Association of tuberculosis risk with genetic polymorphisms of the immune checkpoint genes PDCD1, CTLA-4, and TIM3.

The immune checkpoint proteins were reported to involve to host resistance to Mycobacteria tuberculosis (Mtb). Here, we evaluated 11 single nucleotide polymorphisms (SNPs) in PDCD1, CTLA4, and HAVCR2 genes between participants with and without TB infection. Genomic DNA isolated from 285 patients with TB and 270 controls without TB infection were used to perform the genotyping assay. Odds ratios were used to characterize the association of 11 SNPs with TB risk. In this study, the various genotypes of the 11 SNPs did not differ significantly in frequency between the non-TB and TB groups. When patients were stratified by sex, however, men differed significantly from women in genotype frequencies at HAVCR2 rs13170556. Odds ratios indicated that rs2227982, rs13170556, rs231775, and rs231779 were sex-specifically associated with TB risk. In addition, the combinations of rs2227982/rs13170556 GA/TC in men and the A-C-C haplotype of rs231775-rs231777-rs231779 in women were significantly associated with TB risk. Our results indicate that rs2227982 in PDCD1 and rs13170556 in HAVCR2 are associated with increased TB susceptibility in men and that the CTLA4 haplotype appears protective against TB in women.

Expression patterns of novel immunotherapy targets in intermediate- and high-grade lung neuroendocrine neoplasms.

BACKGROUND: Advancements in immunotherapeutic approaches only had a modest impact on the therapy of lung neuroendocrine neoplasms (LNENs). Our multicenter study aimed to investigate the expression patterns of novel immunotherapy targets in intermediate- and high-grade LNENs. METHODS: The expressions of V-domain Ig suppressor of T cell activation (VISTA), OX40L, Glucocorticoid-induced TNF receptor (GITR), and T cell immunoglobulin and mucin domain 3 (TIM3) proteins were measured by immunohistochemistry in surgically resected tumor samples of 26 atypical carcinoid (AC), 49 large cell neuroendocrine lung cancer (LCNEC), and 66 small cell lung cancer (SCLC) patients. Tumor and immune cells were separately scored. RESULTS: Tumor cell TIM3 expression was the highest in ACs (p < 0.001), whereas elevated tumor cell GITR levels were characteristic for both ACs and SCLCs (p < 0.001 and p = 0.011, respectively). OX40L expression of tumor cells was considerably lower in ACs (vs. SCLCs; p < 0.001). Tumor cell VISTA expression was consistently low in LNENs, with no significant differences across histological subtypes. ACs were the least immunogenic tumors concerning immune cell abundance (p < 0.001). Immune cell VISTA and GITR expressions were also significantly lower in these intermediate-grade malignancies than in SCLCs or in LCNECs. Immune cell TIM3 and GITR expressions were associated with borderline prognostic significance in our multivariate model (p = 0.057 and p = 0.071, respectively). CONCLUSIONS: LNEN subtypes have characteristic and widely divergent VISTA, OX40L, GITR, and TIM3 protein expressions. By shedding light on the different expression patterns of these immunotherapy targets, the current multicenter study provides support for the future implementation of novel immunotherapeutic approaches.

[Relationship Between Tim-3 and Galectin-9 Expression Levels, Clinical Pathological Characteristics, and Prognosis in Patients After Radical Resection of Colorectal Cancer]. / ç»“ç›´è‚ ç™Œæ ¹æ²»æœ¯åŽæ‚£è€ Tim-3、galectin-9è¡¨è¾¾æ°´å¹³ä¸Žå ¶ä¸´åºŠç— ç†ç‰¹å¾åŠé¢„åŽçš„å ³ç³».

Objective: Some colorectal cancer patients still face high recurrence rates and poor prognoses even after they have undergone the surgical treatment of radical resection. Identifying potential biochemical markers and therapeutic targets for the prognostic evaluation of patients undergoing radical resection of colorectal cancer is crucial for improving their clinical outcomes. Recently, it has been reported that the T cell immunoglobulin and mucin domain protein 3 (Tim-3) and its ligand galactose lectin 9 (galectin-9) play crucial roles in immune dysfunction caused by various tumors, such as colorectal cancer. However, their expressions, biological functions, and prognostic value in colorectal cancer are still unclear. This study aims to investigate the relationship between Tim-3 and galectin-9 expression levels and the clinicopathological characteristics and prognosis of patients undergoing radical resection of colorectal cancer. Methods: A total of 171 patients who underwent radical resection of colorectal cancer at Chengdu Fifth People's Hospital between February 2018 and March 2019 were selected. Immunohistochemistry was performed to assess the expression levels of Tim-3 and galectin-9 in the cancer tissue samples and the paracancerous tissue samples of the patients. The relationship between Tim-3 and galectin-9 expression levels and the baseline clinical parameters of the patients was analyzed accordingly. Kaplan-Meier analysis was performed to assess the association between Tim-3 and galectin-9 expression levels and the relapse-free survival (RFS) and the overall survival (OS) of colorectal cancer patients. Cox regression analysis was conducted to identify factors associated with adverse prognosis in the patients. Results: The immunohistochemical results showed that the high expression levels of Tim-3 and galectin-9 were observed in 70.18% (120/171) and 32.16% (55/171), respectively, of the colorectal cancer tissues, whereas the low expression levels were 29.82% (51/171) and 67.84% (116/171), respectively. Furthermore, the expression score of Tim-3 was significantly higher in colorectal cancer tissues than that in the paracancerous tissues, while the expression score of galectin-9 was lower than that in the paracancerous tissues (P<0.05). Further analysis revealed that the expression of Tim-3 and galectin-9 was associated with the depth of tumor infiltration, vascular infiltration, and clinical staging (P<0.05). During the follow-up period of 14-63 months, 7 out of 171 patients were lost to follow-up. Among the remaining patients, 49 and 112 cases presented abnormally low expression of Tim-3 and galectin-9, respectively, whereas 115 and 52 cases presented high expression of Tim-3 and galectin-9, respectively. Kaplan-Meier survival analysis demonstrated that patients with high Tim-3 expression in colorectal cancer tissues had significantly lower RFS and OS than those with low expression did (RFS: log-rank=22.66, P<0.001; OS: log-rank=19.71, P<0.001). Conversely, patients with low galectin-9 expression had significantly lower RFS and OS than those with high expression did (RFS: log-rank=19.45, P<0.001; OS: log-rank=22.24, P<0.001). Cox multivariate analysis indicated that TNM stage Ⅲ (HR=2.26, 95% CI: 1.20-5.68), high expression of Tim-3 (HR=0.80, 95% CI: 0.33-0.91), and low expression of galectin-9 (HR=1.80, 95% CI: 1.33-4.70) were independent risk factors affecting RFS and OS in patients (P<0.05). Conclusion: Aberrant expression of Tim-3 and galectin-9 is observed in colorectal cancer tissues. High expression of Tim-3 and low expression of galectin-9 are closely associated with adverse clinico-pathological characteristics and prognosis. They are identified as independent influencing factors that may trigger adverse prognostic events in patients. These findings suggest that Tim-3 and galectin-9 have potential as new therapeutic targets and clinical indicators.

High frequencies of alpha common cold coronavirus/SARS-CoV-2 cross-reactive functional CD4+ and CD8+ memory T cells are associated with protection from symptomatic and fatal SARS-CoV-2 infections in unvaccinated COVID-19 patients.

Background: Cross-reactive SARS-CoV-2-specific memory CD4+ and CD8+ T cells are present in up to 50% of unexposed, pre-pandemic, healthy individuals (UPPHIs). However, the characteristics of cross-reactive memory CD4+ and CD8+ T cells associated with subsequent protection of asymptomatic coronavirus disease 2019 (COVID-19) patients (i.e., unvaccinated individuals who never develop any COVID-19 symptoms despite being infected with SARS-CoV-2) remains to be fully elucidated. Methods: This study compares the antigen specificity, frequency, phenotype, and function of cross-reactive memory CD4+ and CD8+ T cells between common cold coronaviruses (CCCs) and SARS-CoV-2. T-cell responses against genome-wide conserved epitopes were studied early in the disease course in a cohort of 147 unvaccinated COVID-19 patients who were divided into six groups based on the severity of their symptoms. Results: Compared to severely ill COVID-19 patients and patients with fatal COVID-19 outcomes, the asymptomatic COVID-19 patients displayed significantly: (i) higher rates of co-infection with the 229E alpha species of CCCs (α-CCC-229E); (ii) higher frequencies of cross-reactive functional CD134+CD137+CD4+ and CD134+CD137+CD8+ T cells that cross-recognized conserved epitopes from α-CCCs and SARS-CoV-2 structural, non-structural, and accessory proteins; and (iii) lower frequencies of CCCs/SARS-CoV-2 cross-reactive exhausted PD-1+TIM3+TIGIT+CTLA4+CD4+ and PD-1+TIM3+TIGIT+CTLA4+CD8+ T cells, detected both ex vivo and in vitro. Conclusions: These findings (i) support a crucial role of functional, poly-antigenic α-CCCs/SARS-CoV-2 cross-reactive memory CD4+ and CD8+ T cells, induced following previous CCCs seasonal exposures, in protection against subsequent severe COVID-19 disease and (ii) provide critical insights into developing broadly protective, multi-antigen, CD4+, and CD8+ T-cell-based, universal pan-Coronavirus vaccines capable of conferring cross-species protection.

TIM-3+ CD8 T cells with a terminally exhausted phenotype retain functional capacity in hematological malignancies.

Chronic antigen stimulation is thought to generate dysfunctional CD8 T cells. Here, we identify a CD8 T cell subset in the bone marrow tumor microenvironment that, despite an apparent terminally exhausted phenotype (TPHEX), expressed granzymes, perforin, and IFN-γ. Concurrent gene expression and DNA accessibility revealed that genes encoding these functional proteins correlated with BATF expression and motif accessibility. IFN-γ+ TPHEX effectively killed myeloma with comparable efficacy to transitory effectors, and disease progression correlated with numerical deficits in IFN-γ+ TPHEX. We also observed IFN-γ+ TPHEX within CD19-targeted chimeric antigen receptor T cells, which killed CD19+ leukemia cells. An IFN-γ+ TPHEX gene signature was recapitulated in TEX cells from human cancers, including myeloma and lymphoma. Here, we characterize a TEX subset in hematological malignancies that paradoxically retains function and is distinct from dysfunctional TEX found in chronic viral infections. Thus, IFN-γ+ TPHEX represent a potential target for immunotherapy of blood cancers.

Galectin-9, a pro-survival factor inducing immunosuppression, leukemic cell transformation and expansion.

Leukemia is a malignancy of the bone marrow and blood originating from self-renewing cancerous immature blast cells or transformed leukocytes. Despite improvements in treatments, leukemia remains still a serious disease with poor prognosis because of disease heterogeneity, drug resistance and relapse. There is emerging evidence that differentially expression of co-signaling molecules play a critical role in tumor immune evasion. Galectin-9 (Gal-9) is one of the key proteins that leukemic cells express, secrete, and use to proliferate, self-renew, and survive. It also suppresses host immune responses controlled by T and NK cells, enabling leukemic cells to evade immune surveillance. The present review provides the molecular mechanisms of Gal-9-induced immune evasion in leukemia. Understanding the complex immune evasion machinery driven by Gal-9 expressing leukemic cells will enable the identification of novel therapeutic strategies for efficient immunotherapy in leukemic patients. Combined treatment approaches targeting T-cell immunoglobulin and mucin domain-3 (Tim-3)/Gal-9 and other immune checkpoint pathways can be considered, which may enhance the efficacy of host effector cells to attack leukemic cells.

Deletion of the TMEM30A gene enables leukemic cell evasion of NK cell cytotoxicity.

Natural killer (NK) cell immunotherapy has gained attention as a promising strategy for treatment of various malignancies. In this study, we used a genome-wide CRISPR screen to identify genes that provide protection or susceptibility to NK cell cytotoxicity. The screen confirmed the role of several genes in NK cell regulation, such as genes involved in interferon-γ signaling and antigen presentation, as well as genes encoding the NK cell receptor ligands B7-H6 and CD58. Notably, the gene TMEM30A, encoding CDC50A-beta-subunit of the flippase shuttling phospholipids in the plasma membrane, emerged as crucial for NK cell killing. Accordingly, a broad range of TMEM30A knock-out (KO) leukemia and lymphoma cells displayed increased surface levels of phosphatidylserine (PtdSer). TMEM30A KO cells triggered less NK cell degranulation, cytokine production and displayed lower susceptibility to NK cell cytotoxicity. Blockade of PtdSer or the inhibitory receptor TIM-3, restored the NK cell ability to eliminate TMEM30A-mutated cells. The key role of the TIM-3 - PtdSer interaction for NK cell regulation was further substantiated by disruption of the receptor gene in primary NK cells, which significantly reduced the impact of elevated PtdSer in TMEM30A KO leukemic cells. Our study underscores the potential significance of agents targeting the interaction between PtdSer and TIM-3 in the realm of cancer immunotherapy.

TIM3 activates the ERK1/2 pathway to promote invasion and migration of thyroid tumors.

BACKGROUND: This study aims to study the possible action mechanism of T-cell immunoglobulin and mucin domain 3 (TIM3) on the migratory and invasive abilities of thyroid carcinoma (TC) cells. METHODS: GSE104005 and GSE138198 datasets were downloaded from the GEO database for identifying differentially expressed genes (DEGs). Functional enrichment analysis and protein-protein interaction (PPI) analysis were performed on the common DEGs in GSE104005 and GSE138198 datasets. Subsequently, in order to understand the effect of a common DEG (TIM3) on TC cells, we performed in vitro experiments using FRO cells. The migratory and invasive abilities of FRO cells were detected by wound scratch assay and Transwell assay. Proteins expression levels of the phosphorylated (p)-extracellular signal-regulated kinase (ERK)1/2, matrix metalloproteinase-2 (MMP-2) and MMP-9 were determined via Western blotting after ERK1/2 inhibition in TIM3-NC group and TIM3-mimic group. RESULTS: 316 common DEGs were identified in GSE104005 and GSE138198 datasets. These DEGs were involved in the biological process of ERK1 and ERK2 cascade. TIM3 was significantly up-regulated in TC. In vitro cell experiments showed that TIM3 could promote migration and invasion of TC cells. Moreover, TIM3 may affect the migration, invasive abilities of TC cells by activating the ERK1/2 pathway. CONCLUSION: The above results indicate that TIM3 may affect the migratory and invasive of TC cells by activating the ERK1/2 pathway.

CircPVT1 promotes migration and invasion by regulating miR-490-5p/HAVCR2 axis in osteosarcoma cells.

Circular RNAs (circRNAs) play an important role in the progression of osteosarcoma. However, the precise function of circPVT1 in osteosarcoma remains elusive. This study aims to explore the molecular mechanism underlying the involvement of circPVT1 in osteosarcoma cells. We quantified circPVT1 expression using qRT-PCR in both control and osteosarcoma cell lines. To investigate the roles of circPVT1, miR-490-5p and HAVCR2 in vitro, we separately conducted overexpression and inhibition experiments for circPVT1, miR-490-5p and HAVCR2 in HOS and U2OS cells. Cell migration was assessed through wound healing and transwell migration assays, and invasion was measured via the Matrigel invasion assay. To elucidate the regulatory mechanism of circPVT1 in osteosarcoma, a comprehensive approach was employed, including fluorescence in situ hybridization, qRT-PCR, Western blot, bioinformatics, dual-luciferase reporter assay and rescue assay. CircPVT1 expression in osteosarcoma cell lines surpassed that in control cells. The depletion of circPVT1 resulted in a notable reduction in the in vitro migration and invasion of osteosarcoma cells. Mechanism experiments revealed that circPVT1 functioned as a miR-490-5p sequester, and directly targeted HAVCR2. Overexpression of miR-490-5p led to a significant attenuation of migration and invasion of osteosarcoma cells, whereas HAVCR2 overexpression had the opposite effect, promoting these abilities. Additionally, circPVT1 upregulated HAVCR2 expression via sequestering miR-490-5p, thereby orchestrating the migration and invasion in osteosarcoma cells. CircPVT1 orchestrates osteosarcoma migration and invasion by regulating the miR-490-5p/HAVCR2 axis, underscoring its potential as a promising therapeutic target for osteosarcoma.

The role of TIM-3 in sepsis: a promising target for immunotherapy?

Sepsis remains a significant cause of mortality and morbidity worldwide, with limited effective treatment options. The T-cell immunoglobulin and mucin domain-containing molecule 3 (TIM-3) has emerged as a potential therapeutic target in various immune-related disorders. This narrative review aims to explore the role of TIM-3 in sepsis and evaluate its potential as a promising target for immunotherapy. We discuss the dynamic expression patterns of TIM-3 during sepsis and its involvement in regulating immune responses. Furthermore, we examine the preclinical studies investigating the regulation of TIM-3 signaling pathways in septic models, highlighting the potential therapeutic benefits and challenges associated with targeting TIM-3. Overall, this review emphasizes the importance of TIM-3 in sepsis pathogenesis and underscores the promising prospects of TIM-3-based immunotherapy as a potential strategy to combat this life-threatening condition.

Galectin-9 and Tim-3 are upregulated in response to microglial activation induced by the peptide Amyloid-ß (25-35).

Galectins are a group of ß-galactoside-binding lectins associated with regulating immunological response. In the brains of AD patients and 5xFAD (familial AD) mice, galectin-3 (Gal-3) was highly upregulated and found to be expressed in microglia associated with Aß plaques. However, the participation of other galectins, specifically galectin-9 (Gal-9) and T-cell immunoglobulin and mucin domain 3 (Tim-3) receptors, are unknown in the inflammatory response. The experimental model of the Aß25-35 peptide will allow us to study the mechanisms of neuroinflammation and describe the changes in the expression of the Gal-9 and Tim-3 receptor. This study aimed to evaluate whether Aß25-35 peptide administration into the lateral ventricles of rats upregulated Gal-9 and Tim-3 implicated in the modulation of neuroinflammation. The vehicle or Aß25-35 peptide (1 µg/µL) was bilaterally administered into the lateral ventricles of the rat, and control group. After the administration of the Aß25-35 peptide, animals were tested for learning (day 29) and spatial memory (day 30) in the novel object recognition test (NOR). On day 31, hippocampus was examined for morphological changes by Nilss stain, biochemical changes by NO2 and MDA, immunohistochemical analysis by astrocytes (GFAP), microglia (Iba1), Gal-9 and Tim-3, and western blot. Our results show the administration of the Aß25-35 peptide into the lateral ventricles of rats induce memory impairment in the NOR by increases the oxidative stress and inflammatory response. This result is associated with an upregulation of Gal-9 and Tim-3 predominantly detected in the microglia cells of Aß25-35-treated rats with respect to the control group. Gal-9 and Tim-3 are upregulated in activated microglia that could modulate the inflammatory response and damage in neurodegenerative processes induced by the Aß25-35 peptide. Therefore, we suggest that Gal-9 and Tim-3 participate in the inflammatory process induced by the administration of the Aß25-35 peptide.

Germline HAVCR2/TIM-3 Checkpoint Inhibitor Receptor Deficiency in Recurrent Autoinflammatory Myocarditis.

Myocarditis can be caused by viral infection, drug reaction or general inflammatory condition. To provide understanding on inflammatory myocarditis, we describe clinical, genetic, and immunological properties of a young male patient who suffered from recurrent myocarditis episodes since the age of four years. Electrocardiography, troponin I/T, echocardiography, myocardial magnetic resonance imaging and histological findings were consistent with recurrent myocarditis episodes. Homozygous c.245 A > G p.Tyr82Cys pathogenic variant in Hepatitis A Virus Cellular Receptor 2 (HAVCR2) gene encoding T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) receptor was found. Peripheral blood mononuclear cells were collected when the patient was asymptomatic; CD4+ and CD8+ T lymphoblasts, CD56+ natural killer cells and CD14+ monocytes were negative for surface TIM-3 expression. In vitro, TLR4 mediated interleukin-1ß (IL-1ß) response was high after LPS/ATP stimulation. Clinical symptoms responded to IL-1 receptor antagonist anakinra. TIM-3 p.Tyr82Cys CD4+ and CD8+ T cell proliferation in vitro was unrestrained. Findings on IL-2, interferon gamma, regulatory T cells, signal transducer and activator of transcription (STAT) 1, 3 and 4 phosphorylation, and PD-1 and LAG-3 checkpoint inhibitor receptor analyses were comparable to controls. We conclude that TIM-3 deficiency due to homozygous HAVCR2 c.245 A > G p.Tyr82Cys pathogenic variant in the patient described here is associated with autoinflammatory symptoms limited to early onset recurrent febrile myocarditis. Excessive IL-1ß production and defective regulation of T cell proliferation may contribute to this clinical condition responsive to anakinra treatment.

Evaluation of changes in exhausted T lymphocytes and miRNAs expression in the different trimesters of pregnancy in pregnant women.

BACKGROUND: Throughout the three trimesters of a typical pregnancy, we looked at changes in the expression of miRNAs and exhausted T lymphocytes for this study. METHODS AND RESULTS: Fifty healthy subjects were included in this study. The frequency of exhausted T lymphocytes was measured in isolated PBMCs using flow cytometry. PD-1, TIM-3, and related miRNAs gene expression were assessed using qRT-PCR. The analyses revealed a significant decline in PD-1 and Tim-3 expression in PBMCs from RPL women (p = 0.0003 and p = 0.001, respectively). In addition, PD-1 and TIM-3 expression increased significantly in the 2nd trimester compared with the 1st trimester of healthy pregnant women (p < 0.0001 and p = 0.0002, respectively). PD-1 and TIM-3 expression was down-regulated in the 3rd trimester compared with the 1st and 2nd trimesters. In the present study, we demonstrated that TIM-3+/CD4+, TIM-3+/CD8+, PD-1+/CD4+, and PD-1+/CD8 + exhausted T lymphocytes increased in the circulation of women in the 2nd trimester compared to the 1st and 3rd trimester. In the 3rd trimester, the expression of miR-16-5p increased significantly (p < 0.0001). miR-125a-3p expression was down and upregulated in 2nd (p < 0.0001) and 3rd (p = 0.0007) trimesters compared to 1st trimester, respectively. This study showed a significant elevation of miR-15a-5p in 3rd trimester compared to 1st trimester of pregnant women (p = 0.0002). CONCLUSIONS: Expression pattern of PD-1 and TIM3 in exhausted T lymphocytes is different not only between normal pregnant and RPL women but also in different trimesters of pregnancy. So, our results showed the role of these markers in the modulation lymphocytes activity in different stages of pregnancy.

CXCR5+TIM-3-PD-1+ stem-like cytotoxic CD8+ T cells: elevated in chronic rhinosinusitis and associated with disease severity.

Background: Chronic rhinosinusitis (CRS) is a chronic inflammatory disease with an autoimmune background. Altered expression levels of T cell immunoglobulin and mucin-domain containing-3 (TIM-3), C-X-C chemokine receptor type 5 (CXCR5), and programmed cell death protein 1 (PD-1) are implicated in the progression of inflammatory and autoimmune diseases. Moreover, CXCR5+TIM-3-PD-1+ stem-like cytotoxic T cells function as memory stem cells during chronic disease processes and retain cytotoxicity-related gene networks. Objectives: To explore the expressions of CXCR5, TIM-3, and PD-1 on T cells and their correlation with clinical parameters in CRS. Methods: Flow cytometry was used to assess the expressions and co-expressions of CXCR5, TIM-3, and PD-1 on T cells in the tissues of the paranasal sinus and peripheral blood of patients with CRS as well as healthy controls. Immunofluorescence was used to assess the co-localization of TIM-3, CXCR5, and PD-1 with T cells. The disease severity of our patients with CRS was evaluated using the Lund-Mackay score. A complete blood count was also performed for the patients with CRS. Results: Expression levels of CXCR5 and PD-1 on T cells were significantly increased in the nasal tissues of patients with CRS. Compared with those in healthy controls, patients with CRS had high percentages of CXCR5+TIM-3-PD-1+ CD8+ and CD4+ T cells in nasal tissues, while no significant difference was observed in peripheral blood levels. Patients with CRS had a higher density of nasal CXCR5+TIM-3-PD-1+ T cells than that in healthy controls. CXCR5+TIM-3-PD-1+ CD8+ T cell levels in the nasal polyps of patients with CRS were negatively correlated with the patients' Lund-Mackay scores. The levels of CXCR5+TIM-3-PD-1+ T cells in nasal tissues were also negatively associated with disease duration and positively associated with the chronic inflammatory state of CRS. Conclusions: The level of CXCR5+TIM-3-PD-1+ stem cell-like T cells, especially CXCR5+TIM-3-PD-1+ CD8+ T cells, is increased in CRS. Therefore, inducing CXCR5+TIM-3-PD-1+ T cell exhaustion may be an effective immunotherapy for CRS.

TIM3 and CTLA4 immune checkpoint polymorphisms are associated with acute myeloid leukemia in Saudi Arabia.

BACKGROUND: Immune checkpoints are receptors on the surface of T cells that function crucially in suppressing the immune response, and they are implicated in autoimmunity and cancer diseases. AIM: The present study aimed to investigate the relationship between functional single nucleotide polymorphisms (SNPs) of two immune checkpoint molecules, CTLA-4 and TIM-3, and acute myeloid leukemia (AML) in a Saudi population. METHODS: Two SNPs in CTLA-4 (rs231775, A > G) and TIM-3 (rs10515746, A > C) were genotyped in 229 subjects, including 98 patients and 131 healthy controls, from the Saudi population using TaqMan assay methods. Differential expression of these two genes was performed using in silico analysis. RESULTS: An association was found between polymorphisms in TIM-3 (OR: 6.01; 95% CI: 3.99-9.05, P < 0.0001) and the risk of AML. Inversely, the rs231775 SNP in the CTLA-4 gene was found to protect against AML in allelic, dominant, and additive models (P < 0.05). A significantly higher expression of TIM-3 in the blood of individuals with AML was observed. CONCLUSION: This is the first study focusing on single nucleotide polymorphisms (SNPs) for CTLA-4 and TIM-3 in acute myeloid leukemia patients in a Saudi community and could be a potential new prognostic factor for this disease.

Retrospective Analyses of PD-L1, LAG-3, TIM-3, OX40L Expressions and MSI Status in Gastroenteropancreatic Neuroendocrine Neoplasms.

We investigated expressions of PD-L1, LAG-3, TIM-3, and OX40L as immune checkpoint proteins, and MSI (repetitive short-DNA-sequences due to defective DNA-repair system) status were analyzed with immunohistochemistry from tissue blocks. Of 83 patients, PD-L1 expression was observed in 18.1% (n = 15) of the patients. None of the patients exhibited LAG-3 expression. TIM-3 expression was 4.9% (n = 4), OX40L was 22.9% (n = 19), and 8.4% (n = 7) of the patients had MSI tumor. A low-to-intermediate positive correlation was observed between PD-L1 and TIM-3 expressions (rho: 0.333, p < 0.01). Although PD-L1 expression was higher in grade 3 NET/NEC, MSI status was prominent in grade 1/2 NET.

TIM-3, LAG-3, or 2B4 gene disruptions increase the anti-tumor response of engineered T cells.

Background: In adoptive T cell therapy, the long term therapeutic benefits in patients treated with engineered tumor specific T cells are limited by the lack of long term persistence of the infused cellular products and by the immunosuppressive mechanisms active in the tumor microenvironment. Exhausted T cells infiltrating the tumor are characterized by loss of effector functions triggered by multiple inhibitory receptors (IRs). In patients, IR blockade reverts T cell exhaustion but has low selectivity, potentially unleashing autoreactive clones and resulting in clinical autoimmune side effects. Furthermore, loss of long term protective immunity in cell therapy has been ascribed to the effector memory phenotype of the infused cells. Methods: We simultaneously redirected T cell specificity towards the NY-ESO-1 antigen via TCR gene editing (TCRED) and permanently disrupted LAG3, TIM-3 or 2B4 genes (IRKO) via CRISPR/Cas9 in a protocol to expand early differentiated long-living memory stem T cells. The effector functions of the TCRED-IRKO and IR competent (TCRED-IRCOMP) cells were tested in short-term co-culture assays and under a chronic stimulation setting in vitro. Finally, the therapeutic efficacy of the developed cellular products were evaluated in multiple myeloma xenograft models. Results: We show that upon chronic stimulation, TCRED-IRKO cells are superior to TCRED-IRCOMP cells in resisting functional exhaustion through different mechanisms and efficiently eliminate cancer cells upon tumor re-challenge in vivo. Our data indicate that TIM-3 and 2B4-disruption preserve T-cell degranulation capacity, while LAG-3 disruption prevents the upregulation of additional inhibitory receptors in T cells. Conclusion: These results highlight that TIM-3, LAG-3, and 2B4 disruptions increase the therapeutic benefit of tumor specific cellular products and suggest distinct, non-redundant roles for IRs in anti-tumor responses.

Peptidoglycan from Bacillus anthracis Inhibits Human Macrophage Efferocytosis in Part by Reducing Cell Surface Expression of MERTK and TIM-3.

Bacillus anthracis peptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen-associated molecular pattern contributing to anthrax pathology, including organ dysfunction and coagulopathy. Increases in apoptotic leukocytes are a late-stage feature of anthrax and sepsis, suggesting there is a defect in apoptotic clearance. In this study, we tested the hypothesis that B. anthracis PGN inhibits the capacity of human monocyte-derived macrophages (MΦ) to efferocytose apoptotic cells. Exposure of CD163+CD206+ MΦ to PGN for 24 h impaired efferocytosis in a manner dependent on human serum opsonins but independent of complement component C3. PGN treatment reduced cell surface expression of the proefferocytic signaling receptors MERTK, TYRO3, AXL, integrin αVß5, CD36, and TIM-3, whereas TIM-1, αVß3, CD300b, CD300f, STABILIN-1, and STABILIN-2 were unaffected. ADAM17 is a major membrane-bound protease implicated in mediating efferocytotic receptor cleavage. We found multiple ADAM17-mediated substrates increased in PGN-treated supernatant, suggesting involvement of membrane-bound proteases. ADAM17 inhibitors TAPI-0 and Marimastat prevented TNF release, indicating effective protease inhibition, and modestly increased cell-surface levels of MerTK and TIM-3 but only partially restored efferocytic capacity by PGN-treated MΦ. We conclude that human serum factors are required for optimal recognition of PGN by human MΦ and that B. anthracis PGN inhibits efferocytosis in part by reducing cell surface expression of MERTK and TIM-3.

Comprehensive Co-Inhibitory Receptor (Co-IR) Expression on T Cells and Soluble Proteins in Rheumatoid Arthritis.

Co-inhibitory receptors (Co-IRs) are essential in controlling the progression of immunopathology in rheumatoid arthritis (RA) by limiting T cell activation. The objective of this investigation was to determine the phenotypic expression of Co-IR T cells and to assess the levels of serum soluble PD-1, PDL-2, and TIM3 in Taiwanese RA patients. METHODS: Co-IRs T cells were immunophenotyped employing multicolor flow cytometry, and ELISA was utilized for measuring soluble PD-1, PDL-2, and TIM3. Correlations have been detected across the percentage of T cells expressing Co-IRs (MFI) and different indicators in the blood, including ESR, high-sensitivity CRP (hsCRP), 28 joint disease activity scores (DAS28), and soluble PD-1/PDL-2/TIM3. RESULTS: In RA patients, we recognized elevated levels of PD-1 (CD279), CTLA-4, and TIGIT in CD4+ T cells; TIGIT, HLA-DR, TIM3, and LAG3 in CD8+ T cells; and CD8+CD279+TIM3+, CD8+HLA-DR+CD38+ T cells. The following tests were revealed to be correlated with hsCRP: CD4/CD279 MFI, CD4/CD279%, CD4/TIM3%, CD8/TIM3%, CD8/TIM3 MFI, CD8/LAG3%, and CD8+HLA-DR+CD38+%. CD8/LAG3 and CD8/TIM3 MFIs are linked to ESR. DAS28-ESR and DAS28-CRP exhibited relationships with CD4/CD127 MFI, CD8/CD279%, and CD8/CD127 MFI, respectively. CD4+CD279+TIM3+% was correlated with DAS28-ESR (p = 0.0084, N = 46), DAS28-CRP (p = 0.007, N = 47), and hsCRP (p = 0.002, N = 56), respectively. In the serum of patients with RA, levels of soluble PD-1, PDL-2, and Tim3 were extremely elevated. CD4+ TIM3+% (p = 0.0089, N = 46) and CD8+ TIM3+% (p = 0.0305, N = 46) were correlated with sTIM3 levels; sPD1 levels were correlated with CD4+CD279+% (p < 0.0001, N = 31) and CD3+CD279+% (p = 0.0084, N = 30). CONCLUSIONS: Co-IR expressions on CD4+ and CD8+ T cells, as well as soluble PD-1, PDL-2, and TIM3 levels, could function as indicators of disease activity and potentially play crucial roles in the pathogenesis of RA.

Beyond T cell exhaustion: TIM-3 regulation of myeloid cells.

T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) is an important immune checkpoint molecule initially identified as a marker of IFN-γ-producing CD4+ and CD8+ T cells. Since then, our understanding of its role in immune responses has significantly expanded. Here, we review emerging evidence demonstrating unexpected roles for TIM-3 as a key regulator of myeloid cell function, in addition to recent work establishing TIM-3 as a delineator of terminal T cell exhaustion, thereby positioning TIM-3 at the interface between fatigued immune responses and reinvigoration. We share our perspective on the antagonism between TIM-3 and T cell stemness, discussing both cell-intrinsic and cell-extrinsic mechanisms underlying this relationship. Looking forward, we discuss approaches to decipher the underlying mechanisms by which TIM-3 regulates stemness, which has remarkable potential for the treatment of cancer, autoimmunity, and autoinflammation.