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Aim Methods Results Conclusion Diabetic nephropathy (DN) is the most common complication of diabetes mellitus. We aimed to investigate the role of regulatory T cells (Tregs) and helper T cells 17 (Th17) in the development and progression of DN.A mouse type 2 diabetic nephropathy (T2DN) model was established. Immunohistochemistry was used to detect the expression of HSP70 and Tim‐3 in mouse kidney tissues, and western blotting was used to detect the expression levels of HSP70 and Tim‐3. PAS staining and Masson's trichrome staining were used to detect the degree of kidney injury. Flow cytometry was used to detect the number of Th17 and Treg cells in blood and kidney tissues. The expression levels of interleukin 17 (IL‐17) and interleukin 10 (IL‐10) in the serum were measured via ELISA.The expression of HSP70 was significantly increased while the expression of Tim‐3 was significantly decreased in the kidneys of mice in the T2DN group compared with those in the control (NC) group. Additionally, the inhibition of HSP70 upregulated the expression of Tim‐3 in T2DN mice. The Th17/Treg ratio was significantly greater in the blood and kidneys of the mice in the T2DN group than in those of the NC group, the expression of serum IL‐17 was increased, and the expression of IL‐10 was decreased.Increased HSP70 inhibits Tim‐3 expression in T2DN mouse kidney tissues, and subsequently causes a Th17/Treg imbalance and an inflammatory response, ultimately leading to kidney injury. The inhibition of HSP70 may alleviate the progression of T2DN. [ABSTRACT FROM AUTHOR]

Autoimmune polyglandular syndrome (APS) comprises a complex association of autoimmune pathological conditions. APS Type 1 originates from loss-of-function mutations in the autoimmune regulator (AIRE) gene. APS2, APS3 and APS4 are linked to specific HLA alleles within the major histocompatibility complex, with single-nucleotide polymorphisms (SNPs) in non-HLA genes also contributing to disease. In general, variability in the AIRE locus and the presence of heterozygous loss-of-function mutations can impact self-antigen presentation in the thymus. In this study, whole-exome sequencing (WES) was performed on a sixteen-year-old female APS3A/B patient to investigate the genetic basis of her complex phenotype. The analysis identified two variants (p.Arg111Trp and p.Thr101Ile) of the hepatitis A virus cell receptor 2 gene (HAVCR2) encoding for the TIM-3 (T cell immunoglobulin and mucin domain 3) protein. These variants were predicted, through in silico analysis, to impact protein structure and stability, potentially influencing the patient's autoimmune phenotype. While confocal microscopy analysis revealed no alteration in TIM-3 fluorescence intensity between the PBMCs isolated from the patient and those of a healthy donor, RT-qPCR showed reduced TIM-3 expression in the patient's unfractionated PBMCs. A screening conducted on a cohort of thirty APS patients indicated that the p.Thr101Ile and p.Arg111Trp mutations were unique to the proband. This study opens the pathway for the search of TIM-3 variants possibly linked to complex autoimmune phenotypes, highlighting the potential of novel variant discovery in contributing to APS classification and diagnosis. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitors (ICI) have become the cornerstone of treatment in renal cell carcinoma (RCC), for both metastatic disease and in an adjuvant setting. However, an adaptive resistance from cancer cells may arise during ICI treatment, therefore many studies are focusing on additional immune checkpoint inhibitor pathways. Promising targets of immunotherapeutic agents under investigation include T cell immunoglobulin and ITIM domain (TIGIT), immunoglobulin-like transcript 4 (ILT4), lymphocyte activation gene-3 (LAG-3), vaccines, T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), and chimeric antigen receptor (CAR) T cells. In this review of the literature, we recollect the current knowledge of the novel treatment strategies in the field of immunotherapy that are being investigated in RCC and analyze their mechanism of action, their activity and the clinical studies that are currently underway. [ABSTRACT FROM AUTHOR]

PACAP (pituitary adenylate cyclase activating polypeptide) is a widespread neuropeptide with cytoprotective and anti-inflammatory effects. It plays a role in innate and adaptive immunity, but data are limited about gut-associated lymphoid tissue. We aimed to reveal differences in Peyer's patches between wild-type (WT) and PACAP-deficient (KO) mice. Peyer's patch morphology from young (3-months-old) and aging (12–15-months-old) mice was examined, along with flow cytometry to assess immune cell populations, expression of checkpoint molecules (PD-1, PD-L1, TIM-3, Gal-9) and functional markers (CD69, granzyme B, perforin) in CD3+, CD4+, and CD8+ T cells. We found slight differences between aging, but not in young, WT, and KO mice. In WT mice, aging reduced CD8+ T cell numbers frequency and altered checkpoint molecule expression (higher TIM-3, granzyme B; lower Gal-9, CD69). CD4+ T cell frequency was higher with similar checkpoint alterations, indicating a regulatory shift. In PACAP KO mice, aging did not change cell population frequencies but led to higher TIM-3, granzyme B and lower PD-1, PD-L1, Gal-9, and CD69 expression in CD4+ and CD8+ T cells, with reduced overall T cell activity. Thus, PACAP deficiency impacts immune dysfunction by altering checkpoint molecules and T cell functionality, particularly in CD8+ T cells, suggesting complex immune responses by PACAP, highlighting its role in intestinal homeostasis and potential implications for inflammatory bowel diseases. [ABSTRACT FROM AUTHOR]

Members of the T-cell immunoglobulin and mucin (TIM) family, which is crucial for T-cell function, are implicated in autoimmunity. TIM-1 and -3 play distinct roles in autoimmunity, with TIM-1 acting as a costimulatory molecule and TIM-3 regulating Th1 responses. We investigated the therapeutic potential of anti-TIM-1 (RMT1-10) and anti-TIM-3 (RMT3-23) antibodies in an autoimmune arthritis model. Zymosan A was used to induce arthritis in female SKG mice. The arthritis scores, histology, mRNA expression, cytokine levels, micro-computed tomography, and flow cytometry results were obtained. The application of RMT1-10 reduced the arthritis scores, histological damage, and CD4+ T-cell infiltrations, and it suppressed interleukin (IL)-6 and -17A and reduced TIM-3 mRNA expressions. RMT3-23 also lowered arthritis severity, improved histology, and reduced serum levels of tumor necrosis factor (TNF)-α and IL-17A. RMT3-23 inhibited intracellular TNF-α and IL-6 and early apoptosis. An amelioration of autoimmune arthritis was achieved by blocking the TIM-1 and -3 signaling pathways via RMT1-10 and RMT3-23 administration, leading to a widespread decrease in inflammatory cytokines. Both antibodies exhibited therapeutic effects, suggesting TIM-1 and -3 as potential targets for rheumatoid arthritis. TIM-1 and -3 play distinct roles in autoimmunity, with TIM-1 acting as a costimulatory molecule and TIM-3 regulating Th1 responses. We investigated the therapeutic potential of anti-TIM-1 (RMT1-10) and anti-TIM-3 (RMT3-23) antibodies in an autoimmune arthritis model. An amelioration of autoimmune arthritis was achieved by blocking the TIM-1 and -3 signaling pathways via RMT1-10 and RMT3-23 administration, leading to a widespread decrease in inflammatory cytokines. Both antibodies exhibited therapeutic effects, suggesting TIM-1 and -3 as potential targets for rheumatoid arthritis. Graphical Abstract [ABSTRACT FROM AUTHOR]

Due to the genetic diversity between the mother and the fetus, heightened control over the immune system during pregnancy is crucial. Immunological parameters determined by clinicians in women with idiopathic recurrent spontaneous abortion (RSA) include the quantity and activity of Natural Killer (NK) and Natural Killer T (NKT) cells, the quantity of regulatory T lymphocytes, and the ratio of pro-inflammatory cytokines, which indicate imbalances in Th1 and Th2 cell response. The processes are controlled by immune checkpoint proteins (ICPs) expressed on the surface of immune cells. We aim to investigate differences in the expression of ICPs on T cells, T regulatory lymphocytes, NK cells, and NKT cells in peripheral blood samples collected from RSA women, pregnant women, and healthy multiparous women. We aim to discover new insights into the role of ICPs involved in recurrent pregnancy loss. Peripheral blood mononuclear cells (PBMCs) were isolated by gradient centrifugation from blood samples obtained from 10 multiparous women, 20 pregnant women (11–14th week of pregnancy), and 20 RSA women, at maximum of 72 h after miscarriage. The PBMCs were stained for flow cytometry analysis. Standard flow cytometry immunophenotyping of PBMCs was performed using antibodies against classical lymphocyte markers, including CD3, CD4, CD8, CD56, CD25, and CD127. Additionally, ICPs were investigated using antibodies against Programmed Death Protein-1 (PD-1, CD279), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3, CD366), V-domain Ig suppressor of T cell activation (VISTA), T cell immunoglobulin and ITIM domain (TIGIT), and Lymphocyte activation gene 3 (LAG-3). We observed differences in the surface expression of ICPs in the analyzed subpopulations of lymphocytes between early pregnancy and RSA, after miscarriage, and in women. We noted diminished expression of PD-1 on T lymphocytes (p = 0.0046), T helper cells (CD3CD4 positive cells, p = 0.0165), T cytotoxic cells (CD3CD8 positive cells, p = 0.0046), T regulatory lymphocytes (CD3CD4CD25CD127 low positive cells, p = 0.0106), and NKT cells (CD3CD56/CD16 positive cells, p = 0.0438), as well as LAG-3 on lymphocytes T (p = 0.0225) T helper, p = 0.0426), T cytotoxic cells (p = 0.0458) and Treg (p = 0.0293), and cells from RSA women. Impaired expression of TIM-3 (p = 0.0226) and VISTA (p = 0.0039) on CD8 cytotoxic T and NK (TIM3 p = 0.0482; VISTA p = 0.0118) cells was shown, with an accompanying increased expression of TIGIT (p = 0.0211) on NKT cells. The changes in the expression of surface immune checkpoints indicate their involvement in the regulation of pregnancy. The data might be utilized to develop specific therapies for RSA women based on the modulation of ICP expression. [ABSTRACT FROM AUTHOR]

The immune system plays a pivotal role in controlling chronic myeloid leukemia (CML). CD8+ T cell exhaustion results in reduced effectiveness of T cell-mediated immunity, thereby contributing to disease progression. This study intends to figure out whether the combined blockade of inhibitory molecules TIM-3/PD-1 can affect CD8+ T cell exhaustion in CML. A CML mouse model was established via transplantation of bone marrow cells transduced with BCR-ABL-expressing retrovirus vectors. PD-1 and TIM-3 signaling were blocked using corresponding molecular antibodies. Flow cytometry analysis was conducted to detect cell surface molecules and intracellular cytokines. ELISA was employed for measuring cytokine concentrations in the culture medium. The results showed that TIM-3 and PD-1 were coexpressed on exhausted CD8+ T cells from CML mice. Combined blockade of PD-1/TIM3 synergistically delayed CML progression in mice. Moreover, ex vivo experiments showed that their co-blockade promoted the proliferation and cytokine secretion of CD8+ T cells isolated from CML mice. In conclusion, blocking TIM-3 and PD-1 improves exhausted CD8+ T cell function in CML. [ABSTRACT FROM AUTHOR]

The T cell immunoglobulin domain and mucin domain-containing molecule-3 (TIM-3), an inhibitory checkpoint receptor, has been identified as a crucial regulator of cellular immune responses. TIM-3 has been discovered as a receptor involved in the negative regulation of T cells. Recent studies have demonstrated that TIM-3 is expressed on innate immune cells, including dendritic cells (DCs), even at a higher level than T cells. In the tumor microenvironment, the majority of DCs have a monocytic origin. Models for studying such DCs in vitro are DC cultures generated from monocytes in the presence of growth factors. The present study aimed to investigate the expression of TIM-3 in IFNα-induced monocyte-derived DCs (IFN-DCs) and the impact of DC activation on TIM-3 expression. DCs were obtained by culturing the adherent fraction of mononuclear cells from healthy donors for 4 days in the presence of GM-CSF and IFNα, followed by LPS addition for 24 hours. Human double-stranded DNA (dsDNA, 5 μg/mL) was added as an activation stimulus to intact IFN-DCs at the stage of maturation, along with LPS. Expression of the membrane TIM-3 molecule was determined by flow cytometry, and the level of expression of TIM-3 mRNA – by real-time RT-PCR with reverse transcription. Intact donor IFN-DCs expressed the membrane TIM-3 molecule at a high level (more than 70% of cells). The addition of LPS as a maturation stimulus almost halved the expression of TIM-3 (pW < 0.05) without affecting the expression of HAVCR2/TIM-3 mRNA. Exogenous dsDNA (along with LPS) increased the expression of HAVCR2/TIM-3 mRNA by more than three times (pW = 0.05) with a decrease in the number of TIM-3+DCs (pW = 0.003). Our findings indicate the presence of mechanisms that support expression of this inhibitory checkpoint receptor under conditions of DC activation. Further studies of the regulation of TIM-3 expression by monocyte-derived dendritic cells will expand the understanding of the biological significance of inhibitory receptors on DCs from the point of view of the immune response, as well as, in the future, increase the effectiveness of current approaches in cancer immunotherapy using IFN-DCs and inhibitors of checkpoint molecules.

In multiple myeloma (MM), the content of T lymphocytes expressing “checkpoint” molecules PD-1, TIM-3, LAG-3, etc. is increased. Regulatory T cells (Treg) can suppress antitumor immune response and play a sufficient role in MM pathogenesis. Like effector T lymphocytes, some Tregs express checkpoint receptors PD-1, TIM-3, etc., however, the biological meaning of such expression, as well as the consequences of blockade of these receptors, are not clear. The significance of type I regulatory T cells (Tr1), which produce the immunosuppressive cytokine interleukin-10, in MM also remains unexplored. The purpose of this work was to study the content of PD-1- and TIM-3-expressing Tregs and Tr1 in patients with MM. The study included 36 patients with MM and 24 matched healthy donors. The content of CD4+CD25hiCD127-FoxP3+Tregs and IL-10-producing CD4+IL-10+Tr1 populations expressing PD-1 and TIM-3 was assessed in peripheral blood (PB) and bone marrow (BM) by flow cytometry. The relative content of circulating CD4+CD25hiCD127-FoxP3+Tregs and IL-10-producing CD4+IL-10+Tr1 was significantly higher in MM patients compared to healthy donors. A higher relative content of IL-10-producing T lymphocytes was noted compared to Treg. The relative content of Tregs and Tr1 in BM samples did not differ significantly from PB values. The proportion of Tregs expressing PD-1 and TIM-3 in patients with MM did not differ significantly from the values in healthy donors. The content of PD-1- and TIM-3-positive CD4+IL-10+T cells was significantly higher in PB samples from MM patients compared to donors.IL-10-producing CD4+T cells constitute a significant proportion of T lymphocytes in the PB and BM of patients with MM and may play an important role in the pathogenesis of MM. Their content exceeds that of CD4+CD25hiCD127-FoxP3+Treg. A relatively small number of Tregs express the checkpoint receptors PD-1 and TIM-3, no different from donors. The proportion of PD-1-/TIM-3-positive cells is ~20% of CD4+IL-10+T cells and significantly exceeds the values of healthy individuals.

More than half of the world’s population is infected with the herpes simplex virus. In most cases, infection is not accompanied by symptoms, but in some people the disease occurs as a chronic infection with frequent and severe relapses. One of the most likely reasons for this may be a dysregulation of the immune system. In recent years, the role of checkpoint molecules, in particular PD-1 and Tim-3, in the regulation of the immune response and the functions of immunocompetent cells has been actively studied. Activation of PD-1 and Tim-3 on T cells has previously been shown to suppress the immune response. PD-1 and Tim-3 are also expressed on other immune cells, in particular monocytes. However, the expression of these molecules on monocytes during chronic viral infections has not been previously studied. The study was aimed at assessing the level of PD-1 and Tim-3 expression on various populations of monocytes in patients with chronic often recurrent herpesvirus infection. Twenty-six patients were recruited into the study. All patients received antiviral and immunomodulatory therapy in the immunological department. The number of classical, intermediate, and non-classical monocytes and the expression of PD-1 and Tim-3 on monocytes, were assessed by flow cytometry before and after the therapy. Monocytes were isolated from peripheral blood, and subpopulations were divided according to the level of expression of CD14 and CD16. In patients with herpes, a reduced number of monocytes was observed in comparison with healthy donors. The relative number of PD-1-positive monocytes, the mean fluorescence intensity of PD-1 and Tim-3, and the number of double-positive cells were reduced in herpes patients in all three monocyte subpopulations examined. Three months after therapy, the response to the therapy was assessed; patients who did not have a single recurrence of herpes within 3 months were considered to respond. Responding patients had a lower initial content of double-positive cells among intermediate and non-classical monocytes. The decrease in the level of PD-1 and Tim-3 positive monocytes during herpesvirus infection revealed in the present study may indicate the involvement of monocytes deficient in the expression of checkpoint molecules in the pathogenesis of the disease.

All types of immune cells are involved in the pathogenesis of multiple myeloma (MM). Granulocytic (G-MDSCs) and monocytic myeloid-derived suppressor cells (M-MDSCs) have significant protumor effects. The T cell immune response may be reduced due to the development of T cell exhaustion, characterized by the expression of inhibitory receptors PD-1, TIM-3, etc. Granulocyte colony-stimulating factor (G-CSF) supports the generation and expansion of MDSCs and can influence the functional properties of T cells. The purpose of our work was to investigate the possible effect of stimulation with G-CSF drugs on the induction of PD-1 and TIM-3 expression by T cells in patients with MM. The study included 40 patients with MM who underwent mobilization of hematopoietic progenitor cells with G-CSF drugs (5 mcg/kg/day) for 4-5 days. Content of CD4+PD-1+, CD4+TIM-3+, CD8+PD-1+, CD8+TIM-3+T cells, Lin-HLA-DR-CD33+CD66b+G-MDSCs, and CD14+HLA-DR-M-MDSCs was assessed before the start of a course of G-CSF injections (n = 33), after a course of G-CSF on the first day of separation of hematopoietic progenitor cells (n = 28) and after 3-6 months (n = 40) by flow cytometry. The relative content of G-MDSCs and M-MDSCs was significantly higher in patients with MM after a course of G-CSF. After 3-6 months, the content of G-MDSCs and M-MDSCs decreased to the initial values. After the course of G-CSF, an increase in the content of CD4+PD-1+T cells was noted compared to the values before the study. After 3-6 months, the content of this population did not differ from the initial values. The relative numbers of CD4+TIM-3+, CD8+PD-1+, and CD8+TIM-3+T cells did not change after a course of G-CSF. There were no significant correlations between the content of the populations of MDSCs and T cells expressing PD-1 and TIM-3 after a course of G-CSF.Mobilization of hematopoietic stem cells by G-CSF in patients with MM is accompanied by a transient increase in MM populations and an isolated increase in CD4+PD-1+T cells.

T cell immunoglobulin and mucin domain-3 (TIM-3), a crucial immune checkpoint following PD1 and CTLA4, is widely found in several immune cells. Nonetheless, its performance in recent clinical trials appears disappointing. Ovarian cancer (OC), a malignant tumor with a high mortality rate in gynecology, faces significant hurdles in immunotherapy. The broad presence of TIM-3 offers a new opportunity for immunotherapy in OC. This study reviews the role of TIM-3 in OC and assesses its potential as a target for immunotherapy. The regulatory effects of TIM-3 on the immune microenvironment in OC are discussed, with a focus on preclinical studies that demonstrate TIM-3's modulation of various immune cells in OC. Additionally, the potential therapeutic advantages and challenges of targeting TIM-3 in OC are examined. [ABSTRACT FROM AUTHOR]

T-cell immunoglobulin and mucin domain 3 (TIM-3) belongs to the group of inhibitory checkpoint receptors and has traditionally been of interest in terms of its expression on activated CD4+ and CD8+ T cells. The treatment with TIM-3 inhibitors is considered as a promising strategy in cancer immunotherapy. The review focuses on new data on the expression of TIM-3 on dendritic cells (DCs) that play a key role in initiating the antigen-specific immune response and inducing effector CD8+ T cells. The main hypothesis is that TIM-3 is suggested to act as a negative regulator of DCs. Further studies on TIM-3-mediated DC regulation will improve the effectiveness of current strategies in the treatment of cancer using DCs and checkpoint molecule inhibitors, where the main targets can be not only T cells, but also TIM-3-expressing DCs. [ABSTRACT FROM AUTHOR]

Introduction: Increasing drug resistance and the absence of a cure necessitates exploration of novel treatment strategies for people living with HIV (PLWH). Targeting of soluble co-inhibitory immune checkpoint molecules (sICMs) represents a novel, potentially effective strategy in the management of HIV. Methods: In this retrospective, longitudinal, observational study, the plasma levels of five prominent co-inhibitory sICMs—CTLA-4, LAG-3, PD-1 and its ligand PD-L1, as well as TIM-3—were quantified in 68 PLWH—before and one year after antiretroviral therapy (ART)—and compared with those of 15 healthy control participants. Results: Relative to control participants, PLWH had substantially elevated pre-treatment levels of all five co-inhibitory sICMs (p < 0.0001–p < 0.0657), which, over the 12-month period of ART, remained significantly higher than those of controls (p < 0.0367–p < 0.0001). PLWH with advanced disease, reflected by a CD4+ T cell count <200 cells/mm3 before ART, had the lowest levels of CTLA-4 and LAG-3, while participants with pre-treatment HIV viral loads ≥100,000 copies/mL had higher pre-treatment levels of TIM-3, which also persisted at 12 months. Conclusions: Plasma levels of CTLA-4, LAG-3, PD-1, PD-L1 and TIM-3 were significantly elevated in treatment-naïve PLWH and remained so following one year of virally-suppressive ART, possibly identifying LAG-3 and TIM-3 in particular as potential targets for adjuvant immunotherapy. [ABSTRACT FROM AUTHOR]

Introduction: The current study aims to evaluate the OX40, TIM-3, LAG-3, and PD-L1 targeted pathways in the regulation of T-cell activity in sarcoma patients to determine their relationship with overall survival (OS). Method: This study included one hundred and eleven patients with bone and soft tissue sarcoma diagnosed in two centers between 2010 and 2020. OX40, LAG-3, TIM-3 and PD-L1 expression levels were evaluated immunohistochemically from pathology preparations. Results: PD-L1 staining was detected in tumor cells, OX40, LAG-3, TIM-3 staining was detected in inflammatory cells in tumor tissue. In univariate analysis, no significant relationship was found between OX40, TIM-3, LAG-3, and PD-L1 staining and overall survival (respectively: p = 0.12, p = 0.49, p = 0.31, p = 0.95). When grade and stage at diagnosis, which were found to be significant in univariate analysis, along with OX-40, TIM-3, LAG-3, and PD-L1, were evaluated in multivariate analysis, a positive effect of OX-40 staining on overall survival was determined (p = 0.009). Considering the correlation between PDL-1 and OX40, TIM-3, and LAG-3 staining, a significant positive correlation was found between PDL-1 and TIM-3 and LAG-3 staining (respectively; p = 0.002, p = 0.001). Conclusions: There was no significant relationship between the PDL-1 staining percentage of tumor cells and OX40, TIM-3, and LAG-3 staining in inflammatory cells with the OS of sarcoma patients. However, detecting a significant positive correlation between PDL-1 staining and TIM-3 and LAG-3 staining also holds promise for finding effective targetable combination therapies that can prolong survival in sarcoma patients in the future. [ABSTRACT FROM AUTHOR]

Problem: Endometriosis is a prevalent chronic gynecological disease linked to immune dysfunction. The protein T‐cell immunoglobulin and mucin domain‐containing protein 3 (TIM‐3) plays a crucial role in immune system balance. Malfunction of TIM‐3 may result in excessive immune activation and inflammatory tissue damage. Given TIM‐3's established role in the development of cancer and autoimmune diseases, we decided to study its role in women suffering from endometriosis. Study Method: We included a total of 62 female patients, all of whom had undergone laparoscopic surgery. Of these, 47 had endometriosis and 15 did not. During surgery, we collected peritoneal fluid (PF) and peripheral blood (PB) samples from every patient for analysis using flow cytometry. To mark the samples, we used a panel of monoclonal antibodies and examined TIM‐3 expression in their immune cells. Results: Endometriosis patients in PB demonstrated a significantly lower percentage of CD56+ T cells with TIM‐3 expression. As endometriosis progressed through its stages, this expression lessened. This decrease was particularly notable in women with stage III/IV endometriosis. Additionally, both women diagnosed with intestinal endometriosis and those with recent endometriosis diagnoses showed a significantly reduced percentage of CD56+ T cells expressing TIM‐3. Conclusions: Patients with endometriosis exhibit diminished TIM‐3 expression within circulating T cells. This warrants further investigation to discern whether it contributes to the progression of endometriosis, potentially through the amplification of autoreactive T cells and inflammation. [ABSTRACT FROM AUTHOR]

Mycobacterium avium subspecies paratuberculosis (MAP) infection leads to chronic, persistent granulomatous enteritis, causing prolonged diarrhoea and emaciation. The disease is managed using medications such as antibiotics, live vaccines, mycobacteriophage therapies and other treatments; however, a notable proportion of affected animals do not show improvement with this approach. We hypothesise that immunoinhibitory receptors TIM-3 (T cell immunoglobulin mucin protein-3) and PD-1 (Programmed death receptor 1) may be upregulated on Peripheral blood mononuclear cells (PBMCs) of MAP-seropositive bovines, potentially contributing to immune exhaustion. Samples (blood and faeces) were collected from 32 diarrhoeic bovines suspected of MAP infection; eight apparently healthy buffaloes from the dairy farm at Hisar, Haryana and from 14 cows (suffering from chronic diarrhoea, weakness and emaciation) housed in stray cattle shed. MAP infection was estimated using indigenous ELISA (i-ELISA), faecal IS900 PCR, culture and acid-fast staining. TIM-3 and PD-1 gene expression on PBMCs were determined using qRT-PCR. TIM3 expression was relatively higher (~400-fold, 330-fold, 112-fold, 65-fold and 16-fold) in 5 chronically diarrhoeic PBMCs samples (MAP-seropositive), and higher PD-1 expression (around ~7-fold, 1.75-fold, 2.5-fold, 7.6-fold) was recorded in 4 diarrhoeic MAP-seropositive animals, compared to apparently healthy and other MAP-seronegative diarrhoeic animals. High co-expression of TIM-3 and PD-1 levels was also recorded in chronically diarrhoeic, emaciated stray cattle. Understanding immune responses in field conditions might aid in the therapeutic management of paratuberculosis. [ABSTRACT FROM AUTHOR]

ABSTRACTBackground 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

Introduction: Depending on the microenvironment, γδ T cells may assume characteristics similar to those of Th1, Th2, Th17, regulatory T cells or antigen presenting cells. Despite the wide documentation of the effect of Th1/Th2 balance on pregnancy associated malaria and outcomes, there are no reports on the relationship between γδ T cell phenotype change and Placental Malaria (PM) with pregnancy outcomes. This study sought to investigate the involvement of γδ T cells and its subsets in placental Plasmodium falciparum malaria. Methods: In a case-control study conducted in Yaoundé, Cameroon from March 2022 to May 2023, peripheral, placental and cord blood samples were collected from 50 women at delivery (29 PM negative: PM- and 21 PM positive: PM+; as diagnosed by light microscopy). Hemoglobin levels were measured using hemoglobinometer. PBMCs, IVBMCs and CBMCs were isolated using histopaque-1077 and used to characterize total γδ T cell populations and subsets (Vδ1+, Vδ2+, Vδ1-Vδ2-) by flow cytometry. Results: Placental Plasmodium falciparum infection was associated with significant increase in the frequency of total γδ T cells in IVBMC and of the Vd1+ subset in PBMC and IVBMC, but decreased frequency of the Vδ2+ subset in PBMC and IVBMC. The expression of the activation marker: HLA-DR, and the exhaustion markers (PD1 and TIM3) within total γδ T cells and subsets were significantly up-regulated in PM+ compared to PM- group. The frequency of total γδ T cells in IVBMC, TIM-3 expression within total γδ T cells and subsets in IVBMC, as well as HLA-DR expression within total γδ T cells and Vδ2+ subset in IVBMC were negatively associated with maternal hemoglobin levels. Furthermore, the frequency of total γδ T cells in PBMC and PD1 expression within the Vδ2+ subset in CBMC were negatively associated with birth weight contrary to the frequency of Vδ1-Vδ2- subset in PBMC and HLA-DR expression within the Vδ2+ subset in IVBMC which positively associated with maternal hemoglobin level and birth weight, respectively. Conclusion: The data indicate up-regulation of activated and exhausted γδ T cells in Plasmodium falciparum placental malaria, with effects on pregnancy outcomes including maternal hemoglobin level and birth weight. [ABSTRACT FROM AUTHOR]

Objective: The aberrant mobilization and activation of various T lymphocyte subpopulations play a pivotal role in the pathogenesis of diabetic kidney disease (DKD), yet the regulatory mechanisms underlying these processes remain poorly understood. Our study is premised on the hypothesis that the dysregulation of immune checkpoint molecules on T lymphocytes disrupts kidney homeostasis, instigates pathological inflammation, and promotes DKD progression. Methods: A total of 360 adult patients with DKD were recruited for this study. The expression of immune checkpoint molecules on T lymphocytes was assessed by flow cytometry for peripheral blood and immunofluorescence staining for kidney tissue. Single-cell sequencing (scRNA-seq) data from the kidneys of DKD mouse model were analyzed. Results: Patients with DKD exhibited a reduction in the proportion of CD3+TIM-3 + T cells in circulation concurrent with the emergence of significant albuminuria and hematuria (p=0.008 and 0.02, respectively). Conversely, the incidence of infection during DKD progression correlated with an elevation of peripheral CD3 +TIM-3+ T cells (p=0.01). Both univariate and multivariate logistic regression analysis revealed a significant inverse relationship between the proportion of peripheral CD3+TIM-3+ T cells and severe interstitial mononuclear infiltration (OR: 0.193, 95%CI: 0.040,0.926, p = 0.04). Immunofluorescence assays demonstrated an increase of CD3+, TIM-3+ and CD3+TIM-3+ interstitial mononuclear cells in the kidneys of DKD patients as compared to patients diagnosed with minimal change disease (p=0.03, 0.02 and 0.002, respectively). ScRNA-seq analysis revealed decreased gene expression of TIM3 on T lymphocytes in DKD compared to control. And one of TIM-3's main ligands, Galectin-9 on immune cells showed a decreasing trend in gene expression as kidney damage worsened. Conclusion: Our study underscores the potential protective role of TIM-3 on T lymphocytes in attenuating the progression of DKD and suggests that monitoring circulating CD3+TIM3+ T cells may serve as a viable strategy for identifying DKD patients at heightened risk of disease progression. [ABSTRACT FROM AUTHOR]

Simple Summary: Since TIM-3 on T cells or dendritic cells (DCs) has recently emerged as an attractive target for immunotherapy, we examined its expression on DCs using transcriptomic data from a public database and immunohistochemical evaluations from our cohorts of colorectal cancer. The expression of HAVCR2 (TIM-3) was strongly associated with the infiltration of DCs within the tumor microenvironment, and immunohistochemical staining of clinical tissue samples revealed that tumor-infiltrating DCs expressed TIM-3; however, their number at the tumor-invasive front significantly decreased with stage progression. Among vitro-generated DCs, TIM-3 expression was higher on immature DCs than on mature DCs, while Western blotting showed that STING expression was higher on mature DCs than on immature DCs. TIM-3 was originally identified as a negative regulator of helper T cells and is expressed on dendritic cells (DCs). Since the inhibition of TIM-3 on DCs has been suggested to enhance T cell-mediated anti-tumor immunity, we examined its expression on DCs within the tumor microenvironment (TME) in colorectal cancer (CRC) using transcriptomic data from a public database (n = 592) and immunohistochemical evaluations from our cohorts of CRC (n = 115). The expression of TIM-3 on DCs in vitro was examined by flow cytometry, while the expression of its related molecules, cGAS and STING, on immature and mature DCs was assessed by Western blotting. The expression of HAVCR2 (TIM-3) was strongly associated with the infiltration of DCs within the TME of CRC. Immunohistochemical staining of clinical tissue samples revealed that tumor-infiltrating DCs expressed TIM-3; however, their number at the tumor-invasive front significantly decreased with stage progression. TIM-3 expression was higher on immature DCs than on mature DCs from several different donors (n = 6). Western blot analyses showed that the expression of STING was higher on mature DCs than on immature DCs, which was opposite to that of TIM-3. We demonstrated that TIM-3 was highly expressed on tumor-infiltrating DCs of CRC and that its expression was higher on immature DCs than on mature DCs. [ABSTRACT FROM AUTHOR]

Hemorrhagic fever with renal syndrome (HFRS) is a rodent-borne disease widespread in Europe and Asia. HFRS is caused by negative-sensed single-stranded RNA orthohantaviruses transmitted to humans through inhaling aerosolized excreta of infected rodents. Symptoms of HFRS include acute kidney injury, thrombocytopenia, hemorrhages, and hypotension. The immune response raised against viral antigens plays an important role in the pathogenesis of HFRS. Inhibitory co-receptors are essential in regulating immune responses, mitigating immunopathogenesis, and reducing tissue damage. Our research showed an increased soluble form of inhibitory co-receptors TIM-3, LAG-3, and PD-1 in HFRS patients associated with disease severity. Our study aimed to investigate the impact of HFRS on the concentrations of soluble forms of inhibitory receptors TIM-3, LAG-3, and PD-1 in the patient's serum and the potential correlation with key clinical parameters. Our study aimed to investigate the impact of HFRS on the concentrations of soluble forms of inhibitory receptors TIM-3, LAG-3, and PD-1 in the patient's serum and their possible association with relevant clinical parameters. Using multiplex immunoassay, we found elevated levels of TIM-3, LAG-3, and PD-1 proteins in the serum of HFRS patients. Furthermore, increased levels were associated with creatinine, urea, lactate dehydrogenase concentrations, and platelet count. These findings suggest that these proteins play a role in regulating the immune response and disease progression. [ABSTRACT FROM AUTHOR]

Objective: Investigating T‐cell immunoglobulin and mucin‐domain containing‐3 (TIM‐3), Galectin 9 (Gal‐9), CD160 expression and tumor‐infiltrating lymphocytes (TILs) and correlation with clinicopathological characteristics of salivary adenoid cystic carcinoma (SACC). Methods: Sixty cases of SACC were detected by immunohistochemical staining to evaluate TIM‐3, Gal‐9, and CD160 expression and analyze the correlation between TIM‐3, Gal‐9, CD160 expression and clinicopathologic features by rank‐sum test. The association of TILs with TIM‐3, Gal‐9, and CD160 expression in SACC stromal was done by Chi‐square test. Results: TIM‐3 and CD160 overexpression were correlated with recurrence of SACC (p = 0.029, p = 0.007, respectively). High Gal‐9 expression was correlated with pathological classification (p = 0.018). The average percentage of TILs was 18.2% in SACC and most of TILs were more likely to occur in minor salivary glands (p = 0.038). Pairwise positive correlations were observed between the expression of TIM‐3, Gal‐9, and CD160 in tumor cells as well as in TILs, respectively. Conclusion: Low density of TILs was characteristic of the SACC microenvironment, with upregulation of TIM‐3, Gal‐9, and CD160 all occurring. However, TIM‐3, Gal‐9, and CD160 expression in the stromal dependent on the number of TILs represent potential therapeutic targets in SACC. [ABSTRACT FROM AUTHOR]

Recent studies have demonstrated that β-catenin in dendritic cells (DCs) serves as a key mediator in promoting both CD4 and CD8 T cell tolerance, although the mechanisms underlying how β-catenin exerts its functions remain incompletely understood. Here, we report that activation of β-catenin leads to the up-regulation of inhibitory molecule T-cell immunoglobulin and mucin domain 3 (Tim-3) in type 1 conventional DCs (cDC1s). Using a cDC1-targeted vaccine model with anti-DEC-205 engineered to express the melanoma antigen human gp100 (anti-DEC-205-hgp100), we demonstrated that CD11c-β-cateninactive mice exhibited impaired cross-priming and memory responses of gp100-specific CD8 T (Pmel-1) cells upon immunization with anti-DEC-205-hgp100. Single-cell RNA sequencing (scRNA-seq) analysis revealed that β-catenin in DCs negatively regulated transcription programs for effector function and proliferation of primed Pmel-1 cells, correlating with suppressed CD8 T cell immunity in CD11c-β-cateninactive mice. Further experiments showed that treating CD11c-β-cateninactive mice with an anti-Tim-3 antibody upon anti-DEC-205-hgp100 vaccination led to restored cross-priming and memory responses of gp100-specific CD8 T cells, suggesting that anti-Tim-3 treatment likely synergizes with DC vaccines to improve their efficacy. Indeed, treating B16F10-bearing mice with DC vaccines using anti-DEC-205-hgp100 in combination with anti-Tim-3 treatment resulted in significantly reduced tumor growth compared with treatment with the DC vaccine alone. Taken together, we identified the β-catenin/Tim-3 axis as a potentially novel mechanism to inhibit anti-tumor CD8 T cell immunity and that combination immunotherapy of a DC-targeted vaccine with anti-Tim-3 treatment leads to improved anti-tumor efficacy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

T cell transmembrane, Immunoglobulin, and Mucin (TIM) are important immune system proteins which are especially present in T-cells and regulated the immune system by sensing cell engulfment and apoptotic processes. Their role is exerted by the capacity to detect the presence of phosphatidyl-serine lipid polar head in the outer leaflet of cellular membranes (correlated with apoptosis). In this contribution by using equilibrium and enhanced sampling molecular dynamics simulation we unravel the molecular bases and the thermodynamics of TIM, and in particular TIM-3, interaction with phosphatidyl serine in a lipid bilayer. Since TIM-3 deregulation is an important factor of prooncogenic tumor micro-environment understanding its functioning at a molecular level may pave the way to the development of original immunotherapeutic approaches. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective After kidney transplantation, CD8+ T cells can infiltrate the kidney and cause necrosis, tubulitis, and even transplant rejection. For this reason, control of the T cell response is very important, and T cell immunoglobulin and mucin domain 3 (TIM-3) and programmed death 1 (PD-1) molecules play a role in regulating the T cell response. It is thought that the levels of TIM-3 and PD-1 expressions may be guiding in determining the clinical course after transplantation. This study aimed to determine the relationship between the mRNA levels of PD-1 and TIM-3 genes in peripheral blood samples taken from kidney transplant patients and the clinical conditions of the patients. Material and Method 60 peripheral blood samples were collected from 30 kidney transplant patients, both pre-transplantation (pre-tx) and post-transplantation (post-tx). CD8+ T cells were separated from other lymphocytes by magnetic cell separation system (MACS) and their purity was determined by flow cytometry. Then, RNA was isolated and after cDNA conversion, the expressions of PD-1 and TIM-3 genes were determined by real-time polymerase chain reaction. Results While it was determined that the TIM-3 gene expression level increased in patients with acute tubular necrosis, antibody-mediated rejection and cell-mediated rejection findings (p<0.001), no correlation was found between PD-1 expression levels and the clinical findings of the patients. Conclusion It is thought that comparing TIM-3 mRNA levels before and after kidney transplantation may be a useful tool in evaluating the clinical status of patients. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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Inflammatory bowel disease (IBD) occurring following allogeneic stem cell transplantation (aSCT) is a very rare condition. The underlying pathogenesis needs to be better defined. There is currently no systematic effort to exclude loss- or gain-of-function mutations in immune-related genes in stem cell donors. This is despite the fact that more than 100 inborn errors of immunity may cause or contribute to IBD. We have molecularly characterized a patient who developed fulminant inflammatory bowel disease following aSCT with stable 100% donor-derived hematopoiesis. A pathogenic c.A291G; p.I97M HAVCR2 mutation encoding the immune checkpoint protein TIM-3 was identified in the patient's blood-derived DNA, while being absent in DNA derived from the skin. TIM-3 expression was much decreased in the patient's serum, and in vitro-activated patient-derived T cells expressed reduced TIM-3 levels. In contrast, T cell-intrinsic CD25 expression and production of inflammatory cytokines were preserved. TIM-3 expression was barely detectable in the immune cells of the patient's intestinal mucosa, while being detected unambiguously in the inflamed and non-inflamed colon from unrelated individuals. In conclusion, we report the first case of acquired, "transplanted" insufficiency of the regulatory TIM-3 checkpoint linked to post-aSCT IBD. [ABSTRACT FROM AUTHOR]

T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is a checkpoint protein expressed in exhausted T-cells during cancer scenarios. This exhaustion may end in T-cell effector dysfunction, resulting in suboptimal control of cancers like acute myeloid leukemia (AML). Use of immune checkpoint inhibitors (ICIs) to block checkpoint receptors such as Tim-3 is an emerging, revolutionary concept in the immuno-oncology therapeutic arena; however, ICIs are not effective on myeloid malignancies. Here, a multifaceted approach is utilized to identify novel compounds that target and inhibit Tim-3 with improved efficacy. High-throughput virtual screening of the ChemBridge small molecule library and molecular dynamics simulation yielded a lead molecule C-5401331 predicted to bind with high affinity and inhibit the activity of Tim-3. In vitro evaluations demonstrated the compound to have anti-proliferative effects on Tim-3-positive populations of THP-1 and HC-5401331 AML cells, inducing early and late phase apoptosis. With further development, the lead molecule identified in this work has potential to aid the natural "gatekeeper" functions of the body in immunocompromised AML cancer patients by successfully hampering the binding of Tim-3 to T-cells. [ABSTRACT FROM AUTHOR]

Objective(s): 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).Materials and Methods: 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.Results: 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

Abstract Lung spindle cell carcinoma is an aggressive subtype of pleomorphic lung cancer resistant to cytotoxic chemotherapy. Programmed cell death‐1 (PD‐1) inhibitors have been reported to have clinical effects in patients with spindle cell carcinoma; however, the resistance mechanism to PD‐1 inhibitors is yet to be fully elucidated. Herein, we report the case of an 88‐year‐old man with G‐CSF‐producing spindle cell carcinoma who acquired resistance to PD‐1/PD‐ligand 1 (L1) inhibitor in an early setting after a remarkable response. A histopathological review of the resistant specimen revealed a low count of CD8+ T cells and a predominant presence of M2 and TIM‐3+ macrophages, indicating the presence of an immunosuppressive microenvironment. Our findings suggest a novel resistance mechanism to PD‐1/PD‐L1 inhibitors in G‐CSF‐producing spindle cell carcinoma.

PACAP (pituitary adenylate cyclase activating polypeptide) is a widespread neuropeptide with cytoprotective and anti-inflammatory effects. It plays a role in innate and adaptive immunity, but data are limited about gut-associated lymphoid tissue. We aimed to reveal differences in Peyer’s patches between wild-type (WT) and PACAP-deficient (KO) mice. Peyer’s patch morphology from young (3-months-old) and aging (12–15-months-old) mice was examined, along with flow cytometry to assess immune cell populations, expression of checkpoint molecules (PD-1, PD-L1, TIM-3, Gal-9) and functional markers (CD69, granzyme B, perforin) in CD3+, CD4+, and CD8+ T cells. We found slight differences between aging, but not in young, WT, and KO mice. In WT mice, aging reduced CD8+ T cell numbers frequency and altered checkpoint molecule expression (higher TIM-3, granzyme B; lower Gal-9, CD69). CD4+ T cell frequency was higher with similar checkpoint alterations, indicating a regulatory shift. In PACAP KO mice, aging did not change cell population frequencies but led to higher TIM-3, granzyme B and lower PD-1, PD-L1, Gal-9, and CD69 expression in CD4+ and CD8+ T cells, with reduced overall T cell activity. Thus, PACAP deficiency impacts immune dysfunction by altering checkpoint molecules and T cell functionality, particularly in CD8+ T cells, suggesting complex immune responses by PACAP, highlighting its role in intestinal homeostasis and potential implications for inflammatory bowel diseases.

Immune checkpoint inhibitors (ICIs) are specialized monoclonal antibodies (mAbs) that target immune checkpoints and their ligands, counteracting cancer cell-induced T-cell suppression. Approved ICIs like cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1), its ligand PD-L1, and lymphocyte activation gene-3 (LAG-3) have improved cancer patient outcomes by enhancing anti-tumor responses. However, some patients are unresponsive, and others experience immune-related adverse events (irAEs), affecting organs like the lung, liver, intestine, skin and now the cardiovascular system. These cardiac irAEs include conditions like myocarditis, atherosclerosis, pericarditis, arrhythmias, and cardiomyopathy. Ongoing clinical trials investigate promising alternative co-inhibitory receptor targets, including T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) and T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT). This review delves into the mechanisms of approved ICIs (CTLA-4, PD-1, PD-L1, and LAG-3) and upcoming options like Tim-3 and TIGIT. It explores the use of ICIs in cancer treatment, supported by both preclinical and clinical data. Additionally, it examines the mechanisms behind cardiac toxic irAEs, focusing on ICI-associated myocarditis and atherosclerosis. These insights are vital as ICIs continue to revolutionize cancer therapy, offering hope to patients, while also necessitating careful monitoring and management of potential side effects, including emerging cardiac complications. [ABSTRACT FROM AUTHOR]

To investigate the relationship between serum deoxyribonucleic acid methyltransferase 1 (DNMT1), T cellimmunoglobulin mucin-3(TIM-3) and hepatitis B virus-deoxyribonucleic acid (HBV-DNA) viral load and pregnancy outcome in patientswith pregnancy combined with hepatitis B virus (HBV) infection. 186 pregnant women with HBV infection who were admittedto The First Affiliated Hospital of Nanjing Medical University from January 2021 to January 2022 were selected as HBV infection group, patients were divided into positive group (56 cases) and negative group (130 cases) according to HBV-DNA viral load, patients were di-vided into poor outcome group and good outcome group according to the pregnancy outcome, another 150 healthy pregnant women whounderwent pregnancy tests at The First Affiliated Hospital of Nanjing Medical University during the same period were selected as controlgroup. Serum DNMT1 and TIM-3 levels were detected by enzyme-linked immunosorbent assay. The levels of serum DNMT1 and TIM-3were compared between HBV infection group and control group, positive group and negative group. The influencing factors of adverse pregnancy outcomes in pregnant women with HBV infection were analyzed by Univariate and multivariate logistic regression, the predictive value of serum DNMT1 and TIM-3 levels for adverse pregnancy outcomes in pregnant women with HBV infection were analyzed by receiver operating characteristic (ROC) curve. Compared with control group, the levels of serum DNMT1 and TIM-3 in HBV infection group were increased (P<0.05). Compared with negative group, the levels of serum DNMT1 and TIM-3 in positive group wereincreased (P<0.05). The incidence of adverse pregnancy outcomes in 186 pregnant women with HBV infection was 55.38 % (103/186).Univariate analysis showed that adverse pregnancy outcomes was related to the gestational age of HBV infection, HBV DNA Viral load, aspartate Transaminase (AST), Alanine transaminase (ALT), DNMT1, TIM-3(P<0.05). Multivariate Logistic regression analysis showed that, positive HBV-DNA viral load and DNMT1＞34.94 ng/mL and TIM-3＞18.96 pg/mL were independent risk factors for adverse pregnancy outcomes in pregnant women with HBV infection (P<0.05). ROC curve analysis showed that, the area under the curve of serum DNMT1 and TIM-3 levels alone and in combination to predict adverse pregnancy outcomes in pregnant women with HBV infection by detecting was 0.798, 0.791 and 0.870 respectively. The increase of serum DNMT1 and TIM-3 levels in pregnant women with HBV infection is closely relate to the positive HBV-DNA viral load and adverse pregnancy outcomes, and combination of serum DNMT1 and TIM-3 levels has a good predictive value for pregnancy outcome in pregnant women with HBV infection. [ABSTRACT FROM AUTHOR]

Malignant cells are known to evade immune surveillance by engaging immune checkpoints which are negative regulators of the immune system. By restoring the T-lymphocyte mediated anti-tumor effect, immune checkpoint inhibitors (ICI) have revolutionized the treatment of solid tumors but have met rather modest success in hematological malignancies. Currently, the only FDA approved indications for ICI therapy are in classic hodgkin lymphoma and primary mediastinal B cell lymphoma. Multiple clinical trials have assessed ICI therapy alone and in combination with standard of care treatments in other lymphomas, plasma cell neoplasms and myeloid neoplasms but were noted to have limited efficacy. These trials mostly focused on PD-1/PDL-1 and CTLA-4 inhibitors. Recently, there has been an effort to target other T-lymphocyte checkpoints like LAG-3, TIM-3, TIGIT along with improving strategies of PD-1/PDL-1 and CTLA-4 inhibition. Drugs targeting the macrophage checkpoint, CD47, are also being tested. Long term safety and efficacy data from these ongoing studies are eagerly awaited. In this comprehensive review, we discuss the mechanism of immune checkpoint inhibitors, the key takeaways from the reported results of completed and ongoing studies of these therapies in the context of hematological malignancies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective: We aimed to investigate the plasma levels and cell surface expression of two checkpoint molecules, TIM-3 (T cell immunoglobulin and mucin domain–containing protein 3) and PD-1 (programmed cell death protein 1), in pediatric patients with chronic non-bacterial osteomyelitis (CNO). Methods: Plasma samples of CNO patients were collected at diagnosis or during biologic agent treatment. Plasma levels of TIM-3 and PD-1 were measured using the sandwich enzyme-linked immunosorbent assay method, and the expression of the two immune checkpoint molecules on the cell surface was analyzed by isolating peripheral blood mononuclear cells by density gradient centrifugation technique. Results: Twenty-seven patients with CNO (14 boys, 51.9%) and six healthy controls (3 boys, 50%) were enrolled in the study. There were no age differences between CNO patients and healthy controls (median age 14.5 vs. 13.5 years, respectively, p=0.762). Of the CNO patients, 18 were included at the time of diagnosis while 9 were receiving biologic treatment at enrollment. The median plasma PD-1 levels were significantly lower in the CNO group than in the healthy controls (p=0.011). However, no significant difference was found in the cellular expression of PD-1 and TIM-3 on CD3+CD4+ T cells in patients and healthy controls (p=0.083 and p=0.245, respectively). There was also no statistically significant difference in plasma TIM-3 levels of the patient and control groups (p=0.981). Conclusion: CNO is an autoinflammatory disease, and overall, our results suggest that T cell exhaustion may not be significant in CNO. Further research is needed to find out whether the immune checkpoints are mainly associated with autoimmunity but not autoinflammation. Key Points • The median plasma PD-1 levels were significantly lower in the CNO group than in the healthy controls. • No significant difference was found in the cellular expression of PD-1 and TIM-3 on CD3+CD4+ T cells in patients and healthy controls. • Our results suggest that T cell exhaustion may not be significant in CNO pathogenesis. [ABSTRACT FROM AUTHOR]

Immune checkpoints (ICPs) serve as regulatory switches on immune-competent cells. Soluble ICPs consist of fragments derived from ICP molecules typically located on cell membranes. Research has demonstrated that they perform similar functions to their membrane-bound counterparts but are directly present in the bloodstream. Effective control of the maternal immune system is vital for a successful pregnancy due to genetic differences between the mother and fetus. Abnormalities in the immune response are widely acknowledged as the primary cause of spontaneous abortions. In our research, we introduce a novel approach to understanding the immune-mediated mechanisms underlying recurrent miscarriages and explore new possibilities for diagnosing and preventing pregnancy loss. The female participants in the study were divided into three groups: RSA (recurrent spontaneous abortion), pregnant, and non-pregnant women. The analysis of soluble forms of immune checkpoints and their ligands in the serum of the study groups was conducted using the Luminex method Statistically significant differences in the concentrations of (ICPs) were observed between physiological pregnancies and the RSA group. Among patients with RSA, we noted reduced concentrations of sGalectin-9, sTIM-3, and sCD155, along with elevated concentrations of LAG-3, sCD80, and sCD86 ICPs, in comparison to physiological pregnancies. Our study indicates that sGalectin-9, TIM-3, sLAG-3, sCD80, sCD86, sVISTA, sNectin-2, and sCD155 could potentially serve as biological markers of a healthy, physiological pregnancy. These findings suggest that changes in the concentrations of soluble immune checkpoints may have the potential to act as markers for early pregnancy loss. [ABSTRACT FROM AUTHOR]

Purpose: Immune checkpoint inhibitors (ICI) are then backbone in the therapy of metastatic renal cell carcinoma (RCC). The aim of this analysis was to explore the different expression of the ICI PD-L1, BTLA, and TIM-3 at the different tumor locations of the invasion front and the tumor center. Methods: Large-area sections of the tumor center and invasion front of 44 stage pT1–4 clear cell RCCs were examined immunohistochemically using antibodies against BTLA, TIM-3, and PD-L1 and subsequently correlated with clinicopathologic data. Results: TIM-3 was most strongly expressed at the invasion front (mean ± SD: 84.1 ± 46.6, p = 0.094). BTLA expression was highest in normal tissue, with weak staining in the tumor center and at the invasion front [110.2 vs. 18.6 (p < 0.001) vs. 32.2 (p = 0.248)]. PD-L1 was weakly expressed at the tumor center (n = 5/44) and at the invasion front (n = 5/44). Correlation with clinicopathological parameters revealed significantly higher BTLA expression in ≥ T3 tumors compared to T1/2 tumors (tumor center p = 0.009; invasion front p = 0.005). BTLA-positive tumors at the tumor center correlated with worse CSS (median 48.46 vs. 68.91 months, HR 4.43, p = 0.061). PD-L1 expression was associated with worse CSS (median 1.66 vs. 4.5 years, HR 1.63, p = 0.652). For TIM-3, there were no significant associations with clinicopathological parameters and survival. Conclusion: The present results show heterogeneous intratumoral and intertumoral expression of the investigated checkpoint receptors PD-L1, BTLA, and TIM-3. In the clinical practice tumor sampling should include different tumor locations, and multiple inhibition of different checkpoint receptors seems reasonable to increase the therapeutic success. [ABSTRACT FROM AUTHOR]

Gastric‐type endocervical adenocarcinoma (GEA) is the second most common subtype of endocervical adenocarcinoma and has a poor prognosis. Anti‐programmed death‐1 and anti‐programmed death‐ligand 1 (PD‐L1) inhibitors have emerged as a major treatment option for GEA; however, data on the expression of other immune checkpoints in GEA are limited. We analyzed the expression of T‐cell immunoglobulin and mucin‐domain containing‐3 (TIM‐3) and B7 homolog 3 protein (B7‐H3) in 58 GEA and investigated their prognostic significance as well as association with PD‐L1 expression and other known prognostic factors. Applying the tumor proportion score (TPS) with a cutoff of 1%, B7‐H3 and TIM‐3 were present in 48.3% and 17.2% of cases, respectively. Applying the combined positive score (CPS) with a cutoff of 1, TIM‐3 expression was present in 70.7% of cases. Moreover, the expression of three checkpoints (B7‐H3, TIM‐3, and PD‐L1) was incompletely overlapping. Patients with B7‐H3 positive tumors (by TPS) or TIM‐3 positive tumors (by TPS) had significantly worse recurrence‐free survival (RFS) and overall survival (OS) (log‐rank). Using CPS, patients with TIM‐3 positive tumors showed significantly worse RFS (log‐rank). Similarly, B7‐H3 positivity (by TPS) and TIM‐3 positivity (by TPS) were associated with worse RFS and OS in univariate analysis. TIM‐3 positivity (by CPS) was associated with worse RFS in univariate analysis and the final Cox multivariate analysis. In conclusion, our results show that (1) B7‐H3 and TIM‐3 are frequently expressed in GEA and their expression overlaps incompletely with PD‐L1; and (2) both B7‐H3 and TIM‐3 are independent negative prognostic markers in GEA. [ABSTRACT FROM AUTHOR]

Introduction: Patients with high-risk, triple negative breast cancer (TNBC) often receive neoadjuvant chemotherapy (NAC) alone or with immunotherapy. Various single-cell and spatially resolved techniques have demonstrated heterogeneity in the phenotype and distribution of macrophages and T cells in this form of breast cancer. Furthermore, recent studies in mice have implicated immune cells in perivascular (PV) areas of tumors in the regulation of metastasis and anti-tumor immunity. However, little is known of how the latter change during NAC in human TNBC or their impact on subsequent relapse, or the likely efficacy of immunotherapy given with or after NAC. Methods: We have used multiplex immunofluorescence and AI-based image analysis to compare the immune landscape in untreated and NAC-treated human TNBCs. We quantified changes in the phenotype, distribution and intercellular contacts of subsets of tumor-associated macrophages (TAMs), CD4+ and CD8+ T cells, and regulatory T cells (Tregs) in PV and non-PV various areas of the stroma and tumor cell islands. These were compared in tumors from patients who had either developed metastases or were disease-free (DF) after a three-year follow up period. Results: In tumors from patients who remained DF after NAC, there was a marked increase in stromal CD163+ TAMs, especially those expressing the negative checkpoint regulator, T-cell immunoglobulin and mucin domain 3 (TIM-3). Whereas CD4+ T cells preferentially located to PV areas in the stroma of both untreated and NAC-treated tumors, specific subsets of TAMs and Tregs only did so only after NAC. Distinct subsets of CD4+ and CD8+ T cells formed PV clusters with CD163+ TAMs and Tregs. These were retained after NAC. Discussion: Quantification of stromal TIM-3+CD163+ TAMs in tumor residues after NAC may represent a new way of identifying patients at high risk of relapse. PV clustering of immune cells is highly likely to regulate the activation and function of T cells, and thus the efficacy of T cell-based immunotherapies administered with or after NAC. [ABSTRACT FROM AUTHOR]

Objective(s): 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). Materials and Methods: 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. Results: 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). Conclusion: 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. [ABSTRACT FROM AUTHOR]

IntroductionDepending on the microenvironment, γδ T cells may assume characteristics similar to those of Th1, Th2, Th17, regulatory T cells or antigen presenting cells. Despite the wide documentation of the effect of Th1/Th2 balance on pregnancy associated malaria and outcomes, there are no reports on the relationship between γδ T cell phenotype change and Placental Malaria (PM) with pregnancy outcomes. This study sought to investigate the involvement of γδ T cells and its subsets in placental Plasmodium falciparum malaria.MethodsIn a case-control study conducted in Yaoundé, Cameroon from March 2022 to May 2023, peripheral, placental and cord blood samples were collected from 50 women at delivery (29 PM negative: PM- and 21 PM positive: PM+; as diagnosed by light microscopy). Hemoglobin levels were measured using hemoglobinometer. PBMCs, IVBMCs and CBMCs were isolated using histopaque-1077 and used to characterize total γδ T cell populations and subsets (Vδ1+, Vδ2+, Vδ1-Vδ2-) by flow cytometry.ResultsPlacental Plasmodium falciparum infection was associated with significant increase in the frequency of total γδ T cells in IVBMC and of the Vδ1+ subset in PBMC and IVBMC, but decreased frequency of the Vδ2+ subset in PBMC and IVBMC. The expression of the activation marker: HLA-DR, and the exhaustion markers (PD1 and TIM3) within total γδ T cells and subsets were significantly up-regulated in PM+ compared to PM- group. The frequency of total γδ T cells in IVBMC, TIM-3 expression within total γδ T cells and subsets in IVBMC, as well as HLA-DR expression within total γδ T cells and Vδ2+ subset in IVBMC were negatively associated with maternal hemoglobin levels. Furthermore, the frequency of total γδ T cells in PBMC and PD1 expression within the Vδ2+ subset in CBMC were negatively associated with birth weight contrary to the frequency of Vδ1-Vδ2- subset in PBMC and HLA-DR expression within the Vδ2+ subset in IVBMC which positively associated with maternal hemoglobin level and birth weight, respectively.ConclusionThe data indicate up-regulation of activated and exhausted γδ T cells in Plasmodium falciparum placental malaria, with effects on pregnancy outcomes including maternal hemoglobin level and birth weight.

The T cell immunoglobulin and mucin-domain containing-3 (TIM-3) receptor has gained significant attention as a promising target for cancer immunotherapy. The inhibitory effect of T cells by TIM-3 is mediated through the interaction between TIM-3 and its ligands. Ligand-blocking anti-TIM-3 antibodies possess the potential to reactivate antigen-specific T cells and augment anti-tumor immunity. However, the precise ligand-receptor interactions disrupted by the administration of TIM-3 blocking Abs have yet to be fully elucidated. In this study, we have developed a panel of monoclonal antibodies targeting human TIM-3, namely MsT001, MsT065, MsT229, and MsT286. They exhibited high sensitivities (10 pg/mL) and affinities (3.70 × 10−9 to 4.61 × 10−11 M) for TIM-3. The TIM-3 antibodies recognized distinct epitopes, including linear epitopes (MsT001 and MsT065), and a conformational epitope (MsT229 and MsT286). Additionally, the MsT229 and MsT286 displayed reactivity towards cynomolgus TIM-3. The interactions between TIM-3/Gal-9, TIM-3/HMGB-1, and TIM-3/CEACAM-1 disrupt the binding of MsT229 and MsT286, while leaving the binding of MsT001 and MsT065 unaffected. The inhibitory effect on the interaction between Gal-9 and TIM-3 was found to be dose-dependently in the presence of either MsT229 or MsT286. The findings suggested that the involvement of conformational epitopes in TIM-3 is crucial for its interaction with ligands, and we successfully generated novel anti-TIM-3 Abs that exhibit inhibitory potential. In conclusion, our finding offers valuable insights -on the comprehension and targeting of human TIM-3.

T-cell immunoglobulin domain and mucin domain-3 (Tim-3) is a versatile immunomodulator that protects against intestinal inflammation. Necroptosis is a type of cell death that regulates intestinal homeostasis and inflammation. The mechanism(s) underlying the protective role of macrophage Tim-3 in intestinal inflammation is unclear; thus, we investigated whether specific Tim-3 knockdown in macrophages drives intestinal inflammation via necroptosis. Tim-3 protein and mRNA expression were assessed via double immunofluorescence staining and single-cell RNA sequencing (sc-RNA seq), respectively, in the colonic tissues of patients with inflammatory bowel disease (IBD) and healthy controls. Macrophage-specific Tim3-knockout (Tim-3M−KO) mice were generated to explore the function and mechanism of Tim-3 in dextran sodium sulfate (DSS)-induced colitis. Necroptosis was blocked by pharmacological inhibitors of receptor-interacting protein kinase (RIP)1, RIP3, and reactive oxygen species (ROS). Additionally, in vitro experiments were performed to assess the mechanisms of neutrophil necroptosis induced by Tim-3 knockdown macrophages. Although Tim-3 is relatively inactive in macrophages during colon homeostasis, it is highly active during colitis. Compared to those in controls, Tim-3M−KO mice showed increased susceptibility to colitis, higher colitis scores, and increased pro-inflammatory mediator expression. Following the administration of RIP1/RIP3 or ROS inhibitors, a significant reduction in intestinal inflammation symptoms was observed in DSS-treated Tim-3M−KO mice. Further analysis indicated the TLR4/NF-κB pathway in Tim-3 knockdown macrophages mediates the TNF-α-induced necroptosis pathway in neutrophils. Macrophage Tim-3 regulates neutrophil necroptosis via intracellular ROS signaling. Tim-3 knockdown macrophages can recruit neutrophils and induce neutrophil necroptosis, thereby damaging the intestinal mucosal barrier and triggering a vicious cycle in the development of colitis. Our results demonstrate a protective role of macrophage Tim-3 in maintaining gut homeostasis by inhibiting neutrophil necroptosis and provide novel insights into the pathogenesis of IBD.

Abstract Background Primary biliary cholangitis (PBC) is a chronically progressive liver disease mediated by an autoimmune response. The aetiology and pathogenesis of PBC are not fully understood and may be related to immune disorders caused by genetic factors and their interaction with environmental factors. Immune checkpoints play an important role in preventing the occurrence of autoimmunity. However, the level of immune checkpoints in PBC has not been reported. Here, we aimed to identify the serum levels of soluble checkpoints in patients with PBC. Methods Soluble checkpoint levels were evaluated using enzyme-linked immunosorbent assay in 60 patients with PBC and 20 healthy controls (HCs). The expression of immune checkpoints was compared in liver biopsy tissue samples using immunohistochemistry. Receiver operating characteristic (ROC) curves and area under the curve (AUCs) were used to determine the diagnostic performance of soluble checkpoints and laboratory indexes between patients with PBC and HCs and patients with mild and advanced PBC. A logistic regression was performed for advanced PBC. Results sCD134, sLAG-3, sPD-1, sPD-L1, and sTIM-3 levels were significantly increased in patients with PBC compared with those in healthy controls. Additionally, the levels of sCD134, sPD-1, sPD-L1, and sTIM-3 were positively associated with disease progression. Moreover, soluble checkpoints were correlated with immunoglobulin and liver functions. ROC analyses between patients with PBC and HCs showed that the AUCs of sOX40, sPD-1, and sPD-L1 were 0.967, 0.922, and 0.971, respectively. The optimal cut-off values of sOX40, sPD-1, and sPD-L1 for PBC diagnosis were 89.15, 213.4, and 68, respectively. ROC analyses between mild and advanced patients with PBC revealed that the AUCs of sOX40 and sTIM-3 were 0.767 and 0.765, respectively. The optimal cut-off values for predicting PBC stage ≥ III were 199.45 and 361.5, respectively. In univariate analysis, age, ALB, and sOX40 were associated with advanced PBC. Further, the expression of CD134 and TIM-3 was upregulated in the liver of patients with PBC. Conclusions Our study results indicate that the serum titer of soluble checkpoints is increased in Chinese patients with PBC.