Expression Levels Of Tim-3 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.

Characteristics and clinical correlation of TIM-3 and PD-1/PD-L1 expressions in leukemic cells and tumor microenvironment in newly diagnosed acute myeloid leukemia

Dual targeting of TIM-3 and PD-1/PD-L1 pathways is currently under investigation for cancer immunotherapy. The interaction of these immune checkpoints remains unclear in the leukemic microenvironment of acute myeloid leukemia (AML). We performed an immunophenotypic study of bone marrow in 37 newly diagnosed AML patients. High levels of TIM-3 expression on AML blasts were correlated with first 7 + 3 induction failure (CR 16.2% vs. non-CR 36.4%, p = .038). In contrast, high TIM-3 levels on natural killer (NK) cells were associated with complete remission (CR) status after induction (CR 24.7% vs. non-CR 6.5%, p = .035). Few PD-L1 positive AML blasts and PD-1 or PD-L1 positive NK cells were observed. Although the exhausted PD-1 expressing T cells were detected in 28.3% of T cells, the double positive of PD-1 and TIM-3 T cells were rarely detected. In summary, the TIM-3 levels on AML blasts and NK cells are potentially the prognostic biomarkers in AML.

Genomic cytometry is maturing but has single-cell multiomics hit puberty yet?

Genomic cytometry is maturing but has single-cell multiomics hit puberty yet?

A novel immune-related gene-based prognostic signature to predict biochemical recurrence in patients with prostate cancer after radical prostatectomy.

Accumulating evidences indicates that the immune landscape signature dramatically correlates with tumorigenesis and prognosis of prostate cancer (PCa). Here, we identified a novel immune-related gene-based prognostic signature (IRGPS) to predict biochemical recurrence (BCR) after radical prostatectomy. We also explored the correlation between IRGPS and tumor microenvironment. We identified an IRGPS consisting of seven immune-related genes (PPARGC1A, AKR1C2, COMP, EEF1A2, IRF5, NTM, and TPX2) that were related to the BCR-free survival of PCa patients. The high-risk patients exhibited a higher fraction of regulatory T cells and M2 macrophages than the low-risk BCR patients (P < 0.05) as well as a lower fraction of resting memory CD4 T cells and resting mast cells. These high-risk patients also had higher expression levels of CTLA4, TIGIT, PDCD1, LAG3, and TIM3. Finally, a strong correlation was detected between IRGPS and specific clinicopathological features, including Gleason scores and tumor stage. In conclusion, our study reveals the clinical significance and potential functions of the IRGPS, provides more data for predicting outcomes, and suggests more effective immunotherapeutic target strategies for PCa.

Correlation of Tim-3 expression with chemokine levels for predicting the prognosis of patients with glioblastoma

Glioblastoma (GBM) immunotherapy, which blocks the checkpoint inhibitor molecule T cell immunoglobulin domain and mucin domain-3 (Tim-3), has potential therapeutic applications. However, not all patients do benefit from the targeted therapy. This study aimed to explore Tim-3 expression correlated chemokine profiles and immune cell infiltration and investigate their potential as prognostic markers of glioblastoma (GBM) immunotherapy. We analyzed transcriptional data of GBM from TCGA database, to measure Tim-3 expression by R package DESeq2 analysis and observed differentially expressed genes in GBM samples with high Tim-3 expression levels. We also probed the relative gene enrichment pathways. Tim-3 expression was evident in biological processes including the recruitment of immune cells. We also identified some chemokines related to Tim-3 expression. The expression levels of CCL18, CXCL13 and CCL7 were significantly higher in GBM tissues with high Tim-3 expression than in GBM tissues with low Tim-3 expression. In addition, exploring the relationship between immune cell infiltration and Tim-3 expression suggested that Tim-3 expression was positively related to significant immune cell infiltration.

Low Distribution of TIM-3+ Cytotoxic Tumor-Infiltrating Lymphocytes Predicts Poor Outcomes in Gastrointestinal Stromal Tumors.

There are multiple tumor-infiltrating lymphocytes (TILs) and relevant immune checkpoints existing in gastrointestinal stromal tumor (GIST), which provides opportunities and rationales for developing effective immunotherapies. Recent studies have suggested that checkpoint TIM-3/Gal-9 plays a pivotal role on immune response in multiple tumors, similar to the PD-1/PD-L1, emerging as a potential therapeutic target. However, their functions in GIST are unrevealed. Hence, the expression of immune checkpoints TIM-3 and Gal-9, as well as the infiltration of CD8+ T cells and NK cells, is described in 299 cases of GIST specimens. The results showed that TIM-3 and Gal-9 are mainly expressed in TILs, rarely in tumor cells. Expression levels of TIM-3 and Gal-9 significantly differ in varying risks of GIST and exert opposite distribution trends. Indicated by prognosis analysis, high TIM-3 expression of TILs was associated with improved outcome, while low expression levels of TIM-3 in combination with low amounts of CD8+ and CD56+ TILs predict extremely poor survival. The integrated analysis of TIM-3+, CD8+, and CD56+ TILs as one biomarker is a reliable independent predictor of prognosis. In conclusion, low densities of TIM-3+ TILs are associated with poor survival, and integrated immune biomarkers lead to superior predictors of GIST prognosis.

Significance of Tim-3 in The Imbalance of Th17/Treg in Patients with Multiple Myeloma

To investigate the singnificance of Tim-3 in Th17/Treg balance in patients with multiple myeloma (MM). Fifty-six newly diagnosed MM patients and 30 healthy people were enrolled. Flow cytometry was used to detect the expression of Tim-3 on CD4+T cells, the proportion of Th17 and Treg cell, the Th17/Treg ratio, the expression of Tim-3 in Th17 and Treg cells, and the ratio of Tim-3+Th17/Tim-3+Treg cells in two groups. ELISA was used to detect the level of cytokines IL-17 and IL-10. Moreover, to analyze the relationship between Tim-3+Th17/Tim-3+Treg balance and clinical indicator. Compared with the control group, the expression of Tim-3 on CD4+T cells in peripheral blood of MM patients was increased (P<0.05), the ratio of Th17 cells and Th17/Treg in MM patients were higher than those in control group (P<0.05), and the ratio of Treg cells in MM patients was lower than those in control group, which the difference showed no statistically significant (P>0.05), and the level of cytokines IL-17, IL-10 and the ratio of IL-17/IL-10 in MM patients were significant higher than those in the control group (P<0.05). The expression level of Tim-3+Th17 cells and the ratio of Tim-3+Th17/Tim-3+Treg in MM patients were higher than those in control group (P<0.05), while the expression level of Tim-3+Treg cells in MM patients were lower than that in the control group (P<0.05). The ratio of Tim-3+Th17/Tim-3+Treg in MM patients were positive correlated with ISS staging, DS staging, chromosome abnormality and sFLCR (r=0.635, r=0.501, r=0.449, r=0.587). The ratios of Th17/Treg, IL-17/IL-10 and Tim-3+Th17/Tim-3+Treg increase in MM patients, among which Tim-3+Th17/Tim-3+Treg is correlated with ISS staging, DS staging, chromosomal abnormalities and sFLCR, which suggesting that Tim-3 is involved in the imbalance of Th17/Treg in MM patients.

Immune Cell Landscape in Gastric Cancer.

Background The tumor-infiltrating immune cells are closely associated with the prognosis of gastric cancer (GC). This article is aimed at determining the composition change of immune cells and immune regulatory factors in GC and normal tissues, depicting their prognosis value in GC, and revealing the relationship between them and GC clinical parameters. Methods We used CIBERSORT to calculate the proportion of 22 immune cells in the GC or normal tissues; a t-test was applied to assess the expression difference of immune cells and immune regulatory factors in normal and GC tissues. The relationship of the immune cells, immune regulatory factors, and GC patients' clinical characteristics was assessed by univariate analysis. Results In this study, we found that the proportion of macrophages increased, while plasma cells and monocytes decreased in GC tissues. In these immune fractions, Tregs and naïve B cells were found to be correlated with GC patients' prognosis. Interestingly, the expression of immune regulatory factors was ambiguous with their classical function in GC tissues. For example, TIM-3, FOXP3, and CMTM6 were overexpressed, while CD27 and PD-1 were underexpressed in GC tissues. We also found that IDO1, PD-1, TIGIT, and TIM-3 were highly expressed in high-grade GC tissues, the HERC2 expression level was related to patients' gender, and the TIGIT expression level was sensitive to targeted therapy. Furthermore, our results suggested that the infiltration of Tregs and naive B cells was strongly correlated with the T stage, radiation therapy, targeted molecular therapy, and the expression levels of TIM-3 and FOXP3 in GC. Conclusion The expression pattern of tumor-infiltrating immune cells and immune regulatory factors was systematically depicted in the GC tumor microenvironment, indicating that individualized treatment based on the tumor-infiltrating immune cells and immune regulatory factors may be beneficial to GC patients.

Gene Expression of Inhibitory Receptors and Immune Regulatory Molecules in Polarized Macrophages Derived from Early- and Late-Stage BD Patients

Background: Bipolar disorder (BD) has been associated with increased levels of peripheral inflammatory mediators and neuroinflammation. Previously, we observed different immune responses in macrophages of BD patients at different stages of the disorder. Thus, we aimed to further evaluate the regulation of immune response in BD by quantifying the expression of immune checkpoint receptors and respective ligands, as well as molecules involved in regulation and transcription of inflammatory mediators in polarized macrophages of early and late stages individuals with BD. Methods: qRT-PCR was performed to analyze the expression of TLR1, TLR6, PD-1, NFKB1, PD-L1, PD-L2 and TIM-3 in samples of proinflammatory M1 or M(IFNγ+LPS) and anti-inflammatory M2 or M(IL-4) macrophages derived from peripheral blood mononuclear cells (PBMCs) of euthymic BD patients (n=16), classified as early-stage BD (BD-E, n=9) and late-stage BD (BD-L, n=7) - according to Functional Assessment Short Test (FAST) - and healthy controls (HC, n=10). Results: M(IL-4) from BD-E showed higher expression levels of NFKB1 and PD-L1 in comparison to HC (p<0.05), while BD-L only had higher expression levels of PD-L1 compared to HC (p<0.05). No statistical differences were found between groups for the expression levels of TLR1, PD-L2 and TIM-3 in M(IL-4) phenotype, while expression levels remained unchanged in M(IFNγ+LPS) for all markers. TLR6 and PD-1 did not show PCR amplification in both macrophage phenotypes. Conclusion: Our findings suggest an immunological regulation acting as a compensatory mechanism by M2 anti-inflammatory macrophages at an early stage of BD, although decreased regulation in these cells seems to be observed at late stages of the disorder. We hypothesize that such alterations are possibly due to chronic low-grade inflammation during the course of BD, resulting in the ‘exhaustion’ of macrophage response. However, further investigation is required to comprehend better the role of immune regulatory mechanisms in the disorder. Funding Information: This work was supported by the FIPE-HCPA (Project Number 150396) and National Council for Scientific and Technological Development (CNPq, Brazil; Project Number PQ 302382/2019-4); and BA is a scholarship recipient from CNPq, Brazil. Declaration of Interests: None to declare. Ethics Approval Statement: All participants provided written informed consent before their inclusion, and this study was approved by the Institutional Review Board of Hospital de Clinicas de Porto Alegre (HCPA, Project Number 150396).

TIM-3 levels correlate with enhanced NK cell cytotoxicity and improved clinical outcome in AML patients

ABSTRACT Accumulating evidence indicates that immune checkpoint inhibitors (ICIs) can restore CD8+ cytotoxic T lymphocyte (CTL) functions in preclinical models of acute myeloid leukemia (AML). However, ICIs targeting programmed cell death 1 (PDCD1, best known as PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4) have limited clinical efficacy in patients with AML. Natural killer (NK) cells are central players in AML-targeting immune responses. However, little is known on the relationship between co-inhibitory receptors expressed by NK cells and the ability of the latter to control AML. Here, we show that hepatitis A virus cellular receptor 2 (HAVCR2, best known as TIM-3) is highly expressed by NK cells from AML patients, correlating with improved functional licensing and superior effector functions. Altogether, our data indicate that NK cell frequency as well as TIM-3 expression levels constitute prognostically relevant biomarkers of active immunity against AML.

Clinical-Grade Expanded Regulatory T Cells Are Enriched with Highly Suppressive Cells Producing IL-10, Granzyme B, and IL-35

In the setting of T cell-depleted, full-haplotype mismatched transplantation, adoptive immunotherapy with regulatory T cells (Tregs) and conventional T cells (Tcons) can prevent graft-versus-host disease (GVHD) and improve post-transplantation immunologic reconstitution and is associated with a powerful graft-versus-leukemia effect. To improve the purity and the quantity of the infused Tregs, good manufacturing practices (GMP)-compatible expansion protocols are needed. Here we expanded Tregs using an automated, clinical-grade protocol. Cells were extensively characterized in vitro, and their efficiency was tested in vivo in a mouse model. Tregs were selected by CliniMacs (CD4+CD25+, 94.5 ± 6.3%; FoxP3+, 63.7 ± 11.5%; CD127+, 20 ± 3%; suppressive activity, 60 ± 7%), and an aliquot of 100×106 was expanded for 14 days using the CliniMACS Prodigy System, obtaining 684 ± 279 × 106 cells (CD4+CD25+, 99.6 ± 0.2%; FoxP3+, 82 ± 8%; CD127+, 1.1 ± 0.8%; suppressive activity, 75 ± 12%). CD39 and CTLA4 expression levels increased from 22.4 ± 12% to 58.1 ± 13.3% (P < .05) and from 20.4 ± 6.7% to 85.4 ± 9.8% (P < .01), respectively. TIM3 levels increased from .4 ± .05% to 29 ± 16% (P < .05). Memory Tregs were the prevalent population, whereas naive Tregs almost disappeared at the end of the culture. mRNA analysis displayed significant increases in CD39, IL-10, granzyme B, and IL-35 levels at the end of culture period (P < .05). Conversely, IFNγ expression decreased significantly by day +14. Expanded Tregs were sorted according to TIM3, CD39, and CD62L expression levels (purity >95%). When sorted populations were analyzed, TIM3+ cells showed significant increases in IL-10 and granzyme B (P < .01) .When expanded Tregs were infused in an NSG murine model, mice that received Tcons only died of GVHD, whereas mice that received both Tcons and Tregs survived without GVHD. GMP grade expanded cells that display phenotypic and functional Treg characteristics can be obtained using a fully automated system. Treg suppression is mediated by multiple overlapping mechanisms (eg, CTLA-4, CD39, IL-10, IL-35, TGF-β, granzyme B). TIM3+ cells emerge as a potentially highly suppressive population. © 2020 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

Tim-3 Expression and MGMT Methylation Status Association With Survival in Glioblastoma.

BackgroundA profound understanding of the molecular landscape of glioblastoma multiforme (GBM) will make it possible to develop better and more intelligent therapies directed toward specific molecular targets and may one day yield better prognostic capabilities. Immune checkpoint molecules have inspired the emergence of immune checkpoint-targeting therapeutic strategies. However, the prognostic significance of the immune checkpoint molecule T cell immunoglobulin mucin-3 (Tim-3) on tumor-infiltrating immune cells (TIICs) and O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status has not yet been fully elucidated. We aimed to develop an MGMT promoter methylation status-associated immune prognostic signature for GBM.Patients and MethodsA total of 84 patients with newly diagnosed GBM were included in this study. MGMT promoter methylation status was retrospectively analyzed, and the expression level of Tim-3 was investigated using immunohistochemistry (IHC). The correlation between Tim-3 expression combined with MGMT promoter methylation status and prognosis was explored.ResultsTim-3 expression varied in GBM patients. Mesenchymal expression of Tim-3 in GBM tissues was present 73.81% (62/84) of patients, and these were subdivided into groups based on low 15.48% (13/84), moderate 7.14% (6/84), or strong expression 51.19% (43/84). Forty-eight patients had tumors that tested positive for MGMT promoter methylation, while the remaining 36 patients tested negative.ConclusionsWe profiled the immune status of MGMT promoter methylation in GBM and established a local immune signature for GBM that could independently identify patients with a favorable prognosis, indicating a relationship between prognosis and GBM immune signature. MGMT promoter methylation with lower Tim-3 expression was significantly associated with better survival.

Spatial Distribution and Prognostic Implications of Tumor-Infiltrating FoxP3- CD4+ T Cells in Biliary Tract Cancer.

PurposeThe clinical implications of tumor-infiltrating T cell subsets and their spatial distribution in biliary tract cancer (BTC) patients treated with gemcitabine plus cisplatin were investigated.Materials and MethodsA total of 52 BTC patients treated with palliative gemcitabine plus cisplatin were included. Multiplexed immunohistochemistry was performed on tumor tissues, and immune infiltrates were separately analyzed for the stroma, tumor margin, and tumor core.ResultsThe density of CD8+ T cells, FoxP3− CD4+ helper T cells, and FoxP3+ CD4+ regulatory T cells was significantly higher in the tumor margin than in the stroma and tumor core. The density of LAG3- or TIM3-expressing CD8+ T cell and FoxP3− CD4+ helper T cell infiltrates was also higher in the tumor margin. In extrahepatic cholangiocarcinoma, there was a higher density of T cell subsets in the tumor core and regulatory T cells in all regions. A high density of FoxP3− CD4+ helper T cells in the tumor margin showed a trend toward better progression-free survival (PFS) (p=0.092) and significantly better overall survival (OS) (p=0.012). In multivariate analyses, a high density of FoxP3− CD4+ helper T cells in the tumor margin was independently associated with favorable PFS and OS.ConclusionThe tumor margin is the major site for the active infiltration of T cell subsets with higher levels of LAG3 and TIM3 expression in BTC. The density of tumor margin-infiltrating FoxP3− CD4+ helper T cells may be associated with clinical outcomes in BTC patients treated with gemcitabine plus cisplatin.

Abstract 2194: Inducible MyD88/CD40 (iMC) enhances CAR-T cell expansion and persistence by overcoming T cell exhaustion

Abstract Background: Chimeric antigen receptor (CAR) T cell therapy targeting solid tumors is challenged by inhibitory signals in the tumor microenvironment that may lead to CAR-T exhaustion. Previously, we described a GoCAR platform that uses a rimiducid (Rim)-inducible cytoplasmic costimulatory switch, inducible MyD88/CD40 (iMC), to drive the proliferation, survival, cytokine release and anti-tumor efficacy of T cells expressing a 1st generation CAR. To further understand how iMC signaling improves CAR-T potency, we directly compared iMC-based costimulation to conventional costimulatory domains (i.e., CD28 and 4-1BB) against hematological and solid tumor models. Methods: PBMCs from four donors were activated with anti-CD3/CD28 and transduced with retrovirus encoding 1st generation (CAR.ζ), 2nd generation (CAR.CD28.ζ CAR.4-1BB.ζ or GoCAR (iMC-2A-CAR.ζ) components targeting HER2, CD19 or GD2. Cocultures were established at a 1:1 ratio with antigen-expressing targets and cultures serially passaged weekly against fresh targets for up to six weeks. For in vivo efficacy evaluations NSG mice were engrafted s.c. with 1 × 106 HER2+ OE19-GFPffLuc esophageal carcinoma cells and challenged on day 4 with 5 × 106 CAR-T cells marked with RLuc each monitored by IVIS imaging. Results: 1st generation CAR-T cells produced low levels of IL-2 and IFNγ after the first antigen stimulation and quickly expressed markers of exhaustion including PD-1. 2nd generation CAR-T cells proliferated in early passage but became phenotypically and functionally exhausted following a second stimulation. Conversely, GoCAR-T cells were capable of expanding and sustaining IL-2 production beyond five passages, but only when stimulated with Rim. Lower levels of PD-1, TIM-3, and LAG-3 expression were observed in Rim-treated GoCAR-T cells when compared to 1st gen, 2nd gen and GoCAR-T cells without iMC activation, and PD1 expression remained repressed for six weeks in Rim-treated GoCAR-T cells. In NSG mice engrafted with HER2+ OE19 tumors, Rim treatment drove robust iMC-HER2.ζ CAR-T cell expansion compared to HER2.ζ, HER2.BB.ζ and HER2.28.ζ CAR-T cells over 5 weeks (p &amp;lt; 0.05). iMC-HER2.ζ CAR-T cells significantly enhanced tumor killing with Rim activation relative to vehicle-control and HER2.ζ-expressing T cells days post T cell infusion (tumor radiance = 1.63 × 107 ± 1.7E7 vs 1.40 × 108 ± 5.27E7 vs 1.70 × 108 ± 8.65E7, respectively, p&amp;lt;0.05). When rechallenged with tumor cells at day 35, persistent GoCAR-T cells continued to control tumor growth while 2nd generation CAR-T cells failed to control tumor growth. Furthermore, mice treated with iMC-HER2.ζ CAR-T cells and Rim produced high levels of proinflammatory cytokines including IFN-γ, GM-CSF, and IP-10 consistent with supporting T cell activation and proliferation. Conclusions: iMC activation via Rim infusion provides on-demand control of CAR-T cell signaling to resist CAR-T cell exhaustion, enhance persistence, produce cytokines, and induce antitumor toxicity against solid tumors. Citation Format: MyLinh T. Duong, Aruna Mahendravada, Mary E. Brandt, Kelly L. Sharp, Aaron E. Foster, J. Henri Bayle. Inducible MyD88/CD40 (iMC) enhances CAR-T cell expansion and persistence by overcoming T cell exhaustion [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 2194.

Abstract 5060: Establishment and characterization of humanized mouse NPC-PDX model for testing immunotherapy

Abstract Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) is common in Southern China and South-East Asia. Immune checkpoint blockade (ICB) is being studied in NPC and initial monotherapy ICB studies show early promise. Currently, lack of representative NPC models limit the preclinical studies to evaluate the efficacy and safety of novel ICB and combination regimens. Hence, there is a need to develop and characterize a mouse model with human immune system for testing immunotherapy. Here, we engrafted NPC-PDX in immunodeficient NSG mice, with a successful growth rate of 16.7% (6 out of 36), and humanized mouse NPC-PDX model was established subsequently. Of note, EBV latent membrane protein-1 was retained in the NPC in both NSG and humanized mice as revealed by IHC staining. There was an increase in tumor-infiltrating CD4+ T cells and a decrease in CD8+ cytotoxic T cells in humanized mice, when compared to the immune cells profile in circulation. Intriguingly, the expression levels of various exhaustion markers, namely PD-1, CTLA4 and TIM-3 were upregulated, particularly in the tumor-infiltrating T cells. Therefore, the use of ICB, either alone or in combination, may restore the function of anti-tumor immune effector cells. Taken together, we have established a humanized mouse NPC-PDX model, which plausibly provides a robust platform to test for the efficacy and safety of ICB and combination therapy regimens. Citation Format: Wai Nam LIU, Shin Yie FONG, Wilson Wei Sheng TAN, Sue Yee TAN, Min LIU, Narayanan Gopalakrishna IYER, Darren Wan-Teck LIM, Qingfeng CHEN. Establishment and characterization of humanized mouse NPC-PDX model for testing immunotherapy [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 5060.

Abstract 1608: Adaptive resistance to PD-1 blockade upregulates Tim-3 through STAT3 pathway in regulatory T cells in melanoma

Abstract Tregs play an opposing role in anti-tumour immunity by suppressing effective immunotherapies in melanoma. Thus, understanding the precise mechanisms of Tregs in immune therapy is important. TILs separated from freshly excised melanoma tumours and PBMCs obtained from healthy donors were used to assess the expression levels of Tim-3 and IL-10/TGF-β of Tregs by multi-colour flow cytometry and immunohistochemistry in the presence or absence of an anti-PD-1 antibody or STAT3 inhibitor in vitro and in vivo. The mRNA level of STAT3 gene was closely associated with the expression of PD-1 and Tim-3 in melanoma patients. Tim-3 expression was significantly upregulated on Tregs after PD-1 blockade in mice treated with either induced mouse and human melanoma TILs or healthy donor PBMCs. The levels of IL-10 and TGF-β secretion in the TIL group increased compared with those in the control group, supporting a potential escape mechanism from anti-PD-1 therapy in the tumour microenvironment. Furthermore, in the presence of the STAT3 pathway inhibitor Stattic, Tim-3 and PD-1 expression on Treg was regulated on TILs from respondent mice melanoma, suggesting that STAT3 could mediate Tim-3 and PD-1 expression on Treg in tumor microenvironment. Therefore, our findings reveal the mechanism of PD-1 and Tim-3 expression in a STAT3-dependent manner on Tregs in the TME, providing further support for STAT3 as a potential transient small molecular inhibitor targets for patients suffering from melanoma. Citation Format: Lili Huang, Yu Xu, Juemin Fang, Weixing Liu, Zhuqing Liu, Qing Xu. Adaptive resistance to PD-1 blockade upregulates Tim-3 through STAT3 pathway in regulatory T cells in melanoma [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 1608.

Down-regulation and Clinical Implication of Galectin-9 Levels in Patients with Acute Coronary Syndrome and Chronic Kidney Disease.

In various autoimmune diseases, Galecin-9 (Gal-9) has been shown to regulate the T-cell balance by decreasing Th1 and Th17, while increasing the number of regulatory T cells (Tregs). However, the role of Gal-9 in the patients with acute coronary syndrome (ACS) and chronic kidney disease (CKD) remains unclear. This study aims to measure the Gal-9 levels in serum and peripheral blood mononuclear cells (PBMCs) in patients with ACS plus CKD and examine their clinical implication. The serum levels of Gal-9 were determined by enzyme-linked immunosorbent assay (ELISA), the expression levels of Gal-9, Tim-3, and Foxp3 mRNA in PBMCs were detected by real-time reverse transcription-polymerase chain reaction (RT-PCR), and the expression of Gal-9 on the surface of PBMCs and in PBMCs was analyzed by flow cytometry. Furthermore, the correlation of serum Gal-9 levels with anthropometric and biochemical variables in patients with ACS plus CKD was analyzed. The lowest levels of Gal-9 in serum and PBMCs were found in the only ACS group, followed by the ACS+CKD group, and the normal coronary artery (NCA) group, respectively. Serum Gal-9 levels were increased along with the progression of glomerular filtration rate (GFR) categories of G1 to G4. Additionally, serum Gal-9 levels were negatively correlated with high-sensitivity C-reactive protein (hs-CRP), estimated GFR (eGFR), and lipoprotein(a), but positively with creatinine, age, osmotic pressure, and blood urea nitrogen (BUN). Notably, serum Gal-9 was independently associated with hs-CRP, osmotic pressure, and lipoprotein(a). Furthermore, serum Gal-9 levels were elevated in patients with type 2 diabetes (T2DM) and impaired glucose tolerance (IGT) in ACS group. It was suggested that the levels of Gal-9 in serum and PBMCs were decreased in patients with simple ACS and those with ACS plus CKD, and hs-CRP, eGFR, osmotic pressure and T2DM may have an influence on serum Gal-9 levels.

Genetic variants and expression of the TIM-3 gene are associated with clinical prognosis in patients with epithelial ovarian cancer

ObjectivePolymorphisms of T cell immunoglobulin and mucin domain-containing molecule 3 (TIM-3) were reported to be associated with cancer risk and patients' survival. This study aims to investigate the correlation of TIM-3 polymorphisms with susceptibility to epithelial ovarian cancer (EOC) and patients' outcomes. MethodsA total of 700 EOC patients and 710 healthy controls from North China were included. The polymorphisms (rs10053538, rs10515746 and rs1036199) were genotyped using the polymerase chain reaction/ligase detection reaction (PCR-LDR) method. Survival data were available for 339 patients after cytoreductive surgery. The expression level of TIM-3 was detected by real-time quantitative PCR (RT-qPCR). The prognostic value of TIM3 in EOC patients was assessed using the Kaplan-Meier plotter database. ResultsThe results showed that none of the TIM3 polymorphisms were associated with the risk of developing EOC. Patients with the rs10053538 CA + AA genotype had worse PFS and OS than those with the CC genotype (HR = 1.49, 95% CI = 1.05–2.09, P = 0.024 and HR = 1.57, 95%CI = 1.09–2.26, P = 0.017, respectively). The RT-qPCR results showed that the expression levels of TIM-3 mRNA in EOC tissues with the rs10053538CA + AA genotypes were significantly higher than those with the CC genotype (P = 0.006). Analysis using the Kaplan-Meier plotter database showed that high expression of TIM-3 mRNA was significantly associated with shorter PFS and OS in EOC patients (HR = 1.57, 95%CI = 1.29–1.91, P < 0.001 and HR = 1.31, 95% CI = 1.06–1.63, P = 0.013, respectively). ConclusionsTIM-3 polymorphisms were not associated with risk of developing EOC. Both rs10053538 and the expression level of TIM-3 mRNA may be associated with its clinical outcome in EOC patients.

Prognostic value of immune checkpoint molecules in breast cancer.

Immune checkpoint blockade treatments bring remarkable clinical benefits to fighting several solid malignancies. However, the efficacy of immune checkpoint blockade in breast cancer remains controversial. Several clinical trials of immune checkpoint blockades focused on the effect of CTLA4 and PD1/PDL1 checkpoint inhibitors on breast cancer. Only a small portion of patients benefited from these therapies. Here we systematically investigated the expression of 50 immune checkpoint genes, including ADORA2A, LAG-3, TIM-3, PD1, PDL1, PDL2, CTLA-4, IDO1, B7-H3, B7-H4, CD244, BTLA, TIGIT, CD80, CD86, VISTA, CD28, ICOS, ICOSLG, HVEM, CD160, LIGHT, CD137, CD137L, OX40, CD70, CD27, CD40, CD40LG, LGALS9, GITRL, CEACAM1, CD47, SIRPA, DNAM1, CD155, 2B4, CD48, TMIGD2, HHLA2, BTN2A1, DC-SIGN, BTN2A2, BTN3A1, BTNL3, BTNL9, CD96, TDO, CD200 and CD200R, in different subtypes of breast cancer and assessed their prognostic value. The results showed that the expression patterns of these 50 immune checkpoint genes were distinct in breast cancer. High expression of B7-H3 mRNA was significantly associated with worse overall survival (OS), especially in patients with luminal A and luminal B breast cancer. The mRNA expression levels of TIM-3, ADORA2A, LAG3, CD86, CD80, PD1 and IDO1 had no relationship with OS in breast cancer. High expression levels of CTLA-4 and TIGIT were correlated with favorable prognosis in breast cancer. Interestingly, we observed that B7-H3 expression was negatively correlated with the efficacy of cyclophosphamide (CTX). In summary, our study suggested that B7-H3 has potential prognostic value in breast cancer and is a promising target for immune therapy.

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.