Levels Of Lymphocyte Activation Gene-3 Research Articles

FGL1-LAG3 axis impairs IL-10-Producing regulatory T cells associated with Systemic lupus erythematosus disease activity

BackgroundSystemic Lupus Erythematosus (SLE) is a prototypic autoimmune disease, which is accompanied by liver damage. However, it remains unknown whether liver damage is associated with SLE progression. Methodology: HepG2 and L-02 cells were stimulated with cytokines, and FGL1 mRNA and protein expression levels were determined using Real-time PCR and ELISA, respectively. Regulatory T cells (Treg) isolated from healthy individuals as well as patients with SLE and SLE and liver damage (SLE-LD) were cultured with autologous effector CD4+T cells in the presence of a functional antibody or isotype control. The expression levels of LAG3, CD25, PD-1, CXCR5, ICOS and OX40 were evaluated by flow cytometry. FGL1, IL-10, IL-17a and IL-21 levels in serum or culture supernatants were quantified by ELISA. ResultsPatients with SLE-LD exhibits higher disease activity indices and anti-dsDNA antibody levels. Importantly, fibrinogen-like protein 1 (FGL1), a key factor released from the injured liver, is up-regulated in patients with SLE-LD and is associated with disease activity. FGL1 expression is induced by the inflammatory cytokine IL-6 signaling in hepatocytes. Higher expression of the FGL1 receptor lymphocyte activation gene 3 (LAG3) is detected in Treg cells from patients with SLE-LD. The FGL1-LAG3 signaling axis inhibits Treg cell proliferation and impairs the suppressive activity of Treg cells by limiting IL-10 secretion. Furthermore, FGL1-LAG3 signaling promotes the production of pathogenic IL-17a and IL-21 by CD4+T cells by reducing IL-10 level produced by Treg in patients with SLE. ConclusionsThe FGL1-LAG3 signal axis is a key mechanism that subverts the suppressive function of Treg cells. This may provide a new therapeutic target for SLE and SLE-induced liver damage.

MP56-05 LAG-3/FGL1 AXIS PREDICTS RESPONSE TO IMMUNE CHECKPOINT INHIBITORS IN ADVANCED UROTHELIAL CARCINOMA

You have accessJournal of UrologyCME1 Apr 2023MP56-05 LAG-3/FGL1 AXIS PREDICTS RESPONSE TO IMMUNE CHECKPOINT INHIBITORS IN ADVANCED UROTHELIAL CARCINOMA Takashi Yoshida, Takahiro Nakamoto, Chisato Ohe, Junichi Ikeda, Yoshiki Yasukochi, Naho Atsumi, Ryoichi Saito, Koji Tsuta, and Hidefumi Kinoshita Takashi YoshidaTakashi Yoshida More articles by this author , Takahiro NakamotoTakahiro Nakamoto More articles by this author , Chisato OheChisato Ohe More articles by this author , Junichi IkedaJunichi Ikeda More articles by this author , Yoshiki YasukochiYoshiki Yasukochi More articles by this author , Naho AtsumiNaho Atsumi More articles by this author , Ryoichi SaitoRyoichi Saito More articles by this author , Koji TsutaKoji Tsuta More articles by this author , and Hidefumi KinoshitaHidefumi Kinoshita More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003309.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: PD-1/PDL-1 blockade is standard therapy in advanced urothelial carcinoma (UC), although few cases respond to this therapy. Lymphocyte activation gene-3 (LAG-3), T-cell immunoglobulin and mucin domain-containing-3 (i.e., TIM-3), and T cell immunoreceptor with Ig and ITIM domains (i.e., TIGIT) are major, co-expressed immune checkpoint receptors associated with tumor immune escape. However, the correlation of these receptors and their ligands with immunotherapeutic sensitivity remains unclear in UC. Therefore, we conducted this study to clarify how these checkpoint molecules are associated with response to immunotherapy. METHODS: We retrospectively analyzed a total of 851 patients from the public database and Kansai Medical University database. Clinical and molecular analyses and multiplex immunohistochemistry were performed to characterize immune cell infiltration in UC. RESULTS: Among the three checkpoint receptors, only LAG-3 was associated with the responsiveness of PD-1/PDL-1 blockade. In tumors with high LAG-3 levels, the increased expression of fibrinogen-like protein-1 (FGL-1), not for other LAG-3 ligands, such as major histocompatibility complex II and galectin-3, showed a significantly negative impact on response to anti-PD1/PDL-1 therapy and overall survival. Moreover, although the tumor infiltration of CD8+ T-cells was remarkable regardless of FGL-1 expression, high FGL-1 mRNA levels were linked to the upregulation of CD39 and neuropilin-1, as well as an increase in CD4+ regulatory T-cells and M2-like macrophage gene signatures, which may indicate CD8+ T-cell exhaustion. Further, enrichment analyses suggested that high LAG-3-FGL-1 co-expression was significantly correlated with the activation of epithelial-mesenchymal transition, transforming growth factor-beta, and angiogenesis signaling pathways. CONCLUSIONS: This study indicates that not only high LAG-3 expression but also co-expression of FGL-1 are crucial factors for predicting adverse oncological outcomes, associated with the presence of immunosuppressive contextures in tumors in cases of PD-1/PDL-1 blockade. Emerging combination therapy of anti-LAG3 and PD-1 blockade could be promising management for such UC patients. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e775 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takashi Yoshida More articles by this author Takahiro Nakamoto More articles by this author Chisato Ohe More articles by this author Junichi Ikeda More articles by this author Yoshiki Yasukochi More articles by this author Naho Atsumi More articles by this author Ryoichi Saito More articles by this author Koji Tsuta More articles by this author Hidefumi Kinoshita More articles by this author Expand All Advertisement PDF downloadLoading ...

Implications of LAG3 and CTLA4 immune checkpoints beyond PD-1/PD-L1 as a potential target in determining the prognosis of uveal melanoma patients

BackgroundResponse rate of PD-1/PD-L1 immunotherapeutic blockade agents in uveal melanoma (UM) is poor. Lymphocyte activation gene 3 (LAG3) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) are the two promising immune checkpoint...

LAG-3 expression in tumor microenvironment of triple-negative breast cancer.

This study aimed to evaluate the expression of lymphocyte activation gene-3 (LAG-3) and its relationship with programmed cell death ligand-1 (PD-L1) in triple-negative breast cancer (TNBC). : LAG-3 and PD-L1 was evaluated in tumor-infiltrating lymphocytes (TILs) using immunohistochemistry (IHC). The chi-square test was used to estimate the associations between LAG-3, PD-L1 and clinicopathological characteristics. Correlation between LAG-3 stromal TIL (sTIL), LAG-3 intraepitelial TIL (iTIL) and PD-L1 was assessed with using the Spearman's correlation coefficient. Survival analysis was performed using the Kaplan-Meier method. The percentages of LAG-3 sTIL+, LAG-3 iTIL+, PD-L1+ tumor cells and PD-L1+ inflammatory cells were 52%, 42%, 14% and 70%, respectively. A strong positive correlation between LAG-3 sTIL and LAG-3 iTIL (r = 0.874, p < 0.001) and a moderate positive correlation between LAG-3 sTIL and PD-L1 (r = 0.584, p < 0.001) were found. LAG-3 and PD-L1 status did not significantly affect overall survival (OS) (HR: 0.56 (95% CI: 0.15-2.11) (p = 0.397), HR: 2.70 (95% CI: 0.56-13.02) (p = 0.215), respectively). High levels of LAG-3 and PD-L1 expression were detected in patients with TNBC. Although their contribution to survival could not be determined, the high expression rates of PD-L1 and LAG-3 may help identify the subgroup of TNBC that would benefit from immunotherapy.

Role of tumor infiltrating lymphocytes and spatial immune heterogeneity in sensitivity to PD-1 axis blockers in non-small cell lung cancer

BackgroundTumor infiltrating lymphocytes (TILs) reflect adaptive antitumor immune responses in cancer and are generally associated with favorable prognosis. However, the relationships between TILs subsets and their spatial arrangement with clinical...

Overexpression of LAG3, TIM3, and A2aR in adenoid cystic carcinoma and mucoepidermoid carcinoma.

Adenoid cystic carcinoma (AdCC) and mucoepidermoid carcinoma (MEC) are the two most frequent malignancies of salivary glands. This study aims to explore the expression and migration of LAG3, TIM3, and A2aR in AdCC and MEC, and the potential relationship with oncogenic signaling molecules and immunosuppressive cytokines. Custom made human salivary gland tissue microarrays included 81 AdCCs, 52MECs, 76 normal salivary glands (NSG), and 14 pleomorphic adenoma (PMA) samples. Immunohistochemical analysis of lymphocyte activation gene 3 (LAG3), T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3), adenosine 2a receptor (A2aR), oncogenic phosphorylated S6kinase (p-S6) and ERK1/2 (p-ERK1/2 ), and TGF-β1 was performed with salivary gland tissue microarrays of human samples. The correlation of the immunostaining was analyzed based on a digital pathological system, and data were evaluated by hierarchical cluster. Further in vitro studies of knockdown immune checkpoints LAG3, TIM3, and A2aR were carried out by siRNA transfection. The expression levels of LAG3, TIM3, and A2aR were remarkably increased in AdCC and MEC, compared with NSG and PMA samples, but were independent of pathology grade. They were closely correlated with TGF-β1, slightly related to p-ERK1/2 and p-S6. After the knockdown of immune checkpoints LAG3, TIM3, and A2aR, the migration of SACC-LM cell line was significantly reduced. These results suggested that LAG3, TIM3, and A2aR are overexpressed in AdCC and MEC, may promote migration of SACC-LM cell and correlated with TGF-β1 and oncogenic signaling pathways.

LAG3 and Its Ligands Show Increased Expression in High-Risk Uveal Melanoma.

Simple SummaryUveal melanoma (UM) is a rare type of intraocular malignancy, which often gives rise to metastases. While treatment with immune checkpoint inhibitors is often effective in the treatment of cutaneous melanoma metastases, it is hardly effective in the case of UM metastases. Lymphocyte-activation gene 3 (LAG3) is a recently recognized immune checkpoint; we determined the distribution of LAG3 expression and its ligands in three sets of primary UM. High-risk UM (epithelioid cell type, loss of chromosome 3/BAP1 staining) had a higher expression of LAG3 and its ligands, which correlated with the presence of infiltrating immune cells. We concluded that not only LAG3 but also its ligands Galectin-3 and HLA class II are especially expressed in high-risk UM and may be a target for adjuvant immunotherapy in UM.Uveal melanoma (UM) is a rare ocular malignancy which originates in the uveal tract, and often gives rise to metastases. Potential targets for immune checkpoint inhibition are lymphocyte-activation gene 3 (LAG3) and its ligands. We set out to analyse the distribution of these molecules in UM. The expression of mRNA was determined using an Illumina array in 64 primary UM from Leiden. The T lymphocyte fraction was determined by digital droplet PCR. In a second cohort of 15 cases from Leiden, mRNA expression was studied by Fluidigm qPCR, while a third cohort consisted of 80 UM from TCGA. In the first Leiden cohort, LAG3 expression was associated with the presence of epithelioid cells (p = 0.002), monosomy of chromosome 3 (p = 0.004), and loss of BAP1 staining (p = 0.001). In this Leiden cohort as well as in the TCGA cohort, LAG3 expression correlated positively with the expression of its ligands: LSECtin, Galectin-3, and the HLA class II molecules HLA-DR, HLA-DQ, and HLA-DP (all p < 0.001). Furthermore, ligands Galectin-3 and HLA class II were increased in monosomy 3 tumours and the expression of LAG3 correlated with the presence of an inflammatory phenotype (T cell fraction, macrophages, HLA-A and HLA-B expression: all p < 0.001). High expression levels of LAG3 (p = 0.01), Galectin-3 (p = 0.001), HLA-DRA1 (p = 0.002), HLA-DQA1 (p = 0.04), HLA-DQB2 (p = 0.03), and HLA-DPA1 (p = 0.007) were associated with bad survival. We conclude that expression of the LAG ligands Galectin-3 and HLA class II strongly correlates with LAG3 expression and all are increased in UM with Monosomy 3/BAP1 loss. The distribution suggests a potential benefit of monoclonal antibodies against LAG3 or Galectin-3 as adjuvant treatment in patients with high-risk UM.

Juvenile idiopathic arthritis: lymphocyte activation gene-3 is a central immune receptor in children with oligoarticular subtypes.

We investigated the role of inhibitory receptors (IRs) and especially lymphocyte activation gene-3 (LAG-3) in the pathogenesis of oligoarticular juvenile idiopathic arthritis (o-JIA). Paired samples of synovial fluid (SF) and plasma and peripheral blood (PBMCs) and synovial fluid mononuclear cells (SFMCs) were collected from o-JIA patients along with their clinical data (n = 24). Plasma from healthy controls (n = 14) and paired SF and plasma samples from five non-arthritic juvenile orthopedic patients (n = 5) served as controls. Spontaneously differentiated fibroblast-like synoviocytes (FLSs) from SFMCs were co-cultured with autologous PBMCs/SFMCs and used as ex vivo disease model. Soluble levels and cellular expressions of IRs together with their functional properties in the ex vivo model were analyzed. In patients with o-JIA, soluble levels of LAG-3 and expression of LAG-3 and T cell immunoglobulin mucin03 (TIM-3) on CD3+CD4+CD45RO+ T cells were increased, especially in SF. Major histocompatibility complex (MHC) class II expression was induced on FLSs when these were co-cultured with autologous PBMCs/SFMCs, together with an increased monocyte chemoattractant protein-1 (MCP-1) production. In PBMC and FLS + PBMC co-cultures, neutralizing antibodies to IRs were added. Only anti-LAG-3 antibodies significantly increased MCP-1 secretion. The addition of agonistic LAG-3 antibody resulted in decreased effector cytokine secretion. This is the first report comparing the effects of different IRs in o-JIA and suggests that LAG-3 might contribute to the pathogenesis of this disease. This is the first study addressing the role of different co-IRs in o-JIA. We showed that LAG-3 and TIM-3 seem more important in juvenile arthritis in contrast to adult rheumatoid arthritis, where cytotoxic T-lymphocyte-associated antigen-4 and programmed cell death-1 were reported to be more important. We designed an ex vivo disease model for o-JIA, examined the effects of co-IRs in this model, and demonstrated that they might contribute to the pathogenesis of the disease. LAG-3 might play a role in o-JIA pathogenesis and might be a potential therapeutic option for o-JIA patients.

Lymphocyte activation gene 3 expression on T lymphocyte cell subsets in patients with myelodysplastic syndrome

Objective: To investigate the expression of Lymphocyte activation gene 3 (LAG3) in myelodysplastic syndromes (MDS) patients. Methods: A total of 16 MDS patients newly diagnosed in Hematology Department of Tianjin Medical University were enrolled from January to November 2019. The healthy control (HC) group includes 16 cases of healthy adults. The expression levels of LAG3 on CD8(+)T cells, CD4(+)T cells and regulatory T cells (Treg) in MDS patients and healthy controls were detected by flow cytometry. Results: A total of 16 patients with MDS were included, including 5 males and 11 females, with a median age of 56 (18-80) years old. HC group includes 16 healthy adults, 8 men and 8 women, with a median age of 40 (17-69) years. There was no statistically significant difference in gender and age composition between the two groups (both P>0.05). The expression of LAG3 on CD8(+)T cells in MDS patients (74.45%±22.31%) was significantly higher than that in HC group (58.78%±14.82%, P<0.05). The LAG3 expression on Treg in MDS patients (64.91%±10.32%) were significantly higher than that of HC group (49.09%±13.58%, P<0.05). There was no statistical difference in LAG3 expression on CD4(+)T cells between the two groups. Conclusion: The expression of LAG3 on CD8(+)T cells and Treg increases in MDS patients than that of healthy people.

CD4+ T cell exhaustion revealed by high PD-1 and LAG-3 expression and the loss of helper T cell function in chronic hepatitis B

BackgroundImmune inhibitory receptors play an important role in chronic infections. However, little is known about their role in hepatitis B virus (HBV) infection. Here, we analyzed the relationship between programmed death-1 (PD-1) and lymphocyte activation gene-3 (LAG-3) expression on CD4+ T cells and HBV disease progression.ResultsPD-1 and LAG-3 expression was significantly higher on CD4+ T cells from HBV patients than on those from the HCs. In addition, a significant positive correlation was found between the PD-1 and LAG-3 expression levels and the ALT(alanine aminotransferase) level. CD4+ T cell function was inhibited by high PD-1 and LAG-3 levels, and CD4+ T cells with high PD-1 and LAG-3 expression lost the ability to secrete IFN-γ, IL-2 and TNF-α. Furthermore, blockade of the PD-1 and LAG-3 pathways reversed the damage to CD4+ T cell proliferation and cytokine secretion.ConclusionsCD4+ T cell exhaustion during chronic HBV had high PD-1 and LAG-3 expression and the absence of helper T cell cytokines, including IFN-γ, IL-2 and TNF-α. After blocking PD-L1 and LAG-3, CD4+ T cell function in chronic hepatitis B patients was partially restored.

A first-in-human phase I study of FS118, an anti-LAG-3/PD-L1 bispecific antibody in patients with solid tumors that have progressed on prior PD-1/PD-L1 therapy.

TPS2652 Background:PD-1/PD-L1 checkpoint inhibitors demonstrated remarkable anti-tumor activity, but only a minority of patients achieve full clinical benefit with deep and durable responses. Translational studies suggest resistance to cancer immunotherapy can be mediated by additional immune checkpoints e.g. lymphocyte-activation gene 3 (LAG-3). The combination of LAG-3 and PD-1 mAbs synergistically improved anti-tumor response in murine models and early clinical trials. In a small cohort, TIL LAG-3 expression enriched for responsiveness in PD-1/PD-L1 relapsed/refractory patients. FS118 is a novel bispecific antibody incorporating a LAG-3 binding Fc-region into a PD-L1-specific IgG1 antibody to potentially deliver superior anti-tumor efficacy while limiting immunotherapy-related adverse effects by dual targeting. Methods: The FiH study (NCT03440437) is being conducted in adult patients with solid tumors who failed prior PD-1/PD-L1 treatment. Primary objectives of the study are to determine safety, PK and the maximum tolerated/recommended Phase 2 dose of FS118. Secondary objectives include preliminary evidence of efficacy, immunogenicity, PD profile and exposure/response correlation. 50 subjects from four study sites in the USA are planned to enrol in dose escalation initiated with accelerated titration (5 single subject cohorts) followed by 3+3 design and expansion cohorts. FS118 is administered weekly IV in 21-day treatment cycles until progression, unacceptable toxicity, withdrawal, or death. Patients are followed for safety, overall survival and initiation of subsequent therapy. DLT clearance, dose escalation and cohort expansion (to further characterise safety, PK/PD or clinical efficacy) are supervised by a safety review committee (SRC). Translational studies assess PD-L1/LAG-3 receptor occupancy, soluble PD-L1/LAG-3 levels and the correlation of FS118 exposure with selected PD markers of target engagement and response. Translational endpoints include TIL analysis, transcriptomic profiles and target expression analyses on tumor tissues. Cohorts 1 through 6 have been completed, enrollment in cohort 7 began December 2018. Clinical trial information: NCT03440437.

Blood Levels of Co-inhibitory-Receptors: A Biomarker of Disease Prognosis in Multiple Sclerosis.

Background: The clinical course of multiple sclerosis ranges from benign with little disease progression and minimal disability, to severe disease requiring intensive medical treatment. There are no reliable circulating biomarkers for predicting disease outcome. Co-inhibitory receptors regulate the termination of effective immune responses to infections while limiting autoimmunity and/or immunopathology. Based on this, we studied the potential of circulating co-inhibitory receptor levels as predictive biomarkers of multiple sclerosis outcome.Methods: Co-inhibitory receptor [TIGIT (T cell immunoreceptor with Ig and ITIM domains), TIM-3 (T-cell immunoglobulin and mucin domain–containing 3), LAG-3 (lymphocyte activation gene 3), PD-1 (programmed cell death 1), CTLA-4 (cytotoxic T-lymphocyte–associated protein 4)] expression levels in peripheral blood mononuclear cells (PBMCs) were measured using reverse transcription–PCR in 19 healthy controls and 57 patients with untreated multiple sclerosis. All patients were evaluated for disease outcome and paraclinical measures during the following 9–10 years [progression index, Expanded Disability Status Scale (EDSS) score, number of relapses, number of disease modifying therapies (DMTs), baseline brain magnetic resonance imaging T2 lesion volume, and oligoclonal bands (OCBs)].Results: Patients had significantly lower TIGIT and LAG-3 levels than the controls (P < 0.02 and P < 0.04, respectively). TIM-3 levels were significantly lower in patients with high vs. low disability index and in patients with SPMS diagnosis compared to patients who remained in the relapsing stage of the disease at final visit (both, P < 0.02). LAG-3 levels were significantly higher in patients with low disability index vs. non-low disability index multiple sclerosis (P < 0.05). TIM-3 and LAG-3 expression levels correlated significantly with 1-year progression index (r2 = 0.076, P < 0.05; 0.087, P < 0.04, respectively) and EDSS score at final visit (r2 = 0.31, P < 0.04; 0.320.088, P < 0.04, respectively). Lower LAG-3 levels were associated with higher DMT switching (r2 = 0.67, P < 0.05). Compared to the paraclinical and clinical parameters alone, the combined data of the baseline co-inhibitory receptor expression levels and the paraclinical and clinical parameters were superior for predicting the patients that would progress to secondary progressive multiple sclerosis (SPMS).Interpretation: This is an initial exploration of the utility of CTLA-4, PD-1, TIM-3, LAG-3, and TIGIT expression levels as prognostic indicators in untreated, recently diagnosed multiple sclerosis. Our results support the value of decreased PBMC expression levels of TIM-3 and LAG-3 at diagnosis as an unfavorable prognostic factor, which is to be confirmed in further studies.

LAG-3 expression on tumor-infiltrating T cells in soft tissue sarcoma correlates with poor survival.

ObjectiveTo elucidate the role and prognostic significance of lymphocyte activation-gene-3 (LAG-3) in soft tissue sarcoma (STS).MethodsThe expression of LAG-3 in patient and matched normal blood samples was analyzed by flow cytometry. The localization and prognostic values of LAG-3+ cells in 163 STS patients were analyzed by immunohistochemistry. In addition, the expression of tumor-infiltrating CD3+ T, CD4+ T, and CD8+ T cells and their role in the prognosis of STS were evaluated by immunohistochemistry. The effect of LAG-3 blockade was evaluated in an immunocompetent MCA205 fibrosarcoma mouse model. ResultsPeripheral CD8+ and CD4+ T cells from STS patients expressed higher levels of LAG-3 than those from healthy donors. LAG-3 expression in STS was significantly associated with a poor clinical outcome (P = 0.038 ) and was correlated with high pathological grade (P < 0.001), advanced tumor stage ( P = 0.016). Additionally, LAG-3 expression was highly correlated with CD8+ T-cell infiltration (r = 0.7034, P < 0.001). LAG-3 was expressed in murine tumor-infiltrating lymphocytes, and its blockade decreased tumor growth and enhanced secretion of interferon-gamma by CD8 + and CD4+ T cells. ConclusionsLAG-3 blockade may be a promising strategy to improve the effects of targeted therapy in STS.

Abstract 3652: Combinational activity of LAG3 and PD-1 targeted therapies is significantly enhanced by the addition of phosphatidylserine targeting antibodies and establishes an anti-tumor memory response in murine triple negative breast cancer

Abstract Previous studies utilizing NanoString immune profile analysis demonstrated that intratumoral levels of LAG3 (lymphocyte activation gene 3) mRNA increased in response to phosphatidylserine (PS) and PD-1 targeting antibodies in murine triple negative breast cancers (TNBC). This suggests LAG3 acts to attenuate immune system activation during I/O therapies - and that PD-1 and LAG3 function cooperatively in suppressing immune system activation. Here we show that adding PS targeting antibodies can further enhance the effectiveness of antibodies targeting LAG3 and/or LAG3+PD-1. We first examined expression of LAG3 and PD-1 in the murine TNBC model E0771 and found that tumor associated T-cells (CD4+ and CD8+) have expression of both markers. Mice implanted with TNBC tumors were next treated with antibodies targeting PS, PD-1, and LAG3 alone and in combination with each other. Interestingly, the addition of PS targeting antibodies not only increased the effectiveness anti-PD-1 effectiveness as previously observed, but also enhanced anti-LAG3 treatment, showing that PS targeting antibodies are capable of augmenting additional I/O therapeutic regimens. Comparison of anti-PD-1+LAG3 combination vs. single anti-PD-1 or anti-LAG3 treatments showed moderately more anti-tumor activity than single treatments, however the addition of PS targeting antibodies to either checkpoint inhibitor was as equally effective in inhibiting tumor growth as observed in the anti-LAG3+PD-1 treatment. Further comparison of antibody treatments targeting PD-1+LAG3 vs. PS+PD-1+LAG3 demonstrated that the addition of PS targeting antibodies resulted in a significant decrease in tumor growth with complete tumor regression in 80% of the animals (along with the ability to completely reject secondary TNBC challenge) compared to 0% in the anti-PD-1+LAG3 treatment group. Immunoprofiling showed that the addition of PS targeting antibodies to these checkpoint therapies, including the combination of anti-PD-1+LAG3, resulted in a phenotype associated with enhanced immune system activation and immune-surveillance including increased tumor infiltrating lymphocytes (TILs) with upregulation of T-cell associated activation pathways, increased Th1 to Th2 profile, and enhanced antigen presentation processing /presentation mechanisms along with cytokines associated with immune system activation. Overall our data demonstrate that adding PS targeting antibodies to clinically relevant therapies, including PD-1 and LAG3, may significantly enhance their ability to activate and redirect the host immune system into recognition and elimination of tumor cells compared to single and combinational treatments that lack PS targeting antibodies. Citation Format: Michael J. Gray, Jian Gong, Jeff T. Hutchins, Bruce D. Freimark. Combinational activity of LAG3 and PD-1 targeted therapies is significantly enhanced by the addition of phosphatidylserine targeting antibodies and establishes an anti-tumor memory response in murine triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3652. doi:10.1158/1538-7445.AM2017-3652

Lymphocyte activation gene 3 and coronary artery disease.

BACKGROUND: The lipoprotein scavenger receptor BI (SCARB1) rs10846744 noncoding variant is significantly associated with atherosclerotic disease independently of traditional cardiovascular risk factors. We identified a potentially novel connection between rs10846744, the immune checkpoint inhibitor lymphocyte activation gene 3 (LAG3), and atherosclerosis. METHODS: In vitro approaches included flow cytometry, lipid raft isolation, phosphosignaling, cytokine measurements, and overexpressing and silencing LAG3 protein. Fasting plasma LAG3 protein was measured in hyperalphalipoproteinemic (HALP) and Multi-Ethnic Study of Atherosclerosis (MESA) participants. RESULTS: In comparison with rs10846744 reference (GG homozygous) cells, LAG3 protein levels by flow cytometry (P < 0.001), in lipid rafts stimulated and unstimulated (P = 0.03), and phosphosignaling downstream of B cell receptor engagement of CD79A (P = 0.04), CD19 (P = 0.04), and LYN (P = 0.001) were lower in rs10846744 risk (CC homozygous) cells. Overexpressing LAG3 protein in risk cells and silencing LAG3 in reference cells confirmed its importance in phosphosignaling. Secretion of TNF-α was higher (P = 0.04) and IL-10 was lower (P = 0.04) in risk cells. Plasma LAG3 levels were lower in HALP carriers of the CC allele (P < 0.0001) and by race (P = 0.004). In MESA, race (P = 0.0005), age (P = 0.003), lipid medications (P = 0.03), smoking history (P < 0.0001), and rs10846744 genotype (P = 0.002) were independent predictors of plasma LAG3. In multivariable regression models, plasma LAG3 was significantly associated with HDL-cholesterol (HDL-C) (P = 0.007), plasma IL-10 (P < 0.0001), and provided additional predictive value above the Framingham risk score (P = 0.04). In MESA, when stratified by high HDL-C, plasma LAG3 was associated with coronary heart disease (CHD) (odds ratio 1.45, P = 0.004). CONCLUSION: Plasma LAG3 is a potentially novel independent predictor of HDL-C levels and CHD risk. FUNDING: This work was supported by an NIH RO1 grant (HL075646), the endowed Linda and David Roth Chair for Cardiovascular Research, and the Harold S. Geneen Charitable Trust Coronary Heart Disease Research award to Annabelle Rodriguez. MESA is conducted and supported by the National Heart, Lung, and Blood Institute (NHLBI) in collaboration with MESA investigators. Support for MESA is provided by contracts HHSN268201500003I, N01-HC-95159, N01-HC-95160, N01-HC-95161, N01-HC-95162, N01-HC-95163, N01-HC-95164, N01-HC-95165, N01-HC-95166, N01-HC-95167, N01-HC-95168, N01-HC-95169, UL1-TR-001079, UL1-TR-000040, and DK063491. Cardiometabochip genotyping data for the MESA samples was supported in part by grants and contracts R01HL98077, N02-HL-64278, HL071205, UL1TR000124, DK063491, RD831697, and P50 ES015915.

Abstract PR03: Combinatorial blockade of PD-1, CTLA-4, and LAG-3 pathways inhibits murine ovarian tumor growth.

Abstract We have previously shown that tumor-associated or infiltrating T cells (TALs or TILs) often co-express immune inhibitory receptors lymphocyte activation gene-3 (LAG-3) and programmed cell death 1 (PD-1) in human and murine ovarian tumor microenvironment (TME). Dual antibody blockade or genetic knockout of LAG3 and PD1 significantly enhanced T effector function and delayed ovarian tumor growth. The relative contribution of other immune inhibitory receptors to immune suppression in the ovarian TME, and whether they co-regulate each other are currently unknown. To address this, we analyzed the expression of multiple immune checkpoint proteins and their cognate ligands in ovarian tumor-bearing wild-type (WT), LAG3KO and PD1KO mice. We found that PD-1, cytotoxic T lymphocyte antigen 4 (CTLA-4), LAG-3, T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and CD160 were elevated in TILs from the WT mice. Interestingly, compared with TILs of WT mice, the levels of LAG-3, TIM-3, and CTLA-4 were further significantly elevated in TILs from the tumor-bearing PD1KO mice; while the levels of PD-1, CTLA-4, and CD160 were further increased in the tumor-bearing LAG3KO mice. These results suggest that PD-1 and LAG-3 regulate the expression of multiple inhibitory receptors in the ovarian TME. Furthermore, combinatorial blockade with anti-LAG3 and anti-CTLA4 antibodies in the tumor-bearing PD1KO mice resulted in 30% tumor-rejection as compared to 10% by anti-CTLA4 alone or only delayed tumor growth by anti-LAG3. These data provide a strong basis for rational clinical testing of combinatorial blockade of the PD-1, LAG-3 and CTLA-4 immune inhibitory pathways for ovarian cancer immunotherapy. This abstract is also presented as Poster A51. Citation Format: Ruea-Yea Huang, AJ McGray, Kunle Odunsi. Combinatorial blockade of PD-1, CTLA-4, and LAG-3 pathways inhibits murine ovarian tumor growth. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr PR03.

On the role of the inhibitory receptor LAG-3 in acute and chronic LCMV infection

Chronic viral infections are often characterized by CD8 T-cell responses with poor cytokine secretion potential and limited expansion of the CD8 T-cell pool, collectively referred to as CD8 T-cell exhaustion. Exhaustion of lymphocytic choriomeningitis virus (LCMV)-specific CD8 T cells was shown to be partially regulated by the inhibitory receptor programmed death 1 (PD-1). Here, we demonstrate that exhausted LCMV-specific CD8 T cells also express the negative regulatory receptor lymphocyte activation gene 3 (LAG-3) which is mainly expressed on cells co-expressing the negative regulatory receptors PD-1 and Tim-3. Expression levels of LAG-3 on anti-viral CD8 T cells remain stable over short-term in vitro stimulations in presence of antigenic peptide. Nevertheless, in vitro and in vivo blockade of LAG-3 did not rescue cytokine production by virus-specific CD8 T cells and did not alter the virus titer in various organs. Likewise, chronic LCMV infection of LAG-3-/- mice led to a comparable degree of T-cell exhaustion as observed in C57BL/6 controls and to similar virus titers. Further, LAG-3 did not influence T-cell activation or cell division during chronic LCMV infection. These data suggest that even though LAG-3 is continuously up-regulated on LCMV-specific exhausted CD8 T cells, it alone does not significantly contribute to T-cell exhaustion.

LAG-3 regulates CD8+ T cell accumulation and effector function in murine self- and tumor-tolerance systems

Lymphocyte activation gene-3 (LAG-3) is a cell-surface molecule with diverse biologic effects on T cell function. We recently showed that LAG-3 signaling is important in CD4+ regulatory T cell suppression of autoimmune responses. Here, we demonstrate that LAG-3 maintains tolerance to self and tumor antigens via direct effects on CD8+ T cells using 2 murine systems. Naive CD8+ T cells express low levels of LAG-3, and expression increases upon antigen stimulation. Our data show increased levels of LAG-3 protein on antigen-specific CD8+ T cells within antigen-expressing organs or tumors. In vivo antibody blockade of LAG-3 or genetic ablation of the Lag-3 gene resulted in increased accumulation and effector function of antigen-specific CD8+ T cells within organs and tumors that express their cognate antigen. Most notably, combining LAG-3 blockade with specific antitumor vaccination resulted in a significant increase in activated CD8+ T cells in the tumor and disruption of the tumor parenchyma. A major component of this effect was CD4 independent and required LAG-3 expression by CD8+ T cells. Taken together, these data demonstrate a direct role for LAG-3 on CD8+ T cells and suggest that LAG-3 blockade may be a potential cancer treatment.