Tim-3 Expression Research Articles

Identification of TIM-3 Expression in Acute Myeloid Leukemia: Clinical and Laboratory Correlation

Abstract Background Acute myeloid leukemia (AML) is an aggressive highly heterogenous hematological malignancy associated with poor prognosis and high mortality rate. Both cancer cells and immune cells participate in tumor initiation, growth and progression and might affect clinical outcomes. Indeed, leukemic cells exploit a variety of mechanisms to evade immune response, leading to disease progression and relapse. T-cell immunoglobulin and mucin domain 3 (TIM-3), an immune checkpoint molecule, is expressed not only on immune cells but also on leukemic stem cells (LSCs) in AML. Studies relating to TIM-3 function in AML are limited, and the role of TIM3 in maintaining leukemic stem cells and contributing to the suppression of the anti-tumor immune response is still poorly understood. Aim of the Work To investigate TIM-3 expression in patients with AML and explore the impact of its expression on the clinico-laboratory characteristics and prognostic behavior of denovo-AML patients. Methods A total of 45 patients newly diagnosed AML patients were tested for TIM-3 expression by flowcytometry at diagnosis. Results TIM-3 was found to be over expressed in patients with AML with significant association with the different FAB subtypes (p = 0.010). TIM-3 positivity was negatively correlated with CD34 expression (p = 0.019.). However, no significant association between TIM-3 expression and extramedullary disease (EMD) as well as cytogenetic risk groups. Conclusion TIM-3 is overexpressed in AML patients. The prognostic value of TIM3 in AML is not clear and remains to be further explored.

Immune regulation by activation markers at feto-maternal interface in infection-associated spontaneous preterm birth

BackgroundSpontaneous preterm birth (sPTB) is a global health concern. Studies reveal infections are majorly responsible for sPTB and immune activation markers play a role in regulation of maternal immune responses against pathogens during sPTB. AimTo study the mRNA expression and correlation of activation markers (CD66a, ICAM1, ITGB1, TIM3, CD25, CD95) and associated cytokines (IL-1β and IL-17)/prostaglandin receptors (EP2 and IP) in the placenta of Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum-infected sPTB women. MethodsPlacental samples were collected from 160 sPTB and 160 term birth women. PCR was used for the detection of C. trachomatis, M. hominis, U. urealyticum. The mRNA expression of activation markers, cytokines and prostaglandin receptors was evaluated by real-time qPCR. ResultsThe fold-change expression of CD66a, ICAM1, TIM3, CD25 and CD95 was 2.89, 5.5, 4.95, 6.44 and 6.95-fold (p < 0.001), respectively; while for cytokines- IL-1β and IL-17 was 5.41 and 4.71-fold (p < 0.001), respectively and for prostaglandin receptors- EP2 and IP was 5.5 and 5-fold (p < 0.001) upregulated, respectively in infected sPTB women. Significant positive correlation was obtained among ICAM-1 and IL-1β/EP2/IL-17, TIM3 and IP/IL-17. Significant negative correlation was obtained between CD66a and EP2/IL-17, CD25 and IL-1β/EP2, CD95 and IL-1β/EP2 in infected sPTB women. ConclusionsCD66a, ICAM1 and TIM3 may play role in inflammation and have potential for the clinical beginning of preterm labour during infection while CD25 and CD95 are possibly involved in immunotolerance at feto-maternal interface during C. trachomatis, M. hominis and U. urealyticum infection.

Expression and Clinical Significance of Peripheral Blood IL-17A, IL-22, Tim-3, and gal-9 in Children with Infectious Mononucleosis.

To investigate the expression and clinical significance of peripheral blood interleukin (IL)-17A, IL-22, T cell immunoglobulin molecule-3 (Tim-3), and galectin-9 (gal-9) in children with infectious mononucleosis (IM) caused by the Epstein-Barr virus (EBV). Peripheral blood of 54 children with IM (case group) was collected and divided into a liver damage group and a non-liver damage group. During the same period, 20 healthy children were in the control group. IL-17A and IL-22 were measured by enzyme-linked immunosorbent assay. Real-time quantitative polymerase chain reaction was used to measure the mRNA expression of Tim-3 and gal-9. Their correlation with clinical indicators was then analyzed. The IL-17A expression level was higher in the case group than in the control group, while Tim-3, gal-9, and IL-22 were lower than those in the control group. Tim-3 was positively correlated with gal-9, but negatively correlated with IL-17A. Tim-3 and gal-9 were positively correlated with CD4+/CD8+ cells. Conversely, they were negatively correlated with CD3+, CD3+CD8+, white blood cell, lymphocyte (L), alanine transaminase (ALT), aspartate transaminase (AST), glutamyl transpeptidase (GGT), and lactate dehydrogenase (LDH). In the case group, IL-17A was positively correlated with L, GGT, and LDH, but negatively correlated with the natural killer (NK) cell count. IL-17A and IL-22 were positively correlated with CD3+, CD3+CD8+, ALT, and AST, but they were negatively correlated with the ratio of CD4+/CD8+. In the liver damage group, IL-17A, IL-22, CD3+, CD3+CD8+, immunoglobulin A (IgA), IgG, IgM, L, ALT, AST, GGT, LDH, and α-hydroxybutyrate levels were higher than those in the non-liver damage group. However, Tim-3, gal-9, the ratio of CD4+/CD8+, and NK were lower than those in the non-liver damage group. IL-17A, IL-22, Tim-3, and gal-9 are involved in the immune pathogenesis of IM caused by EBV infection in children, which may be related to immune liver injury.

Characterization of TCF-1 and its relationship between CD8+ TIL densities and immune checkpoints and their joint influences on prognoses of lung adenocarcinoma patients.

T cell factor-1 (TCF-1) + stem-like tumor-infiltrating lymphocytes (stem-like TILs) are important memory cells in the tumor microenvironment. However, their relationship with clinicopathological features, CD8+ TIL densities, immune checkpoint inhibitors (ICs), and prognostic values remain unknown for lung adenocarcinomas (LUADs). In this study, we aimed to characterize TCF-1+ TILs and their prognostic significance in patients with surgically resected LUADs. Expression of TCF-1, CD8, and ICs including programmed death-1 (PD-1), lymphocyte activating-3 (LAG-3), and T cell immunoglobulin and mucin-domain containing-3 (TIM-3) in TILs were estimated using immunohistochemistry of resected LUADs. The association between TCF-1 expressions and clinicopathological characteristics of patient prognoses were analyzed. Positive TCF-1 expression significantly correlated with advanced pathological stage, tumor grade, CD8+ TILs density, TIM-3 expression, LAG-3 expression, and PD-1 expression. TCF-1 positivity was significantly associated with a better recurrence-free survival (RFS), and overall survival (OS). Subgroup analysis revealed that the TCF-1+/CD8+ group had the best RFS and OS, while the TCF-1-/CD8- group had the worst RFS and OS. Similarly, patients with TCF-1 + PD-1- had the best prognoses and patients with TCF-1-PD-1+ had the worst prognoses. TCF-1 had relatively high positive expression and special clinicopathological features in patients with LUAD. TCF-1+ TILs were related to CD8 density, TIM-3 expression, LAG-3 expression, and PD-1 expression, and were associated with better prognoses in LUAD patients. A combination of TCF-1 and CD8 densities or PD-1 expression further stratified patients into different groups with distinct prognoses.

Tumor-infiltrating gamma delta T-cells reveal exhausted subsets with remarkable heterogeneity in colorectal cancer.

The γδT-cells recognize infected or transformed cells. However, unlike αβT-cells, γδT-cells are innate-like immune cells, with no major histocompatibility complex restriction requirements. γδT-cells are the main population of intestinal intraepithelial lymphocytes (IELs) and are associated with the antitumor immune response, particularly in colorectal cancer (CRC). Although CD8+ T-cells exhibit dysfunction and even exhaustion in the tumor microenvironment (TME), which contributes to tumor immune escape, whether the same applies to tumor-infiltrating (TI)-γδT-cells is not completely understood. Here, we sought to investigate the expression pattern of inhibitory receptors and functional state of TI-γδT-cells, and reveal the features of exhausted TI-γδT-cells in the CRC TME. We demonstrated that TI-γδT-cells exhibited exhaustion phenotypes and displayed more severe functional exhaustion than TI-CD8+ T-cells or NK-cells in the TME of CRC. In addition, scRNA-seq analysis of TI-γδT-cells revealed three exhausted subsets with remarkable heterogeneity. The presence of three heterogeneous exhausted γδT-cell (Tex) populations, including Texprog , Textran and Texterm were further confirmed by flow cytometry, on the basis of PD-1 and TIM-3 expression. Finally, we revealed that c-Maf not only contributed to γδT-cell exhaustion via upregulation of inhibitory receptors, but also involved in the exhaustion of CD8+ T and NK-cells. c-Maf may also be an important contributor to γδT-cell exhaustion in CRC patients. These findings indicated that TI-γδT-cells exhibit phenotypic and functional exhaustion in the CRC TME. The revealed features of exhausted TI-γδT-cells may provide help for understanding the mechanisms and the association of γδT-cell exhaustion with tumor development and pathogenesis.

IL8 derived from macrophages inhibits CD8+ T-cell function by downregulating TIM3 expression through IL8-CXCR2 axis in patients with advanced colorectal cancer.

T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) is a vital immune checkpoint that regulates the immune response. However, the specific role of TIM3 in patients with colorectal cancer (CRC) have rarely been studied. In this study, we investigated the effect of TIM3 on CD8+ T cells in CRC and explored the mechanism of TIM3 regulation in tumor microenvironment (TME). Peripheral blood and tumor tissues of patients with CRC were collected to evaluate TIM3 expression using flow cytometry. Cytokines in the serum of healthy donors and patients with early- and advanced-stage CRC were screened using a multiplex assay. The effects of interleukin-8 (IL8) on TIM3 expression on CD8+ T cells were analyzed using cell incubation experiments in vitro. The correlation between TIM3 or IL8 and prognosis was verified using bioinformatics analysis. TIM3 expression on CD8+ T cells was obviously reduced in patients with advanced-stage CRC, whereas a lower TIM3 expression level was associated with poorer prognosis. Macrophage-derived IL8, which could inhibit TIM3 expression on CD8+ T cells, was significantly increased in the serum of patients with advanced CRC. In addition, the function and proliferation of CD8+ and TIM3+CD8+ T cells were inhibited by IL8, which was partly depending on TIM3 expression. The inhibitory effects of IL8 were reversed by anti-IL8 and anti-CXCR2 antibodies. In summary, macrophages-derived IL8 suppresses TIM3 expression on CD8+ T cells through CXCR2. Targeting the IL8/CXCR2 axis may be an effective strategy for treating patients with advanced CRC.

Role of Tim-3 in COVID-19: a potential biomarker and therapeutic target.

T cell immunoglobulin and mucin domain containing protein 3 (Tim-3), an immune checkpoint, is important for maintaining immune tolerance. There is increasing evidence that Tim-3 is aberrantly expressed in patients with COVID-19, indicating that it may play an important role in COVID-19. In this review, we discuss the altered expression and potential role of Tim-3 in COVID-19. The expression of Tim-3 and its soluble form (sTim-3) has been found to be upregulated in COVID-19 patients. The levels of Tim-3 on T cells and circulating sTim-3 have been shown to be associated with the severity of COVID-19, suggesting that this protein could be a potential biomarker of COVID-19. Moreover, this review also highlights the potential of Tim-3 as a therapeutic target of COVID-19.

Increased TIM-3 and galectin-9 serum levels in patients with advanced systemic mastocytosis

Systemic mastocytosis is characterized by expansion of clonal mast cells in various tissues. Several biomarkers with diagnostic and therapeutic potential have recently been characterized in mastocytosis, such as the serum marker tryptase and the immune checkpoint molecule PD-L1. We aimed to investigate whether serum levels of other checkpoint molecules are altered in systemic mastocytosis and whether these proteins are expressed in mastocytosis infiltrates in the bone marrow. Levels of different checkpoint molecules were analyzed in serum of patients with different categories of systemic mastocytosis and healthy controls and correlated to disease severity. Bone marrow biopsies from patients with systemic mastocytosis were stained to confirm expression. Serum levels of TIM-3 and galectin-9 were increased in systemic mastocytosis, particularly in advanced subtypes, compared with healthy controls. TIM-3 and galectin-9 levels were also found to correlate with other biomarkers of systemic mastocytosis, such as serum tryptase and KIT D816V variant allele frequency in the peripheral blood. Moreover, we observed expression of TIM-3 and galectin-9 in mastocytosis infiltrates in bone marrow. Together, our results demonstrate for the first time that serum levels of TIM-3 and galectin-9 are increased in advanced systemic mastocytosis. Moreover, TIM-3 and galectin-9 are expressed in bone marrow infiltrates in mastocytosis. These findings provide a rationale for exploring TIM-3 and galectin-9 as diagnostic markers and eventually therapeutic targets in systemic mastocytosis, particularly in advanced forms.

The effectiveness of a novel treatment of TIM-3(-) NK cells infusion in murine models of immune-mediated bone marrow failure.

T-cell immunoglobulin and mucin-containing domain (TIM)-3 exerts its inhibitory effect on NK cells and participates in the immune pathogenesis of SAA. In this study, we aimed to explore a novel treatment method of TIM-3(+) NK or TIM-3(-) NK cell infusion in combination with immunosuppressive therapy for bone marrow failure (BMF)/aplastic anemia (AA) mice. BMF/AA mouse model was constructed. The TIM-3 expression and functional molecules on TIM-3(+) and TIM-3(-) NK cells of the BMF group, total body irradiation (TBI) group, and normal control (NC) group mice were detected by flow cytometry. After treatment, the general condition, whole blood cell and bone marrow cell (BMC) count, and immune condition of mice from each group were compared. TIM-3 expression in the peripheral blood NK cells of BMF mice was significantly lower than that of the TBI and NC group mice. TIM-3(-) NK cells expressed more NKG2D receptors than TIM-3(+) NK cells. The levels of P-Akt and PI3K in TIM-3(-) NK cells were higher than those in TIM-3(+) NK cells. On the 17th day after BMF induction, the weight, peripheral whole blood cell count, and BMC count of BMF mice decreased significantly compared with that of the NC group mice. The therapeutic effect in the TIM-3(-) NK cell treatment group was better than that in the TIM-3(+) NK cell treatment and CsA treatment groups. Concurrently, the ratio of CD4+ T and CD8+ T cells of BMF mice was significantly lower than that of the NC group mice. The therapeutic effect in CsA + TIM-3(-) NK group was more significant than that of the CsA treatment and the CsA + TIM-3(+) NK groups. In this study, we found that the general condition, peripheral whole blood cell and BMC count, and immune status of BMF mice improved significantly after CsA + TIM-3(-) NK cell treatment. These results may provide further insights into the immune pathogenesis of SAA and novel therapeutic ideas for improving SAA treatment.

Efferocytes release extracellular vesicles to resolve inflammation and tissue injury via prosaposin-GPR37 signaling

Macrophages release soluble mediators following efferocytic clearance of apoptotic cells to facilitate intercellular communication and promote the resolution of inflammation. However, whether inflammation resolution is modulated by extracellular vesicles (EVs) and vesicular mediators released by efferocytes is not known. We report that efferocyte-derived EVs express prosaposin, which binds to macrophage GPR37 to increase expression of the efferocytosis receptor Tim4 via an ERK-AP1-dependent signaling axis, leading to increased macrophage efferocytosis efficiency and accelerated resolution of inflammation. Neutralization and knockdown of prosaposin or blocking GRP37 abrogates the pro-resolution effects of efferocyte-derived EVs invivo. Administration of efferocyte-derived EVs in a murine model of atherosclerosis is associated with an increase in lesional macrophage efferocytosis efficiency and a decrease in plaque necrosis and lesional inflammation. Thus, we establish a critical role for efferocyte-derived vesicular mediators in increasing macrophage efferocytosis efficiency and accelerating the resolution of inflammation and tissue injury.

Tim-3 protects against cisplatin nephrotoxicity by inhibiting NF-κB-mediated inflammation

The impact of Tim-3 (T cell immunoglobulin and mucin domain-containing protein 3) on cisplatin-induced acute kidney injury was investigated in this study. Cisplatin-induced Tim-3 expression in mice kidney tissues and proximal tubule-derived BUMPT cells in a time-dependent manner. Compared with wild-type mice, Tim-3 knockout mice have higher levels of serum creatinine and urea nitrogen, enhanced TUNEL staining signals, more severe 8-OHdG (8-hydroxy-2’ -deoxyguanosine) accumulation, and increased cleavage of caspase 3. The purified soluble Tim-3 (sTim-3) protein was used to intervene in cisplatin-stimulated BUMPT cells by competitively binding to the Tim-3 ligand. sTim-3 obviously increased the cisplatin-induced cell apoptosis. Under cisplatin treatment conditions, Tim-3 knockout or sTim-3 promoted the expression of TNF-α (tumor necrosis factor-alpha) and IL-1β (Interleukin-1 beta) and inhibited the expression of IL-10 (interleukin-10). NF-κB (nuclear factor kappa light chain enhancer of activated B cells) P65 inhibitor PDTC or TPCA1 lowed the increased levels of creatinine and BUN (blood urea nitrogen) in cisplatin-treated Tim-3 knockout mice serum and the increased cleavage of caspase 3 in sTim-3 and cisplatin-treated BUMPT cells. Moreover, sTim-3 enhanced mitochondrial oxidative stress in cisplatin-induced BUMPT cells, which can be mitigated by PDTC. These data indicate that Tim-3 may protect against renal injury by inhibiting NF-κB-mediated inflammation and oxidative stress.

TIM-3 signaling contributes to the suppressive capacity of Tregs from people with HIV on antiretroviral therapy.

TIM-3 expression is increased on peripheral regulatory T cells (Tregs) of virally suppressed persons with HIV-1 on antiretroviral therapy (PWH-ART). However, the relevance of TIM-3 expression in this setting is unclear. We used flow cytometry to evaluate the suppressive phenotype and signaling pathways in peripheral TIM-3- vs TIM-3+ Tregs in PWH-ART. TIM-3+ Tregs showed increased expression of IL-10 compared with persons without HIV-1. In addition, TIM-3+ Tregs displayed elevated signaling and activation, relative to TIM-3- Tregs from the same PWH-ART. Dramatically, TIM-3 blockade restrained the in vitro suppressive capacity of peripheral Tregs. Therefore, our data demonstrate not only that TIM-3 expression by Tregs is associated with an immunosuppressive response among PWH-ART, but also that TIM-3 contributes directly to the enhanced suppressive activity of Tregs in this setting.

Depletion of Gr1+ myeloid cells attenuates high-fat-diet-aggravated esophageal squamous cell carcinoma in mice.

Obesity is a leading cause of multiple cancers, but whether it promotes esophageal squamous cell carcinoma (ESCC) and its underlying cancer-promoting mechanism remains unclear. To evaluate the effect of a high-fat diet (HFD) on ESCC and explore the role and mechanism of myeloid-derived suppressor cells (MDSCs) in HFD-induced ESCC, C57BL/6J mice were treated with 4-nitroquinoline 1-oxide (4NQO) to induce ESCC and randomly assigned to an HFD or a normal-fat diet. An anti-Gr1 monoclonal antibody was used to deplete MDSCs in the context of experimental diets and ESCC induction. The expression of MDSC markers CD11b and Gr1 and immune checkpoints (ICs) PD1, TIM3, and VISTA in lesions were detected by immunohistochemistry. The correlation between myeloid cell markers (CD11b and CD33) and ICs and their relationship with ESCC patient prognosis were further analyzed using the The Cancer Genome Atlas dataset. The results showed that HFD accelerated esophageal carcinogenesis, induced MDSC expansion, and upregulated IC expression, whereas depletion of Gr1+ myeloid cells significantly suppressed tumor growth, decreased the number of MDSCs, and downregulated IC expression in HFD mice. PD1, TIM3, and VISTA expressions were positively correlated with myeloid cell marker expression in human ESCC. Moreover, the high expression of IC molecules was associated with poor survival in patients with ESCC. These data indicate that HFD promotes the initiation and development of ESCC. Gr1+ myeloid cell targeting significantly inhibited ESCC formation in HFD mice, which may be associated with IC downregulation.

Development and validation of a MUC16 mutation-associated immune prognostic model for lung adenocarcinoma.

Mucin 16 (MUC16) mutation ranks third among all common mutations in lung adenocarcinoma (LUAD), and it has a certain effect on LUAD development and prognostic outcome. This research aimed to analyze the effects of MUC16 mutation on LUAD immunophenotype regulation and determine the prognostic outcome using an immune prognostic model (IPM) built with immune-related genes. The MUC16 mutation status and mRNA expression profiles were analyzed using diverse platforms and among several LUAD patients (n = 691). An IPM was then constructed using differentially expressed immune-related genes (DEIRGs) in MUC16MUT LUAD cases, and the data were compared with those of MUC16WT LUAD cases. The IPM's performance in distinguishing high-risk cases from low-risk ones among 691 LUAD cases was verified. Additionally, a nomogram was built and applied in the clinical setting. Furthermore, a comprehensive IPM-based analysis of how MUC16 mutation affected the tumor immune microenvironment (TIME) of LUAD was performed. MUC16 mutation decreased the immune response in LUAD. As revealed by functional annotation, the DEIRGs in the IPM were most significantly enriched in the humoral immune response function and the immune system disease pathway. Moreover, high-risk cases were associated with increased proportions of immature dendritic cells, neutrophils, and B-cells; enhanced type I interferon T-cell response; and increased expression of PD-1, CTLA-4, TIM-3, and LAG3 when compared with low-risk cases. MUC16 mutation shows potent association with TIME of LUAD. The as-constructed IPM displays high sensitivity to MUC16 mutation status and can be applied to discriminate high-risk LUAD cases from low-risk ones.

Association of TIM-3 with anterior uveitis and associated systemic immune diseases: a Mendelian randomization analysis.

We aimed to investigate the causal association between TIM-3, an immune checkpoint inhibitor, and anterior uveitis (AU), as well as associated systemic immune diseases. We performed two-sample Mendelian randomization (MR) analyses to estimate the causal effects of TIM-3 on AU and three associated systemic diseases, namely ankylosing spondylitis (AS), Crohn's disease (CD), and ulcerative colitis (UC). Single-nucleotide polymorphisms (SNPs) associated with AU, AS, CD, and UC were selected as the outcomes: AU GWAS with 2,752 patients with acute AU accompanied with AS (cases) and 3,836 AS patients (controls), AS GWAS with 968 cases and 336,191 controls, CD GWAS with 1,032 cases and 336,127 controls, and UC GWAS with 2,439 cases and 460,494 controls. The TIM-3 dataset was used as the exposure (n = 31,684). Four MR methods, namely, inverse-variance weighting (IVW), MR-Egger regression, weighted median, and weighted mode, were used in this study. Comprehensive sensitivity analyses were conducted to estimate the robustness of identified associations and the potential impact of horizontal pleiotropy. Our studies show that TIM-3 is significantly associated with CD using the IVW method (OR = 1.001, 95% CI = 1.0002-1.0018, P-value = 0.011). We also found that TIM-3 may be a protective factor for AU although these results lacked significance (OR = 0.889, 95% CI = 0.631-1.252, P-value = 0.5). No association was observed between the genetic predisposition to particular TIM-3 and susceptibility to AS or UC in this study. No potential heterogeneities or directional pleiotropies were observed in our analyses. According to our study, a small correlation was observed between TIM-3 expression and CD susceptibility. Additional studies in different ethnic backgrounds will be necessary to further explore the potential roles and mechanisms of TIM-3 in CD.

DIPG-04. TUMOR CELL-INTRINSIC TIM-3 PROMOTES DIFFUSE INTRINSIC PONTINE GLIOMA TUMORIGENESIS

Abstract Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor and the leading cause of pediatric death caused by cancer. Despite great strides in the understanding of this disease, the median overall survival is still dismal. TIM-3 is emerging as an alternative to the classic immune checkpoints CTLA-4 and PD-1, and its expression regulates by various mechanisms the function of innate and adaptative immunity. In our work, we demonstrated that TIM-3 is expressed in murine and human DIPG tumor cell lines in vitro and in vivo. TIM-3 knockout using CRISPR/Cas9 was lethal in all human DIPG cell lines evaluated. However, a doxycycline-inducible shRNA-TIM-3 significantly reduced the proliferation of DIPG human cell lines (100% control vs. 25% shRNA TIM-3). Additionally, TIM-3KO murine cell lines displayed a significant decrease in clonogenicity, proliferation, and migration in vitro compared with parental DIPG murine cell lines. Interestingly, loss of TIM-3 expression did not result in cell death, as shown by the evaluation of apoptosis. In vivo evaluation of TIM-3KO cell lines tumorigenicity demonstrated that mice bearing TIM3-KO cells had an overall median survival (35 days) significantly higher than the ones bearing the control guide cells (16 days). RNAseq data indicated that TIM-3 plays a critical role in DIPG murine cells tumorigenesis and survival through the MAPK signaling pathway. Finally, single-cell sequencing and immunochemistry of human tumor samples uncovered TIM-3 expression in patients. TIM-3 expression in DIPG tumor cells correlated with genes positively involved in regulating ion transport, IL-6 production, and response to IFN-γ pathways. We also observed enrichment in genes regulating MAPK and ERK1/ERK2 cascades in these patients. Collectively, these data suggest that TIM-3 expression might have a role in the survival and tumorigenesis of DIPG cells.

Dynamics of the inhibitory immune checkpoint TIM-3 in mouse pulmonary phagocytes after silica exposure

Long-term inhalation of silica particles in the workplace causes silicosis, which is incurable and seriously endangers the health of workers. It is believed that silicosis is caused by an imbalance of the pulmonary immune microenvironment, in which pulmonary phagocytes play a crucial role. As an emerging immunomodulatory factor, it is unclear whether T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) participate in silicosis by modulating pulmonary phagocytes function. The purpose of this study was to investigate the dynamic changes of the TIM-3 in pulmonary macrophages, dendritic cells (DCs), and monocytes during the development of silicosis in mice. The plasma levels of soluble TIM-3 in silicosis patients were also examined. Flow cytometry was used to identify alveolar macrophages (AMs), interstitial macrophages (IMs), CD11b+ DC, CD103+ DC, Ly6C+, and Ly6C- monocytes in mouse lung tissues, and further analyses were conducted on the expression of TIM-3. Results showed that soluble TIM-3 was significantly elevated in plasma of silicosis patients, and the level of which was higher in stage II and III patients than that in stage I. In silicosis mice, the protein and mRNA levels of TIM-3 and Galectin9 were significantly upregulated in lung tissues. Specific to pulmonary phagocytes, silica exposure affected TIM-3 expression in a cell-specific and dynamic manner. In macrophages, TIM-3 expression upregulated in AM after 28 days and 56 days of silica instillation, while the expression of TIM-3 in IM decreased at all observation time points. In DCs, silica exposure only caused a decrease of TIM-3 expression in CD11b+ DCs. In monocytes, TIM-3 dynamics in Ly6C+ and Ly6C- monocytes were generally consistent during silicosis development, which significant decrease after 7 and 28 days of silica exposure. In conclusion, TIM-3 may mediate the development of silicosis by regulating pulmonary phagocytes.

High-dimensional single-cell analysis unveils distinct immune signatures of peripheral blood in patients with pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with poor response to immune checkpoint inhibitors. The mechanism of such poor response is not completely understood. We assessed T-cell receptor (TCR) repertoire and RNA expression at the single-cell level using high-dimensional sequencing of peripheral blood immune cells isolated from PDAC patients and from healthy human controls. We validated RNA-sequencing data by performing mass cytometry (CyTOF) and by measuring serum levels of multiple immune checkpoint proteins. We found that proportions of T cells (CD45+CD3+) were decreased in PDAC patients compared to healthy controls, while proportion of myeloid cells was increased. The proportion of cytotoxic CD8+ T cells and the level of cytotoxicity per cell were increased in PDAC patients, with reduced TCR clonal diversity. We also found a significantly enriched S100A9+ monocyte population and an increased level of TIM-3 expression in immune cells of peripheral blood in PDAC patients. In addition, the serum level of soluble TIM-3 (sTIM-3) was significantly higher in PDAC patients compared to the non-PDAC participants and correlated with worse survival in two independent PDAC cohorts. Moreover, sTIM-3 exhibited a valuable role in diagnosis of PDAC, with sensitivity and specificity of about 80% in the training and validation groups, respectively. We further established an integrated model by combining sTIM-3 and carbohydrate antigen 19- 9 (CA19-9), which had an area under the curve of 0.974 and 0.992 in training and validation cohorts, respectively. Our RNA-seq and proteomic results provide valuable insight for understanding the immune cell composition of peripheral blood of patients with PDAC.

CD56 and Tim-3 molecule expression in different monocyte subsets in physiological pregnancy

Monocytes play an important role in the systemic immune defense against pathogens and maintaining physiological pregnancy. During pregnancy peripheral monocytes migrate into the decidua and form the pool of decidual macrophages which participate in the formation and development of placental tissues. The population of peripheral blood monocytes is phenotypically and functionally heterogeneous. In humans, there are different monocyte subsets depending on the expression of CD14 and CD16. CD56-positive monocytes are found in healthy women. Their number is positively correlated with body mass index, body fat. Tim-3 (T cell Ig and mucin domain-containing protein 3) expression is observed in peripheral monocytes during pregnancy. It is known that peripheral monocyte functions effectively change at pregnancy to form the immune tolerance at the maternal-fetal interface and the systemic immune defense against pathogens. However, the monocyte phenotype shift during pregnancy remain poorly understood. Therefore, the aim of the study was to evaluate the CD56 and Tim-3 expressions in monocyte subsets in human pregnancy. Peripheral blood mononuclear cells were isolated from peripheral blood of pregnant women (gestational age 29 weeks (28-31) by density gradient centrifugation and analyzed by flow cytometry. Peripheral blood of healthy non-pregnant fertile women (in follicular phase of the menstrual cycle) aged 21-29 years was studied as control. Pregnant women had a lower percentage of classical CD14hi/CD16- monocytes in comparison with non-pregnant. The percentages of intermediate (CD14hi/CD16+) and non-classical (CD14low/CD16+) monocytes did not change. The CD56 molecule expression was observed in all monocyte subsets in pregnant and non-pregnant women. Pregnant women had a higher percentage of CD56-positive classical (CD14hiCD16-) and non-classical (CD14lowCD16+) monocytes than non-pregnant. The percentage of CD56-positive intermediate (CD14hiCD16+) monocytes did not change. The percentages of double-positive CD56+Tim-3+ classical (CD14hiCD16-) and non-classical (CD14lowCD16+) monocytes were increased in pregnant women. The numbers of double-positive CD56+Tim-3+intermediate (CD14hiCD16+) monocytes did not change. Thus, the CD56 and Tim-3 expressions in different monocyte subsets were changed in human pregnancy.

Upregulating the Expression of F-CAR-T Cells by Increasing theAmount of Tcm Cells in F-CAR-T Cells

CAR-T cells are most known for protecting one’s immune system by attacking cancer cells. Usually, it is infused intothe patient’s body to provide treatment. When comparing F-CAR-T cells with C-CAR-T cells, F-CAR-T cells typicallyhave more Tscm cells. Thus, this study investigates the effect of upregulating Tscm cells in F-CAR-T cells using FACSfor CD45RO- and CD62L+. The experiments will use the expression of Tim3, LAG3, and PD1 and measure if F-CAR-Tcells can kill CD19-expressing RAJI cells by FACS for CD19 and Annexin V/PI through various durations and withvarious numbers of injected CarT. Then Tscm cells are measured by FACS for CD45RO- and CD62L+. Moreover,two special diets will be used as an experimental control to regulate the increase and decrease of Tscm cells. There aretwo most possible results: (1) Both the Tscm receptor and the diet will have a positive effect in terms of upregulatingTscm cells in F-CAR-T cells; (2) Both the mouse peritoneal macrophage and the diet will inhibit the growth of Tscmcells. The result of our study will provide important information for the future clinical trial of Tscm cell application andimprove CAR-T cells’ persistence. Future studies should focus on shortening the CAR-T cells manufacturing processesas well as exploring more applicable usages of Tscm cells in detail