Role Of Lymphocyte Activation Gene-3 Research Articles

Monoclonal Antibody against Porcine LAG3 Inhibits Porcine Reproductive and Respiratory Syndrome Virus Infection

Lymphocyte activation gene 3 (LAG3) is an inhibitory receptor and the interaction between fibrinogen-like protein 1 and LAG3 can inhibit the anti-tumor effect of T cells both in vivo and in vitro, which was regarded as a new immune evasion mechanism. Porcine reproductive and respiratory syndrome (PRRS), caused by PRRSV, is an infectious disease characterized by reproductive disorders in pregnant sows and gilts and respiratory problems in pigs of all ages, seriously impacting the pig industry worldwide. In this study, monoclonal antibodies (mAbs) against porcine LAG3 (pLAG3) were developed, and one mAb (1C2) showed good reactivity with pLAG3 on PHA-activated porcine peripheral blood lymphocytes. Epitope mapping showed the epitope recognized by mAb 1C2 was located at amino acid residues 214–435 of pLAG3. LAG3 expression in the tissues of PRRSV-infected pigs was detected, using mAb 1C2 as the primary antibody, and the results revealed that PRRSV infection caused a marked increase in LAG3 expression compared to the control group. Interference of LAG3 expression on PHA-activated lymphocytes promoted PRRSV replication in the co-culture system of monocyte-derived dendritic cells and lymphocytes, whereas overexpression of LAG3 or blocking of the LAG3 signal with mAb 1C2 inhibited PRRSV replication, indicating that PRRSV infection activates the LAG3-signaling pathway, suggesting that this pathway plays an important role in PRRSV pathogenesis. The results obtained lay the foundation for subsequent research on the role of LAG3 in PRRS and other diseases with persistent infection characteristics.

Shedding light on the role of LAG-3 in hepatocellular carcinoma: unraveling immunomodulatory pathways

Hepatocellular carcinoma (HCC) stands as a primary malignant liver tumor characterized by chronic inflammation and complex alterations within the tumor microenvironment (TME). Lymphocyte activation gene 3 (LAG-3), also known as CD223, has gained prominence as a potential next-generation immune checkpoint, maintaining continuous expression in response to persistent antigen exposure within the TME, warranting our attention. In patients with HCC, LAG-3 expression on T cells, regulatory T cells (Tregs), and natural killer (NK) cells contributes to immune evasion, while high expression of LAG-3 leads to increased angiogenesis and poor prognosis. By interacting with major histocompatibility complex class II molecules, LAG-3 promotes T cell exhaustion and suppresses antitumor responses, often in collaboration with other immune checkpoints like programmed cell death protein 1 (PD-1), while on Tregs and NK cells, LAG-3 modulates their suppressive functions, indirectly facilitating tumor immune escape. LAG-3 expression may offer prognostic insights, correlating with disease progression and outcomes in HCC patients, while various preclinical studies highlight the potential of LAG-3-targeted therapies in reinvigorating immune responses against HCC, with a few combination approaches targeting LAG-3 alongside other checkpoints demonstrating synergistic effects in restoring T cell function. Therefore, harnessing LAG-3 as a therapeutic target holds promise for enhancing antitumor immunity and potentially improving HCC treatment outcomes. Our narrative review aims to delve into the full spectrum of LAG-3 signaling in HCC, with the goal of a better understanding of the pathophysiological and immunological basis of its use to arrest HCC growth and development.

Upregulation of LAG3 modulates the immune imbalance of CD4+ T-cell subsets and exacerbates disease progression in patients with alveolar echinococcosis and a mouse model.

Infection with the cestode Echinococcus multilocularis (E. multilocularis) causes alveolar echinococcosis (AE), a tumor-like disease predominantly affecting the liver but able to spread to any organ. T cells develop functional defects during chronic E. multilocularis infection, mostly due to upregulation of inhibitory receptors such as T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) and programmed death-1 (PD-1). However, the role of lymphocyte activation gene-3 (LAG3), an inhibitory receptor, in AE infection remains to be determined. Here, we discovered that high expression of LAG3 was mainly found in CD4+ T cells and induced regulatory T cells (iTregs) in close liver tissue (CLT) from AE patients. In a mouse model of E. multilocularis infection, LAG3 expression was predominantly found in T helper 2 (Th2) and Treg subsets, which secreted significantly more IL-4 and IL-10, resulting in host immune tolerance and disease progression at a late stage. Furthermore, LAG3 deficiency was found to drive the development of effector memory CD4+ T cells and enhance the type 1 CD4+ T-cell immune response, thus inhibiting metacestode growth in vivo. In addition, CD4+ T cells from LAG3-deficient mice produced more IFN-γ and less IL-4 when stimulated by E. multilocularis protoscoleces (EmP) antigen in vitro. Finally, adoptive transfer experiments showed that LAG3-knockout (KO) CD4+ T cells were more likely to develop into Th1 cells and less likely to develop into Tregs in recipient mice. Our work reveals that high expression of LAG3 accelerates AE disease progression by modulating the immune imbalance of CD4+ T-cell subsets. These findings may provide a novel immunotherapeutic strategy against E. multilocularis infection.

Diminished LAG3+ B cells correlate with exacerbated rheumatoid arthritis

Background Lymphocyte activation gene-3 (LAG3) positive B cells have been identified as a novel regulatory B cell subset, while the role of LAG3+ B cells in the pathogenesis of rheumatoid arthritis (RA) remains elusive. Materials and methods Peripheral blood mononuclear cells (PBMCs) from RA, osteoarthritis (OA) patients and healthy volunteers were collected for flow cytometry staining of LAG3+ B cells. Their correlation with RA patient clinical and immunological features were analyzed. Moreover, the frequencies of LAG3+ B cells in collagen-induced arthritis (CIA) mice and naive mice were also detected. Results A significant decrease of LAG3+ B cells was observed in RA patients as compared with healthy individuals and OA patients. Notably, the frequencies of LAG3+ B cells were negatively correlated with tender joint count (r = −0.4301, p = .0157) and DAS28-ESR (r = −0.4018, p = .025) in RA patients. In CIA mice, LAG3+ B cell frequencies were also decreased and negatively correlated with the CIA arthritis score. Conclusions Impairment of LAG3+ B cells potentially contributes to RA development. Reconstituting LAG3+ B cells might provide novel therapeutic strategies for the persistent disease. Key messages LAG3+ B cells have been identified as a novel regulatory B cell subset. However, its role in the pathogenesis of RA remains unknown. This study revealed the decreased frequency of LAG3+ B cells in RA patients. Notably, LAG3+ B cells were negatively correlated with RA disease activity including the tender joint count and DAS28-ESR. In CIA mice, LAG3+ B cell frequencies were also decreased and negatively correlated with the CIA arthritis score. Reconstitution of LAG3+ B cells might provide novel therapeutic strategies for disease perpetuation.

404.5: The Role of LAG3 in Antibody Responses to Kidney Transplantation

The functions of coinhibitory receptor lymphocyte activation gene-3 (LAG3) in T cells are well studied, however its role in humoral immune responses remains poorly characterized. The goal of this study was to test the role of recipient LAG3 in a mouse model of renal allograft rejection. Murine kidney transplant surgeries were performed from C3H (H-2Dk) donors to B6 WT, LAG3-/-, CD4CreLAG3fl/fl & CD19CreLAG3fl/fl recipients (H-2Db) after bilateral nephrectomy. Grafts were evaluated by immunistochemistry. WT recipients were treated with anti-mCD20 or anti-CD8 mAbs to deplete B cells or CD8 T cells, respectively. Immune responses were assessed by flow cytometry, ELISPOT assay, and serum levels of MHC-I and MHC-II-reactive IgG donor specific antibody (DSA) were determined by ELISA. Compared to WT animals, naïve B6.LAG3-/- mice have elevated numbers of CD44hi memory T cells, CXCR5hi follicular T cells, and B220+CD138+ plasma cells, and increased frequencies of memory T cells reactive to H-2Dd, H-2Ds, H-2Dq and H-2Dk (Figure 1A) and increased levels of serum IgG antibodies against H-2Dd, H-2Dk, I-Ad and I-Ak alloantigens (Figure 1B). All C3H kidney allografts survived for > 60d (n=5) in WT recipients, whereas recipient LAG3 deficiency led to rapid allograft rejection (MST of 14d, n=5) (Figure 2A) and elevated serum creatinine levels at d14 posttransplant. Compared to WT, LAG3-/- recipients had elevated frequencies of anti-donor IFNγ producing T cells and increased levels of DSA against MHC-I and MHC-II (Figure 2B). Graft histology at rejection revealed minimal T cell infiltration, diffuse C4d staining, atrophic peritubular capillaries, endothelial swelling and edema characteristic of antibody mediated rejection (AMR) (Figure 2C). Recipient CD8 T cell depletion did not alter rejection kinetics in LAG3-/- recipients (MST of 16d) (Figure 3A&B), and graft histological findings (Figure 3C) mirrored those of unmanipulated LAG3 deficient recipients (Figure 2C), demonstrating hallmark characterstics of AMR. B cell depletion significantly extended C3H kidney allograft survival (MST of >30d) (Figure 3D&E), suggesting the predominant role of alloantibody rather than T cell mediated rejection in these mice. However, neither T nor B cell conditional knockout recipients rejected the allograft demonstrating LAG3 expression on both cell types is necessary to mediate rejection.These findings demonstrate that LAG3 regulates both T and B cell functions in response to kidney allografts, and is an attractive therapeutic target for the prevention of AMR.

The Role of LAG3 in Antibody Responses To Kidney Transplantation

Abstract The role of lymphocyte activation gene-3 (LAG3) in T cell functions is well studied, however its role in humoral immune responses remains poorly characterized. The goal of this study was to test the role of recipient LAG3 in a mouse model of renal allograft rejectionNaive B6.LAG3−/− mice have elevated numbers of follicular and memory T cells, and plasma cells compared to WT mice, as well as increased frequencies of memory T cells reactive to H-2Dd, H-2Ds, H-2Dq and H-2Dk alloantigens. When kidneys were transplanted from C3H donors to B6 WT and LAG3−/− mice, all C3H kidney allografts survived for > 60d in WT recipients, whereas LAG3−/− recipients rapidly reject allografts and have elevated serum creatinine levels at d14 posttransplant. Graft histology at rejection revealed minimal T cell infiltration, diffuse C4d staining, atrophic peritubular capillaries, endothelial swelling and edema characteristic of antibody mediated rejection (AMR). Compared to WT, LAG3−/− recipients had elevated frequencies of anti-donor IFNγ producing T cells and increased levels of donor specific antibody (DSA) against MHC-I and MHC-II. Depletion of CD8 T cells in LAG3−/− recipients did not alter rejection kinetics, while B cell depletion significantly extended C3H kidney allograft survival, suggesting the predominant role of alloantibody rather than T cell mediated rejection in these mice. However, neither T nor B cell conditional knockout recipients rejected the allograft demonstrating LAG3 expression on both cell types is necessary to mediate rejection. These findings demonstrate that LAG3 regulates both T and B cell functions in response to kidney allografts, and is an attractive therapeutic target for the prevention of AMR. Supported by grants from NIH (P01AI087586-10)

Lymphocyte activation gene-3 is associated with programmed death-ligand 1 and programmed cell death protein 1 in small cell lung cancer.

BackgroundIn recent years, immunotherapy has achieved notable success in cancer treatment. Indeed, the novel immune checkpoint lymphocyte activation gene-3 (LAG3) has shown promising therapeutic efficacy in non-small cell lung cancer. However, it is unclear about the role of LAG3 in immunotherapy and survival in small cell lung cancer (SCLC).MethodsThe expression of LAG3 in SCLC was evaluated in four public datasets. The association of LAG3 with programmed death-ligand 1 (PD-L1), programmed cell death protein 1 (PD-1), and overall survival (OS) was investigated. The LAG3-related biological processes and pathways were identified by functional analyses.ResultsLAG3 expression was detected in SCLC tumor tissues. In the cBioPortal dataset with 81 clinical SCLC samples, LAG3 expression was markedly associated with PD-1 and PD-L1 expression (both P<0.050). In addition, Patients with high LAG3 expression had a trend toward a better OS (P=0.073). A similar survival trend was also observed in the GSE60052 dataset. Significantly, LAG3 expression was related to immune-related biological processes, such as immune response, antigen processing and presentation, and T cell co-stimulation (all P<0.001).ConclusionsThis study demonstrated that LAG3 is an important immune checkpoint that is closely associated with PD-1/PD-L1. LAG3 may be a promising novel immunotherapy target for SCLC.

Lymphocyte-activation gene 3 expression associates with poor prognosis and immunoevasive contexture in Epstein-Barr virus-positive and MLH1-defective gastric cancer patients.

Lymphocyte activation gene 3 (LAG-3) is a transmembrane immune checkpoint that facilitates immune escape via suppressing T-cell-mediated anti-tumor immunity. The role of LAG-3 in gastric cancer is little known. Consequently, we assessed the clinical significance of LAG-3 in gastric cancer. In our study, patients with gastric cancer from Zhongshan Hospital (n = 464) and data from the Asian Cancer Research Group (n = 300) were analyzed. LAG-3+ cell infiltration and other immune contexture in gastric cancer were detected by immunohistochemistry. Kaplan-Meier curves and log-rank test were used for survival analyses. Intratumoral LAG-3+ cells mainly accumulated in Epstein-Barr virus (EBV)-positive (EBV subtype) and MLH1-defective (dMLH1 subtype) gastric cancer. Furthermore, LAG-3+ cell infiltration was strongly associated with inferior clinical outcomes in patients with these two subtypes of gastric cancer. Moreover, we found intratumoral LAG-3+ cell high infiltration was associated with an immunoevasive contexture featured by decreased IFN-γ+ cells and perforin-1+ cells, but increased regulatory T cells and M2-like macrophages in EBV/dMLH1 subtype of gastric cancer. LAG-3 was a poor prognostic factor and might be a potential immunotherapeutic target in EBV-positive and MLH1-defective gastric cancer.

Lymphocyte-Activation Gene 3 (LAG3) Protein as a Possible Therapeutic Target for Parkinson's Disease: Molecular Mechanisms Connecting Neuroinflammation to α-Synuclein Spreading Pathology.

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder without any objective biomarker available to date. Increasing evidence highlights the critical role of neuroinflammation, including T cell responses, and spreading of aggregated α-synuclein in PD progression. Lymphocyte-activation gene 3 (LAG3) belongs to the immunoglobulin (Ig) superfamily expressed by peripheral immune cells, microglia and neurons and plays a key role in T cell regulation. The role of LAG3 has been extensively investigated in several human cancers, whereas until recently, the role of LAG3 in the central nervous system (CNS) has been largely unknown. Accumulating evidence highlights the potential role of LAG3 in PD pathogenesis, mainly by binding to α-synuclein fibrils and affecting its endocytosis and intercellular transmission, which sheds more light on the connection between immune dysregulation and α-synuclein spreading pathology. Serum and cerebrospinal fluid (CSF) soluble LAG3 (sLAG3) levels have been demonstrated to be potentially associated with PD development and clinical phenotype, suggesting that sLAG3 could represent an emerging PD biomarker. Specific single nucleotide polymorphisms (SNPs) of the LAG3 gene have been also related to PD occurrence especially in the female population, enlightening the pathophysiological background of gender-related PD clinical differences. Given also the ongoing clinical trials investigating various LAG3-targeting strategies in human diseases, new opportunities are being developed for PD treatment research. In this review, we discuss recent preclinical and clinical evidence on the role of LAG3 in PD pathogenesis and biomarker potential, aiming to elucidate its underlying molecular mechanisms.

CD4+CD25−LAG3+ regulatory T cells in humoral immunity

AbstractRegulatory T cells (Tregs) are necessary for the maintenance of peripheral immune tolerance. These Tregs are divided into two main subsets: thymus‐derived Tregs and peripherally‐derived Tregs. As a thymus‐derived Treg subset, CD4+CD25+ Tregs (CD25+ Tregs) that express the transcription factor, Forkhead box P3 (Foxp3), have been extensively studied. Type 1 regulatory T cells are representative Foxp3‐independent peripherally‐derived Tregs that produce a large amount of the inhibitory cytokine, interleukin (IL)‐10, and suppress immune responses through IL‐10 production. Accumulating evidence has shown that lymphocyte activation gene 3 (LAG3) is a reliable cell surface marker for type 1 regulatory T cells. Peripherally‐derived CD4+CD25−LAG3+ Tregs (LAG3+ Tregs) produce a large amount of IL‐10 and suppress colitis in a mouse model in an IL‐10‐dependent manner. LAG3+ Tregs characteristically express transcriptional factor early growth response gene 2 (Egr2), and ectopic expression of Egr2 conferred a suppressive function to CD4+ T cells. Intriguingly, polymorphism of the EGR2 gene is associated with susceptibility to systemic lupus erythematosus characterized by a wide spectrum of auto‐antibodies, and mice lacking Egr2 specifically in lymphocytes develop a lupus‐like autoimmune disease. We recently reported that Egr2‐expressing LAG3+ Tregs effectively suppress humoral immune responses in a transforming growth factor β3‐dependent manner. In addition, transforming growth factor β3 and IL‐10 synergistically regulate Toll‐like receptor‐mediated humoral immune responses. In the present review, we focus on the role of LAG3+ Tregs in humoral immunity, and also discuss its therapeutic potential for the treatment of autoimmune diseases.

Overexpression of Lymphocyte Activation Gene-3 Inhibits Regulatory T Cell Responses in Osteoarthritis.

Lymphocyte activation gene-3 (LAG-3) is a CD4 homologue expressed on the surface of activated conventional T cells and regulatory T (Treg) cells. In conventional T cells, LAG-3 acts as an inhibitory receptor of T cell inflammation. However, the role of LAG-3 in Treg cells remains controversial. In this study, we investigated the effect of LAG-3 on Tregs in osteoarthritis (OA). We observed that the proportion of LAG-3-expressing cells in CD4+CD25+/high T cells and Foxp3+CD4+CD25+/high T cells were significantly upregulated in OA patients. The level of LAG-3-to-Foxp3 ratio was further increased in synovial fluid. Several aspects of Treg responses in LAG-3+ and LAG-3- Treg cells were then examined. First, LAG-3+ Treg cells demonstrated significantly lower proliferation than LAG-3- Treg cells. Second, the increase in interleukin (IL)-10 and transforming growth factor (TGF)-β expression was significantly lower in LAG-3+ Treg cells than that in LAG-3- Treg cells. Third, LAG-3+ Treg cells were less capable of inhibiting the proliferation of CD4+CD25- conventional T cells than LAG-3- Treg cells. This study suggests that Treg cells in OA may be enriched with a LAG-3-expressing population that exhibits functional impairment, which limits their capacity in suppressing inflammation.

The upregulation of LAG-3 on T cells defines a subpopulation with functional exhaustion and correlates with disease progression in HIV-infected subjects.

T cells develop functional defects during HIV-1 infection, partially due to the upregulation of inhibitory receptors such as programmed death-1 (PD-1) and CTLA-4. However, the role of lymphocyte activation gene-3 (LAG-3; CD223), also known as an inhibitory receptor, in HIV infection remains to be determined. In this study, we revealed that LAG-3 on T cells delivers an inhibitory signal to downregulate T cell functionality, thereby playing an immunoregulatory role during persistent HIV-1 infection. We observed that HIV-1 infection results in a significant increase in LAG-3 expression in both the peripheral blood and the lymph nodes. The upregulation of LAG-3 is dramatically manifested on both CD4(+) and CD8(+) T cells and is correlated with disease progression. As expected, prolonged antiretroviral therapy reduces the expression of LAG-3 on both CD4(+) and CD8(+) T cells. The ex vivo blockade of LAG-3 significantly augments HIV-specific CD4(+) and CD8(+) T cell responses, whereas the overexpression of LAG-3 in T cells or the stimulation of LAG-3 on T cells leads to the reduction of T cell responses. Furthermore, most LAG-3 and PD-1 are expressed in different T cell subsets. Taken together, these data demonstrate that the LAG-3/MHC class II pathway plays an immunoregulatory role, thereby providing an important target for enhancing immune reconstitution in HIV-infected patients. Additionally, the LAG-3/MHC class II pathway may synergize with PD-1/PD ligand to enhance T cell-mediated immune responses.

Role of Lymphocyte Activation Gene-3 (Lag-3) in Conventional and Regulatory T Cell Function in Allogeneic Transplantation

Lag-3 has emerged as an important molecule in T cell biology. We investigated the role of Lag-3 in conventional T cell (Tcon) and regulatory T cell (Treg) function in murine GVHD with the hypothesis that Lag-3 engagement diminishes alloreactive T cell responses after bone marrow transplantation. We demonstrate that Lag-3 deficient Tcon (Lag-3−/− Tcon) induce significantly more severe GVHD than wild type (WT) Tcon and that the absence of Lag-3 on CD4 but not CD8 T cells is responsible for exacerbating GVHD. Lag-3−/− Tcon exhibited increased activation and proliferation as indicated by CFSE and bioluminescence imaging analyses and higher levels of activation markers such as CD69, CD107a, granzyme B, and Ki-67 as well as production of IL-10 and IFN-g early after transplantation. Lag-3−/− Tcon were less responsive to suppression by WT Treg as compared to WT Tcon. The absence of Lag-3, however, did not impair Treg function as both Lag-3−/− and WT Treg equally suppress the proliferation of Tcon in vitro and in vivo and protect against GVHD. Further, we demonstrate that allogeneic Treg acquire recipient MHC class II molecules through a process termed trogocytosis. As MHC class II is a ligand for Lag-3, we propose a novel suppression mechanism employed by Treg involving the acquisition of host MHC-II followed by the engagement of Lag-3 on T cells. These studies demonstrate for the first time the biologic function of Lag-3 expression on conventional and regulatory T cells in GVHD and identify Lag-3 as an important regulatory molecule involved in alloreactive T cell proliferation and activation after bone marrow transplantation.

The negative regulatory function of the lymphocyte‐activation gene‐3 co‐receptor (CD223) on human T cells

Accumulating evidence indicates that the CD4 homologue lymphocyte activation gene-3 (LAG-3) plays a down-regulatory role on T-cell responses. However, the role of LAG-3/major histocompatibility complex (MHC) class II interactions on primary human T-cell responses, as well as the mechanism by which down-regulation occurs, are not clear. Here, we show that LAG-3 colocalized with CD3, CD4 or CD8 in areas of cholesterol-rich raft aggregation during this primary response, as well as in the clustered raft region formed between T cells and antibody-coated beads. Addition of a blocking LAG-3-specific monoclonal antibody to both CD4 and CD8 primary resting T cells activated under conditions of antigen-presenting cell-driven stimulation and low antigen concentrations augments CD69 activation antigen expression, T-cell expansion and T helper 1 (Th1, but not Th2) cytokine production. Blocking LAG-3/MHC class II interactions leads to an increase in the number of cells entering division at these low concentrations of antigen and to more rounds of divisions with an accumulation of cells in the S-phase of the cell cycle. These results indicate that LAG-3 signalling inhibits early events in primary activation of human CD4 and CD8 T cells and further support a role for LAG-3 signalling in regulating the expansion of activated effector or memory T cells, either directly or indirectly through Treg suppressor activity.