T-cell Immunoglobulin Mucin-3 Expression Research Articles

Identification and validation of a prognostic risk-scoring model based on the level of TIM-3 expression in acute myeloid leukemia

Acute myeloid leukemia (AML) is characterized by an unfavorable prognosis due to the presence of self-renewing leukemic stem cells (LSCs). The presence of T-cell immunoglobulin mucin-3 (TIM-3) on the surface of LSCs has been observed in various types of human AML, exerting an impact on the prognostic outcome. Exploring the hub genes associated with varying levels of TIM-3 expression offers a valuable approach to enhance our understanding of the underlying mechanisms involving TIM-3 and to identify potential prognostic indicators in AML. Nevertheless, to date, no research studies have reported a prognostic model that relies on the level of TIM-3 expression. In our study, we screen the hub-genes based on different expression level of TIM-3 through WGCNA. The prognostic risk-scoring model was constructed based on hub-genes. The results show the risk prognostic model has extraordinary ability to predict prognosis in both the training and validation sets. The high-risk group present poor prognosis with mutation of NPM1, TP53 (Multiple Hit) and FLT3(multiple hit), while IDH2 (Missense Mutation), MUC16 (Multiple Hit/Missense Mutation) occur mutation in low-risk group presenting favorite prognosis than high-risk group. Leukocyte cell–cell adhesion, regulation of T cell activation and I-κB kinase/NF-κB signaling enriched in high-risk group, involving in HSCs or LSCs anchoring to BM, which implicated in LSCs survival and chemotherapy resistance. B7-H3 (CD276) and CD276 would be the potential immune targets in high-risk group. The risk score model may help in distinguishing immune and molecular characteristics, predicting patient outcomes.

Expression of Galectin-9-related immune checkpoint receptors in B-cell acute lymphoblastic leukemia.

Exhausted CD8+ T-cells over-express immune checkpoint receptors (ICRs), which interact with their ligands on malignant cells. However, some ICRs have been reported to be expressed on both T-cells and tumor cells, including V-domain immunoglobulin suppressor of T cell activation (VISTA), Galectin-9, and T-cell immunoglobulin mucin-3 (TIM-3). We aimed to evaluate the mRNA expression of VISTA, Galectin-9, and TIM-3 on CD8+ T-cells and leukemic cells in B-cell acute lymphoblastic leukemia (B-ALL). Samples were obtained from 26 untreated B-ALL patients and 25 control subjects. CD8+ T-cells were isolated using Magnetic Activated Cell Sorting (MACS). Relative gene expression was then evaluated by qRT-PCR with specific primers for VISTA, Galectin-9, and TIM-3. Also, the mRNA expression profile and clinical data of 154 B-ALL patients were obtained from the TARGET. mRNA expression of Galectin-9 on CD8+ T-cells in B-ALL patients was significantly lower than those in the control group (P=0.043), while VISTA expression was not significantly different between the two study groups (P=0.259). Besides, TIM-3 expression was significantly higher in B-ALL patients than in the control group (P<0.001). Also, data obtained from TARGET showed that the relapse incidence was not significantly different between patients with high and low expression of Galectin-9 and TIM-3 in leukemic cells (P=0.360 and P=0.655, respectively). Collectively, gene expression results suggest an important role for TIM-3, but not VISTA and Galectin-9, in B-ALL and it seems that TIM-3 could be a candidate for immune checkpoint therapy.

TIM-3 signaling hijacks the canonical Wnt/β-catenin pathway to maintain cancer stemness in human acute myeloid leukemia

Acute myeloid leukemia (AML) is one of the most common hematologic malignancies derived from self-renewing and highly propagating leukemic stem cells (LSCs). We have previously identified T-cell immunoglobulin mucin-3 (TIM-3) as an AML LSC-specific surface molecule by comparing the gene expression profiles of LSCs and hematopoietic stem cells (HSCs). TIM-3 expression clearly discriminates LSCs from HSCs within the CD34+CD38- stem cell fraction. Furthermore, AML cells secrete galectin-9 (Gal-9, a TIM-3 ligand) in an autocrine manner, resulting in constitutive TIM-3 signaling, which maintains LSC self-renewal capacity through β-catenin accumulation. In this study, we investigated the LSC-specific mechanisms of TIM-3 signaling. We found that TIM-3 signaling drove the canonical Wnt pathway, which was independent of Wnt ligands, to maintain cancer stemness in LSCs. Gal-9 ligation activated the cytoplasmic Src homology 2 (SH2) binding domain of TIM-3 to recruit hematopoietic cell kinase (HCK), a Src family kinase that is highly expressed in LSCs. HCK phosphorylated p120-catenin to promote the formation of the LDL receptor-related protein 6 (LRP6) signalosome, hijacking the canonical Wnt pathway. This TIM-3/HCK/p120-catenin axis was employed principally in immature LSCs compared to TIM-3-expressing exhausted T-cells.

Differential expression of HAVCR2 gene in pan-cancer: A potential biomarker for survival and immunotherapy.

T-cell immunoglobulin mucin 3 (TIM-3) has emerged as a promising immune checkpoint target in cancer therapy. However, the profile of the hepatitis A virus cellular receptor 2 (HAVCR2) gene, encoding TIM-3 expression, is still obscure, along with its role in cancer immunity and prognosis. This study comprehensively analyzed HAVCR2 expression patterns in pan-cancer and underlined its potential value for immune checkpoint inhibitor-based immunotherapy. Our results displayed that HAVCR2 was differentially expressed and closely corresponded to survival status in pan-cancer. More importantly, the HAVCR2 expression level was also significantly related to cancer immune infiltration, immune checkpoint genes, and immune marker genes. Enrichment analyses implicated HAVCR2-associated terms in cancer, including immunity, metabolism, and inflammation. Our study demonstrated that HAVCR2 could participate in differing degrees of immune infiltration in tumorigenesis. The highlights of the HAVCR2 pathway revealed that TIM-3 could function as both a biomarker and clinical target to improve the therapeutic efficacy of immunotherapy.

Prognostic significance of the expression levels of T-cell immunoglobulin mucin-3 and its ligand galectin-9 for relapse-free survival in triple-negative breast cancer.

T-cell immunoglobulin mucin-3 (TIM-3) expressed at the T-cell surface acts as an immune checkpoint when bound by its ligand galectin-9. Blockade of immunosuppression by the TIM3/galectin-9 signalling pathway may offer novel therapeutic approaches for cancer immunotherapy. Consistent with this, TIM-3 expression is associated with poorer prognosis in several different types of cancer, possibly as a result of suppression of anticancer immunosurveillance. A number of studies have now documented some effectiveness of immune checkpoint blockade even in triple-negative breast cancer (TNBC), which is highly aggressive. However, clinical responses are relatively weak, suggesting that several different pathways may be involved. In this context, the role of the TIM-3/galectin-9 checkpoint in TNBC is not clear. The present study aimed to determine the clinicopathological significance of TIM-3 and galectin-9 expression in this cancer. To this end, 62 patients with TNBC undergoing surgery at Kansai Medical University Hospital (Hirakata, Japan), but not given neoadjuvant chemotherapy, were examined. Tissue microarrays were employed for immunohistochemistry to analyse associations of TIM-3 and galectin-9 expression and their impact on relapse-free survival relative to other poor prognostic risk factors. Galectin-9 expression was detected in 49 of 62 patient samples (79%), and TIM-3 in 30 of them (48.4%). Tumour cell galectin-9 expression was associated with a more favourable prognosis (P=0.027) as was TIM-3 expression on tumour-infiltrating lymphocytes (P=0.007). Multivariate analysis indicated that galectin-9- and TIM-3-double-positivity was significantly associated with a more favourable prognosis compared with galectin-9 and/or TIM-3 negativity (P=0.044). Thus, the TIM-3/galectin-9 signalling pathway may impact anticancer immune reactions in the tumour microenvironment of patients with TNBC. Further investigation will be necessary to determine the molecular mechanisms underlying these relationships.

The characteristics and novel clinical implications of CD4+CXCR5+Foxp3+ follicular regulatory T cells in breast cancer.

BackgroundFollicular regulatory T cells (Tfr) are a subset of regulatory T cells (Tregs) that suppress the humoral immune response in the germinal center. They are associated with increased rates of disease stabilization and decreased autoantibody levels in a variety of tumor and autoimmune diseases. The binding of T-cell immunoglobulin mucin 3 (TIM-3) and its ligand on the surface of Tfr cells could result in the depletion of T lymphocytes and the termination of the immune response mediated by helper T cell 1. However, the role of Tfr cells in breast cancer (BC) remains unclear.MethodsIn this study, we detected the expression of CD4+CXCR5+Foxp3+Tfr cells in the peripheral blood of 35 BC patients and 30 healthy control patients by flow cytometry, and analyzed the relationship between Tfr cells and the clinical characteristics of patients. In addition, the expression of TIM-3 on the surface of Tfr cells in 6 triple-negative BC (TNBC) patients was further investigated using mass spectrometry.ResultsWe found a significant increase in Tfr cells in BC patients compared to healthy control patients (23.47%±9.70% vs. 10.99%±4.68%; P=0.001). Notably, the increase was more significant in early stage than advanced stage TNBC patients (28.52%±10.75% vs. 18.69%±5.19%; P=0.006), and there was a negative correlation between Tfr cells and serum lactate dehydrogenase (LDH) in early stage TNBC patients (r=−0.585; P=0.008). Additionally, we found that the expression of Tfr cells was higher in TNBC patients than luminal BC patients (28.25%±10.11% vs. 18.5%±8.15%; P=0.028); however, there was no significant difference in expression in hormone receptor positive (HR+) BC and hormone receptor negative (HR−) BC (P=0.141) patients. Notably, the surface of Tfr cells of TNBC patients had higher levels of TIM-3 expression than those of healthy control patients (3.93±0.92 vs. 2.65±0.15, respectively; t=−3.02; P<0.05), which the mass spectrometry showed were positively correlated with the intracellular Foxp3 expression of Tfr cells (r=0.82; P=0.036).ConclusionsOur results suggest that circulating Tfr cells and the expression of TIM-3 were significantly increased in BC patients, which were related to stage and histological type, and may be involved in the pathogenesis of BC.

Immune checkpoint markers and anti-CD20-mediated NK cell activation.

Anti-CD20 mAb is an effective therapy for most B-cell malignancies. Checkpoint blockade has been used to enhance T-cell-mediated antitumor response. Little is known about the biologic significance of immune checkpoints expressed by NK cells in anti-CD20-based therapy. To investigate the role of checkpoints in anti-CD20-mediated NK cell biology, Raji B-cell lymphoma cells, and PBMCs from normal donors were cocultured with rituximab (RTX), obinutuzumab (OBZ), or trastuzumab as a control mAb for between 20h and 9 d. RTX and OBZ induced a dose-dependent NK cell up-regulation of T-cell immunoreceptor with Ig and ITIM domain (TIGIT) and T-cell immunoglobulin mucin-3 (TIM3), but not PD1, CTLA4, or LAG3. Resting CD56dim NK had higher TIGIT and TIM3 expression than resting CD56bright NK although TIGIT and TIM3 were up-regulated on both subsets. NK cells with the CD16 158VV single nucleotide polymorphism had greater TIM3 up-regulation than did NK from VF or FF donors. TIGIT+ and TIM3+ NK cells degranulated, produced cytokines, and expressed activation markers to a greater degree than did TIGIT- or TIM3- NK cells. Blockade of TIGIT, TIM3, or both had little impact on RTX-induced NK cell proliferation, degranulation, cytokine production, or activation. Taken together, TIGIT and TIM3 can serve as markers for anti-CD20-mediated NK cell activation, but may not serve well as targets for enhancing the anti-tumor activity of such therapy.

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' (&gt; 90 %, n=7), 'intermediate' (61-90 %, n=26), and 'low' (&lt; 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&lt;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&lt;0.001)) in addition to pre-SCT disease status (non-hCR vs hCR: P&lt;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&lt;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.

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

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.

Germline TIM-3 Mutations Characterize Sub-Cutaneous Panniculitis T-Cell Lymphomas with Hemophagocytic Lymphohistiocytic Syndrome

IntroductionSub-cutaneous panniculitis T-cell lymphomas (SPTCL), a rare non-Hodgkin lymphoma, can be associated with hemophagocytic lymphohistiocytosis (HLH), a life-threatening activation of the immune system which adversely impacts survival. T-cell immunoglobulin mucin 3 (TIM-3) is a modulator of immune responses expressed on subgroups of T- and innate immune cells. In this work we describe the first germline variants associated with SPTCL, which are in the TIM-3 gene.MethodsWe sequenced 27 SPTCL cases to identify gene variants. We performed TIM-3 functional analysis on immune cells from patients and HEK293 cells engineered to overexpress wild-type or mutant TIM-3.ResultsWe identified homozygous, germline, loss-of-function, missense variants in highly conserved residues of TIM-3, namely p.Y82C and p.I97M in about 60% (16/27) of SPTCL cases. These samples were drawn from cases series across 3 continents. Patients with bi-allelic TIM-3 mutations were younger at diagnosis, and several had life-threatening HLH and severe disease course. TIM-3 mutations show specific geographic distribution. Y82C TIM-3 mutations occur on a founder chromosome in patients with East-Asian and Polynesian ancestry, while I97M TIM-3 is observed in Caucasians. Both variants induce protein misfolding and cytoplasmic retention of TIM-3. Loss of TIM-3 membrane expression in TIM-3 mutants abrogates the PD-1/PDL-1 checkpoint and prevents the termination of a Th1-immune response. In HEK293 cells, mutant TIM-3 was not expressed on the cell surface. Defective TIM-3 expression leads to persistent immune activation with increased production of inflammatory cytokines including TNF-alpha and IL-1beta by innate immune cells.ConclusionOur findings highlight HLH/SPTCL as a new genetic entity where loss of the TIM-3 immune checkpoint is associated with T-cell infiltration of adipose tissue and inflammasome activation. This is the first causative germline defect identified in SPTCL. While our findings indicate that TIM-3-mutant HLH/SPTCL benefit from immunomodulation, therapeutic repression of the TIM-3 checkpoint could have serious adverse consequences. DisclosuresPrince:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Evaluation of TIM-3+ LSCs Is a Highly Sensitive MRD Monitoring Strategy to Predict AML Relapse after Allogenic Stem Cell Transplantation

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 normal and malignant 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.50 AML patients who had been detected CD34+CD38-TIM-3+ LSCs in BM sample at least once before SCT were analyzed. All 50 patients underwent allo-SCT at Fukuoka Blood and Marrow Transplantation Group (FBMTG)-related hospitals from July 2015 to May 2018. 43 out of 50 patients achieved complete donor chimerism confirmed by short tandem repeat PCR (STR-PCR) at the time of engraftment (day 15-36), and these 43 patients was prospectively evaluated the frequencies of residual CD34+CD38-TIM-3+ LSCs using multi-color flow cytometry. Median age of the evaluated 43 AML patients at SCT was 44.8 years (28-67). 26 patients underwent SCT on disease and 17 patients at hematological CR. Relapse-free survival (RFS) was calculated from the time of SCT until last follow-up or documentation of relapse.In each timing (at diagnosis, relapse, engraftment, etc.), we tracked the frequencies of TIM-3+ cells within CD34+CD38- fraction in BM. Of note, the proportion of CD34+CD38- fraction was 0.016 [0.0056 - 0.0267] % of BMMNC at engraftment. Then, according to the frequency of TIM-3+ cells within CD34+CD38- fraction, we classified patients into 3 groups; ‘high’ (> 90 % of CD34+CD38- cells were positive for TIM-3, n=5), ‘intermediate’ (61-90 % of CD34+CD38- cells were positive for TIM-3, n=11), and ‘low’ (< 60 % of CD34+CD38- cells were positive for TIM-3, n=27). This classification revealed that the recurrence rate within the observation period (median 334.4 days) was 100 % (5 of 5) in ‘high’ group, 45.4 % (5 of 11) in ‘intermediate’ group and 14.8 % (4 of 27) in ‘low’ group. Relapse-free survival was 83 (72-92) days in ‘high’ group, 368 (56-869) days in ‘intermediate’ group and 482.6 (45-1029) days in ‘low’ group, respectively (p<0.01) (Figure 1). Of note, all of 5 patients in ‘high’ group exhibited hematological relapse within 100 days. Thus, our new classification using TIM-3 as LSC-specific marker could isolate the patients at high risk for early relapse in allo-SCT settings even if they were considered as hematological CR and maintained complete donor chimerism assessed by clinically available methods (three-color FACS, STR-PCR, FLT3-ITD status and fusion gene specific qPCR) at the same timepoint. It suggests that monitoring of LSCs using TIM-3 should be a more sensitive and versatile strategy to predict early relapse of AML than aberrant surface markers monitoring by multi-color FACS because conventional FACS did not detect MRD population at engraftment while our strategy detected (we could detect LSCs (CD34+CD38-TIM-3+ fraction) within BMMNC in 0.0053 [0 - 0.012] %), and, the cases whose aberrant markers could be traced were only 28 cases of 43 in this study.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 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 are involved in relapse. Evaluation of TIM-3+ LSCs by multi-color FACS should be a highly sensitive strategy to predict relapse in clinical allo-SCT settings, and might enable us to appropriately intervene to overcome the poor clinical outcomes of AML. [Display omitted] DisclosuresAkashi:Taiho Pharmaceutical: Research Funding; Novartis pharma: Research Funding; Pfizer: Research Funding; Kyowa Hakko Kirin: Research Funding, Speakers Bureau; Eisai: Research Funding; Celgene: Research Funding, Speakers Bureau; Astellas Pharma: Research Funding; sanofi: Research Funding; MSD: Research Funding; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Chugai Pharma: Research Funding; Eli Lilly Japan: Research Funding; Ono Pharmaceutical: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi-kasei: Research Funding.

The effects of resiquimod in an ovalbumin-induced allergic rhinitis model

Growing evidence indicates that the Toll-like receptor7/8(TLR7/8) agonist resiquimod (R848) is a potential inhibitor of type-2 immunity. However, the mechanisms mediating its therapeutic effects are not fully understood. This study investigated the effects of R848 on OVA-induced allergic rhinitis(AR) mice and the expression of IL-25, IL-33, TSLP, T-cell immunoglobulin mucin1 (TIM1) and T-cell immunoglobulin mucin3 (TIM3). BALB/c mice were intranasally sensitized and challenged with ovalbumin (OVA), and R848 was intraperitoneally injected into AR mice. Histological changes in the nasal mucosa were evaluated by hematoxylin and eosin (H & E) and Periodic Acid-Schiff (PAS) staining; cytokine levels in serum were measured with enzyme-linked immunosorbent assays (ELISAs);the mRNA expression levels of IFN-γ, IL-17 and Foxp3 in the spleen determined by quantitative real-time RT-PCR (qRT-PCR); the proportions of Th1, Th2, Th17, Treg and TIM3 + IFN-γ + Th1 cells in the spleen were assessed with flow cytometry; TIM1, TIM3 and IL-33 expression levels in the nasal mucosa were evaluated with immunofluorescence staining(IF).R848 alleviated the nasal allergic symptoms; reduced eosinophil cell infiltration, goblet cell hyperplasia in the nasal mucosa; reduced IL-13, IL-17, IL-25 and IL-33 levels in serum; upregulated the relative mRNA expression of IFN-γ and Foxp3, and downregulated the relative mRNA expression of IL-17 in the spleen; decreased Th2, Th17 and TIM3 + IFN-γ + Th1 cells ratios, increased the proportion of Th1 and Treg cells in the spleen; suppressed TIM1 and TIM3,but increased IL-33 expression in the nasal mucosa in OVA-induced AR mice. R848 suppresses IL-25, IL-33 released and TIM1, TIM3 expression, which may contribute to its anti-allergic effects.

T-cell immunoglobulin mucin-3 expression in invasive ductal breast carcinoma: Clinicopathological correlations and association with tumor infiltration by cytotoxic lymphocytes.

As a negative regulatory molecule, T-cell immunoglobulin and mucin domain-3 (Tim-3) is closely associated with tumor immunological tolerance. The aim of this study was to investigate Tim-3 expression in invasive ductal breast cancer (IDC), its effect on clinicopathological parameters and its association with cytotoxic lymphocyte infiltration. Tim-3 protein expression was measured in 150 paraffin-embedded IDC specimens and 100 paired normal breast tissue specimens by immunohistochemistry. It was demonstrated that the infiltration of the tumor by CD8+ T cells was significantly higher compared with that of normal tissue, and the Tim-3 expression on CD8+ T cells was higher in IDC tissue compared with that in normal tissue; the differences were statistically significant (both P-values=0.000). The median expression level of Tim-3 on tumor cells was significantly associated with clinicopathological parameters such as age, axillary lymph node metastasis and TNM stage (P=0.015, 0.001 and 0.027, respectively). The expression of Tim-3 on CD8+ T cells was correlated with lymph node metastasis, World Health Organization (WHO) grade and molecular classification (P=0.000, 0.004 and 0.000, respectively). Additionally, the number of tumor-infiltrating CD8+ T cells was associated with primary tumor size, lymph node metastasis, WHO grade, Ki-67 and molecular classification (P=0.017, 0.002, 0.007, 0.003 and 0.000, respectively). Thus, Tim-3 may promote the development and progression of breast cancer and affect the tumor microenvironment; thus, it may be used as an independent prognostic factor for IDC patients.

Decreased TIM-3 expression of peripheral blood natural killer cells in patients with severe aplastic anemia

Severe aplastic anemia (SAA) is an autoimmune disease characterized by severe pancytopenia and bone marrow failure. In our previous studies, we found natural killer (NK) cells were aberrant in SAA patients. T cell immunoglobulin mucin-3 (TIM-3), an important regulator of immunity, is widely detected on NK cells and may contribute as a marker of activation and maturation of NK cells. In this study, we found that SAA untreated patients had lower TIM-3 expression on NK cells and CD56dim NK subsets compared with normal controls, and were correlated with the severity of pancytopenia of SAA. After immunosuppressive therapy (IST), TIM-3 expression recovered to normal level. Moreover, the TIM-3 mRNA levels in NK cells significantly increased in SAA remission patients after IST. We inferred that low expression of TIM-3 on NK cells might lead to NK cells dysfunction and involve in the progress of bone marrow failure in SAA.

T-cell immunoglobulin mucin 3 blockade drives an antitumor immune response in head and neck cancer.

T‐cell immunoglobulin mucin 3 (TIM3) contributes to immune suppression during progression of many cancers, but the precise role of TIM3 in head and neck squamous cell carcinoma (HNSCC) is not clearly understood. In this study, we report that TIM3 expression was significantly up‐regulated in patients with HNSCC and associated with lymph node metastasis. Additionally, TIM3 expression was increased in patients with recurrent HNSCC and patients with preradiotherapy or prechemotherapy. We also characterized CD8+ T cells and CD11b+ CD33+ myeloid‐derived suppressor cells (MDSCs) in human HNSCC, and found that their expression was positively correlated with TIM3 expression. To determine the underlying mechanism of TIM3 in immune response during HNSCC progression, we utilized the Tgfbr1/Pten 2cKO HNSCC mouse model with TIM3 overexpression. Treatment with anti‐TIM3 monoclonal antibody effectively suppressed tumor growth through restoring effector T‐cell function by targeting CD4+ TIM3+ cells and CD8+ TIM3+ cells and decreasing MDSCs. Our findings demonstrate TIM3 expression in patients with HNSCC and suggest anti‐TIM3 immunotherapy as a novel therapeutic approach for effective treatment of HNSCC.

Combination Therapy with Anti-PD-1, Anti-TIM-3, and Focal Radiation Results in Regression of Murine Gliomas.

Checkpoint molecules like programmed death-1 (PD-1) and T-cell immunoglobulin mucin-3 (TIM-3) are negative immune regulators that may be upregulated in the setting of glioblastoma multiforme. Combined PD-1 blockade and stereotactic radiosurgery (SRS) have been shown to improve antitumor immunity and produce long-term survivors in a murine glioma model. However, tumor-infiltrating lymphocytes (TIL) can express multiple checkpoints, and expression of ≥2 checkpoints corresponds to a more exhausted T-cell phenotype. We investigate TIM-3 expression in a glioma model and the antitumor efficacy of TIM-3 blockade alone and in combination with anti-PD-1 and SRS. C57BL/6 mice were implanted with murine glioma cell line GL261-luc2 and randomized into 8 treatment arms: (i) control, (ii) SRS, (iii) anti-PD-1 antibody, (iv) anti-TIM-3 antibody, (v) anti-PD-1 + SRS, (vi) anti-TIM-3 + SRS, (vii) anti-PD-1 + anti-TIM-3, and (viii) anti-PD-1 + anti-TIM-3 + SRS. Survival and immune activation were assessed. Dual therapy with anti-TIM-3 antibody + SRS or anti-TIM-3 + anti-PD-1 improved survival compared with anti-TIM-3 antibody alone. Triple therapy resulted in 100% overall survival (P < 0.05), a significant improvement compared with other arms. Long-term survivors demonstrated increased immune cell infiltration and activity and immune memory. Finally, positive staining for TIM-3 was detected in 7 of 8 human GBM samples. This is the first preclinical investigation on the effects of dual PD-1 and TIM-3 blockade with radiation. We also demonstrate the presence of TIM-3 in human glioblastoma multiforme and provide preclinical evidence for a novel treatment combination that can potentially result in long-term glioma survival and constitutes a novel immunotherapeutic strategy for the treatment of glioblastoma multiforme. Clin Cancer Res; 23(1); 124-36. ©2016 AACR.

Decreased TIM-3 Expression Level of Peripheral Blood Natural Killer Cells in Severe Aplastic Anemia

Severe aplastic anemia (SAA) is a life threatening disease characterized by severe pancytopenia and bone marrow failure. Natural killer (NK) cells are large granular lymphocytes which are one important component of the innate immune system and play a core role in regulation of adaptive immunity. T cell immunoglobulin mucin-3 (TIM-3), a member of the TIM family, appears to play an important negative regulatory role in T cells initial. Now, TIM-3 is widely detected on NK cells, and may contribute as a marker for activation and maturation of NK cells. Our previous studies have confirmed that the decrease of total NK cells, and CD56bright, CD56dim NK cell subsets and the higher expressions of NKp46 and perforin on NK cells may cause the over-function of T lymphocytes and thus lead to hematopoiesis failure in SAA. But, the expression of TIM-3 on NK cells in patients with SAA was still unknown.In this study, we investigated the expression of TIM-3 and its mRNA on NK cells in peripheral blood of untreated and recovered SAA patients by flow cytometry and Real-time PCR. Results showed that the expression of TIM-3 on peripheral blood NK cells in SAA patients before IST was (63.57±12.14) %, which was significantly decreased than that in normal controls (85.62±9.03) % ( p<0.01). The expression of TIM-3 on CD56dim NK cells was (66.41±11.74) % and (83.83±1.59) % separately in SAA patients before IST and normal controls. TIM-3 expressed in SAA patients before IST was lower than that in normal controls ( p<0.01). We also measured TIM-3 expressed on the surface of CD56bright NK cells, but the result showed that there was no statistical difference between SAA patients before IST (61.11±24.99%) and normal controls (62.64±12.06%) (p>0.05). More interesting, the expression of TIM-3 on NK cells was (75.88±12.83) % in SAA patients after IST, which was significantly increased than that in SAA patients before IST, and was no difference with normal controls. As well, we found TIM-3 expression on both CD56dim NK cells and CD56bright NK cells in SAA patients after IST also has a rising trend compared with SAA patients before IST. However, these differences had no statistical significance. Further, we detected TIM-3 mRNA expression in NK cells isolated from peripheral blood. TIM-3 mRNA expression in peripheral blood NK cells from newly diagnosis SAA patients, recovering SAA patients and normal controls was evaluated, respectively. The relative TIM-3 mRNA expression was significantly increased in NK cells in SAA patients after IST compared with SAA patients before IST and normal controls, and this difference had statistical significance. Meanwhile, the relative TIM-3 mRNA expression was lower in NK cells in SAA patients before IST compared with normal controls. However, the difference had no statistical significance. In SAA patients, the expression of TIM-3 on NK cells was positively correlated with the level of WBC (r=0.685, p=0.000), proportion of neutrophil (r=0.825, p=0.000), and proportion of reticulocyte in peripheral blood (r=0.465, p=0.029). And it was negatively correlated with the proportion of lymphocyte in peripheral blood (r=-0.802, p=0.000).According to our series studies, we hypothesize that the lower numbers and dysfunction of NK cells induce the failure of suppressing the over function of DC cells and T cells, that lead damage to healthy hematopoietic stem cells and the onset of SAA. DisclosuresNo relevant conflicts of interest to declare.

Ovarian carcinoma-infiltrating regulatory T cells were more potent suppressors of CD8(+) T cell inflammation than their peripheral counterparts, a function dependent on TIM3 expression.

Ovarian carcinoma is one of the most severe cancers in women, with a high relapse rate and limited secondary treatment options. To assist research in novel treatment technologies, including CD8(+) T cell-base immunotherapy, we examined the effect of tumor-infiltrating regulatory T cells (Tregs) in inhibiting CD8(+) T cell inflammation. We found that compared to their peripheral blood counterparts, tumor-infiltrating Tregs exhibited more potent inhibitory function, which was associated with higher interleukin 10 (IL-10) production in tumor-infiltrating Tregs. Blockade of T cell immunoglobulin mucin 3 (TIM3), a regulatory molecule overrepresented on tumor-infiltrating Tregs, had significantly reverted Treg-mediated suppression. Moreover, expression of TIM3 on tumor-infiltrating Tregs was directly correlated with tumor size. Together, our results demonstrated that ovarian tumor-infiltrating Treg cells were more immunosuppressive than their peripheral blood counterparts in a TIM3-dependent fashion.

Prognostic implication of TIM-3 in clear cell renal cell carcinoma

Clear cell renal carcinoma (ccRCC) is the most common tumor of the kidney.in adults. The prognosis of ccRCC remains unsatisfactory. Recently, T cell immunoglobulin mucin-3 (TIM-3), a novel transmembrane protein, has been implicated in tumor biology. Its role in ccRCC remains unknown. The aim of this study was to investigate the roles of TIM-3 as a prognostic marker in patients with ccRCC.TIM-3 protein expression was determined by immunohistochemistry and Western blot from 137 ccRCC tumor samples and adjacent normal renal tissues. We performed also the cell proliferation assay using 3-(4,5-dimethylthiazol -2yl)-2,5-diphenyl-2H tetrazolium bromide (MTT) and cell invasion assay. The effects of small interfering RNA (siRNA)-mediated knockdown of TIM-3 (TIM-3 siRNA) in two human ccRCC cell lines were evaluated. TIM-3 expression was higher in ccRCC tissue than in the adjacent normal renal tissue (P<0.001). High TIM-3 expression was an independent predictor of both cancer-specific survival and progression-free survival. TIM-3 protein was expressed in both ccRCC cell lines. Knockdown of TIM-3 suppressed the proliferation and invasion capacity of ccRCC cell lines.TIM-3 expression was associated with poor prognosis in ccRCC. Taken together, TIM-3 is a potential prognostic marker in ccRCC.