Lactococcus lactis subsp. cremoris C60 Upregulates Macrophage Function by Modifying Metabolic Preference in Enhanced Anti-Tumor Immunity.

Lactococcus lactis subsp. cremoris C60 is a probiotic strain of lactic acid bacteria (LAB) which induces various immune modifications in myeloid lineage cells. These modifications subsequently regulate T cell function, resulting in enhanced immunity both locally and systemically. Here, we report that C60 suppresses tumor growth by enhancing macrophage function via metabolic alterations, thereby increasing adenosine triphosphate (ATP) production in a murine melanoma model. Intragastric (i.g.) administration of C60 significantly reduced tumor volume compared to saline administration in mice. The anti-tumor function of intratumor (IT) macrophage was upregulated in mice administered with C60, as evidenced by an increased inflammatory phenotype (M1) rather than an anti-inflammatory/reparative (M2) phenotype, along with enhanced antigen-presenting ability, resulting in increased tumor antigen-specific CD8+ T cells. Through this functional modification, we identified that C60 establishes a glycolysis-dominant metabolism, rather than fatty acid oxidation (FAO), in IT macrophages, leading to increased intracellular ATP levels. To address the question of why orally supplemented C60 exhibits functions in distal places, we found a possibility that bacterial cell wall components, which could be distributed throughout the body from the gut, may induce stimulatory signals in peripheral macrophages via Toll-like receptors (TLRs) signaling activation. Thus, C60 strengthens macrophage anti-tumor immunity by promoting a predominant metabolic shift towards glycolysis upon TLR-mediated stimulation, thereby increasing substantial energy production.

The components of the AhR-molecular chaperone complex differ depending on whether the ligands are toxic or non-toxic.

The aryl hydrocarbon receptor (AhR) forms a complex with the HSP90-XAP2-p23 molecular chaperone when the cells are exposed to toxic compounds. Recently, 1,4-dihydroxy-2-naphthoic acid (DHNA) was reported to be an AhR ligand. Here, we investigated the components of the molecular chaperone complex when DHNA binds to AhR. Proteins eluted from the 3-Methylcolanthrene-affinity column were AhR-HSP90-XAP2-p23 complex. The AhR-molecular chaperone complex did not contain p23 in the eluents from the DHNA-affinity column. In 3-MC-treated cells, AhR formed a complex with HSP90-XAP2-p23 and nuclear translocation occurred within 30 min, while in DHNA-treated cells, AhR formed a complex with AhR-HSP90-XAP2, and translocation was slow from 60 min. Thus, the AhR activation mechanism may differ when DHNA is the ligand compared to toxic ligands.

Coccomyxa subellipsoidea KJ Components Enhance the Expression of Metallothioneins and Th17 Cytokines during Human T Cell Activation.

Coccomyxa subellipsoidea KJ (C-KJ) is a green alga with unique immunoregulatory characteristics. Here, we investigated the mechanism underlying the modification of T cell function by C-KJ components. The water-soluble extract of C-KJ was fractionated into protein (P) and sugar (S) fractions acidic (AS), basic (BS), and neutral (NS). These fractions were used for the treatment of peripheral blood mononuclear cells stimulated with toxic shock syndrome toxin-1. Transcriptome analysis revealed that both P and AS enhanced the expression of the genes encoding metallothionein (MT) family proteins, inflammatory factors, and T helper (Th) 17 cytokine and suppressed that of those encoding Th2 cytokines in stimulated T cells. The kinetics of MT1 and MT2A gene expression showed a transient increase in MT1 and maintenance of MT2A mRNA after T cell stimulation in the presence of AS. The kinetics of Th17-related cytokine secretion in the early period were comparable to those of MT2A mRNA. Furthermore, our findings revealed that static, a STAT-3 inhibitor, significantly suppressed MT2A gene expression. These findings suggest that the expression of MTs is involved in the immune regulatory function of C-KJ components, which is partially regulated by Th17 responses, and may help develop innovative immunoregulatory drugs or functional foods.

Probiotic lactic acid bacteria promote anti-tumor immunity through enhanced major histocompatibility complex class I-restricted antigen presentation machinery in dendritic cells.

Lactic acid bacteria (LAB) possess the ability to argument T cell activity through functional modification of antigen presenting cells (APCs), such as dendritic cells (DCs) and macrophages. Nevertheless, the precise mechanism underlying LAB-induced enhancement of antigen presentation in APCs remains incompletely understood. To address this question, we investigated the detailed mechanism underlying the enhancement of major histocompatibility complex (MHC) class I-restricted antigen presentation in DCs using a probiotic strain known as Lactococcus lactis subsp. Cremoris C60. We found that Heat-killed-C60 (HK-C60) facilitated the processing and presentation of ovalbumin (OVA) peptide antigen OVA257-264 (SIINFEKL) via H-2Kb in bone marrow-derived dendritic cells (BMDCs), leading to increased generation of effector CD8+ T cells both in vitro and in vivo. We also revealed that HK-C60 stimulation augmented the activity of 20S immunoproteasome (20SI) in BMDCs, thereby enhancing the MHC class I-restricted antigen presentation machinery. Furthermore, we assessed the impact of HK-C60 on CD8+ T cell activation in an OVA-expressing B16-F10 murine melanoma model. Oral administration of HK-C60 significantly attenuated tumor growth compared to control treatment. Enhanced Ag processing and presentation machineries in DCs from both Peyer's Patches (PPs) and lymph nodes (LNs) resulted in an increased tumor antigen specific CD8+ T cells. These findings shed new light on the role of LAB in MHC class-I restricted antigen presentation and activation of CD8+ T cells through functional modification of DCs.

Proteomic Analysis Reveals Changes in Tight Junctions in the Small Intestinal Epithelium of Mice Fed a High-Fat Diet.

The impact of a high-fat diet (HFD) on intestinal permeability has been well established. When bacteria and their metabolites from the intestinal tract flow into the portal vein, inflammation in the liver is triggered. However, the exact mechanism behind the development of a leaky gut caused by an HFD is unclear. In this study, we investigated the mechanism underlying the leaky gut related to an HFD. C57BL/6J mice were fed an HFD or control diet for 24 weeks, and their small intestine epithelial cells (IECs) were analyzed using deep quantitative proteomics. A significant increase in fat accumulation in the liver and a trend toward increased intestinal permeability were observed in the HFD group compared to the control group. Proteomics analysis of the upper small intestine epithelial cells identified 3684 proteins, of which 1032 were differentially expressed proteins (DEPs). Functional analysis of DEPs showed significant enrichment of proteins related to endocytosis, protein transport, and tight junctions (TJ). Expression of Cldn7 was inversely correlated with intestinal barrier function and strongly correlated with that of Epcam. This study will make important foundational contributions by providing a comprehensive depiction of protein expression in IECs affected by HFD, including an indication that the Epcam/Cldn7 complex plays a role in leaky gut.

Female reproductive tract-organ axes.

The female reproductive tract (FRT) and remote/versatile organs in the body share bidirectional communication. In this review, we discuss the framework of the "FRT-organ axes." Each axis, namely, the vagina-gut axis, uterus-gut axis, ovary-gut axis, vagina-bladder axis, vagina-oral axis, uterus-oral axis, vagina-brain axis, uterus-brain axis, and vagina-joint axis, is comprehensively discussed separately. Each axis could be involved in the pathogenesis of not only gynecological diseases but also diseases occurring apart from the FRT. Although the microbiota is clearly a key player in the FRT-organ axes, more quantitative insight into the homeostasis of the microbiota could be provided by host function measurements rather than current microbe-centric approaches. Therefore, investigation of the FRT-organ axes would provide us with a multicentric approach, including immune, neural, endocrine, and metabolic aspects, for understanding the homeostatic mechanism of women's bodies. The framework of the FRT-organ axes could also provide insights into finding new therapeutic approaches to maintain women's health.

Trend of anticoagulant therapy in elderly patients with atrial fibrillation considering risks of cerebral infarction and bleeding.

The introduction of direct oral anticoagulants (DOACs) has greatly changed the use of anticoagulant therapy in patients with non-valvular atrial fibrillation (Af). Therefore, this study aimed to examine changes in the proportions of oral anticoagulant prescriptions in patients with non-valvular Af aged ≥ 65 years, taking into consideration the risk of cerebral infarction and bleeding. Anticoagulant prescriptions in outpatients aged ≥ 65 years with Af were temporally analyzed using the nationwide claims database in Japan. Trends in anticoagulant prescriptions were examined according to cerebral infarction and bleeding risk. The proportion of anticoagulant prescriptions for 12,076 Af patients increased from 41% in 2011 to 56% in 2015. An increase in DOAC prescriptions was accompanied by an increase in the proportion of anticoagulant prescriptions in each group according to the CHA2DS2-VASc and HAS-BLED scores. The proportion of anticoagulant prescriptions for patients with a high risk of developing cerebral infarction and bleeding showed a marked increase. Trends in anticoagulant prescriptions in Af patient with a CHA2DS2-VASc score ≥ 2 and HAS-BLED scores ≥ 3 showed a marked increase in DOAC prescriptions. The widespread use of DOACs greatly changes the profile the prescription of anticoagulant therapy in patients with Af.

Astaxanthin Attenuates Nonalcoholic Steatohepatitis with Downregulation of Osteoprotegerin in Ovariectomized Mice Fed Choline-Deficient High-Fat Diet.

BACKGROUND: Postmenopausal estrogen decline increases the risk of developing nonalcoholic steatohepatitis (NASH), and it might accelerate progression to cirrhosis and hepatocellular carcinoma. AIMS: This study aimed to investigate a novel therapy for postmenopausal women who are diagnosed with NASH. METHODS: Seven-week-old female C57BL/6 J mice were divided into three experimental groups as follows: (1) sham operation (SHAM group), (2) ovariectomy (OVX group), and (3) ovariectomy + 0.02% astaxanthin (OVX + ASTX group). These three groups of mice were fed a choline-deficient high-fat (CDHF) diet for 8 weeks. Blood serum and liver tissues were collected to examine liver injury, histological changes, and hepatic genes associated with NASH. An in vitro study was performed with the hepatic stellate cell line LX-2. RESULTS: The administration of ASTX significantly improved pathological NASH with suppressed steatosis, inflammation, and fibrosis, in comparison with those in the OVX-induced estrogen deficiency group. As a result, liver injury was also attenuated with reduced levels of alanine aminotransferase and aspartate transaminase. In addition, our study found that ASTX supplementation decreased hepatic osteoprotegerin (OPG) in vivo, a possible factor that contributes to NASH development. In vitro, this study further confirmed that ASTX has an inhibitory effect on the secretion of OPG in LX-2 human hepatic stellate cells. CONCLUSIONS: Our findings suggest that ASTX alleviates CDHF-OVX-induced pathohistological NASH with downregulated OPG, possibly via suppression of the transforming growth factor beta pathway. ASTX could has promise for use in postmenopausal women diagnosed with NASH.

Lymphocyte antigen 6 complex locus G6D downregulation is a novel parameter for functional impairment of neutrophils in aged mice.

Immunological aging is a critical event that causes serious functional impairment in the innate immune system. However, the identification markers and parameters are still poorly understood in immunological aging of myeloid lineage cells. Here, we show that a downregulation of lymphocyte antigen 6 complex locus G6D (Ly-6G) observed in aged mouse neutrophils could serve as a novel marker for the prediction of age-associated functional impairment in the neutrophils. Ly-6G expression was significantly downregulated in the bone marrow (BM) neutrophils of aged mice compared to young mice confirmed by flow cytometry analysis. In vitro experiments using BM-isolated neutrophils showed significant downregulations in their activities, such as phagocytosis, reactive oxygen species (ROS) production, interleukin (IL)-1ß production, neutrophil extracellular trap (NET) formation, and migration as well as bacterial clearance, in the aged mouse neutrophils compared to those of young mice counterparts. Interestingly, the magnitudes of functional parameters were strongly correlated with the Ly-6G expression in the neutrophils. Thus, our results suggest that downregulation of Ly-6G reflects the age-associated functional attenuation of the neutrophils.

Creatine supplementation enhances immunological function of neutrophils by increasing cellular adenosine triphosphate.

Creatine is an organic compound which is utilized in biological activities, especially for adenosine triphosphate (ATP) production in the phosphocreatine system. This is a well-known biochemical reaction that is generally recognized as being mainly driven in specific parts of the body, such as the skeletal muscle and brain. However, our report shows a novel aspect of creatine utilization and ATP synthesis in innate immune cells. Creatine supplementation enhanced immune responses in neutrophils, such as cytokine production, reactive oxygen species (ROS) production, phagocytosis, and NETosis, which were characterized as antibacterial activities. This creatine-induced functional upregulation of neutrophils provided a protective effect in a murine bacterial sepsis model. The mortality rate in mice challenged with Escherichia coli K-12 was decreased by creatine supplementation compared with the control treatment. Corresponding to this decrease in mortality, we found that creatine supplementation decreased blood pro-inflammatory cytokine levels and bacterial colonization in organs. Creatine supplementation significantly increased the cellular ATP level in neutrophils compared with the control treatment. This ATP increase was due to the phosphocreatine system in the creatine-treated neutrophils. In addition, extracellular creatine was used in this ATP synthesis, as inhibition of creatine uptake abolished the increase in ATP in the creatine-treated neutrophils. Thus, creatine is an effective nutrient for modifying the immunological function of neutrophils, which contributes to enhancement of antibacterial immunity.

Lactococcus lactis subsp. cremoris C60 induces macrophages activation that enhances CD4+ T cell-based adaptive immunity.

Lactococcus lactis subsp. cremoris C60 is a probiotic strain that induces diverse functional modifications in immune cells. In this report, as a novel effect of C60 on myeloid lineage cells, we show that C60 enhances the immunological function of macrophages that consequently promotes CD4+ T cell activity in an antigen-dependent manner. Heat-killed (HK) C60 induced the production of pro-inflammatory cytokines in thioglycolate-elicited peritoneal macrophages (TPMs) much stronger than Toll-like receptor (TLR) ligand stimulation. The HK-C60 treatment also augmented the expression of antigen-presenting and co-stimulatory molecules, such as major histocompatibility complex (MHC) class II, CD80, and CD86, as well as antigen uptake in TPMs. These HK-C60-mediated functional upregulations in TPMs resulted in the promotion of CD4+ T cell activation in an antigen-dependent manner. Interestingly, the TPMs that originated from the mice fed the HK-C60 diet showed pre-activated characteristics, which was confirmed by the upregulation of cytokine production and antigen presentation-related molecule expression under lipopolysaccharide (LPS) stimulation. Furthermore, the antigen-dependent CD4+ T cell activation was also enhanced by the TPMs. This implied that antigen presentation activity was enhanced in the TPMs that originated from the HK-C60 diet mice. Thus, C60 effectively upregulates the immunological function of macrophages that directly connects to CD4+ T cell-based adaptive immunity.

Microbiome, fibrosis and tumor networks in a non-alcoholic steatohepatitis model of a choline-deficient high-fat diet using diethylnitrosamine.

BACKGROUND & AIMS: Hepatocellular carcinoma in nonalcoholic steatohepatitis is caused by the complex factors of inflammation, fibrosis and microbiomes. We used network analysis to examine the interrelationships of these factors. METHODS: C57Bl/6 mice were categorized into groups: choline-sufficient high-fat (CSHF, n = 8), choline-deficient high-fat (CDHF, n = 9), and CDHF+ diethylnitrosamine (DEN, n = 8). All mice were fed CSHF or CDHF for 20 weeks starting at week 8, and mice in the CDHF + DEN group received one injection of DEN at 3 weeks of age. Bacterial gene was isolated from feces and analyzed using Miseq. RESULTS: The CSHF group had less fibrosis than the other groups. Tumors were found in 22.2% and 87.5% of the CDHF group and CDHF + DEN groups, respectively. Gene expression in the liver of Cdkn1a (p21: tumor-suppressor) and c-jun was highest in the CDHF group. Bacteroides, Roseburia, Odoribacter, and Clostridium correlated with fibrosis. Streptococcus and Dorea correlated with inflammation and tumors. Akkermansia and Bilophila were inversely correlated with fibrosis and Bifidobacterium was inversely correlated with tumors. CONCLUSIONS: DEN suppressed the overexpression of p21 caused by CDHF. Some bacteria formed a relationship networking associated with their progression and inhibition for tumors and fibrosis.

Lactococcus lactis subsp. Cremoris C60 restores T Cell Population in Small Intestinal Lamina Propria in Aged Interleukin-18 Deficient Mice.

Lactic acid bacteria (LAB), a major commensal bacterium in the small intestine, are well known beneficial bacteria which promote establishment of gut-centric immunity, such as anti-inflammation and anti-infection. In this report, we show that a LAB strain Lactococcus lactis subsp. Cremoris C60 possess an ability to activate antigen presenting cells, such as dendritic cells (DCs), and intestinal T cells which possibly support to maintain healthy intestinal immunological environment in aging process. We found that CD4+ T cells in the small intestine are dramatically decreased in aged Interleukin-18 knock out (IL-18KO) mice, associated with the impairment of IFN-γ production in the CD4+ T cells, especially in small intestinal lamina propria (LP). Surprisingly, heat killed-C60 (HK-C60) diet completely recovered the CD4+ T cells population and activity in SI-LP and over activated the population in Peyer's patches (PPs) of IL-18KO mice. The HK-C60 diet was effective approach not only to restore the number of cells, but also to recover IFN-γ production in the CD4+ T cell population in the small intestine of IL-18-deficient mice. As a possible cause in the age-associated impairment of CD4+ T cells activity in IL-18KO mice, we found that the immunological activity was downregulated in the IL-18-deficient DCs. The cytokines production and cellular activation markers expression were downregulated in the IL-18-deficient bone marrow derived dendritic cells (BMDCs) at the basal level, however, both activities were highly upregulated in HK-C60 stimulation as compared to those of WT cells. Antigen uptake was also attenuated in the IL-18-deficient BMDCs, and it was significantly enhanced in the cells as compared to WT cells in HK-60 stimulation. An in vitro antigen presentation assay showed that IFN-γ production in the CD4+ T cells was significantly enhanced in the culture of IL-18-deficient BMDCs compared with WT cells in the presence of HK-C60. Thus, we conclude that HK-C60 diet possesses an ability to restore T cells impairment in the small intestine of IL-18-deficient environment. In addition, the positive effect is based on the immunological modification of DCs function which directory influences into the promotion of effector CD4+ T cells generation in the small intestine.

A nanoscale metal organic frameworks-based vaccine synergises with PD-1 blockade to potentiate anti-tumour immunity.

Checkpoint blockade therapy has provided noteworthy benefits in multiple cancers in recent years; however, its clinical benefits remain confined to 10-40% of patients with extremely high costs. Here, we design an ultrafast, low-temperature, and universal self-assembly route to integrate immunology-associated large molecules into metal-organic-framework (MOF)-gated mesoporous silica (MS) as cancer vaccines. Core MS nanoparticles, acting as an intrinsic immunopotentiator, provide the niche, void, and space to accommodate antigens, soluble immunopotentiators, and so on, whereas the MOF gatekeeper protects the interiors from robust and off-target release. A combination of MOF-gated MS cancer vaccines with systemic programmed cell death 1 (PD-1) blockade therapy generates synergistic effects that potentiate antitumour immunity and reduce the effective dose of an anti-PD-1 antibody to as low as 1/10 of that for PD-1 blockade monotherapy in E.G7-OVA tumour-bearing mice, with eliciting the robust adaptive OVA-specific CD8+ T-cell responses, reversing the immunosuppressive pathway and inducing durable tumour suppression.

A Double-Blind, Randomized, Placebo-Controlled Trial of Heat-Killed Pediococcus acidilactici K15 for Prevention of Respiratory Tract Infections among Preschool Children.

Although some probiotic bacteria have been reported to prevent infections in children, there are few well-designed double-blind studies. Here we evaluated the effects of a probiotic strain of lactic acid bacteria (LAB), Pediococcus acidilactici K15, on viral respiratory tract infections in preschool children. A four-month, randomized, double-blind, placebo-controlled study was performed in 172 healthy children aged 3 to 6 years. Subjects were administered dextrin alone or dextrin including heat-killed K15 (5 × 1010 bacteria). The number of febrile days was the primary outcome. The number of absent days from preschools and the influenza incidence were secondary outcomes. Secretory IgA (sIgA) concentrations in saliva were measured as an exploratory outcome. The primary and secondary outcomes were not significantly different between both groups. Analyses in children with little intake of fermented foods including LAB showed that the duration of a fever significantly decreased by K15 intake. The salivary sIgA level in the K15 group was maintained significantly higher than it was in the placebo group. The effects of K15 on preventing viral respiratory tract infections were not observed without the restriction of fermented foods intake. However, K15 supported anti-infectious immune systems in children who took less fermented foods and the maintenance of salivary sIgA levels in all subjects.

Biochemical properties of human full-length aryl hydrocarbon receptor (AhR).

The aryl hydrocarbon receptor (AhR) is a very unstable protein. AhR binds to the molecular chaperone complex (HSP90-p23-XAP2) to maintain a stable structure in the cytoplasm. After binding to ligands, such as dioxin, AhR translocates from the cytoplasm to the nucleus with a molecular chaperone complex. The protein forms a heterodimer with Arnt after nuclear transfer, functions as a transcription factor by binding to a xenobiotic responsive element (XRE), and induces the cytochrome P450 1A1 (CYP1A1). Because of the unstable protein, expression of the full-length AhR in the E. coli expression system is very difficult. Many studies investigated AhR using AhR domains in vitro. We expressed and purified the human full-length AhR in E. coli expression system. Furthermore, specific antibodies were prepared. Purified full-length AhR could bind to ligand. In the presence of ligand, α-helix and random coil of AhR increased and ß-sheet decreased on CD spectrum. Full-length AhR could bind to HSP90, XAP2 and p23 in the presence or absence of ligand. We now show the biochemical properties of full-length AhR.

Propolis induces Ca2+ signaling in immune cells.

Propolis possesses several immunological functions. We recently generated a conditional Ca2+ biosensor yellow cameleon (YC3.60) transgenic mouse line and established a five-dimensional (5D) (x, y, z, time, and Ca2+ signaling) system for intravital imaging of lymphoid tissues, including Peyer's patches (PPs). To assess the effects of propolis on immune cells, we analyzed Ca2+ signaling in vitro and in vivo using CD11c-Cre/YC3.60flox transgenic mice, in which CD11c+ dendritic cells (DCs) specifically express YC3.60. We found that propolis induced Ca2+ signaling in DCs in the PPs. Intravital imaging of PPs also showed that an intraperitoneal injection of propolis augmented Ca2+ signaling in CD11c+ cells, suggesting that propolis possesses immune-stimulating activity. Furthermore, CD11c+ cells in PPs in mice administrated propolis indicated an increase in Ca2+ signaling. Our results indicate that propolis induces immunogenicity under physiological conditions.

Rod-Scale Design Strategies for Immune-Targeted Delivery System toward Cancer Immunotherapy.

Strengthening the antitumor immune response to surpass the activation energy barrier associated with the immunosuppressive tumor microenvironment is an active area of cancer immunotherapy. Emerging evidence suggests that delivery of immunostimulatory molecules with the aid of a carrier system is essential for cancer immunotherapy. However, the size-dependent effect of the delivery system on immune-targeted sites and anticancer immune responses is yet to be comprehensively understood. Herein, to clarify the size-dependent effect of the delivery system on the underlying anticancer immune mechanism, rod-shaped hydroxyapatite (HA) particles with lengths from 100 nm to 10 µm are designed. HA rods stimulate anticancer immunity in a size-dependent manner. Shorter HA rods with lengths ranging from 100 to 500 nm promote antigen cellular uptake, dendritic cell (DC) maturation, and lymph node targeting antigen. In contrast, longer HA rods with lengths ranging from 500 nm to 10 µm prolong antigen retention and increase DC accumulation. Medium-sized HA rods with a length of 500 nm, taking advantage of both short and long rods, show optimized antigen release and uptake, increased DCs accumulation and maturation, highest CD4+ and CD8+ T cell population, and the best anticancer immunity in vivo. The present study provides a rod-scale design strategy for an immune-targeted delivery system toward cancer immunotherapy in the future.

High TNFRSF14 and low BTLA are associated with poor prognosis in Follicular Lymphoma and in Diffuse Large B-cell Lymphoma transformation.

The microenvironment influences the behavior of follicular lymphoma (FL) but the specific roles of the immunomodulatory BTLA and TNFRSF14 (HVEM) are unknown. Therefore, we examined their immunohistochemical expression in the intrafollicular, interfollicular and total histological compartments in 106 FL cases (57M/49F; median age 57-years), and in nine relapsed-FL with transformation to DLBCL (tFL). BTLA expression pattern was of follicular T-helper cells (TFH) in the intrafollicular and of T-cells in the interfollicular compartments. The mantle zones were BTLA+ in 35.6% of the cases with similar distribution of IgD. TNFRSF14 expression pattern was of neoplastic B lymphocytes (centroblasts) and "tingible body macrophages". At diagnosis, the averages of total BTLA and TNFRSF14-positive cells were 19.2%±12.4STD (range, 0.6%-58.2%) and 46.7 cells/HPF (1-286.5), respectively. No differences were seen between low-grade vs. high-grade FL but tFL was characterized by low BTLA and high TNFRSF14 expression. High BTLA correlated with good overall survival (OS) (total-BTLA, Hazard Risk=0.479, P=0.022) and with high PD-1 and FOXP3+Tregs. High TNFRSF14 correlated with poor OS and progression-free survival (PFS) (total-TNFRSF14, HR=3.9 and 3.2, respectively, P<0.0001), with unfavorable clinical variables and higher risk of transformation (OR=5.3). Multivariate analysis including BTLA, TNFRSF14 and FLIPI showed that TNFRSF14 and FLIPI maintained prognostic value for OS and TNFRSF14 for PFS. In the GSE16131 FL series, high TNFRSF14 gene expression correlated with worse prognosis and GSEA showed that NFkB pathway was associated with the "High-TNFRSF14/dead-phenotype".In conclusion, the BTLA-TNFRSF14 immune modulation pathway seems to play a role in the pathobiology and prognosis of FL.