐4 and IL‐6 double high expression showed the worst prognosis. IL‐6 promoted TIM‐4 expression in NSCLC cells depending on NF‐κB signal pathway. Both TIM‐4 and IL‐6 promoted migration, invasion and EMT of NSCLC cells. Interestingly, TIM‐4 knockdown reversed the role of IL‐6 in NSCLC and IL‐6 promoted metastasis of NSCLC by up‐regulating TIM‐4 via NF‐κB.ConclusionsTIM‐4 involves in IL‐6 promoted migration, invasion and EMT of NSCLC.

Soluble T-Cell Immunoglobulin Mucin Domain-3 Is Associated With Hepatitis C Virus Coinfection and Low-Grade Inflammation During Chronic Human Immunodeficiency Virus Infection

BackgroundIn well treated human immunodeficiency virus infection (HIV), there is a residual immune activation and immune exhaustion that may contribute to increased risk of comorbidities. T-cell immunoglobulin mucin domain-3 (Tim-3) is an inhibitory molecule involved in HIV-associated T-cell dysfunction. The Tim-3 can be cleaved to soluble Tim-3 (sTim-3) that may serve as a soluble marker of immune exhaustion.MethodsWe measured sTim-3 with enzyme-linked immunosorbent assay DuoSets in a cross-sectional cohort of 1010 people with HIV (PWH) on antiretroviral therapy (ART), and 76 controls from the Copenhagen Co-Morbidity in HIV Infection (COCOMO) study, and in a longitudinal cohort of 60 PWH before and during ART.ResultsIn the cross-sectional cohort, levels of sTim-3 were elevated in PWH on ART compared with controls, especially in hepatitis C virus (HCV)-coinfected individuals, and were associated with HCV viremia and inflammation. In the longitudinal cohort, pretreatment sTim-3 correlated with HIV viral load and decreased after ART initiation. Pretreatment sTim-3 correlated inversely with CD4 counts, but it did not predict immunological response in multivariable analyses.ConclusionsLevels of sTim-3 decreased after ART initiation. In a cross-sectional cohort, levels of sTIM-3 were higher in PWH than in controls and were independently associated with HCV coinfection and high-sensitivity C-reactive protein, representing a potential link between immune exhaustion, inflammation, and risk of comorbidities.

Identification of the immune checkpoint signature of multiple myeloma using mass cytometry-based single-cell analysis.

ObjectivesNew targets or strategies are needed to increase the success of immune checkpoint‐based immunotherapy for multiple myeloma (MM). However, immune checkpoint signals in MM microenvironment have not been fully elucidated. Here, we aimed to have a broad overview of the different immune subsets and their immune checkpoint status, within the MM microenvironment, and to provide novel immunotherapeutic targets to treat MM patients.MethodsWe performed immune checkpoint profiling of bone marrow (BM) samples from MM patients and healthy controls using mass cytometry. With high‐dimensional single‐cell analysis of 30 immune proteins containing 10 pairs of immune checkpoint axes in 0.55 million of BM cells, an immune landscape of MM was mapped.ResultsWe identified an abnormality of immune cell composition by demonstrating a significant increase in activated CD4 T, CD8 T, CD8+ natural killer T‐like and NK cells in MM BM. Our data suggest a correlation between MM cells and immune checkpoint phenotypes and expand the view of MM immune signatures. Specifically, several critical immune checkpoints, such as programmed cell death 1 (PD‐1)/PD ligand 2, galectin‐9/T‐cell immunoglobulin mucin‐3, and inducible T‐cell costimulator (ICOS)/ICOS ligand, on both MM and immune effector cells and a number of activated PD‐1+ CD8 T cells lacking CD28 were distinguished in MM patients.ConclusionA clear interaction between MM cells and the surrounding immune cells was established, leading to immune checkpoint dysregulation. The analysis of the immune landscape enhances our understanding of the MM immunological milieu and proposes novel targets for improving immune checkpoint blockade‐based MM immunotherapy.

Effect of Dihydrotestosterone on Costimulatory Molecules in a Mouse Model of Graves' Disease

Objective Graves' disease is the most common autoimmune thyroid disease and its prevalence and clinical manifestations are disparate between females and males. Costimulatory molecules play an essential role in regulating autoimmune responses. The objective of this study was to determine if expression of inhibitory molecules was correlated with treatment by dihydrotestosterone (DHT) in an in vivo BALB/c mouse model of experimental autoimmune Graves' disease.Methods Female BALB/c mice were immunized three times with thyroid stimulating hormone receptor A-subunit encoded by adenovirus to establish a Graves' disease model. Three different doses of DHT or a matching placebo were administered by implantation of slow-release pellets a week before the first immunization. Four weeks after the third immunization, the mice were euthanatized, and then the spleen and thymus were removed. Total thyroxine and free thyroxine levels in serum of mice were detected using a radioimmunoassay kit. Real-time polymerase chain reaction was performed to estimate the expression of costimulatory molecules in lymphocytes from the spleen and thymus. Flow cytometry was used to analyze the percentage of CD4+ T cells in splenic lymphocytes. Quantitative data were compared with unpaired t-tests. Correlation between two variables was analyzed using Analysis of Variance.Results Treatment with DHT can dramatically reduce total thyroxine and free thyroxine levels. Higher expression of programmed death-1 was found in the spleen of Graves' disease mice receiving 5 mg of DHT treatment (0.635±0.296 vs. 0.327±0.212; t=2.714, P=0.014), similarly, T-cell immunoglobulin domain and mucin domain 3 (TIM-3) in both the spleen (1.004±0.338 vs. 0.646±0.314; t=2.205, P=0.022) and the thymus (0.263±0.127 vs. 0.120±0.076; t=3.221, P=0.004) also increased after 5 mg of DHT treatment compared with the parallel placebo model mice. Moreover, the percentage of CD4+ T cells declined in the splenic lymphocytes of Graves' disease mice treated with 5 mg of DHT (19.90%±3.985% vs. 24.05%±2.587%; t=2.804, P=0.012). A significant negative association was observed between expression of TIM-3 in the spleen and serum levels of total thyroxine (r=-0.7106, P=0.014) as well as free thyroxine (r=-0.6542, P=0.029).Conclusion This study demonstrates that DHT can ameliorate experimental autoimmune Graves' disease, which may occur by up-regulating expression of programmed death-1 and TIM-3 and inhibiting development of CD4+ T cells.

Proteomic Analysis of Differentially Expressed Proteins in the Placenta of Anticardiolipin Antibody‐ (ACA‐) Positive Pregnant Mice after Anzi Heji Treatment

Anzi Heji (AZHJ) has been used to treat anticardiolipin antibody- (ACA-) positive pregnant women at risk of spontaneous abortion for many years. The aim of this study was to investigate the protective mechanism of AZHJ in a mouse model of ACA-positive pregnancy at risk of spontaneous abortion using label-free quantitative proteomics. Mice were divided into three groups: normal pregnant mice (control group), ACA-positive pregnant mice administered normal saline (model group), and ACA-positive pregnant mice administered AZHJ (AZHJ group). The model was established by injecting β2-glycoprotein I (GPI) into mice for 18 days. The DEPs and their functions were analyzed by label-free quantitative proteomic and bioinformatic analyses. The levels of IL-6, IL-10, ACA, and TNF-α in the serum and placentas of the mice were measured by enzyme-linked immunosorbent assays (ELISAs). Proteomic data were validated by western blot analysis. The abnormal serum and placental levels of IL-6, ACA, and TNF-α in the model group were reversed by AZHJ. There were 39 upregulated and 10 downregulated DEPs in the AZHJ group relative to the model group. Bioinformatic analysis revealed that the DEPs were mainly involved in nucleic acid binding, signal conduction, and posttranslational modification. The placental levels of T-cell immunoglobulin mucin 3 (Tim-3) and Toll-like receptor 4 (TLR4) expression and AKT phosphorylation in the three groups were consistent with the proteomic findings. Tim-3/AKT signaling is involved in maternal-fetal immune tolerance, while TLR4 is associated with inflammatory responses. Collectively, these results indicate that AZHJ may exert its protective effect in ACA-positive pregnant mice by regulating the maternal-fetal immune tolerance and inflammatory response.

Overexpression of TIM-3 in Macrophages Aggravates Pathogenesis of Pulmonary Fibrosis in Mice.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disorder and lacks effective treatments because of unclear mechanisms. Aberrant function of alveolar macrophages is directly linked to pulmonary fibrosis. Here, we show TIM-3 (T-cell immunoglobulin domain and mucin domain-3), a key regulator of macrophage function, aggravates pulmonary fibrosis. TIM-3 mRNA of patients with IPF was analyzed based on the Gene Expression Omnibus and Array Express databases. Lung pathology and profibrotic molecules were assessed in a bleomycin (BLM)-induced pulmonary fibrosis model using wild-type (WT) and TIM-3 transgenic (TIM-3-TG) mice. Macrophage cells, RAW264.7, were then applied to investigate the effect of macrophage TIM-3 under BLM exposure in vitro. Macrophage depletion and adoptive-transfer experiments were finally performed to examine lung morphology and profibrotic molecules. TIM-3 expression was increased both in patients with IPF and in our BLM-induced mouse model. TIM-3-TG mice developed more serious pathological changes in lung tissue and higher expressions of TGF-β1 (transforming growth factor-β1) and IL-10 than WT mice. After BLM treatment, TGF-β1 and IL-10 expression was significantly decreased in RAW264.7 cells after TIM-3 knock-out, whereas it was increased in TIM-3-TG peritoneal macrophages. The scores of pulmonary fibrosis in WT and TIM-3-TG mice were significantly reduced, and there was no difference between them after macrophage depletion. Furthermore, WT mice receiving adoptive macrophages from TIM-3-TG mice also had more serious lung fibrosis and increased expression of TGF-β1 and IL-10 than those receiving macrophages from WT mice. Our findings revealed that overexpressed TIM-3 in alveolar macrophages aggravated pulmonary fibrosis.

TIM-4 interference in Kupffer cells against CCL4-induced liver fibrosis by mediating Akt1/Mitophagy signalling pathway.

ObjectivesT‐cell immunoglobulin domain and mucin domain‐4 (TIM‐4) is selectively expressed on antigen‐presenting cells (APCs) and modulates various immune responses. However, the role of TIM‐4 expressed by Kupffer cells (KCs) in liver fibrosis remains unclear. The present study aimed to explore whether and how TIM‐4 expressed by KCs is involved in liver fibrosis.Materials and MethodsMice chronic liver fibrosis models were established and divided into the olive‐induced control group, CCL4‐induced control group, olive‐induced TIM‐4 interference group and CCL4‐induced TIM‐4 interference group. Different techniques were used to monitor the fibrotic effects of TIM‐4, including histopathological assays, Western blotting, ELISA and transmission electron microscopy. Additionally, mice liver transplant models were established to determine the fibrotic effects of TIM‐4 on fibrosis after liver transplantation (LT).ResultsWe found that the induction of liver fibrosis by CCL4 was associated with TIM‐4 expression in KCs. TIM‐4 interference essentially contributed to liver fibrosis resolution. KCs from the TIM‐4 interference group had decreased levels of pro‐fibrotic markers, reduced TGF‐β1 secretion and inhibited hepatic stellate cell (HSC) differentiation into myofibroblast‐like cells. In addition, we used GdCl3 to verify that KCs are the primary source of TGF‐β1 during fibrosis progression. Moreover, KCs from CCL4‐induced mice showed increased ROS production, mitophagy activation and TGF‐β1 secretion. However, TIM‐4 interference in the KCs inhibited Akt1‐mediated ROS production, resulting in the suppression of PINK1, Parkin and LC3‐II/I activation and the reduction of TGF‐β1 secretion during liver fibrosis. Additionally, TIM‐4 interference potentially attenuated development of fibrosis after LT.ConclusionsOur findings revealed the underlying mechanisms of TIM‐4 interference in KCs to mitigate liver fibrosis.

Evaluation of TIM-3-Positive LSCs Post Allo-SCT Is a Highly Sensitive Strategy to Predict AML Relapses

Relapse of acute myeloid leukemia (AML) is a critical problem in clinics. Particularly, the prognosis of patients who relapsed after allogeneic stem cell transplantation (allo-SCT) is still poor. Self-renewing leukemic stem cells (LSCs), which are mainly enriched within CD34+CD38-fraction, cause re-growth of leukemia and eventually lead to recurrence. Therefore, evaluation of residual LSCs is crucial to estimate the efficacy of therapies including allo-SCT. As we previously described, T-cell immunoglobulin mucin-3 (TIM-3) is a useful marker for discrimination of LSCs from normal HSCs. We, therefore, prospectively evaluated the frequencies of LSCs at multiple time points and assessed the validity of TIM-3 as a minimal residual disease (MRD) marker in the setting of allo-SCT. We analyzed 63 allo-SCT from 57 patients who had been detected over 20% of the frequency of TIM-3+cells within CD34+CD38-fraction in BM sample at least once before SCT. All 63 allo-SCT were undergone at Fukuoka Blood and Marrow Transplantation Group (FBMTG)-related hospitals from July 2015 to July 2019. In all SCTs, patients achieved complete donor chimerism at the time of engraftment (day 15-36). Median age of the 57 patients was 45.8 [19-70]. Pre-SCT disease status in 58.6% of patients were refractory/relapse. We tracked residual LSCs marked by surface TIM-3 using multicolor FACS, and defined the frequency of TIM-3+cells within CD34+CD38- fraction as TIM-3+ LSC(%). Of note, the proportion in BM mononuclear cells of CD34+CD38-fraction was 0.029 [0.002 - 0.088] % at engraftment. And then, according to the level of TIM-3+ LSC(%) at engraftment, we classified into 3 groups; 'high' (> 90 %, n=7), 'intermediate' (61-90 %, n=26), and 'low' (< 60 %, n=29). Relapse-free survival (RFS) after allo-SCT was 77.7 [49-92] days in 'high' group, 358 [56-1238] days in 'intermediate' group and 619.4 [55-1398] days in 'low' group, respectively (p<0.01). Of note, all 7 patients in 'high' group relapsed within 100 days. In competing risk analysis for RFS, TIM-3+ LSC(%) at engraftment was a significant risk factor ('high-intermediate' group vs 'low' group: hazard ratio (HR), 3.72; 95% CI, 1.71-8.75; P<0.001)) in addition to pre-SCT disease status (non-hCR vs hCR: P<0.001), number of SCT (2nd SCT vs 1st SCT: P=0.032) and cytogenetic risk (Adverse vs Intermediate/favorable: P=0.017), whereas, other variables including age, gender, FLT3-ITD status, intensity of conditioning regimen and donor source did not have significant effect on RFS. For multivariate analysis, variables were included in a multivariate model if P<0.05. As results, TIM-3+LSC(%) at engraftment was independent predictor of post-SCT RFS (HR, 2.81; 95% CI, 1.27-6.76; P=0.010) as well as pre-SCT disease status (P=0.028) and cytogenetic risk (P=0.041). We next evaluated the correlation of TIM-3 and Leukemia-associated immunophenotype (LAIP) expression by multicolor FACS using single tube (including CD34, CD38, TIM-3 and case-specific LAIP markers). In 29 cases of this study cohort, LAIP was detectable as aberrant cross-lineage expression. As results, in the hematological CR samples, the majority of LAIP-MRD expressed TIM-3 on the surface. Inversely, TIM-3+ LSCs did not express the LAIP because LAIP-MRD were barely distributed in CD34+CD38- fraction. These results indicate TIM-3+LSCs should be different cell population from LAIP-MRD. Considering LSCs is enriched in CD34+CD38- fraction, our method might be superior to detection of "cross-lineage" LAIP-MRD in terms of identification of cells responsible for relapse. We also confirmed that the identical driver mutations were detected at both the initial diagnosis and relapse using whole exome sequencing (WES) of purified LSCs. As a typical example, WES detected CEBPA mutation (c.11dupG, 72.7 % and 50.0 %) and WT1 mutation (c.1091_1092insTTGTACGGTC, 41.9 % and 39.5 %) in a single patient. Additionally, we also validated that purified TIM-3+LSCs at engraftment harbored the identical mutations by amplicon sequencing. It indicates that, in terms of genetic background, CD34+CD38-TIM-3+cells should represent LSCs throughout the clinical course, from diagnosis to relapse. In summary, TIM-3 expression represents the clones of functional LSCs responsible for relapse. Evaluation of TIM-3+LSCs by multicolor FACS should be a highly sensitive strategy to predict relapse in clinical allo-SCT settings. Disclosures Akashi: Sumitomo Dainippon, Kyowa Kirin: Consultancy; Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding.

Phase Ib Study of the Anti-TIM-3 Antibody MBG453 in Combination with Decitabine in Patients with High-Risk Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML)

Phase Ib Study of the Anti-TIM-3 Antibody MBG453 in Combination with Decitabine in Patients with High-Risk Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML)

A Randomized, Double-Blind, Placebo-Controlled, Phase II Study of MBG453 Added to Hypomethylating Agents (HMAs) in Patients (pts) with Intermediate, High, or Very High Risk Myelodysplastic Syndrome (MDS): Stimulus-MDS1

A Randomized, Double-Blind, Placebo-Controlled, Phase II Study of MBG453 Added to Hypomethylating Agents (HMAs) in Patients (pts) with Intermediate, High, or Very High Risk Myelodysplastic Syndrome (MDS): Stimulus-MDS1

TMIC-43. ROLE OF EXTRACELLULAR FERRITIN HEAVY CHAIN PROTEIN IN GLIOBLASTOMA MULTIFORME

Abstract Increased expression of Ferritin heavy chain (FHC) protein has been associated with poor prognosis in Glioblastoma Multiforme (GBM) which is one of the most aggressive and common types of brain cancer. GBM patients have also been found to have increased extracellular ferritin levels, in their serum and cerebrospinal fluid (CSF), which are lowered once the source/tumor has been resected. Extracellular FHC can function as an iron delivery protein, and increasing amount of iron has been known to contribute to tumor initiation and proliferation. To study the effect of extracellular FHC in GBM cells we used patient derived GBM, CD133+ cancer stem cells (GSCs) from the pro-neural (T3691) and mesenchymal (T387) subtypes. Using recombinant FHC, conjugated with quantum dots (QD), we observed significant increase in cellular viability and intracellular uptake of FHC by the GSCs in a dose dependent manner. Our lab has previously shown that extracellular FHC interacts with T-Cell Immunoglobulin Mucin Receptor 1 (Tim-1) in the human oligodendrocytes. In order to determine if GSCs express the Tim-1 receptor we first confirmed its expression on GSCs using immunoblotting and immunocytochemistry. To test if FHC interacts with Tim-1, we performed knockdown of Tim-1 using siRNAs. However, the siRNA was not able to downregulate the Tim-1 receptors. Next, we exposed the GSCs to Sema4A, which has been shown in our previous studies to interact with Tim-1 receptor on human oligodendrocytes and is toxic to oligodendrocytes. The GSCs however were not affected by the saturable concentration of Sema4A. Thus, through this study we have shown the expression of potential FHC receptors on GSCs and a robust effect of H-ferritin on GSCs proliferation. Further experiments are warranted in this direction to understand this extracellular FHC uptake pathway and its role in GBM cell proliferation.

T-cell immunoglobulin mucin-3 expression in Acute Myeloid Leukemia: a cross-sectional study

T-cell immunoglobulin mucin-3 expression in Acute Myeloid Leukemia: a cross-sectional study

Molecular and clinical characterization of Galectin-9 in glioma through 1,027 samples.

In recent years, research on glioma immunotherapy have grown rapidly. However, the autoimmune‐like side effects that are caused by blocking immunological checkpoints hinder their clinical application in gliomas currently. Galectin‐9, a ligand for T‐cell immunoglobulin mucin 3, has shed a new light on the treatment of malignant glioma. However, the potential mechanism of Galectin‐9 is still under discussion. In this study, first, we methodically gathered 1,027 glioma patients with RNA‐seq and 986 patients with survival data to explore the role and mechanism of Galectin‐9 in gliomas. Second, we analyzed glioma samples from 50 patients in the Department of Neurosurgery, Tianjin Medical University General Hospital. Finally, we found that Galectin‐9 was strongly upregulated in glioblastoma multiforme compared with normal brain tissues and lower‐grade glioma. Patients with Galectin‐9 overexpression had a significantly shorter overall survival. Moreover, the tissue microarray data displayed that the expression of Galectin‐9 in the core of tumor is higher than that in the border and was correlated with the shorter survival in glioma patients. Galectin‐9 is more highly expressed in the mesenchymal subtype of glioblastoma multiforme than in the other subtypes. Simultaneously, Galectin‐9 was closely associated with the immune response and lymphocyte activation, especially T‐cell activation. To further determine the underlying role of Galectin‐9 in the immune response, we selected seven immune metagenes. Through cluster analysis and correlation analysis, we discovered that Galectin‐9 was highly correlated with immune checkpoint molecules and M2 tumor‐associated macrophages. In summary, Galectin‐9 serves as a potential therapeutic target to treat glioblastoma multiforme.

Cyclin-dependent kinase 9 expression and its association with CD8+ T cell infiltration in microsatellite-stable colorectal cancer.

Programmed death 1 (PD-1)-targeted therapy has benefited patients with microsatellite instability-high metastatic colorectal cancer (mCRC). However, the efficacy of PD-1-targeted therapy is poor in patients with microsatellite-stable (MSS) mCRC. Therefore, it is imperative to explore additional co-inhibitory molecular signalling pathways to improve the efficacy of immunotherapy in MSS mCRC treatment. In the present study, the association between cyclin-dependent kinase 9 (CDK9) expression and the survival of patients with CRC was analysed using RNA sequencing data from 605 patients, including 121 cases of mortality, from human cancer datasets. Furthermore, 35 clinical MSS stage III–IV CRC specimens were collected to assess CDK9 protein expression by immunohistochemistry, and the frequency of tumor-infiltrating CD8+ T cells was assessed by flow cytometry. The human cancer datasets demonstrated that upregulation CDK9 significantly shortened the survival of patients with stage II–IV colon cancer. Additionally, CDK9 mRNA expression was positively correlated with the expression levels of genes associated with immune evasion in the tumor. Notably, CDK9 was expression was upregulated in stage IV CRC compared with para-cancerous tissues and early-stage tumors. Interestingly, CDK9 expression was negatively associated with the infiltration of CD8+ T cells at the tumor site. In addition, the expression levels of T-cell immunoglobulin mucin family member 3 and CD39, proteins associated with exhaustion, on tumor-infiltrating CD8+ T cells were significantly elevated in patients with abnormal CDK9 expression levels. The present study demonstrated that CDK9 expression was negatively associated with CD8+ T cell infiltration and positively associated with CD8+ T cell exhaustion in MSS mCRC. In conclusion, CDK9 may be utilized to evaluate the prognosis and the immune-type of the tumor microenvironment in patients with MSS mCRC.

CHARACTERISTIC OF TIM-3 EXPRESSION IN T CELL SUBSETS IN PATIENTS WITH IMMUNE THROMBOCYTOPENIC PURPURA

T cell dysregulation is a common event involved in immune thrombocytopenic purpura (ITP). Recent findings indicated that aberrant expression of immune checkpoints may be related to autoimmune disease pathogenesis as well as in ITP. However, the distribution of Tim-3 and its co-expression with exhausted phenotype in T cell subsets remain unclear. In this study, the frequency of T-cell immunoglobulin mucin 3 (TIM-3) expression and co-expression with CD244 and CD57 in CD3+, CD4+ and CD8+ T cells were evaluated in peripheral blood (PB) from 16 cases with ITP and 21 healthy individuals (HI). Overall, the frequency of Tim-3+CD3+, CD4+ and CD8+T cells in ITP is similar to HI. However, Tim-3+CD244+CD8+ and Tim-3+CD57+CD8+T cells were increased in ITP in compared with HI group. While the percentage of CD244+ and CD57+CD3+, CD4+ or CD8+T cells was similar between ITP and HI groups. Moreover, the Tim-3+CD244+CD3+ and Tim-3+CD244+CD8+ T cells in newly diagnosed ITP group were higher than that in relapsed ITP group, similar trends showed in Tim-3+CD57+CD3+T cells. Obviously, relative higher Tim-3+T cells in most T cell subsets were detected in a case with acute ITP with viral infection. The distribution of Tim-3+ T cells in different subsets was also compared between PB and bone marrow (BM). High trends of Tim-3+T cells was detected in BM group. In conclusion, we firstly characterized T cell immune disorder with higher level of Tim-3+exhausted+CD8+T cells in ITP, the effect and mechanism of such alteration in ITP is needed further investigation.

Abstract 4973: The immunosuppressive tumor microenvironment of metastatic osteosarcoma inhibits the cytotoxic effect of tumor-infiltrating lymphocytes

Abstract Background: Patients with metastatic osteosarcoma (OS) have a 5 year overall survival of <25%. We aim to understand the immune architecture of the tumor microenvironment (TME) of OS, with the goal of harnessing the immune system as a major therapeutic strategy for the treatment of patients with OS. Methods: Immunohistochemistry (IHC) slides from 66 formalin-fixed paraffin-embedded OS tissue blocks were analyzed. Laser capture microdissection and RNA extraction was performed on 13 OS specimens, and gene expression profiles of the tumor interior vs. tumor interface region were compared utilizing RNA seq. Tumor infiltrating lymphocytes (TILs) were isolated from 25 freshly obtained OS specimens and analyzed by multiparameter flow cytometry (MFC). Results: Digital image analysis of IHC specimens revealed significantly higher immune cell infiltration (CD3+ and CD8+ cells) in the pulmonary metastases compared to primary bone tumors, particularly at the interface between the OS lesion and normal lung tissue. There is increased expression at the interface of the immune checkpoint molecules programmed cell death 1 (PD-1), T cell immunoglobulin mucin-3 (TIM-3) and lymphocyte activation gene 3 (LAG-3). Gene expression profiling showed increased CD8 T cell and resting CD4 memory T cell signature at the interface compared to the tumor interior, and a strong M2 and M0 macrophage signature in both regions. TILs isolated from pulmonary metastases and analyzed by MFC had higher expression of PD-1 and other immune checkpoint molecules, and these TILs were more capable of producing the effector molecules IFN-gamma and granzyme B. Conclusions: Pulmonary metastatic OS lesions are more highly infiltrated with immune cells than primary bone lesions, particularly at the interface. The fact that these immune cells are shown via MFC to be capable of producing cytotoxic cytokines and proteases suggests that these TILs may be tumor-specific lymphocytes capable of acting against the tumor if they were not being inhibited by multiple immune checkpoint molecules. The inability of these immune cells to penetrate further into the tumor interior may represent an adaptive immune response by the tumor through a combination of repellant cytokines and inhibition of TILs via upregulation of PD-1, TIM-3 and LAG-3. Treatment with a combination of anti-PD-1, anti-TIM-3 or anti-LAG-3 directed treatments may unleash these immune cells and allow them to destroy OS tumor cells. Citation Format: John A. Ligon, Teniola F. Oke, Woonyoung Choi, Megan H. Fong, Adam Levin, Daniel S. Rhee, Carol D. Morris, Nicholas Siegel, Emily H. Hsieu, Christian F. Meyer, David J. McConkey, Robert A. Anders, Drew M. Pardoll, Nicolas J. Llosa. The immunosuppressive tumor microenvironment of metastatic osteosarcoma inhibits the cytotoxic effect of tumor-infiltrating lymphocytes [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 4973.

Evaluation of Silencing Effect of miR-133a-5p Mimic on TIM-3 Expression in AML (HL-60) Cell Line.

Acute myelogenous leukemia (AML) is a complex blood malignancy leading to immature leukemic stem cells (LSCs) proliferation. T cell immunoglobulin mucin-3 (TIM-3) is known as a biomarker of AML LSCs. Several microRNAs (miRNAs) can affect gene expression in AML. In this study, the silencing effect of miR-133a-5p on TIM-3 expression in AML cell lineage (HL-60) was investigated. It's been hypothesized that miR-133a-5p may suppress the TIM-3 expression in AML cell line. Initially, miRNA-TIM-3 prediction, enrichment, and network analysis were done. Then, miR-133a-5p mimic was transfected into HL-60 cells. The TIM-3 protein and gene expression were measured by flow cytometry analysis and real-time PCR, respectively. MTT assay was also carried out. Based on the Bioinformatics predictions, miR-133a-5p was able to silence TIM-3 expression. Also, significant pathways pertained to miR-133a-5p were obtained using enrichment analysis. According to this, miR-133a-5p was mainly engaged in the MAPK signaling pathway and Nicotine addiction pathway using the KEGG database. The TIM-3 protein expression of the transfected cells was measured as 17.15 ± 8.87% (p = 0.001). A 52.48% significant gene silencing in mRNA level was obtained in comparison to the negative control. Despite of down regulation of TIM-3, HL-60 cell viability has not been significantly changed. It has been finally confirmed that miR-133a-5p could strongly suppress TIM-3 expression in AML cell line. Presumably, down regulation of TIM-3 could affect MAPK and Nicotine addiction signaling pathways.

The appropriate frequency and function of decidual Tim-3+CTLA-4+CD8+ T cells are important in maintaining normal pregnancy

Maternal decidual CD8+ T (dCD8+ T) cells must integrate the antithetical demands of maternal–fetal tolerance and anti-viral immunity to establish a successful pregnancy. T-cell immunoglobulin mucin-3 (Tim-3) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) are two important co-inhibitory molecules that regulating CD8+ T cells responses during infection and tumor. In the present study, we examined the co-expression of Tim-3 and CTLA-4 on CD8+ T cells during pregnancy and found the higher frequency of Tim-3+CTLA-4+dCD8+ T cells in response to trophoblasts. This Tim-3+CTLA-4+dCD8+ T cells subset showed an active status and produced more anti-inflammatory cytokines. Furthermore, the decreased number and altered function of Tim-3+CTLA-4+dCD8+ T cells correlated to miscarriage. Combined blocking Tim-3 and CTLA-4 pathways were highly effective in inhibiting the production of anti-inflammatory cytokines and were detrimental to the maintenance of pregnancy. Together, these findings supported that Tim-3 and CTLA-4 pathways might play positive roles in the establishment and/or maintenance of maternal–fetal tolerance so to promote the maintenance of normal pregnancy. So the reproductive safety must be considered, especially when anti-Tim-3/CTLA-4 antibody (and other immune checkpoint inhibitors) are used in pregnancy.

Altered frequency and function of spleen CTLA-4+Tim-3+ T cells are associated with miscarriage†.

Normal pregnancy is associated with several immune adaptations in both systemic and local maternal-fetal interface to allow the growth of semi-allogeneic conceptus. A failure in maternal immune tolerance to the fetus may result in abnormal pregnancies, such as recurrent spontaneous abortion. The regulation of T-cell homeostasis during pregnancy has important implications for maternal tolerance and immunity. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) and T-cell immunoglobulin mucin-3 (Tim-3) are important negative immune regulatory molecules involved in viral persistence and tumor metastasis. Here we described the lower frequency of splenic T cells co-expressing CTLA-4 and Tim-3 accompanied by higher levels of proinflammatory but lower anti-inflammatory cytokines production in abortion-prone mouse model. Blockade of CTLA-4 and Tim-3 pathways leaded to the dysfunction of splenic T cells. By the higher expression during normal pregnancy, CTLA-4 and Tim-3 co-expression on splenic T cells linked to immunosuppressive phenotype. As the spleen is an important site for peripheral immune activation, our data suggest potential noninvasive biomarkers and therapeutic targets for miscarriage.