Lymphocyte Activation Gene-3 Expression Research Articles

First-Line Nivolumab and Relatlimab Plus Chemotherapy for Gastric or Gastroesophageal Junction Adenocarcinoma: The Phase II RELATIVITY-060 Study

PURPOSE Open-label phase II study (RELATIVITY-060) to investigate the efficacy and safety of first-line nivolumab, a PD-1–blocking antibody, plus relatlimab, a lymphocyte-activation gene 3 (LAG-3)–blocking antibody, plus chemotherapy in patients with previously untreated advanced gastric cancer (GC) or gastroesophageal junction cancer (GEJC). METHODS Patients with unresectable, locally advanced or metastatic GC/GEJC were randomly assigned 1:1 to nivolumab + relatlimab (fixed-dose combination) + chemotherapy or nivolumab + chemotherapy. The primary end point was objective response rate (ORR; per RECIST v1.1 by blinded independent central review [BICR]) in patients whose tumors had LAG-3 expression ≥1%. RESULTS Of 274 patients, 138 were randomly assigned to nivolumab + relatlimab + chemotherapy and 136 to nivolumab + chemotherapy. Median follow-up was 11.9 months. In patients with LAG-3 expression ≥1%, BICR-assessed ORR (95% CI) was 48% (38 to 59) in the nivolumab + relatlimab + chemotherapy arm and 61% (51 to 71) in the nivolumab + chemotherapy arm; median progression-free survival (95% CI) by BICR was 7.0 months (5.8 to 8.4) versus 8.3 months (6.9 to 12.1; hazard ratio [HR], 1.41 [95% CI, 0.97 to 2.05]), and median overall survival (95% CI) was 13.5 months (11.9 to 19.1) versus 16.0 months (10.9 to not estimable; HR, 1.04 [95% CI, 0.70 to 1.54]), respectively. Grade 3 or 4 treatment-related adverse events (TRAEs) occurred in 69% and 61% of all treated patients, and 42% and 36% of patients discontinued because of any-grade TRAEs in the nivolumab + relatlimab + chemotherapy and nivolumab + chemotherapy arms, respectively. CONCLUSION RELATIVITY-060 did not meet its primary end point of improved ORR in patients with LAG-3 expression ≥1% when relatlimab was added to nivolumab + chemotherapy compared with nivolumab + chemotherapy. Further studies are needed to address whether adding anti–LAG-3 to anti–PD-1 plus chemotherapy can benefit specific GC/GEJC patient subgroups.

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.

Inhibition of USP7 enhances CD8+ T cell activity in liver cancer by suppressing PRDM1-mediated FGL1 upregulation.

Lymphocyte activation gene 3 (LAG3), an immune checkpoint molecule expressed on activated T cells, functions as a negative regulator of immune responses. Persistent antigen exposure in the tumor microenvironment results in sustained LAG3 expression on T cells, contributing to T cell dysfunction. Fibrinogen-like protein 1 (FGL1) has been identified as a major ligand of LAG3, and FGL1/LAG3 interaction forms a novel immune checkpoint pathway that results in tumor immune evasion. In addition, ubiquitin-specific peptidase 7 (USP7) plays a crucial role in cancer development. In this study we investigated the role of USP7 in modulation of FGL1-mediated liver cancer immune evasion. We showed that knockdown of USP7 or treatment with USP7 inhibitor P5091 suppressed liver cancer growth by promoting CD8+ T cell activity in Hepa1-6 xenograft mice and in HepG2 or Huh7 cells co-cultured with T cells, whereas USP7 overexpression produced the opposite effect. We found that USP7 upregulated FGL1 in HepG2 and Huh7 cells by deubiquitination of transcriptional factor PR domain zinc finger protein 1 (PRDM1), which transcriptionally activated FGL1, and attenuated the CD8+ T cell activity, leading to the liver cancer growth. Interestingly, USP7 could be transcriptionally stimulated by PRDM1 as well in a positive feedback loop. P5091, an inhibitor of USP7, was able to downregulate FGL1 expression, thus enhancing CD8+ T cell activity. In an immunocompetent liver cancer mouse model, the dual blockade of USP7 and LAG3 resulted in a superior antitumor activity compared with anti-LAG3 therapy alone. We conclude that USP7 diminishes CD8+ T cell activity by a USP7/PRDM1 positive feedback loop on FGL1 production in liver cancer; USP7 might be a promising target for liver cancer immunotherapy.

Neoadjuvant nivolumab or nivolumab plus LAG-3 inhibitor relatlimab in resectable esophageal/gastroesophageal junction cancer: a phase Ib trial and ctDNA analyses

Gastroesophageal cancer dynamics and drivers of clinical responses with immune checkpoint inhibitors (ICI) remain poorly understood. Potential synergistic activity of dual programmed cell death protein 1 (PD-1) and lymphocyte-activation gene 3 (LAG-3) inhibition may help improve immunotherapy responses for these tumors. We report a phase Ib trial that evaluated neoadjuvant nivolumab (Arm A, n = 16) or nivolumab–relatlimab (Arm B, n = 16) in combination with chemoradiotherapy in 32 patients with resectable stage II/stage III gastroesophageal cancer together with an in-depth evaluation of pathological, molecular and functional immune responses. Primary endpoint was safety; the secondary endpoint was feasibility; exploratory endpoints included pathological complete (pCR) and major pathological response (MPR), recurrence-free survival (RFS) and overall survival (OS). The study met its primary safety endpoint in Arm A, although Arm B required modification to mitigate toxicity. pCR and MPR rates were 40% and 53.5% for Arm A and 21.4% and 57.1% for Arm B. Most common adverse events were fatigue, nausea, thrombocytopenia and dermatitis. Overall, 2-year RFS and OS rates were 72.5% and 82.6%, respectively. Higher baseline programmed cell death ligand 1 (PD-L1) and LAG-3 expression were associated with deeper pathological responses. Exploratory analyses of circulating tumor DNA (ctDNA) showed that patients with undetectable ctDNA post-ICI induction, preoperatively and postoperatively had a significantly longer RFS and OS; ctDNA clearance was reflective of neoantigen-specific T cell responses. Our findings provide insights into the safety profile of combined PD-1 and LAG-3 blockade in gastroesophageal cancer and highlight the potential of ctDNA analysis to dynamically assess systemic tumor burden during neoadjuvant ICI that may open a therapeutic window for future intervention. ClinicalTrials.gov registration: NCT03044613.

Synergistic combination therapy using cowpea mosaic virus intratumoral immunotherapy and Lag-3 checkpoint blockade

Immune checkpoint therapy (ICT) for cancer can yield dramatic clinical responses; however, these may only be observed in a minority of patients. These responses can be further limited by subsequent disease recurrence and resistance. Combination immunotherapy strategies are being developed to overcome these limitations. We have previously reported enhanced efficacy of combined intratumoral cowpea mosaic virus immunotherapy (CPMV IIT) and ICT approaches. Lymphocyte-activation gene-3 (LAG-3) is a next-generation inhibitory immune checkpoint with broad expression across multiple immune cell subsets. Its expression increases on activated T cells and contributes to T cell exhaustion. We observed heightened efficacy of a combined CPMV IIT and anti-LAG-3 treatment in a mouse model of melanoma. Further, LAG-3 expression was found to be increased within the TME following intratumoral CPMV administration. The integration of CPMV IIT with LAG-3 inhibition holds significant potential to improve treatment outcomes by concurrently inducing a comprehensive anti-tumor immune response, enhancing local immune activation, and mitigating T cell exhaustion.

Expression of lymphocyte activation gene-3 on CD4+T cells is regulated by cytokine interleukin-18 in myasthenia gravis

Myasthenia gravis (MG) is a T cell-dependent, B cell-mediated, and complement-dependent autoimmune disease. Lymphocyte activation gene-3 (LAG-3; CD223) is an immune checkpoint protein that plays an important role in maintaining autoimmune tolerance and homeostasis. To investigate the cytokine-regulated expression pattern of LAG-3, CD4+T cells were sorted from the peripheral blood of healthy volunteers by density gradient centrifugation and stimulated with various cytokines in vitro. The expression of membrane LAG-3 (mLAG-3), membrane a disintegrin and metallopeptidase domain10 (mADAM10) and membrane ADAM17 (mADAM17) on CD4+T cells was detected by flow cytometry; the concentration of soluble LAG-3 (sLAG-3) was detected by ELISA; and the relative expression of genes at the transcriptional level was detected by fluorescence quantitative RT-PCR (qRT-PCR). sLAG-3 levels were significantly increased in the peripheral plasma of AChR Ab-positive patients with MG compared to healthy volunteers, while the percentage of mLAG-3 expression on CD4+T lymphocytes in the peripheral blood of patients with MG was significantly reduced. IL-18 inhibited mLAG-3 levels on CD4+T cells in a concentration-dependent manner. Additionally, the concentration of sLAG-3 in the supernatant increased. After PHA and IL-18 stimulation, ADAM10 and ADAM17 also increased compared to those in the PHA-active group. Moreover, there were significant differences in the expression of mADAM10 and mADAM17 in CD4+T lymphocytes between patients with MG and healthy volunteers. These results suggest that IL-18 may regulate the expression pattern of mLAG-3 in CD4+T cells and sLAG-3 via ADAM10- and ADAM17-mediated pathways, thus affecting the immune effects of CD4+T cells. This study provides a preliminary exploration of the upstream regulatory molecules of the LAG-3 and IL-18/LAG-3 signalling pathways for potential targeted therapy of autoimmune diseases in the future.

Integrative analysis of LAG3 immune signature and identification of a LAG3-related genes prognostic signature in kidney renal clear cell carcinoma.

Immune checkpoint blockade (ICB) therapy has resulted in improved overall survival in kidney renal clear cell carcinoma (KIRC), but most treated patients fail to show durable clinical responses. Lymphocyte activation gene-3 (LAG3) is a novel inhibitory immune checkpoint, but its expression pattern, prognostic value, and immunological role in KIRC remain unknown. In this study, we utilized TCGA_KIRC RNA-sequencing data to analyze the relationship between LAG3 expression and clinical features. The single-cell sequencing data and tissue immunofluorescence are employed to investigate the subcellular localization of LAG3 in KIRC. Kaplan-Meier plotter, TIMER, and TISIDB were used to assess the association between LAG3 expression and prognosis, as well as its correlation with immune-related components. We constructed the LAG3 interaction network by using STRING, GeneMANIA, BioGRID, and HitPredict databases. We found that LAG3 is upregulated and correlates with poor prognostic phenotype in KIRC. LAG3 is predominantly expressed on exhausted CD8+ T cells and shows strong co-expression with PDCD1 in KIRC. Moreover, our findings indicated that LAG3 not only inhibits T cell activation but also potentially regulates cell adhesion in KIRC. In conclusion, our study implies that LAG3 can serve as a potential prognostic biomarker for KIRC. Furthermore, blocking both LAG3 and PDCD1 may alleviate resistance to anti-PDCD1 therapy, providing novel insights for immunotherapy decision-making in KIRC patients.

Development of LAG-3/FGL1 blocking peptide and combination with radiotherapy for cancer immunotherapy

Aside from antibodies, peptides show great potential as immune checkpoint inhibitors (ICIs) due to several advantages, such as better tumor penetration and lower cost. Lymphocyte-activation gene 3 (LAG-3) is an immune checkpoint which can induce T cell dysfunction through interaction with its soluble ligand fibrinogen like protein-1 (FGL1). Here, we found that LAG-3 expression was higher than programmed cell death protein 1 (PD-1) in multiple human cancers by TCGA databases, and successfully identified a LAG-3 binding peptide LFP-6 by phage display bio-panning, which specifically blocks the interaction of LAG-3/FGL1 but not LAG-3/MHC-II. Subsequently, d-amino acids were introduced to substitute the N- and C-terminus of LFP-6 to obtain the proteolysis-resistant peptide LFP-D1, which restores T cell function in vitro and inhibits tumor growth in vivo. Further, a bispecific peptide LFOP targeting both PD-1/PD-L1 and LAG-3/FGL1 was designed by conjugating LFP-D1 with PD-1/PD-L1 blocking peptide OPBP-1(8–12), which activates T cell with enhanced proliferation and IFN-γ production. More importantly, LFOP combined with radiotherapy significantly improve the T cell infiltration in tumor and elevate systemic antitumor immune response. In conclusion, we developed a novel peptide blocking LAG-3/FGL1 which can restore T cell function, and the bispecific peptide synergizes with radiotherapy to further enhance the antitumor immune response.

A combined analysis of multi-omics data reveals the prognostic values and immunotherapy response of LAG3 in human cancers

Lymphocyte-activation gene 3 (LAG3) is a highly anticipated immune checkpoint in the context of cancer, exerting regulatory control over immune cell proliferation and function to reinforce the advancement of cancers. However, the comprehensive functional analysis of LAG3 across various cancer types remains undisclosed; thus, this study aims to investigate the pan-cancer expression profile of LAG3. We have investigated the expression profile, prognostic significance, and genetic alterations of LAG3 in various cancers while elucidating its characteristic in immune response regulation. Our findings demonstrated that elevated LAG3 expression is significantly associated with favorable prognosis in patients with cutaneous melanoma (SKCM), and it may be a potential biomarker for SKCM. Furthermore, multiple immune algorithms have highlighted the important regulatory role of LAG3 for the tumor-infiltrating immune cells including CD8 + T cells, B cells, dendritic cells (DCs), macrophages, and natural killer (NK) cells. We also examined the distribution of LAG3 at the single-cell level and explored its functional significance. A comprehensive and systematic analysis of LAG3 would facilitate a comprehensive evaluation of LAG3 in cancer biology and provide valuable insights for cancer management.

Soluble LAG3: A Potential Marker for the Development and Activity of Acute Graft-Versus-Host Disease

Acute graft-versus-host disease (aGVHD), usually developing within 100 days after hematopoietic stem cell transplantation (HSCT), is mainly attributed to donor T cell pro-inflammatory responses against mismatched host histocompatibility antigens. The persistence of increased effector T cell responses following HSCT is partially related to an altered function of T regulatory cells (Tregs) and their failure to prevent GVHD. Certain immune checkpoint molecules, such as cytotoxic T-lymphocyte antigen 4, programmed cell death protein 1 (PD1) and lymphocyte activation gene-3 (LAG3), highly expressed on Tregs, are involved in maintaining immune tolerance and preventing immune-mediated inflammation. LAG3 is a newly identified inhibitory receptor, found on most immune cells, including CD4+ and CD8+ T cells, Tregs, and dendritic cells. Previous studies have demonstrated a role of LAG3 expression on T cells in GVHD suppression. Its soluble isoform sLAG3, released from the cell membrane, has been recently reported to also contribute to enhanced immune-mediated responses in solid tumors and autoimmune diseases, such as anti-PD1-resistant melanoma, rheumatoid arthritis (RA), and ANCA-associated vasculitis. Given this evidence, the current study aimed to explore a potential role of sLAG3 in GVHD development. Serum samples of 25 patients (≥18 years old) who underwent allogeneic HSCT at Rambam from 01.2021 to 01.2023 were drawn on days -7, +100, +180 of transplant. Twelve serum samples obtained from the Biobank were used as healthy control (HC). All samples were assessed for the sLAG3 levels using a commercial ELISA kit. The median level measured in HC (56pg/mL) was considered a cutoff to define the “low and high level sLAG3 groups”. Twenty-three patients were included in the analysis [median age at transplant - 59.5 years (range 19-75); 52% males]. Seventeen patients had acute myeloid leukemia, 3 - myelodysplastic syndrome, 2 - acute lymphoblastic leukemia and 1 had primary myelofibrosis. Fourteen patients had a matched unrelated donor, 6- matched related donors, and 3 - haploidentical donors. Nineteen patients received calcineurin inhibitor (CNI) and methotrexate-based GVHD prophylaxis (13 with ATG); 4 patients received post-transplant cytoxan-based prophylaxis, with CNI and mycophenolate-mofetil. Overall, 17/23 (74%) patients developed GVHD: 9 - aGVHD and 8 - chronic GVHD (cGVHD). A low pre-transplant sLAG3 level (<56pg/mL) was found in 8/23 patients, while it was high (>56pg/mL) in 15/23. Notably, 9/15 (60%) patients in the “high sLAG3 group”, but none in the “low sLAG3 group” were diagnosed with aGVHD, suggesting a high sLAG3 level as a possible indicator for patient-pre-transplant susceptibility to aGVHD development, irrespective of transplant factors, such as conditioning regimen, donor type, etc. Four patients in each group developed cGVHD. Furthermore, there was a significant increase in sLAG3 levels between the baseline and GVHD diagnosis in patients who developed aGVHD (mean: from 156.1±172.8, to 323.9±201.3), but not in those who either had cGVHD (mean: from 119.71±90.5 to 150.1±147.1) or did not have GVHD (mean: from 49.7±36 to 52.1±22.2). Overall, 11/23 patients were in complete remission (CR) at the time of HSCT; 9/11 were in the high-sLAG3 group and 6 of them developed aGVHD. In the low-sLAG3 group, most patients (6/8) had some degree of active disease (persistent disease or minimal-residual disease). These findings may reflect high T-effector cell and/or low Treg activity, possibly contributing to both anti-leukemic effect and GVHD development. In conclusion: The current study demonstrates, for the first time, that the pre-transplant sLAG3 level is significantly increased and continues to further increase in patients who develop aGVHD following HSCT compared to those who don't. These findings suggest a potential role of sLAG3 as a predicting factor for aGVHD development as well as a marker of treatment response that could guide therapeutic decisions in this patient setting.

LAG-3 expression in microglia regulated by IFN-γ/STAT1 pathway and metalloproteases.

Microglia are resident innate immune cells in the central nervous system (CNS) and play important roles in the development of CNS homeostasis. Excessive activation and neurotoxicity of microglia are observed in several CNS disorders, but the mechanisms regulating their activation remain unclear. Immune checkpoint molecules are expressed on activated immune cells and regulate their activation in peripheral immunity. However, the expression mechanism of immune checkpoint molecules in activated microglia is still unknown. Here, we analyzed the expression of immune checkpoint molecules in activated microglia using the mouse microglial cell line BV2 and primary cultured microglia. The expression of lymphocyte activation gene-3 (LAG-3), a type of immune checkpoint molecule, was increased in microglia activated by IFN-γ. IFN-γ-induced LAG-3 expression in microglia was suppressed by transfection of siRNA targeting STAT1. LAG-3 has two forms, membrane and soluble, and both forms were upregulated in microglia activated by IFN-γ. The production of soluble LAG-3 was suppressed by treatment with inhibitors of metalloproteinases such as ADAM10 and ADAM17. IFN-γ administration into cisterna magna of mice increased LAG-3 expression in spinal microglia. Furthermore, LAG-3 knockdown in microglia promoted nitric oxide production by IFN-γ. Our results demonstrate that LAG-3 expression in microglia is induced by the IFN-γ-STAT1 pathway and soluble LAG-3 production is regulated via cleavage of membranous LAG-3 by metalloproteinases including ADAM10 and ADAM17.

Lag-3 expression and clinical outcomes in metastatic melanoma patients treated with combination anti-lag-3 + anti-PD-1-based immunotherapies

ABSTRACT Lymphocyte-activation gene-3 (LAG-3), an immune checkpoint receptor, negatively regulates T-cell function and facilitates immune escape of tumors. Dual inhibition of LAG-3 and programmed cell death receptor-1 (PD-1) significantly improved progression-free survival (PFS) in metastatic melanoma patients compared to anti-PD-1 therapy alone. Investigating the utility of LAG-3 expression as a biomarker of response to anti-LAG-3 + anti-PD-1 immunotherapy is of great clinical relevance. This study sought to evaluate the association between baseline LAG-3 expression and clinical outcomes following anti-LAG-3 and anti-PD-1-based immunotherapy in metastatic melanoma. LAG-3 immunohistochemistry (clone D2G4O) was performed on pre-treatment formalin-fixed, paraffin-embedded metastatic melanoma specimens from 53 patients treated with combination anti-LAG-3 + anti-PD-1-based therapies. Eleven patients had received prior anti-PD-1-based treatment. Patients were categorized as responders (complete/partial response; n = 36) or non-responders (stable/progressive disease; n = 17) based on the Response Evaluation Criteria in Solid Tumours (RECIST). Tumor-infiltrating lymphocytes (TILs) were scored on hematoxylin and eosin-stained sections. LAG-3 expression was observed in 81% of patients, with staining in TILs and dendritic cells. Responders displayed significantly higher proportions of LAG-3+ cells compared to non-responders (P = .0210). LAG-3 expression positively correlated with TIL score (P < .01). There were no significant differences in LAG-3 expression between different sites of metastases (P > .05). Patients with ≥ 1% LAG-3+ cells in their tumors had significantly longer PFS compared to patients with < 1% LAG-3 expression (P = .0037). No significant difference was observed in overall survival between the two groups (P = .1417). Therefore, the assessment of LAG-3 expression via IHC warrants further evaluation to determine its role as a predictive marker of response and survival in metastatic melanoma.

Positive TIGIT and VISTA Expression Predict Worse Prognosis in Cervical Cancer Patients Treated with (Chemo)Radiotherapy

Immune checkpoint inhibitors combined with (chemo)radiotherapy could be an attractive treatment strategy for patients with cervical cancer (CC), but the expression of some immune checkpoint proteins in cervical cancer and their impact on patient survival remains largely unknown. Here, we investigated the predictive value of T cell immunoreceptor with Ig and ITIM domain (TIGIT), V-domain Ig suppressor of T cell activation (VISTA), and lymphocyte-activation gene-3 (LAG-3) expression in pathological tissues of CC patients treated with (chemo)radiotherapy. We enrolled 175 CC patients who received (chemo)radiotherapy and collected their pre-treatment tumor tissue sections for the immunohistochemical stain of TIGIT, VISTA, and LAG-3. The Kaplan-Meier method was used to calculate progression-free survival (PFS) and overall survival (OS) after (chemo)radiotherapy. Univariate and multivariate COX proportional hazards regression models were employed to analyze potential risk factors for patient survival. Kaplan-Meier survival analyses showed that the PFS and OS of patients with positive expression of TIGIT and VISTA were significantly shorter than those patients with negative expression of the proteins (all p<0.05). However, we did not reach the same conclusion in the analysis of LAG-3 (both p>0.05). Univariate COX regression analysis showed that the positive expression of TIGIT and VISTA are related to poor PFS and OS (both HR>1.0 and p<0.05). Multivariate COX regression analysis showed that TIGIT positive and VISTA positive patients have shorter PFS and OS (both HR>1.0 and p<0.05). There is no significant correlation between LAG-3 expression and PFS or OS in these CC patients treated with (chemo)radiotherapy. We revealed that positive TIGIT and VISTA expression could predict worse prognosis in cervical cancer patients treated with (chemo)radiotherapy, which may help to refine the treatment strategies of combining immune checkpoint inhibitors and radiotherapy.

Blockade of PD-1 and LAG-3 expression on CD8+ T cells promotes the tumoricidal effects of CD8+ T cells.

The diffuse large B-cell lymphoma (DLBCL) has the highest incidence of all lymphomas worldwide. To investigate the functions of lymphocyte activation gene 3 (LAG-3) and programmed cell death 1 (PD-1) in tissues and peripheral blood of patients with DLBCL, the expression of LAG-3 and PD-1 genes in DLBCL-TCGA were analyzed. LAG-3 and PD-1 mRNA levels in DLBCL were analyzed using data from The Cancer Genome Atlas (TCGA) database. Utilize the Genotype-Tissue Expression (GTEx) database for assessing the variance in the expression of LAG-3, PD-1, and other associated factors between the tissues of DLBCL patients and healthy individuals. Immunohistochemistry was applied to detect the expression of LAG-3 and PD-1 levels in 137 cases of DLBCL tissues and 20 cases of reactive lymphoid hyperplasia. The prognostic value of LAG-3 and PD-1 were assessed using the Kaplan-Meier curve. The Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE) and ssGSEA algorithm were used to explore the immune microenvironment of DLBCL. Additionally, the expression and co-expression of LAG-3 and PD-1 were detected on CD4 and CD8 T cells in peripheral blood samples from 100 cases of DLBCL tissues and 30 cases of healthy individuals using flow cytometry. According to TCGA database, LAG-3 and PD-1 gene expression levels were significantly up-regulated in DLBCL tissues. LAG-3 and PD-1 levels were also strongly positively correlated with those of most infiltrating immune cells. Overall survival of patients with high LAG-3 and PD-1 co-expression was significantly shorter than that of patients with low co-expression. In DLBCL patients, LAG-3 and PD-1 were highly expressed in peripheral blood CD8+ T cells. In addition, LAG-3 was highly expressed in CD4+ T cells, while the expression of PD-1 in CD4+ T cells of DLBCL patients showed no significant difference compared to healthy individuals. Additionally, CD8+ T cells and SU-DHL6/OCI-LY3 from patients with DLBCL were co-cultured in vitro; after addition of LAG-3 and/or PD-1 inhibitors alone, an increased perforin and granzyme B secretion levels by CD8+ T cells were detected, as well as an increase in the overall proportion of tumor cells undergoing apoptosis. High LAG-3 and PD-1 levels significantly inhibit CD8+ T cell function, resulting in weakened ability to kill tumor cells. Combined LAG-3 and PD-1 blockade can restore CD8+ T cell function and provides a potential avenue for development of personalized cellular immunotherapy for DLBCL.

Prognostic value and immune landscapes of TERT promoter methylation in triple negative breast cancer.

Treatment options for patients with triple-negative breast cancer (TNBC) remain limited to mainstay therapies owing to a lack of efficacious therapeutic targets. Accordingly, there is an urgent need to discover and identify novel molecular targets for the treatment and diagnosis of this disease. In this study, we analyzed the correlation of telomerase reverse transcriptase (TERT) methylation status with TERT expression, prognosis, and immune infiltration in TNBC and identified the role of TERT methylation in the regulation TNBC prognosis and immunotherapy. Data relating to the transcriptome, clinicopathological characteristics and methylation of TNBC patients were obtained from The Cancer Genome Atlas (TCGA) database. TERT expression levels and differential methylation sites (DMSs) were detected. The correlations between TERT expression and DMSs were calculated. Kaplan-Meier curves was plotted to analyze the relationship between the survival of TNBC patients and the DMSs. The correlations of DMSs and TERT expression with several immunological characteristics of immune microenvironment (immune cell infiltration, immunomodulators, immune-related biological pathways, and immune checkpoints) were assessed. The results were validated using 40 TNBC patients from Sun Yat-sen University Cancer Center (SYSUCC). Six DMSs were identified. Among them, four sites (cg11625005, cg07380026, cg17166338, and cg26006951) were within the TERT promoter, in which two sites (cg07380026 and cg26006951) were significantly related to the prognosis of patients with TNBC. Further validation using 40 TNBC samples from SYSUCC showed that the high methylation of the cg26006951 CpG site was associated with poor survival prognosis (P=0.0022). TERT expression was significantly correlated with pathological N stage and clinical stage, and cg07380026 were significantly associated with pathological T and N stages in the TCGA cohort. Moreover, the methylation site cg26006951, cg07380026 and TERT expression were significantly correlated with immune cell infiltration, common immunomodulators, and the level of the immune checkpoint receptor lymphocyte activation gene 3 (LAG-3) in TNBC patients. TERT promotertypermethylation plays an important role in TERT expression regulation and tumor microenvironment in TNBC. It is associated with overall survival and LAG-3 expression. TERT promoter hypermethylation may be a potential molecular biomarker for predicting response to the TERT inhibitors and immune checkpoint inhibitors in TNBC.

The effect of blocking immune checkpoints LAG-3 and PD-1 on human invariant Natural Killer T cell function

Invariant Natural Killer T (iNKT) cells undergo immune exhaustion during chronic activation caused by cancer and viral infections, such as HIV. Exhaustion is marked by cell dysfunction and increased expression of immune checkpoint proteins programmed cell-death-1 (PD-1) and lymphocyte-activation-gene-3 (LAG-3). We hypothesize that blockade of PD-1 and/or LAG-3 will enhance iNKT cell function. Utilizing peripheral blood mononuclear cells from healthy donors, LAG-3 and PD-1 expression on iNKT cells was assessed using flow cytometry following in vitro stimulation with iNKT-specific stimulant α-galactosylceramide (n = 4). Efficacy of anti-LAG-3 and/or anti-PD-1 antibody blockades in enhancing iNKT cell function was assessed by determining proliferative capacity and IFN-γ production (n = 9). LAG-3 and PD-1 expression on iNKT cells peaked at Day 4 (98.8%; p ≤ 0.0001 and 98.8%; p = 0.005, respectively), followed by steep decrease by Day 10, coinciding with peak iNKT cell proliferation. In a 10-day blocking assay, both the anti-PD-1 alone and dual anti-PD-1 and anti-LAG-3 significantly increased iNKT proliferation (6 and 6.29 log2 fold-change respectively) compared to the no blockade control (ANOVA-p = 0.0005) with the dual blockade system being more effective (t-test-p = 0.013). This provides proof-of-concept for LAG-3 and PD-1 as immunotherapeutic targets to enhance human iNKT cell function, with the long-term goal of addressing immune exhaustion.

Development of a high-throughput TR-FRET screening assay for LAG-3/FGL1 interaction.

Lymphocyte activation gene 3 (LAG-3) is a negative immune checkpoint and a key regulator of immunehomeostasiswith multiple biological activities related to T-cell functions. Fibrinogen-likeprotein 1(FGL1) is a major LAG-3 functional ligand that is upregulated in various human cancers. LAG-3 positive T cells bind FGL1 expressed by cancer cells, which inhibits T-cell activation and cytokine secretion via indirect blocking of T cell receptor (TCR) signaling. High expression of LAG-3 and FGL1 in patients with solid tumors is associated with drug resistance and decreased survival in response to FDA-approved immune checkpoint inhibitors. Therefore, targeting the LAG-3/FGL1 pathway represents a promising therapeutic strategy to maximize the number of patients benefiting from checkpoint blockade therapy. However, there are no small molecules in existence that target LAG-3/FGL1 interaction. Herein, we report a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to evaluate the ability of small molecules to inhibit LAG-3/FGL1 interaction. We further demonstrate the implementation of the developed assay in screening chemical libraries of small molecules from the NCI Diversity Set VII, FDA-approved drugs, and a focused library of NF-κB modulators. This work will pave the way for drug discovery efforts focused on therapeutic targeting of LAG-3/FGL1 interaction using small molecules.

Lymphocyte-activation gene 3 protein expression in tumor-infiltrating lymphocytes is associated with a poor prognosis of ovarian clear cell carcinoma

BackgroundHistological analysis has revealed the need for new treatment techniques for epithelial ovarian cancer. Immune checkpoint inhibitors may be a new therapeutic strategy for ovarian clear cell carcinoma (OCCC). Lymphocyte-activation gene 3 (LAG-3), an immune checkpoint, is a poor prognostic factor and a new therapeutic target for several malignancies. In this study, we demonstrated the correlation between LAG-3 expression and the clinicopathological features of OCCC. We evaluated LAG-3 expression in tumor-infiltrating lymphocytes (TILs) via immunohistochemical analysis using tissue microarrays containing surgically resected specimens from 171 patients with OCCC.ResultsThe number of LAG-3-positive cases was 48 (28.1%), whereas the number of LAG-3-negative cases was 123 (71.9%). LAG-3 expression significantly increased in patients with advanced stages (P = 0.036) and recurrence (P = 0.012); however, its expression did not correlate with age (P = 0.613), residual tumor (P = 0.156), or death (P = 0.086). Using the Kaplan − Meier method, LAG-3 expression was found to be correlated with poor overall survival (P = 0.020) and progression-free survival (P = 0.019). Multivariate analysis revealed LAG-3 expression (hazard ratio [HR] = 1.86; 95% confidence interval [CI], 1.00 − 3.44, P = 0.049) and residual tumor (HR = 9.71; 95% CI, 5.13 − 18.52, P < 0.001) as independent prognostic factors.ConclusionOur study demonstrated that LAG-3 expression in patients with OCCC may be a useful biomarker for the prognosis of OCCC and could serve as a new therapeutic target.

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.

LAG-3 transcriptomic expression patterns across malignancies: Implications for precision immunotherapeutics.

Lymphocyte activation gene 3 (LAG-3) or CD223 is a transmembrane protein that serves as an immune checkpoint which attenuates T-cell activation. Many clinical trials of LAG-3 inhibitors have had modest effects, but recent data indicate that the LAG-3 antibody relatlimab, together with nivolumab (anti-PD-1), provided greater benefit than nivolumab alone in patients with melanoma. In this study, the RNA expression levels of 397 genes were assessed in 514 diverse cancers at a clinical-grade laboratory (OmniSeq: https://www.omniseq.com/). Transcript abundance was normalized to internal housekeeping gene profiles and ranked (0-100 percentile) using a reference population (735 tumors; 35 histologies). A total of 116 of 514 tumors (22.6%) had high LAG-3 transcript expression (≥75 percentile rank). Cancers with the greatest proportion of high LAG-3 transcripts were neuroendocrine (47% of patients) and uterine (42%); colorectal had among the lowest proportion of high LAG-3 expression (15% of patients) (all p < 0.05 multivariate); 50% of melanomas were high LAG-3 expressors. There was significant independent association between high LAG-3 expression and high expression of other checkpoints, including programmed death-ligand 1 (PD-L1), PD-1, and CTLA-4, as well as high tumor mutational burden (TMB) ≥10 mutations/megabase, a marker for immunotherapy response (all p < 0.05 multivariate). However, within all tumor types, there was inter-patient variability in LAG-3 expression level. Prospective studies are therefore needed to determine if high levels of the LAG-3 checkpoint are responsible for resistance to anti-PD-1/PD-L1 or anti-CTLA-4 antibodies. Furthermore, a precision/personalized immunotherapy approach may require interrogating individual tumor immunograms to match patients to the right combination of immunotherapeutic agents for their malignancy.