Dissociation of LAG-3 inhibitory cluster from TCR microcluster by immune checkpoint blockade.

Lymphocyte activation gene (Lag)-3 is an inhibitory co-receptor and target of immune checkpoint inhibitor (ICI) therapy for cancer. The dynamic behavior of Lag-3 was analyzed at the immune synapse upon T-cell activation to elucidate the Lag-3 inhibitory mechanism. Lag-3 formed clusters and co-localized with T-cell receptor microcluster (TCR-MC) upon T-cell activation similar to PD-1. Lag-3 blocking antibodies (Abs) inhibited the co-localization between Lag-3 and TCR-MC without inhibiting Lag-3 cluster formation. Lag-3 also inhibited MHC-II-independent stimulation and Lag-3 Ab, which did not block MHC-II binding could still block Lag-3's inhibitory function, suggesting that the Lag-3 Ab blocks the Lag-3 inhibitory signal by dissociating the co-assembly of TCR-MC and Lag-3 clusters. Consistent with the combination benefit of PD-1 and Lag-3 Abs to augment T-cell responses, bispecific Lag-3/PD-1 antagonists effectively inhibited both cluster formation and co-localization of PD-1 and Lag-3 with TCR-MC. Therefore, Lag-3 inhibits T-cell activation at TCR-MC, and the target of Lag-3 ICI is to dissociate the co-localization of Lag-3 with TCR-MC.

Inhibitory co-receptor Lag3 supports Foxp3+ regulatory T cell function by restraining Myc-dependent metabolic programming.

Lymphocyte activation gene 3 (Lag3) is an inhibitory co-receptor expressed on activated T cells and has been proposed to regulate regulatory T (Treg) cell function. However, its precise modality and mechanisms remain elusive. We generated Treg cell-specific Lag3-mutant mouse models and found that Lag3 was essential for Treg cell control of autoimmunity. RNA sequencing analysis revealed that Lag3 mutation altered genes associated with metabolic processes, especially Myc target genes. Myc expression in Lag3-mutant Treg cells was increased to the level seen in conventional T helper (Th)1-type effector cells and directly correlated with their metabolic profiles and in vivo suppressive functions. The phosphatidylinositol 3-kinase (PI3K)-Akt-Rictor pathway was activated in Lag3-mutant Treg cells, and inhibiting PI3K, Rictor, or lactate dehydrogenase A (Ldha), a key Myc target enzyme converting pyruvate to lactate, was sufficient to restore normal metabolism and suppressive function in Lag3-mutant Treg cells. These findings indicate that Lag3 supports Treg cell suppression partly by tuning Myc-dependent metabolic programming.

Baseline sLAG-3 levels in Caucasian and African-American breast cancer patients.

BACKGROUND: Worse survival persists for African-Americans (AA) with breast cancer compared to other race/ethnic groups despite recent improvements for all. Unstudied in outcomes disparities to date is soluble LAG-3 (sLAG-3), cleaved from the LAG-3 immune checkpoint receptor which is a proposed target for deactivation in emerging immunotherapies due to its prominent immunosuppressive function in the tumoral microenvironment. A prior study has found that lower sLAG-3 baseline level was associated with poor outcomes. METHODS: In a cross-sectional study of 95 patients with primary breast cancer (n = 58 Caucasian, n = 37 AA), we measured sLAG-3 (ELISA pg/ml) in pre-treatment blood samples using the non-parametric Mann-Whitney u-Test for independent samples, and, calculated Pearson r correlation coefficients of sLAG-3 with circulating cytokines by race. RESULTS: Mean sLAG-3 level was lower in AA compared to Caucasian patients (1377.6 vs 3690.3, P = .002), and in patients with triple-negative breast cancer (TNBC) compared to those with non-TNBC malignancies (P = .02). When patients with TNBC tumors were excluded from analyses, the difference in sLAG-3 level between AA (n = 21) and Caucasian patients (n = 40) substantially remained (1937.4 vs 4182.4, P = .06). Among Caucasian patients, sLAG-3 was correlated with IL-6, IL-8 and IL-10 (r = .69, P < .001; r = .70, P < .001; and, r = .46, P = .01; respectively). For AA patients, sLAG-3 was correlated only with IL-6 (r = .37, P = .03). CONCLUSIONS: We present the first report that African-American breast cancer patients might have comparatively low pre-treatment sLAG-3 levels, independent of TNBC status, along with reduced co-expression with circulating cytokines. The mechanistic and prognostic role of cleaved LAG-3, particularly in disparate outcomes, remains to be elucidated.

Unlocking therapeutic potential: Targeting lymphocyte activation Gene-3 (LAG-3) with fibrinogen-like protein 1 (FGL1) in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) represents an autoimmune disorder that affects multiple systems. In the treatment of this condition, the focus primarily revolves around inflammation suppression and immunosuppression. Consequently, targeted therapy has emerged as a prevailing approach. Currently, the quest for highly sensitive and specifically effective targets has gained significant momentum in the context of SLE treatment. Lymphocyte activation gene-3 (LAG-3) stands out as a crucial inhibitory receptor that binds to pMHC-II, thereby effectively dampening autoimmune responses. Fibrinogen-like protein 1 (FGL1) serves as the principal immunosuppressive ligand for LAG-3, and their combined action demonstrates a potent immunosuppressive effect. This intricate mechanism paves the way for potential SLE treatment by targeting LAG-3 with FGL1. This work provides a comprehensive summary of LAG-3's role in the pathogenesis of SLE and elucidates the feasibility of leveraging FGL1 as a therapeutic approach for SLE management. It introduces a novel therapeutic target and opens up new avenues of therapeutic consideration in the clinical context of SLE treatment.

Evaluation of immune-checkpoint molecules in dMMR/pMMR colorectal cancer by multiplex immunohistochemistry.

PURPOSE: Colorectal cancer is the most common malignancy worldwide. A number of pathological and molecular genetic criteria are currently used as predictors of the disease. They include assessment of MMR deficiency or MSI/MSS status, which among others, determine the immunogenicity of the tumor. In this regard, the evaluation of PD-L1, CTLA-4, and LAG-3 immune checkpoint molecules in different tumor compartments according to MMR status deserves special attention. METHODS: Multiplex immunohistochemistry was used to evaluate the expression of immune checkpoint molecules in the tumor core and at the invasive margin. RESULTS: Data analysis showed the predominance of PD-L1 (p = 0.011), CTLA-4 (p = 0.004), and LAG-3 (p = 0.013) expression at the invasive margin of dMMR carcinomas compared to pMMR samples. Quantitative analysis of TILs population in the tumor core and at the invasive margin allowed establishment of the predominance of CD3+ and CD8+ lymphocytes at the invasive margin of dMMR carcinomas. Study of the CD163+ macrophages population in the same tumor compartments revealed the predominance of the studied TAMs in the core and at the invasive margin of dMMR carcinomas and the predominance of CD163+ macrophages with PD-L1-phenotype in the tumor stroma. CONCLUSION: This study revealed a significant predominance of PD-L1, CTLA-4, LAG-3, and CD 3+ ,CD8+ lymphocytes in dMMR colorectal carcinomas. Further research on the immune landscape in different tumor compartments will likely have high prognostic value for CRC patients, as it might expand the criteria for prescribing immunotherapy.

Advances in understanding the role of immune checkpoint LAG-3 in tumor immunity: a comprehensive review.

Lymphocyte activation gene 3 (LAG-3), also known as CD223, is an emerging immune checkpoint that follows PD-1 and CTLA-4. Several LAG-3 targeting inhibitors in clinical trials and the combination of relatlimab (anti-LAG-3) and nivolumab (anti-PD-1) have been approved for treating - unresectable or metastatic melanoma. Despite the encouraging clinical potential of LAG-3, the physiological function and mechanism of action in tumors are still not well understood. In this review, we systematically summarized the structure of LAG-3, ligands of LAG-3, cell-specific functions and signaling of LAG-3, and the current status of LAG-3 inhibitors under development.

TIGIT expression in renal cell carcinoma infiltrating T cells is variable and inversely correlated with PD-1 and LAG3.

PURPOSE: Immune checkpoint inhibitors have revolutionized the treatment of renal cell carcinoma (RCC), but many patients do not respond to therapy and the majority develop resistant disease over time. Thus, there is increasing need for alternative immunomodulating agents. The co-inhibitory molecule T-cell immunoglobulin and ITIM domain (TIGIT) may play a role in resistance to approved immune checkpoint inhibitors and is being investigated as a potential therapeutic target. The purpose of this study was to quantify TIGIT positivity in tumor-infiltrating T cells in RCC. METHODS: We employed tissue microarrays containing specimens from primary RCC tumors, adjacent normal renal tissue, and RCC metastases to quantify TIGIT within tumor-infiltrating CD3+ T cells using quantitative immunofluorescent analysis. We also compared these results to TIGIT+ CD3+ levels in four other tumor types (melanoma, non-small cell lung, cervical, and head and neck cancers). RESULTS: We did not observe significant differences in TIGIT positivity between primary RCC tumors and patient-matched metastatic samples. We found that the degree of TIGIT positivity in RCC is comparable to that in lung cancer but lower than that in melanoma, cervical, and head and neck cancers. Correlation analysis comparing TIGIT positivity to previously published, patient-matched spatial proteomic data by our group revealed a negative association between TIGIT and the checkpoint proteins PD-1 and LAG3. CONCLUSION: Our findings support careful evaluation of TIGIT expression on T cells in primary or metastatic RCC specimens for patients who may be treated with TIGIT-targeting antibodies, as increased TIGIT positivity might be associated with a greater likelihood of response to therapy.

Immunological Profiling of CD8+ and CD8- NK Cell Subpopulations and Immune Checkpoint Alterations in Early-Onset Preeclampsia and Healthy Pregnancy.

Despite the numerous studies on the clinical aspects of early-onset preeclampsia, our understanding of the immunological consequences of inadequate placenta development remains incomplete. The Th1-predominance characteristic of early-onset preeclampsia significantly impacts maternal immunotolerance, and the role of immune checkpoint molecules in these mechanisms is yet to be fully elucidated. Our study aims to fill these crucial knowledge gaps. A total of 34 pregnant women diagnosed with early-onset preeclampsia and 34 healthy pregnant women were enrolled in this study. A mononuclear cell fragment from the venous blood was separated and frozen. The CD8+ and CD8- NK cell subpopulations were identified and compared to their immune checkpoint molecule expressions using multicolor flow cytometry. The serum CD226 levels were measured by ELISA. Based on our measures, the frequency of the CD8- subpopulation was significantly higher than that of the CD8+ counterpart in both the NKdim and NKbright subsets. Significantly lower CD226 surface expressions were detected in the preeclamptic group compared to healthy women in all the investigated subpopulations. However, while no difference was observed in the level of the soluble CD226 molecule between the two groups, the CD112 and CD155 surface expressions were significantly different. Our study's findings underscore the significant role of the CD8+ and CD8- NK subpopulations in the Th1-dominated immune environment. This deepens our understanding of early-onset preeclampsia and suggests that each subpopulation could contribute to the compensation mechanisms and the restoration of the immunological balance in this condition, a crucial step toward developing effective interventions.

LAG-3 and PD-1 synergize on CD8+ T cells to drive T cell exhaustion and hinder autocrine IFN-γ-dependent anti-tumor immunity.

Overcoming immune-mediated resistance to PD-1 blockade remains a major clinical challenge. Enhanced efficacy has been demonstrated in melanoma patients with combined nivolumab (anti-PD-1) and relatlimab (anti-LAG-3) treatment, the first in its class to be FDA approved. However, how these two inhibitory receptors synergize to hinder anti-tumor immunity remains unknown. Here, we show that CD8+ T cells deficient in both PD-1 and LAG-3, in contrast to CD8+ T cells lacking either receptor, mediate enhanced tumor clearance and long-term survival in mouse models of melanoma. PD-1- and LAG-3-deficient CD8+ T cells were transcriptionally distinct, with broad TCR clonality and enrichment of effector-like and interferon-responsive genes, resulting in enhanced IFN-γ release indicative of functionality. LAG-3 and PD-1 combined to drive T cell exhaustion, playing a dominant role in modulating TOX expression. Mechanistically, autocrine, cell-intrinsic IFN-γ signaling was required for PD-1- and LAG-3-deficient CD8+ T cells to enhance anti-tumor immunity, providing insight into how combinatorial targeting of LAG-3 and PD-1 enhances efficacy.

Indirect suppression of CD4 T cell activation through LAG-3-mediated trans-endocytosis of MHC class II.

Blockade of immune checkpoint receptors has shown outstanding efficacy for tumor immunotherapy. Promising treatment with anti-lymphocyte-activation gene-3 (LAG-3) has already been recognized as the next efficacious treatment, but there is still limited understanding of the mechanism of LAG-3-mediated immune suppression. Here, utilizing high-resolution molecular imaging, we find a mechanism of CD4 T cell suppression via LAG-3, in which LAG-3-bound major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs) gather at the central region of an immunological synapse and are trans-endocytosed by T cell receptor-driven internalization motility toward CD4 and CD8 T cells expressing LAG-3. Downregulation of MHC class II molecules on APCs thus results in the attenuation of their antigen-presentation function and impairment of CD4 T cell activation. From these data, anti-LAG-3 treatment is suggested to have potency to directly block the inhibitory signaling via LAG-3 and simultaneously reduce MHC class II expression on APCs by LAG-3-mediated trans-endocytosis for recovery from T cell exhaustion.

Intranasal LAG3 antibody infusion induces a rapid antidepressant effect via the hippocampal ERK1/2-BDNF signaling pathway in chronically stressed mice.

The decline of microglia in the dentate gyrus is a new phenomenon that may explain the pathogenesis of depression, and reversing this decline has an antidepressant effect. The development of strategies that restore the function of dentate gyrus microglia in under stressful conditions is becoming a new focus. Lymphocyte-activating gene-3 (LAG3) is an immune checkpoint expressed by immune cells including microglia. One of its functions is to suppress the expansion of immune cells. In a recent study, chronic systemic administration of a LAG3 antibody that readily penetrates the brain was reported to reverse chronic stress-induced hippocampal microglia decline and depression-like behaviors. We showed here that a single intranasal infusion of a LAG3 antibody (In-LAG3 Ab) reversed chronic unpredictable stress (CUS)-induced depression-like behaviors in a dose-dependent manner, which was accompanied by an increase in brain-derived neurotrophic factor (BDNF) in the dentate gyrus. Infusion of an anti-BDNF antibody into the dentate gyrus, construction of knock-in mice with the BDNF Val68Met allele, or treatment with the BDNF receptor antagonist K252a abolished the antidepressant effect of In-LAG3 Ab. Activation of extracellular signal-regulated kinase1/2 (ERK1/2) is required for the reversal effect of In-LAG3 Ab on CUS-induced depression-like behaviors and BDNF decrease in the dentate gyrus. Moreover, both inhibition and depletion of microglia prevented the reversal effect of In-LAG3 Ab on CUS-induced depression-like behaviors and impairment of ERK1/2-BDNF signaling in the dentate gyrus. These results suggest that In-LAG3 Ab exhibits an antidepressant effect through microglia-mediated activation of ERK1/2 and synthesis of BDNF in the dentate gyrus.

LAG-3 sustains TOX expression and regulates the CD94/NKG2-Qa-1b axis to govern exhausted CD8 T cell NK receptor expression and cytotoxicity.

Exhausted CD8 T (Tex) cells in chronic viral infection and cancer have sustained co-expression of inhibitory receptors (IRs). Tex cells can be reinvigorated by blocking IRs, such as PD-1, but synergistic reinvigoration and enhanced disease control can be achieved by co-targeting multiple IRs including PD-1 and LAG-3. To dissect the molecular changes intrinsic when these IR pathways are disrupted, we investigated the impact of loss of PD-1 and/or LAG-3 on Tex cells during chronic infection. These analyses revealed distinct roles of PD-1 and LAG-3 in regulating Tex cell proliferation and effector functions, respectively. Moreover, these studies identified an essential role for LAG-3 in sustaining TOX and Tex cell durability as well as a LAG-3-dependent circuit that generated a CD94/NKG2+ subset of Tex cells with enhanced cytotoxicity mediated by recognition of the stress ligand Qa-1b, with similar observations in humans. These analyses disentangle the non-redundant mechanisms of PD-1 and LAG-3 and their synergy in regulating Tex cells.

Tumor antigen­specific interleukin­10­producing T­cell response in patients with head and neck squamous cell carcinoma.

Interleukin-10 (IL-10) is a highly pleiotropic cytokine that regulates immunological homeostasis through anti-inflammatory and/or immunostimulatory functions. Moreover, IL-10 is well known to exert diverse roles in tumor immunology and immunotherapy. The present study investigated the presence of circulating tumor antigen-specific IL-10-producing T cells in patients with head and neck squamous cell carcinoma (HNSCC), and determined factors that may influence the immunodynamics of IL-10-producing T cells. In vitro, peripheral blood mononuclear cells (PBMCs) stimulated with the tumor antigens p53 and MAGE-A4 were evaluated for interferon (IFN)-γ/IL-10 production using the IFN-γ/IL-10 double-color enzyme-linked immunosorbent spot assay. The proportion of T cells expressing immune checkpoint molecules in PBMCs was analyzed using flow cytometry. Of the 18 patients with HNSCC, 2 (11.1%) and 9 (50.0%) exhibited p53-specific IFN-γ and IL-10 production, respectively. Meanwhile, MAGE-A4-specific IFN-γ and IL-10 production was detected in 4 (28.6%) and 7 (50.0%) of 14 patients. In the p53-specific responses, IL-10-producing T cells were observed in significantly more patients than IFN-γ producing T cells (P=0.0275). In both CD4+ and CD8+ T cells, the proportion of T cells expressing lymphocyte activation gene-3 (Lag-3) was significantly lower in patients with p53-specific IL-10 production than in those without. In certain patients, Lag-3 blockade enhanced tumor antigen-specific IL-10. Taken together, the present study successfully demonstrated that tumor antigen-specific IL-10-producing T cells exist in the peripheral blood of patients with HNSCC and that Lag-3+ T cells may serve an important role in modulating IL-10-producing T cells. These findings provide novel insights into the roles of IL-10 and Lag-3 in mediating antitumor immune responses.

Blockade of LAG-3 and PD-1 leads to co-expression of cytotoxic and exhaustion gene modules in CD8+ T cells to promote antitumor immunity.

Relatlimab (rela; anti-LAG-3) plus nivolumab (nivo; anti-PD-1) is safe and effective for treatment of advanced melanoma. We designed a trial (NCT03743766) where advanced melanoma patients received rela, nivo, or rela+nivo to interrogate the immunologic mechanisms of rela+nivo. Analysis of biospecimens from this ongoing trial demonstrated that rela+nivo led to enhanced capacity for CD8+ T cell receptor signaling and altered CD8+ T cell differentiation, leading to heightened cytotoxicity despite the retention of an exhaustion profile. Co-expression of cytotoxic and exhaustion signatures was driven by PRDM1, BATF, ETV7, and TOX. Effector function was upregulated in clonally expanded CD8+ T cells that emerged after rela+nivo. A rela+nivo intratumoral CD8+ T cell signature was associated with a favorable prognosis. This intratumoral rela+nivo signature was validated in peripheral blood as an elevated frequency of CD38+TIM3+CD8+ T cells. Overall, we demonstrated that cytotoxicity can be enhanced despite the retention of exhaustion signatures, which will inform future therapeutic strategies.

Targeting PD-1/PD-L1 in tumor immunotherapy: Mechanisms and interactions with host growth regulatory pathways.

Tumor immunotherapy has garnered considerable attention, emerging as a new standard of care in cancer treatment. The conventional targets, such as VEGF and EGFR, have been extended to others including BRAF and PD-1/PD-L1, which have shown significant potential in recent cancer treatments. This review aims to succinctly overview the impact and mechanisms of therapies that modulate PD-1/PD-L1 expression by targeting VEGF, EGFR, LAG-3, CTLA-4 and BRAF. We investigated how modulation of PD-1/PD-L1 expression impacts growth factor signaling, shedding light on the interplay between immunomodulatory pathways and growth factor networks within the tumor microenvironment. By elucidating these interactions, we aim to provide insights into novel potential synergistic therapeutic strategies for cancer immunotherapy.

IL-35 promotes IL-35+IL-10+ Bregs and Conventional LAG3+ Tregs in the lung tissue of OVA-Induced Asthmatic Mice.

AIMS: This study aimed to investigate the effect of interleukin-35 (IL-35) on inflamed lung tissue in a murine model of asthma. IL-35 was examined for its potential to induce regulatory lymphocytes during ovalbumin (OVA)-induced acute lung injury. METHODS: Female BALB/c mice sensitized with OVA and were treated with recombinant IL-35 (rIL-35) via intranasal or intraperitoneal routes and were administered 4 h before OVA challenge. The effects of rIL-35 treatment on the lung and blood levels of regulatory B cells (Bregs) and regulatory T cells (Tregs), as well as their production of immunosuppressive cytokines, were determined using flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: Treatment of OVA-sensitized asthmatic mice with rIL-35, whether administered intranasally or intraperitoneally, resulted in reduced lung inflammation and injury. This reduction was accompanied by an increase in the frequency of IL-35 producing Bregs, IL-35 and IL-10 producing Bregs, and conventional LAG3+ Tregs in the lung tissues and blood. This increase was more pronounced with intranasal rIL-35. Furthermore, there was a positive correlation between the levels of these regulatory cells and lung gene expression of IL-35 and IL-10, and an inverse correlation with both lung gene expression and plasma level of IL-17. CONCLUSIONS: The results of this study suggest that IL-35, through its ability to increase Bregs and Tregs, is effective in reversing lung inflammation in the context of asthma. Since the increase was more pronounced with intranasal administration, this highlights the therapeutic potential of its local intrapulmonary application in managing asthma-related inflammation.

PD-1/LAG-3 co-signaling profiling uncovers CBL ubiquitin ligases as key immunotherapy targets.

Many cancer patients do not benefit from PD-L1/PD-1 blockade immunotherapies. PD-1 and LAG-3 co-upregulation in T-cells is one of the major mechanisms of resistance by establishing a highly dysfunctional state in T-cells. To identify shared features associated to PD-1/LAG-3 dysfunctionality in human cancers and T-cells, multiomic expression profiles were obtained for all TCGA cancers immune infiltrates. A PD-1/LAG-3 dysfunctional signature was found which regulated immune, metabolic, genetic, and epigenetic pathways, but especially a reinforced negative regulation of the TCR signalosome. These results were validated in T-cell lines with constitutively active PD-1, LAG-3 pathways and their combination. A differential analysis of the proteome of PD-1/LAG-3 T-cells showed a specific enrichment in ubiquitin ligases participating in E3 ubiquitination pathways. PD-1/LAG-3 co-blockade inhibited CBL-B expression, while the use of a bispecific drug in clinical development also repressed C-CBL expression, which reverted T-cell dysfunctionality in lung cancer patients resistant to PD-L1/PD-1 blockade. The combination of CBL-B-specific small molecule inhibitors with anti-PD-1/anti-LAG-3 immunotherapies demonstrated notable therapeutic efficacy in models of lung cancer refractory to immunotherapies, overcoming PD-1/LAG-3 mediated resistance.

Expression of immune checkpoint molecules TIGIT and TIM-3 by tumor-infiltrating lymphocytes predicts poor outcome in sinonasal mucosal melanoma.

BACKGROUND: Sinonasal mucosal melanoma (SNMM) is a rare but aggressive tumor with a poor prognosis. The co-inhibitory receptors T cell immunoglobulin and mucinodomain containing-3 (TIM-3), lymphocyte activation gene-3 (LAG-3) and T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) are promising new targets in anti-cancer immunotherapy. The expression profiles of these immune checkpoint molecules (ICMs) and potential prognostic implications have not been characterized in SNMM yet. METHODS: Immunohistochemical staining for TIGIT, LAG-3 and TIM-3 was performed on tumor tissue samples from 27 patients with primary SNMM. Associations between ICM expression and demographic parameters, AJCC tumor stage, overall survival, and recurrence-free survival were retrospectively analyzed. RESULTS: SNMM patients with low numbers of TIGIT+ and TIM-3+ tumor infiltrating lymphocytes (TILs) in the primary tumor survived significantly longer than patients with a high degree of TIGIT+ and TIM-3+ TILs. High infiltration with TIM-3+ or TIGIT+ lymphocytes was associated with the higher T4 stage and decreased 5-year survival. CONCLUSION: We identified high densities of TIM-3+ and TIGIT+ TILs as strong negative prognostic biomarkers in SNMM. This suggests that TIM-3 and TIGIT contribute to immunosuppression in SNMM and provides a rationale for novel treatment strategies based on this next generation of immune checkpoint inhibitors. Prospective studies with larger case numbers are warranted to confirm our findings and their implications for immunotherapy.

Exhausted Lag-3+ CD4+ T cells are increased in pediatric Inflammatory Bowel Disease.

T cells are one of the main drivers of inflammatory bowel diseases (IBD). Infliximab (IFX) is used in the treatment of IBD as an anti-inflammatory drug to induce remission by neutralizing TNFα. We determined the individual chemokine/homing receptor and cytokine profile in pediatric IBD patients before and during IFX therapy to identify predictive biomarkers for therapy success. Peripheral blood CD4+ cells from pediatric patients with IBD were immunomagnetically isolated and either directly analyzed by FACS for cell distribution and chemokine/homing receptor expression or evaluated for cytokine production after in-vitro-stimulation. 21 responders (RS) and 21 non-responders (NRS) were recruited. Before IFX therapy, flow cytometry revealed decreased percentages of naïve conventional T cells in pediatric IBD patients. The proportions of CD62-L+ T cells were decreased in both CD and UC therapy responders. The cytokine profile of T cells was highly altered in IBD patients compared to healthy controls (HC). During IFX therapy, the frequencies of conventional memory and regulatory memory T cells expanded in both cohorts. IFX response was marked by a decrease of α4ß7+ and IFNγ+ memory T cells in both CD and UC. In contrast, frequencies of Lag-3+ T cells proved to be significantly increased in NRS. These observations were irrespective of the underlying disease. T cells of pediatric IBD patients display an activated and rather Th1/Th17 shifted phenotype The increased expression of the checkpoint molecule Lag-3 on T cells of NRS resembles a more exhausted phenotype than in RS and HC which appeared to be a relevant predictive marker for therapy failure.

A novel inhibitory pathway of synovial inflammation exerted by glucocorticoids and tumor necrosis factor inhibitors via lymphocyte activation gene-3 up-regulation: an ex-vivo study.

OBJECTIVE: To investigate the impact of glucocorticoids (GCs) and anti-rheumatic drugs on the lymphocyte activation gene-3 (LAG-3) and on programmed cell death-1 (PD-1) expression on synovial and peripheral cells ex-vivo. METHODS: Synovial fluid mononuclear cells (SFMCs) from psoriatic arthritis (PsA, n = 26) and rheumatoid arthritis (RA, n = 13) patients, SFCs from osteoarthritis (OA, n = 5) patients and peripheral blood mononuclear cells (PBMCs) of healthy donors (n = 14) were co-cultured with GCs, glucocorticoid receptor antagonist RU486, methotrexate (MTX) and biologics. LAG-3 and PD-1 expressions on immune subsets were analyzed by flow cytometry. RESULTS: GCs in PsA inhibited SFMCs growth vs medium (2.3 ± 0.4X105  vs 5.3 ± 0.7X105, respectively, p < 0.01) and markedly upregulated CD14+LAG-3+ cells (11.7 ± 2.4% vs 0.8 ± 0.3%, p < 0.0001, respectively), but not CD3+LAG-3+ and CD14+PD-1+ cells. MTX had no effect on CD14+LAG-3+ cells (0.7 ± 0.3%). The TNFi inhibitors, infliximab (IFX) and etanercept, but not IL-12/23i, upregulated CD14+LAG-3+ cells vs medium (2.0 ± 0.6% and 1.6 ± 0.4% vs 0.5 ± 0.1%, p < 0.03, respectively). SFMCs growth inhibition in both PsA and RA correlated with CD14+LAG-3+ cell upregulation (r = 0.53, p = 0.03). RU486 inhibited GC-induced CD14+LAG-3+ cell up-regulation in a dose-dependent manner compared with GC alone (5µM 5.3 ± 1.2% and 50µM 1.3 ± 0.5% vs 7.0 ± 1.4%, p < 0.003), but had no significant effect on CD14+LAG-3+ cells co-cultured with IFX. GCs in healthy donors' PBMCs upregulated the immune subsets CD3+LAG-3+, CD14+LAG-3+ and CD14+PD-1+ cells. CONCLUSION: This study proposes a novel regulatory mechanism of GCs and of TNFi mediated by LAG-3 upregulation in synovial monocytes and PBMCs. LAG-3 modulation may be a promising target for development of novel therapies for inflammatory arthritis.