Tumor-associated macrophages impair NK cell IFN-γ production and contribute to tumor progression in clear cell renal cell carcinoma.

Tumor-associated macrophages (TAM) are abundant in several tumor types and usually correlate with poor prognosis. Previously, we demonstrated that anti-inflammatory macrophages (M2) inhibit NK cell effector functions. Here, we explored the impact of TAM on NK cells in the context of clear-cell renal cell carcinoma (ccRCC). Bioinformatics analysis revealed that an exhausted NK cell signature strongly correlated with an M2 signature. Analysis of TAM from human ccRCC samples confirmed that they exhibited an M2-skewed phenotype and inhibited IFN-γ production by NK cells. Moreover, human M0 macrophages cultured with conditioned media from ccRCC cell lines generated macrophages with an M2-skewed phenotype (TAM-like), which alike TAM, displayed suppressive activity on NK cells. Moreover, TAM depletion in the mouse Renca ccRCC model resulted in delayed tumor growth and reduced volume, accompanied by an increased frequency of IFN-γ-producing tumor-infiltrating NK cells that displayed heightened expression of T-bet and NKG2D and reduced expression of the exhaustion-associated co-inhibitory molecules PD-1 and TIM-3. Therefore, in ccRCC, the tumor microenvironment polarizes TAM toward an immunosuppressive profile that promotes tumor-infiltrating NK cell dysfunction, contributing to tumor progression. In addition, immunotherapy strategies targeting TAM may result in NK cell reinvigoration, thereby counteracting tumor progression.

The level of Tim-3+CD8+ T cells can serve as a potential marker for evaluating the severity of acute graft-versus-host disease after haplo-PBSCT.

Early and accurate diagnosis of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation is crucial for the prognosis of patients. This study identified a potential biomarker for the severity of aGVHD after human leukocyte antigen (HLA)-haploidentical peripheral blood hematopoietic stem cell transplantation (haplo-PBSCT). We included 20 healthy subjects and 57 patients who underwent haplo-PBSCT. Of these patients, 22 developed aGVHD after haplo-PBSCT. The results showed that patients with aGVHD had significantly increased levels of Tim-3+/Perforin+/Granzyme B+CD8+ T cells, but significantly decreased Galectin-9. The differences in Galectin-9 and Tim-3+/Granzyme B+CD8+ T cells between grade I-II aGVHD and III-IV aGVHD were also significant. In vitro, the apoptosis of CD8+ T cells from aGVHD patients was significantly increased after Tim-3/Galectin-9 pathway activation, which decreased Granzyme B secretion. As revealed by univariate analysis, the level of Tim-3+CD8+ T cells was a risk factor for severe aGVHD. ROC analysis demonstrated that high levels of Tim-3+CD8+ T cells had a significant diagnostic value for severe aGVHD, with an area under the curve of 0.854 and cut-off value of 14.155%. In conclusion, the binding of Tim-3 with exogenous Galectin-9 can promote apoptosis of CD8+ T cells and affect the secretion of Granzyme B. Tim-3+CD8+ T cells have the potential to serve as immunological markers for assessing the severity of aGVHD after haplo-PBSCT and identifying patients at a higher risk for severe aGVHD.

Tim-3 Is Differentially Expressed during Cell Activation and Interacts with the LSP-1 Protein in Human Macrophages.

T-cell Immunoglobulin and Mucin Domain 3 (TIM-3) is an immune checkpoint receptor known to regulate T-cell activation and has been targeted for immunotherapy in cancer and other diseases. However, its expression and function in other cell types, such as macrophages, are poorly understood. This study investigated TIM-3 expression in human macrophages polarized to M1 (stimulated with IFN-γ and LPS) and M2 (stimulated with IL-4 and IL-13) phenotypes using an in vitro model. Our results show that M1 macrophages have a lower frequency of TIM-3+ cells compared to M2 macrophages at 48 and 72 hr poststimulation. Additionally, we observed differential levels of soluble ADAM 10, an enzyme responsible for TIM-3 release, in the supernatants of M1 and M2 macrophages at 72 hr. We also found that the TIM-3 intracellular tail might associate with lymphocyte-specific protein 1 (LSP-1), a protein implicated in cell motility and podosome formation. These findings enhance our understanding of TIM-3 function in myeloid cells such as macrophages and may inform the development of immunotherapies with reduced immune-related adverse effects.

CXCR5 and TIM-3 expressions define distinct exhausted T cell subsets in experimental cutaneous infection with Leishmania mexicana.

T-cell exhaustion is a key stage in chronic infections since it limits immunopathology, but also hinders the elimination of pathogens. Exhausted T (Tex) cells encompass dynamic subsets, including progenitor cells that sustain long-term immunity through their memory/stem like properties, and terminally-differentiated cells, resembling the so-called Tex cells. The presence of Tex cells in chronic leishmaniasis has been reported in humans and murine models, yet their heterogeneity remains unexplored. Using flow cytometry, we identified Tex cells subtypes based on PD-1, CXCR5 and TIM-3 expressions in draining lymph nodes (dLNs) and lesion sites of C57BL/6 mice infected with L. mexicana at 30-, 60- and 90-days post-infection. We showed that infected mice developed a chronic infection characterized by non-healing lesions with a high parasite load and impaired Th1/Th2 cytokine production. Throughout the infection, PD-1+ cells were observed in dLNs, in addition to an enhanced expression of PD-1 in both CD4+ and CD8+ T lymphocytes. We demonstrated that CD4+ and CD8+ T cells were subdivided into PD-1+CXCR5+TIM-3- (CXCR5+), PD-1+CXCR5+TIM-3+ (CXCR5+TIM-3+), and PD-1+CXCR5-TIM-3+ (TIM-3+) subsets. CXCR5+ Tex cells were detected in dLNs during the whole course of the infection, whereas TIM-3+ cells were predominantly localized in the infection sites at day 90. CXCR5+TIM-3+ cells only increased at 30 and 60 days of infection in dLNs, whereas no increase was observed in the lesions. Phenotypic analysis revealed that CXCR5+ cells expressed significantly higher levels of CCR7 and lower levels of CX3CR1, PD-1, TIM-3, and CD39 compared to the TIM-3+ subset. CXCR5+TIM-3+ cells expressed the highest levels of all exhaustion-associated markers and of CX3CR1. In agreement with a less exhausted phenotype, the frequency of proliferating Ki-67 and IFN-γ expressing cells was significantly higher in the CXCR5+ subset within both CD4+ and CD8+ T cells compared to their respective TIM-3+ subsets, whereas CD8+CXCR5+TIM-3+ and CD8+TIM-3+ subsets showed an enhanced frequency of degranulating CD107a+ cells. In summary, we identified a novel, less-differentiated CXCR5+ Tex subset in experimental cutaneous leishmaniasis caused by L. mexicana. Targeting these cells through immune checkpoint inhibitors such as anti-PD-1 or anti PD-L1 might improve the current treatment for patients with the chronic forms of leishmaniasis.

Expression of Inhibitory Receptors TIGIT, TIM-3, and LAG-3 on CD4+ T Cells from Patients with Different Clinical Forms of Chronic Chagas Disease.

T cells are central to the adaptive immune response against Trypanosoma cruzi infection. In chronic Chagas disease (CCD), circulating parasite-specific memory T cells show reduced functionality and increased expression of inhibitory receptors as a result of persistent antigenic stimulation. This phenotype has been linked to progression of cardiac pathology, whereas the presence of polyfunctional T cells shows association with therapeutic success. In this study, we demonstrate that T. cruzi-specific human CD4+ T cells can be identified by their expression of OX40 and CD25 upon in vitro stimulation. We characterized the expression of the inhibitory receptors T cell immunoreceptor with Ig and ITIM domains (TIGIT), T cell Ig and mucin-domain containing-3 (TIM-3), and lymphocyte activation gene 3 (LAG-3) in CD4+ T cells from CCD patients with and without cardiac alterations. Our results show that, independently of their clinical stage, CCD patients present an increased frequency of CD4+ T cells expressing TIGIT in comparison with non-T. cruzi-infected donors. Exposure to parasite Ags increases the expression of TIM-3 in CD4+ T cells from CCD patients, especially in those with cardiac compromise. Upregulation of LAG-3 was also detected in CCD individuals without cardiac manifestations, predominantly within the subpopulation of cells that did not become activated upon stimulation. Further differences were found between groups in the coexpression of these receptors. Blockade of each individual receptor did not affect activation or the production of IFN-γ and IL-10 by CD4+ T cells in response to parasite Ags. Our results suggest a role for TIGIT, TIM-3, and LAG-3 in the modulation of inflammatory phenomena thought to ultimately lead to tissue damage and cardiac pathology.

Immunophenotypic Analysis of T Lymphocytes and Cytokine Production in Elderly Practicing Physical Activities and Its Relationship with Quality of Life and Depression.

Aging is a complex process often associated with a chronic inflammatory profile that alters several biological functions, including the immune system and cognitive and physical capacity. The practice of physical activity is increasingly gaining popularity as a method of preventing infections, depression, and other disorders that affect the quality of life of the elderly. Thus, this work analyzes the profile of cytokines and molecular markers expressed in immune cells of elderly people who practice physical activities or not, evaluating their impacts on the immune system and quality of life. For this, 48 individuals were recruited, and peripheral blood samples were collected for hemogram analysis, cytokine determination, and immunophenotyping. Elderly people were separated into two groups: practitioners with low-intensity physical activity and non-practitioners. Quality of life was assessed using the Whoqol-Old instrument, and depression was assessed using the Beck II Depression Inventory. When comparing the scores of the Whoqol-Old and Beck questionnaires, we observed a significant negative correlation between these two factors. The perception of a higher quality of life was present in the elderly who exercised and was related to greater autonomy and sensory abilities, whereas the presence of depression was lower. In the hemogram, we observed higher basophil and segmented counts in the sedentary elderly, whereas lymphocytes and monocytes had lower counts. Elderly practitioners of physical activities had higher levels of IFN-γ, IL-4, and IL-10; increased expression of CD69, PD1, and TIM-3 in CD4+ T lymphocytes and increased CD14+CD80+ and CD14+CD86+ monocytes. Elderly people with an increased perception of quality of life had higher levels of IFN-γ, higher expression of CD14+CD80+CD86+, and decreased levels of TRAIL. An increase in TRAIL was observed in individuals with depression, in addition to an increased expression of CD14+CD86+. These results show a clear correlation between the quality of life, level of depression, physical activity, and immune system function. Although some cytokines with a typical proinflammatory profile (IFN-γ) were observed, the results point to a protective state with benefits reflected in the general well-being of the elderly who exercise.

Is IFN expression by NK cells a hallmark of severe COVID-19?

Natural Killer cells (NK) are crucial in host defense against viruses. There are many unanswered questions about the immune system in COVID-19, especially the mechanisms that contribute to the development of mild or severe forms of the disease. Although NK cells may have an essential role in the pathogenesis of COVID-19, the mechanisms involved in this process are not yet fully elucidated. Here, we demonstrate that CD3-CD56+ NK cells frequency in the volunteers who recovered from mild COVID-19 (Mild CoV) presented a significant increase compared to the healthy control (HC) and individuals recovering from severe COVID-19 (Severe CoV) groups. Furthermore, distinct IFN profiles in recovered COVID-19 patients with mild or severe clinical forms of the disease were observed in the total NK cells (CD3-CD56+). In the first group, NK cells express increased levels of IFN-α compared to the severe CoV, while higher production of IFN-γ in severe CoV was found. Moreover, NK cells in mild CoV express more cytolytic granules depicted by granzyme B and perforin. Compared to HC, PBMCs from mild CoV presented higher Ki-67 and TIM-3 production after Pool CoV-2 and Pool Spike CoV-2 peptides stimulus. In addition, non-stimulated PBMCs in the mild CoV group had higher NK TIM-3+ frequency than severe CoV. In the mild CoV group, Pool Spike CoV-2 and Pool CoV-2 peptides stimuli elicited higher granzyme B and perforin coexpression and IFN-α production by PBMCs. However, in severe CoV, Pool Spike CoV-2 reduced the coexpression of granzyme B, perforin, and CD107a suggesting a decrease in the cytotoxic activity of NK cells. Therefore, our study shows that NK cells may have a crucial role in COVID-19 with the involvement of IFN-α and cytotoxic properties that aid in developing qualified immune responses. Furthermore, the data suggest that higher amounts of IFN-γ may be linked to the severity of this disease.

Circulating Levels of PD-L1, TIM-3 and MMP-7 Are Promising Biomarkers to Differentiate COVID-19 Patients That Require Invasive Mechanical Ventilation.

Background: COVID-19 is an infectious disease caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Many COVID-19 patients require invasive mechanical ventilation (IMV) while others, even with acute respiratory failure, do not (NIMV). Therefore, we aimed to evaluate serum levels of MMP-7 and molecules related to exhausted T-cells as potential biomarkers to differentiate between IMV and NIMV patients. Methods: 105 patients diagnosed with COVID-19 and confirmed by RT-PCR for SARS-CoV-2 were divided into two groups according to the requirement for IMV. Serum levels of sPD-L1, sPD-L2, sTIM-3, sGal-9 and sMMP-7 were quantified by ELISA and correlated with clinical data. Twelve patients were followed up after eight months to compare the levels of the biomarkers between acute disease and post-COVID-19. Results: IMV patients experienced a lower PaO2/FiO2 (p < 0.0001) and a longer hospital stay (p < 0.0001), and exhibited higher levels of sPD-L1 (p < 0.05), sTIM-3 (p < 0.01) and sMMP-7 (p < 0.0001) when compared with NIMV patients. According to a ROC analysis, sMMP-7 had the highest sensitivity (78%) and specificity (76%) with a cut point of 4.5 ng/mL, followed by sTIM-3 and sPD-L1. Eight months post-COVID-19, IMV patients displayed a significant decrease in the initially high levels of sPD-L1, sTIM-3 and sGal-9, while sPD-L2 was increased, and sMMP-7 was unchanged. Conclusion: Circulating levels of sPD-L1, sTIM-3 and sMMP-7 are potential biomarkers of disease severity to distinguish patients requiring IMV. MMP-7 could also be a marker for the persistence of lung lesions post-COVID-19.

PD1/PDL1 expression is associated with increased TIM3 expression and tumor-infiltrating T lymphocytes in fibroblastic tumors.

PURPOSE: The combined therapy of inhibiting T cell immunoglobulin domain and mucin domain 3 (TIM3) and programmed cell death 1/programmed death-ligand 1 (PD1/PDL1) has shown encouraging therapeutic effects in some solid tumors. However, the expression of PD1/PDL1 and TIM3 in fibroblastic tumors is ill defined, which has limited the application of these immune checkpoint inhibitors in such tumors. METHODS: Immunostaining of 68 tissue microarray cores of fibroblastic tumors, including intermediate dermatofibrosarcoma protuberans and malignant myxofibrosarcoma and adult-type fibrosarcoma, was used to determine the expression of PD1, PDL1 and TIM3, as well as their relationship with the accumulation of tumor-infiltrating T lymphocytes (TILs). RESULTS: Both PD1 and PDL1 expression was only observed in a small proportion of fibroblastic tumors, whereas TIM3 was expressed in almost all tumors. However, only the positive expression of PDL1 was related to tumors with high grade and staging. A considerable number of TILs, including CD4- and CD8A-positive T cells and a small group of FoxP3-positive T cells, was also observed in most tumors. The density of TIM3 was positively correlated with that of TILs. Furthermore, higher densities of TIM3, CD4, CD8A and FoxP3 were observed in PD1 and PDL1 double-positive fibroblastic tumors. CONCLUSIONS: This study indicates that TILs with high expression of TIM3 may contribute to immunosuppression in the tumor microenvironment of fibroblastic tumors. Patients with fibroblastic tumors with high expression of PD1/PDL1 and TIM3 may therefore benefit from combination therapy with PD1/PDL1 and TIM3 inhibitors.

Alterations in regulatory T cells and immune checkpoint molecules in pancreatic cancer patients receiving FOLFIRINOX or gemcitabine plus nab-paclitaxel.

PURPOSE: This pilot study aimed on generating insight on alterations in circulating immune cells during the use of FOLFIRINOX and gemcitabine/nab-paclitaxel in pancreatic ductal adenocarcinoma (PDAC). PATIENTS AND METHODS: Peripheral blood mononuclear cells were isolated before and 30 days after initiation of chemotherapy from 20 patients with advanced PDAC. Regulatory T cells (FoxP3+) and immune checkpoints (PD-1 and TIM-3) were analyzed by flow cytometry and immunological changes were correlated with clinical outcome. RESULTS: Heterogeneous changes during chemotherapy were observed in circulating T-cell subpopulations with a pronounced effect on PD-1+ CD4+/CD8+ T cells. An increase in FoxP3+ or PD-1+ T cells had no significant effect on survival. An increase in TIM3+/CD8+ (but not TIM3+/CD4+) T cells was associated with a significant inferior outcome: median progression-free survival in the subgroup with an increase of TIM-3+/CD8+ T cells was 6.0 compared to 14.0 months in patients with a decrease/no change (p = 0.026); corresponding median overall survival was 13.0 and 20.0 months (p = 0.011), respectively. CONCLUSIONS: Chemotherapy with FOLFIRNOX or gemcitabine/nab-paclitaxel induces variable changes in circulating T-cell populations that may provide prognostic information in PDAC.

Bispecific and split CAR T cells targeting CD13 and TIM3 eradicate acute myeloid leukemia.

Chimeric antigen receptor (CAR) T cells have radically improved the treatment of B cell-derived malignancies by targeting CD19. The success has not yet expanded to treat acute myeloid leukemia (AML). We developed a Sequentially Tumor-Selected Antibody and Antigen Retrieval (STAR) system to rapidly isolate multiple nanobodies (Nbs) that preferentially bind AML cells and empower CAR T cells with anti-AML efficacy. STAR-isolated Nb157 specifically bound CD13, which is highly expressed in AML cells, and CD13 CAR T cells potently eliminated AML in vitro and in vivo. CAR T cells bispecific for CD13 and TIM3, which are upregulated in AML leukemia stem cells, eradicated patient-derived AML, with much reduced toxicity to human bone marrow stem cells and peripheral myeloid cells in mouse models, highlighting a promising approach for developing effective AML CAR T cell therapy.

TIM-3 genetic variants and risk of Behçet disease in the Iranian population.

BACKGROUND: Behçet disease is a prototypical systemic autoimmune disease, caused by a complex interplay between environmental and genetic factors. The transmembrane immunoglobulin and mucin domain-3 (TIM-3) is a distinct member of the TIM family that is preferentially expressed on Th1 cells and plays a role in Th1-mediated autoimmune or inflammatory diseases, such as Behçet disease. OBJECTIVE: The aim of this study was to test the potential association between TIM-3 gene polymorphisms and Behçet disease. METHODS: Two single-nucleotide polymorphisms of TIM-3 (rs9313439 and rs10515746) were genotyped in 212 patients with Behçet disease and 200 healthy controls. Typing of the polymorphisms was performed using multiplex PCR amplification. RESULTS: There were no significant differences in allele and genotype frequencies between the Behçet disease patients and controls who were successfully genotyped. Similar results were also found after stratification by gender, age, or clinical features. STUDY LIMITATIONS: Lack of studies on various racial or ethnic groups and small sample size. CONCLUSION: This study failed to demonstrate any association between the tested TIM-3 polymorphisms and Behçet disease.

Peripheral changes in immune cell populations and soluble mediators after anti-PD-1 therapy in non-small cell lung cancer and renal cell carcinoma patients.

Patients with non-small cell lung cancer (NSCLC) and renal cell carcinoma (RCC) have shown benefit from anti-PD-1 therapies. However, not all patients experience tumor shrinkage, durable responses or prolonged survival, demonstrating the need to find response markers. In blood samples from NSCLC and RCC patients obtained before and after anti-PD-1 treatment, we studied leukocytes by complete blood cell count, lymphocyte subsets using flow cytometry and plasma concentration of nine soluble mediators, in order to find predictive biomarkers of response and to study changes produced after anti-PD-1 therapy. In baseline samples, discriminant analysis revealed a combination of four variables that helped differentiate stable disease-response (SD-R) from progressive disease (PD) patients: augmented frequency of central memory CD4+ T cells and leukocyte count was associated with response while increased percentage of PD-L1+ natural killer cells and naïve CD4+ T cells was associated with lack of response. After therapy, differential changes between responders and non-responders were found in leukocytes, T cells and TIM-3+ T cells. Patients with progressive disease showed an increase in the frequency of TIM-3 expressing CD4+ and CD8+ T cells, whereas SD-R patients showed a decrease in these subsets. Our findings indicate that a combination of immune variables from peripheral blood (PB) could be useful to distinguish response groups in NSCLC and RCC patients treated with anti-PD-1 therapy. Frequency of TIM-3+ T cells showed differential changes after treatment in PD vs SD-R patients, suggesting that it may be an interesting marker for monitoring progression during therapy.

TIM-3 genetic variants and risk of Behçet disease in the Iranian population

Abstract: Background: Behçet disease is a prototypical systemic autoimmune disease, caused by a complex interplay between environmental and genetic factors. The transmembrane immunoglobulin and mucin domain-3 (TIM-3) is a distinct member of the TIM family that is preferentially expressed on Th1 cells and plays a role in Th1-mediated autoimmune or inflammatory diseases, such as Behçet disease. Objective: The aim of this study was to test the potential association between TIM-3 gene polymorphisms and Behçet disease. Methods: Two single-nucleotide polymorphisms of TIM-3 (rs9313439 and rs10515746) were genotyped in 212 patients with Behçet disease and 200 healthy controls. Typing of the polymorphisms was performed using multiplex PCR amplification. Results: There were no significant differences in allele and genotype frequencies between the Behçet disease patients and controls who were successfully genotyped. Similar results were also found after stratification by gender, age, or clinical features. Study limitations: Lack of studies on various racial or ethnic groups and small sample size. Conclusion: This study failed to demonstrate any association between the tested TIM-3 polymorphisms and Behçet disease.

Cancer immunotherapy using anti-TIM3/PD-1 bispecific antibody: a patent evaluation of EP3356411A1.

Introduction: TIM3 and PD-1 are checkpoint inhibitors in cancer that coordinate the downregulation of the proliferation of antigen-specific lymphocytes. There is a great need to discover and develop new therapies focused on inhibiting the action of TIM3 and PD-1 and consequently improving the immune response in the various types of cancer. The authors of patent EP3356411A1 propose several anti-TIM3/anti-PD-1 bispecific antibodies, as well as the method for producing them and their pharmacological application in the treatment of cancer. Areas covered: Patent EP3356411A1 describes a method by producing anti-TIM3/anti-PD-1 bispecific antibodies and their potential in cancer treatment. Expert opinion: Data supporting the patent demonstrate the ability by producing anti-TIM3/anti-PD-1 bispecific antibodies. Although the proposed methodology is very interesting and promising, further studies are necessary to assess the clinical applicability of the inventions on cancer.

Non-invasive messenger RNA transcriptional evaluation in human kidney allograft dysfunction.

The aim of the present study was to evaluate messenger RNA expression in kidney allograft recipients. Forty-four kidney transplant recipients were evaluated up to three months after grafting. After transplantation, peripheral blood samples were drawn sequentially for real-time polymerase chain reaction analyses of perforin and TIM-3 genes. Biopsies were obtained to evaluate acute graft dysfunction and interpreted according to the Banff classification. Eight patients presented episodes of acute rejection. Recipients with rejection had significantly higher levels of TIM-3 mRNA transcripts compared to those without rejection (median gene expression 191.2 and 36.9 mRNA relative units, respectively; P<0.0001). Also, perforin gene expression was higher in patients with rejection (median gene expression 362.0 and 52.8 mRNA relative units; P<0.001). Receiver operating characteristic curves showed that the area under the curve (AUC) for the TIM-3 gene was 0.749 (95%CI: 0.670-0.827). Perforin gene mRNA expression provided an AUC of 0.699 (95%CI: 0.599 to 0.799). Overall accuracy of gene expression was 67.9% for the TIM-3 gene and 63.6% for the perforin gene. Combined accuracy was 76.8%. Negative predictive values were 95.3% for the TIM-3 gene, 95.5% for the perforin gene, and 95.4% in the combined analyses. Gene expression was significantly modulated by rejection treatment decreasing 64.1% (TIM-3) and 90.9% (perforin) compared to the median of pre-rejection samples. In conclusion, the longitudinal approach showed that gene profiling evaluation might be useful in ruling out the diagnosis of acute rejection and perhaps evaluating the efficacy of treatment.

Receptors That Inhibit Macrophage Activation: Mechanisms and Signals of Regulation and Tolerance.

A variety of receptors perform the function of attenuating or inhibiting activation of cells in which they are expressed. Examples of these kinds of receptors include TIM-3 and PD-1, among others that have been widely studied in cells of lymphoid origin and, though to a lesser degree, in other cell lines. Today, several studies describe the function of these molecules as part of the diverse mechanisms of immune tolerance that exist in the immune system. This review analyzes the function of some of these proteins in monocytes and macrophages and as well as their participation as inhibitory molecules or elements of immunological tolerance that also act in innate defense mechanisms. We chose the receptors TIM-3, PD-1, CD32b, and CD200R because these molecules have distinct functional characteristics that provide examples of the different regulating mechanisms in monocytes and macrophages.

Non-invasive messenger RNA transcriptional evaluation in human kidney allograft dysfunction

The aim of the present study was to evaluate messenger RNA expression in kidney allograft recipients. Forty-four kidney transplant recipients were evaluated up to three months after grafting. After transplantation, peripheral blood samples were drawn sequentially for real-time polymerase chain reaction analyses of perforin and TIM-3 genes. Biopsies were obtained to evaluate acute graft dysfunction and interpreted according to the Banff classification. Eight patients presented episodes of acute rejection. Recipients with rejection had significantly higher levels of TIM-3 mRNA transcripts compared to those without rejection (median gene expression 191.2 and 36.9 mRNA relative units, respectively; P<0.0001). Also, perforin gene expression was higher in patients with rejection (median gene expression 362.0 and 52.8 mRNA relative units; P<0.001). Receiver operating characteristic curves showed that the area under the curve (AUC) for the TIM-3 gene was 0.749 (95%CI: 0.670-0.827). Perforin gene mRNA expression provided an AUC of 0.699 (95%CI: 0.599 to 0.799). Overall accuracy of gene expression was 67.9% for the TIM-3 gene and 63.6% for the perforin gene. Combined accuracy was 76.8%. Negative predictive values were 95.3% for the TIM-3 gene, 95.5% for the perforin gene, and 95.4% in the combined analyses. Gene expression was significantly modulated by rejection treatment decreasing 64.1% (TIM-3) and 90.9% (perforin) compared to the median of pre-rejection samples. In conclusion, the longitudinal approach showed that gene profiling evaluation might be useful in ruling out the diagnosis of acute rejection and perhaps evaluating the efficacy of treatment.

Role of TIM-3 in ovarian cancer.

Evidences have suggested that immunotherapy for ovarian cancer is effective. Immune checkpoints have emerged in the field of cancer immunotherapy. Multiple studies have shown negative regulation of TIM-3 expression on CD4+ and CD8+ T cells and other immunocytes. Overexpression of TIM-3 in innate immune cells has been found in certain types of tumor. The blockade of TIM-3 leads to sustained anti-tumor reactions. TIM-3 plays an inhibitive role for immunity in ovarian cancer. TIM-3 is involved in the development of various subtypes of ovarian cancer and thus has the potential to be a therapeutic target for treatment of ovarian cancer.

Tim-3 facilitates osteosarcoma proliferation and metastasis through the NF-κB pathway and epithelial-mesenchymal transition.

The aim of this study was to investigate the expression of T-cell immunoglobulin mucin domain molecule-3 (Tim-3) in osteosarcoma tissues, and analyze its effect on cell proliferation and metastasis in an osteosarcoma cell line. Tim-3 mRNA and protein expression in osteosarcoma tissue was detected by reverse transcriptase-polymerase chain reaction and immunohistochemistry, respectively. Additionally, the cell viability, apoptosis rate, and invasive ability of the osteosarcoma cell line MG-63 were tested using the methyl thiazolyl tetrazolium assay, Annexin V-propidium iodide flow cytometry, and a Transwell assay, respectively, following Tim-3 interference using small interfering RNA (siRNA). We also analyzed the expression of Snail, E-cadherin, vimentin, and nuclear factor (NF)-kB in the cells by western blot. We observed that Tim-3 mRNA and protein was significantly overexpressed in osteosarcoma tissues, compared to the adjacent normal tissue (P < 0.01). Moreover, MG-63 cells transfected with the Tim-3 siRNA presented lower cell viability, a greater number of apoptotic cells, and decreased invasive ability (P < 0.01), compared to control cells. Additionally, we observed a decrease in Snail and vimentin expression, an increase in the E-cadherin level, and an increase in NF-kB p65 phosphorylation (P < 0.01) in Tim-3 siRNA-transfected MG-63 cells. Based on these results, we concluded that Tim-3 is highly expressed in osteosarcoma tissue. Moreover, we speculated that interfering in Tim-3 expression could significantly suppress osteosarcoma cell (MG-63) proliferation and metastasis via the NF-kB/Snail signaling pathway and epithelial-mesenchymal transition.