B And T Lymphocyte Attenuator Research Articles

Hepatic expansion of virus-specific CD8+BTLA+ T cells with regulatory properties in chronic hepatitis B virus infection

Similar to programmed death-1 (PD-1), B and T lymphocyte attenuator (BTLA) is a co-inhibitory molecule of the CD28 family. PD-1 is involved in T cell exhaustion during chronic viral infection. However, the role of BTLA in virus-specific T cells is poorly defined. Here we investigated the expression and function of BTLA in T cells from patients with chronic hepatitis B virus (HBV) infection. The phenotype of peripheral and intrahepatic HBV-specific T cells from 43 patients with chronic HBV infection was assessed by flow cytometry. Functional evaluation was analyzed by T cell expansion and cytokine secretion after different treatments. In chronic HBV patients, a subset of inefficient interferon-γ producing antigen-specific CD8+ T cells recruited to the liver expressed high BTLA levels. The BTLA+ HBV-specific CD8+ T cell suppressive function was antigen-specific, at least in the induction phase, because they were only activated by a pool of HBV peptides but not with a pool of unrelated peptides. Suppression of T cell responses was restored by a BTLA signaling blockade and neutralizing IL-10, indicating that BTLA signaling-mediated IL-10 secretion plays a key role in suppression. This study provides important evidence that there is a subset of liver infiltrated virus-specific CD8+BTLA+ regulatory T cells in patients with chronic HBV infection. This subset of cells plays a pivotal role in controlling hepatic effector CD8+ T cell responses through BTLA signaling mediated regulatory factor IL-10 production.

BTLA-expressing CD11c antigen presenting cells in patients with active tuberculosis exhibit low capacity to stimulate T cell proliferation

Despite past extensive studies on B and T lymphocyte attenuator (BTLA)-mediated negative regulation of T cell activation, the role of BTLA in antigen presenting cells (APCs) in patients with active pulmonary tuberculosis (ATB) remains poorly understood. Here, we demonstrate that BTLA expression on CD11c APCs increased in patients with ATB. Particularly, BTLA expression in CD11c APCs was likely associated with the attenuated stimulatory capacity on T cells (especially CD8+ T cell) proliferation. BTLA-expressing CD11c APCs showed lower antigen uptake capacity, lower CD86 expression, higher HLA-DR expression, and enhanced IL-6 secretion, compared to counterpart BTLA negative CD11c APCs in healthy controls (HC). Interestingly, BTLA-expressing CD11c APCs from ATB patients displayed lower expression of HLA-DR and less IL-6 secretion, but higher expression of CD86 than those from HC volunteers. Mixed lymphocyte reaction suggests that BTLA expression is likely associated with positive rather than conventional negative regulation of CD11c APCs stimulatory capacity. This role is impaired in ATB patients manifested by low expression of HLA-DR and low production of IL-6. This previous unappreciated role for BTLA may have implications in the prevention and treatment of patients with ATB.

Loss of the HVEM Tumor Suppressor in Lymphoma and Restoration by Modified CAR-T Cells

The HVEM (TNFRSF14) receptor gene is among themost frequently mutated genes in germinal center lymphomas. We report that loss of HVEM leads tocell-autonomous activation of B cell proliferationand drives the development of GC lymphomasinvivo. HVEM-deficient lymphoma B cells also induce a tumor-supportive microenvironment marked by exacerbated lymphoid stroma activation and increased recruitment of T follicular helper (TFH) cells. These changes result from the disruption of inhibitory cell-cell interactions between the HVEM and BTLA (B and T lymphocyte attenuator) receptors. Accordingly, administration of the HVEM ectodomain protein (solHVEM(P37-V202)) binds BTLA and restores tumor suppression. To deliver solHVEM to lymphomas invivo, we engineered CD19-targeted chimeric antigen receptor (CAR) Tcells that produce solHVEM locally and continuously. These modified CAR-T cells show enhanced therapeutic activity against xenografted lymphomas. Hence, the HVEM-BTLA axis opposes lymphoma development, and our study illustrates the use of CAR-T cells as "micro-pharmacies" able to deliver an anti-cancer protein.

Modulation of cytotoxic responses by targeting CD160 prolongs skin graft survival across major histocompatibility class I barrier

CD160 is a glycosylphosphatidylinositol-anchored protein of the immunoglobulin superfamily. It exhibits a pattern of expression coincident in humans and mice thatis mainly restricted to cytotoxic cells and to all intestinal intraepithelial Tlymphocytes. B- and T-lymphocyte attenuator (BTLA) and CD160 interact with cysteine-rich domain 1 of the extracellular region of Herpesvirus entry mediator (HVEM). CD160 engagement by HVEM can deliver inhibitory signals to a small subset of human CD4 Tcells and attenuate its proliferation and cytokine secretion, but can also costimulate natural killer cells or intraepithelial lymphocytes. In turn, CD160 and BTLA can also function as agonist ligands being capable of costimulating Tcells through membrane HVEM. Based on the restricted pattern of CD160 expression in cytotoxic cells, we postulated that CD160 may represent a suitable target for immune intervention in the setting of transplantation to modulate allogeneic cytotoxic responses. We demonstrated that invivo administration of anti-CD160 antibody in combination with anti-CD40L antibody to limit CD4 T-cell help modulated cytotoxic responses in a major histocompatibility complex class I mismatched model of allogeneic skin graft transplantation (bm1 donor to C57BL/6 recipient) and significantly prolonged graft survival. The implementation of this strategy in transplantation may reinforce current immunosuppression protocols and contribute to a better control of CD8 T-cell responses.

Effects of clostridium butyricum and bifidobacterium on BTLA expression on CD4+ T cells and lymphocyte differentiation in late preterm infants

Background & aimsProbiotics is recognized to promote growth performance and immune function via balancing the intestinal microflora. Live clostridium butyricum and bifidobacterium combined powder (LCBBCP) has been widely to treat intestinal dysbacteriosis in newborns in China. This study was undertaken to investigate the effects of the combined probiotics on the expression of B and T lymphocyte attenuator (BTLA) on CD4+ T cells and the differentiation of lymphocyte subsets in late preterm infants. MethodsEighty eligible late preterm infants were equally randomized into LCBBCP therapy group (oral LCBBCP dissolved in formula milk before intake) and control group (treated with simple formula milk for preterm infants) by random digit table. Flow cytometry was used to determine the expression level of BTLA on CD4+ T cells and the percentage of individual subpopulation of lymphocytes in peripheral-blood mononuclear cells (PBMCs) obtained from the late preterm infants in both groups. ResultsBTLA protein expression on CD4+ T cells showed no significant change in LCBBCP therapy group before and after intervention, yet was rapidly and significantly down-regulated in the controls. The percentage of increased CD4+ T cells, decreased CD8+ T cells and increased ratio of CD4+/CD8+ T cell proportion were seen in both groups after treatment, yet the increasing or decreasing extent in LCBBCP therapy group was more obvious than in control group. The proportion of NK cells and B lymphocytes remained no significant difference between the two groups before and after therapy. ConclusionsLCBBCP appears capable of facilitating the activation, proliferation and differentiation of T lymphocytes, which is beneficial to improving immunity in late preterm infants. The continuous high expression of BTLA on CD4+ T cells in LCBBCP therapy group may be involved in the inhibiting of excessive activation of T lymphocytes. Our findings may lay a basis for further clinical evaluation of the efficacies and wider clinical recommendation of probiotics containing live clostridium butyricum and bifidobacterium for late preterm infants.

Knockdown of HVEM, a Lymphocyte Regulator Gene, in Ovarian Cancer Cells Increases Sensitivity to Activated T Cells.

Ovarian cancer is highly malignant with a gradually increasing incidence and a high mortality rate. Immunosuppression is induced in ovarian cancer, although the mechanism detail is not clear. It has been indicated that HVEM (herpesvirus entry mediator) B- and T-lymphocyte attenuator (BTLA) negatively regulates the immune responses of T lymphocytes. Here, HVEM mRNA was found to be elevated in ovarian cancer tissue samples and primary ovarian cancer cells in comparison with benign tissue samples. We then knocked down HVEM expression in an ovarian cancer cell line, OVCAR3, by lentivirus-based small hairpin RNA (shRNA). Cell Counting Kit-8 (CCK-8) assay and flow cytometry analysis showed that HVEM-shRNA had no effect on the proliferation, early apoptosis, or cell cycle distribution of OVCAR3. We then isolated activated T cells and performed coculture experiments in Transwell. Remarkably, HVEM-silenced ovarian cancer cells (primary ovarian cancer cells and OVCAR3) increased the number of T cells and the secretion of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), while activated T cells promoted the apoptosis of HVEM-silenced ovarian cancer cells. The current study partially explains the immune escape mechanism of ovarian cancer cells and provides a possible target for immunotherapy.

Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases.

The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. Here we report the discovery of a highly potent (IC50 = 0.071 μM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS–ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.

Regulatory T Cell Dysfunction Acquiesces to BTLA+ Regulatory B Cells Subsequent to Oral Intervention in Experimental Autoimmune Encephalomyelitis.

Regulatory T cells (Tregs) induced during autoimmunity often become quiescent and unable to resolve disease, suggesting inadequate activation. Resolution of established experimental autoimmune encephalomyelitis (EAE) can be achieved with myelin oligodendrocyte glycoprotein (MOG) fused to reovirus protein σ1 (MOG-pσ1), which activates Tregs, restoring protection, but requiring other regulatory cells to revitalize them. B cells have a dichotomous role in both the pathogenesis and recovery from EAE. Although inflammatory B cells contribute to EAE's pathogenesis, treatment of EAE mice with MOG-pσ1, but not OVA-pσ1, resulted in an influx of IL-10-producing B220(+)CD5(+) B regulatory cells (Bregs) enabling Tregs to recover their inhibitory activity, and in turn, leading to the rapid amelioration of EAE. These findings implicate direct interactions between Bregs and Tregs to facilitate this recovery. Adoptive transfer of B220(+)CD5(-) B cells from MOG-pσ1-treated EAE or Bregs from PBS-treated EAE mice did not resolve disease, whereas the adoptive transfer of MOG-pσ1-induced B220(+)CD5(+) Bregs greatly ameliorated EAE. MOG-pσ1-, but not OVA-pσ1-induced IL-10-producing Bregs, expressed elevated levels of B and T lymphocyte attenuator (BTLA) relative to CD5(-) B cells, as opposed to Tregs or effector T (Teff) cells, whose BTLA expression was not affected. These induced Bregs restored EAE Treg function in a BTLA-dependent manner. BTLA(-/-) mice showed more pronounced EAE with fewer Tregs, but upon adoptive transfer of MOG-pσ1-induced BTLA(+) Bregs, BTLA(-/-) mice were protected against EAE. Hence, this evidence shows the importance of BTLA in activating Tregs to facilitate recovery from EAE.

B and T Lymphocyte Attenuator is a Target of miR-155 during Naïve CD4+ T Cell Activation.

MicroRNA-155 (miR-155) is upregulated during T cell activation, but the exact mechanisms by which it influences CD4+ T cell activation remain unclear. To examine whether the B and T lymphocyte attenuator (BTLA) is a target of miR-155 during naïve CD4+ T cell activation. Firefly luciferase reporter plasmids pEZX-MT01-wild-type-BTLA and pEZX-MT01-mutant-BTLA were constructed. Lymphocytes were nucleofected with miR-155 inhibitor or negative control (NC). Then, naïve CD4+ CD62L+ helper T cells purified from lymphocytes were stimulated with immobilized antibody to CD3 and soluble antibody to CD28. miR-155 and BTLA expression were examined by real-time RT-PCR. Cell surface CD69 expression and IL-2 secretion were measured by ELISA and flowcytometry, respectively. Luciferase reporter assay showed that miR-155 targeted the BTLA 3'UTR region. Compared with non-stimulated condition, both miR-155 and BTLA mRNA expression were upregulated after T cell activation. Similar results were observed for BLTA protein expression. Compared with NC, the miR-155 inhibitor decreased miR-155 by about 45%, but did not influence BTLA mRNA expression. Compared with NC, the miR-155 inhibitor decreased the surface BTLA expression by about 60%. Upregulation of BTLA in miR-155 knockdown CD4+ T cells did not influence the cell surface expression of CD69, an early activation marker (p=0.523). Similarly, IL-2 production was not changed. miR-155 is involved in the inhibition of BTLA during CD4+ T cell activation. These results might serve as a basis for an eventual therapeutic manipulation of this pathway to treat inflammatory and autoimmune diseases.

Association of 3′ nearby gene BTLA polymorphisms with the risk of renal cell carcinoma in the Polish population

ObjectiveT cells play an important role in antitumor immunity, and molecules regulating T-cell activity could influence cancer susceptibility. The distinct role of coinhibitory receptors in immunosurveillance has been considered. B- and T-lymphocyte attenuator (BTLA) is one of these receptors, which negatively regulate immune responses. The aim of this study was to investigate the association between BTLA gene polymorphisms and susceptibility to renal cell carcinoma (RCC) in the Polish population. MethodsAltogether 282 patients with RCC and 480 healthy subjects were genotyped for the following polymorphisms: rs2705511, rs1982809, rs9288952, rs16859633, rs9288953, rs2705535, and rs1844089 using the TaqManSNP Genotyping Assays. ResultsHere, we found that the presence of rs1982809G allele (genotype GG+AG) is associated with increased risk of RCC (odds ratio = 1.38; 95% CI: 1.03–1.86; P = 0.03). In patients with clear-cell RCC (ccRCC) with high-grade (3 and 4) tumors, the frequency of rs1982809[GG] genotype was significantly higher as compared to those with low-grade (1 and 2) tumors and to the controls (0.14 vs. 0.06, P = 0.05 and 0.14 vs. 0.06, P = 0.04, respectively). Moreover, we have noticed the trend for overrepresentation of carriers of rs2705511C allele in patients with RCC as compared with the controls (0.51 vs. 0.44, P = 0.08)Haplotype rs2705511C/rs1982809G/rs9288952A/rs9288953T/rs2705535C/rs1844089G (CGATCG) increased the risk of RCC of 46% (odds ratio = 1.46; 95% CI: 1.08–1.96; Pcorrected = 0.05). ConclusionOur results indicate that polymorphisms rs1982809 situated in 3′ UTR nearby region of BTLA gene might be considered as low-penetrating risk factor for RCC, but results have to be confirmed in further studies.

Pathogenic Function of Herpesvirus Entry Mediator in Experimental Autoimmune Uveitis by Induction of Th1- and Th17-Type T Cell Responses.

Herpesvirus entry mediator (HVEM), a member of the TNFR superfamily, serves as a unique molecular switch to mediate both stimulatory and inhibitory cosignals, depending on its functions as a receptor or ligand interacting with multiple binding partners. In this study, we explored the cosignaling functions of HVEM in experimental autoimmune uveitis (EAU), a mouse model resembling human autoimmune uveitis conditions such as ocular sarcoidosis and Behcet disease. Our studies revealed that EAU severity significantly decreased in HVEM-knockout mice compared with wild-type mice, suggesting that stimulatory cosignals from the HVEM receptor are predominant in EAU. Further studies elucidated that the HVEM cosignal plays an important role in the induction of both Th1- and Th17-type pathogenic T cells in EAU, including differentiation of IL-17-producing αβ(+)γδ(-) conventional CD4(+) T cells. Mice lacking lymphotoxin-like, inducible expression, competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes : LIGHT), B- and T-lymphocyte attenuator (BTLA) or both LIGHT and BTLA are also less susceptible to EAU, indicating that LIGHT-HVEM and BTLA-HVEM interactions, two major molecular pathways mediating HVEM functions, are both important in determining EAU pathogenesis. Finally, blocking HVEM cosignals by antagonistic anti-HVEM Abs ameliorated EAU. Taken together, our studies revealed a novel function of the HVEM cosignaling molecule and its ligands in EAU pathogenesis through the induction of Th1- and Th17-type T cell responses and suggested that HVEM-related molecular pathways can be therapeutic targets in autoimmune uveitis.

Decreased B and T lymphocyte attenuator in Behcet's disease may trigger abnormal Th17 and Th1 immune responses.

Behcet’s disease (BD) is a chronic, systemic and recurrent inflammatory disease associated with hyperactive Th17 and Th1 immune responses. Recent studies have shown that B and T lymphocyte attenuator (BTLA) negatively regulates the immune response. In this study, we investigated whether BTLA activation could be exploited to inhibit the development of abnormal immune responses in BD patients. BTLA expression in PBMCs and CD4+ T cells was significantly decreased in active BD patients. Decreased BTLA level was associated with increased Th17 and Th1 responses. Activation of BTLA inhibited the abnormal Th17 and Th1 responses and IL-22 expression in both patients and controls. Addition of an agonistic anti-BTLA antibody remarkably inhibited DC-induced Th17 and Th1 cell responses, resulted in decreased production of the Th17 and Th1-related cytokines IL-1beta, IL-6, IL-23 and IL-12p70 and reduced CD40 expression in DCs. In conclusion, decreased BTLA expression in ocular BD may lead to inappropriate control of the Th17 and Th1 immune responses and DC functions. Therefore, BTLA may be involved in the development and recurrence of this disease. Agonistic agents of BTLA may represent a potential therapeutic approach for the treatment of BD and other inflammatory diseases mediated by abnormal Th17 and Th1 immune responses.

Use of the dendritic cell marker, B and T lymphocyte attenuator, to identify functionally distinct subsets of human CD1c+ dendritic cells

BackgroundDendritic cells specialise in initiating adaptive immune responses. The CD1c+ subset in human beings is proposed to excel at CD4+ T cell priming. An equivalent population in mice has been shown to be heterogeneous and contain functionally distinct monocyte-related and conventional dendritic cell-related subpopulations. We aimed to examine whether analogous diversity exists in human beings using the conserved conventional dendritic cell marker, B and T lymphocyte attenuator (BTLA), which exhibits bimodal expression on CD1c+ dendritic cells. MethodsBTLA–CD1c+ and BTLA+CD1c+ dendritic cells were flow-sorted from peripheral blood. Gene expression (NanoString, Seattle, WA, USA), cytokine production after stimulation with lipopolysaccharide and R848 (Luminex, eBioscience, San Diego, CA, USA), and naive CD4+ T cell polarisation capacity (allogeneic mixed lymphocyte reaction assay) were compared. Their potential to differentiate into Langerhans cells (by culturing in granulocyte macrophage colony stimulating factor [GM-CSF] plus BMP7) and osteoclasts (by culturing in macrophage colony stimulating factor plus RANKL) in vitro was assessed. Their relative proportions in healthy blood and rheumatoid arthritis synovial fluid were compared by flow cytometry. FindingsCD1c+BTLA– dendritic cells had higher expression than CD1c+BTLA+ dendritic cells of typical monocyte genes (CD14, FCGR2A, S100A8, S100A9); they preferentially promoted interferon γ (mean 17·79% [SE 3·69] vs 7·21 [1·20], p=0·0094), interleukin (IL) 17 (5·10% [0·96] vs 2·30 [0·77], p=0·0015), and GM-CSF-producing T cells (34·39% [8·65] vs 18·31 [7·16], p=0·0066) and produced more IL1β (2517 pg/mL [241] vs 702·6 [206·4], p<0·0001) and IL18 (71·7 pg/mL [4·97] vs 48·07 [3·11], p=0·0016). CD1c+BTLA+ dendritic cells had higher expression than CD1c+BTLA– dendritic cells of conventional dendritic cell genes (CLEC9A, CD24, KIT) and preferentially promoted IL4-producing T cells (15·7% [2·06%] vs 11·7 [1·65], p=0·0059) and CD25hiFOXP3+ regulatory T cells (Tregs) (15·09% [4·18] vs 9·26 [2·48], p=0·0346). CD1c+BTLA– dendritic cells had greater potential to differentiate into tartrate-resistant acid phosphatase positive multinucleated osteoclasts whereas CD1c+BTLA+ dendritic cells preferentially differentiated into langerin+CD1a+ Langerhans cells. The ratio of CD1c+BTLA– to CD1c+BTLA+ dendritic cells was significantly higher in rheumatoid arthritis synovial fluid than in peripheral blood (3·76 [0·64] vs 1·11 [0·10], p<0·0001). InterpretationCD1c-expressing dendritic cells have been regarded as a single population but these data demonstrate underlying complexity. CD1c+BTLA– dendritic cells support T helper (Th) 1 cell and Th17 cell responses and possess osteoclast potential. CD1c+BTLA+ dendritic cells support Th2 cell and Treg responses and possess Langerhans cell potential. The bulk CD1c+ population has previously been manipulated to produce vaccine and tolerogenic dendritic cell therapies in cancer and autoimmunity, but more refined targeting could potentially improve their efficacy. FundingWellcome Trust.

Enhanced Innate Inflammation Induced by Anti-BTLA Antibody in Dual Insult Model of Hemorrhagic Shock/Sepsis.

Sepsis following hemorrhagic shock is a common clinical condition, in which innate immune system suffers from severe suppression. B and T lymphocyte attenuator (BTLA) is an immune-regulatory coinhibitory receptor expressed not only on adaptive, but also on innate immune cells. Our previous data showed that BTLA gene deficient mice were protected from septic mortality when compared with wild-type control C57BL/6 mice. Here, we extended our study by treating C57BL/6 mice with an anti-BTLA monoclonal antibody (clone 6A6; reported to have the ability to neutralize or agonize/potentiate BTLA signaling) in a mouse model of hemorrhagic shock (Hem) followed by sepsis induced by cecal ligation and puncture (CLP); positing initially that if BTLA engagement was neutralized, like gene deficiency, an anti-BTLA mAb would have the similar effects on the inflammatory response/morbidity in these mice after such insults. Here, we report that BTLA expression is elevated on innate immune cells after Hem/CLP. However, anti-BTLA antibody treatment increased cytokine (TNF-α, IL-12, IL-10)/chemokine (KC, MIP-2, MCP-1) levels and inflammatory cells (neutrophils, macrophages, dendritic cells) recruitment in the peritoneal cavity, which in turn aggravated organ injury and elevated these animals' mortality in Hem/CLP. When compared with the protective effects of our previous study using BTLA gene deficient mice in a model of lethal septic challenge, we further confirmed BTLA's contribution to enhanced innate cell recruitment, elevated IL-10 levels, and reduced survival, and that engagement of antibody with BTLA potentiates/exacerbates the pathophysiology in Hem/sepsis.

Expression of B and T Lymphocyte Attenuator in Patients with Severe Community-Acquired Pneumonia and the Effect of Steroid Therapy in a Mouse Model.

Severe community-acquired pneumonia (CAP) remains a clinical problem, and the identification of new therapeutic targets may increase the rate of successfully treating patients with severe CAP. B and T lymphocyte attenuator (BTLA) is an immunoglobulin-related membrane protein that may play a vital role in the pathogenesis of infection. However, the effects of BTLA in related to severe CAP involved are unknown. In this study, we investigated potential changes in BTLA expression on lymphocytes in patients with severe CAP and determined how BTLA expression affects a model of LPS-induced acute lung inflammation. The percentages of circulating BTLA+CD4+ lymphocytes were determined in patients with severe CAP and in an LPS-induced acute lung inflammation model. Bronchoalveolar lavage fluid (BALF) was collected from mice and analyzed for leukocyte counts and by enzyme-linked immunosorbent assay (ELISA). Lung tissue samples were collected and assessed via Western blotting, immunohistochemistry and HE staining. The percentages of circulating BTLA+CD4+ lymphocytes were significantly higher in patients with severe CAP and in mice with LPS-induced acute lung inflammation than in the control groups. After treatment with either dexamethasone or the agonistic anti-BTLA antibody 6A6, BTLA expression was significantly higher than that in the control mice with LPS-induced acute lung inflammation. Moreover, both dexamethasone and the agonistic anti-BTLA antibody 6A6 attenuated inflammatory responses and nuclear factor (NF)-κB pathway activation. These results demonstrated that BTLA may be a therapeutic target for the treatment of severe CAP and that BTLA expression may reflect the body's immune status and guide decisions regarding steroid therapy for treating severe CAP.

Expression of B and T Lymphocyte Attenuator (BTLA) Correlates with CNS Metastasis and Adverse Prognosis in Activated B-Cell Lymphoma and Acute Lymphoblastic Leukemia

Background. The genetic factors that contribute to dissemination and relapse in aggressive lymphomas and acute lymphoblastic leukemia (ALL) are incompletely defined. We tested the hypothesis that shared molecular pathways contribute to drug resistance and metastasis in three distinct types of lymphoid neoplasms: large B-cell lymphoma, primary central nervous system lymphoma (PCNSL, DLBCL) and ALL.Methods. We used a variety of approaches involving high-resolution array-based comparative genomic hybridization, patient-derived cell lines, murine tumor models, diagnostic tumor specimens, and clinical outcome data sets to interrogate the biological significance of candidate mediators of relapse and CNS metastasis.Results. We first tested the hypothesis that genomic aberrations may contribute to the refractory phenotype in aggressive B-cell lymphoma. To pursue this, we compared DNA copy number aberrations in relapsed large B-cell lymphoma that had metastasized to the brain (3 cases, each isolated by resection) with the genomic changes identified in large B-cell lymphoma at diagnosis (15 cases: 12 PCNSL and 3 nodal lymphomas; all DLBCL). We identified a focal recurrent copy number gain at chromosome 3q encoding two candidate proteins: SIDT1, a mediator of microRNA transport, and BTLA, a member of the CD28 superfamily and modifier of both B-cell receptor signaling as well as T-cell responses. Elevated BTLA transcript and protein expression in relapsed specimens was confirmed. We also demonstrated by immunohistochemistry that tumor cell expression of BTLA but not SIDT1 correlated with short overall survival in an independent set of 40 patients with PCNSL treated uniformly with an immunochemotherapy protocol. Using independent databases of systemic DLBCL (n=203 and 69 respectively, Lenz et al. PNAS 2008; Shaknovich et al. Blood 2010), we determined that expression of both BTLA and SIDT1 were significantly higher in ABC compared to GCB DLBCL (p<0.0001) and, in an independent cohort of 73 ABC DLBCL cases, high BTLA expression was associated with a trend towards shorter overall survival (P=0.059). Finally, high BTLA transcript levels, but not SIDT1, correlated with significantly shorter overall survival in both ALL and Ph+ALL in ECOG E2993 (n=215, Geng el al. Cancer Discovery 2012), and with minimal residual disease in COG P9906 (n=207, Harney et al. Blood 2010). In addition, we used flow-cytometry to demonstrate that BTLA was strongly expressed by patient-derived cell lines of primary and secondary CNS lymphoma as well as Ph+ALL. In a murine model of spontaneous CNS metastasis involving patient-derived ABC DLBLC cells, we demonstrated that tumor cell clones that homed to the CNS meninges after flank implantation displayed higher BTLA expression compared to lymphoma cells that metastasized to spleen. Finally, we demonstrated that in a murine model of ALL, genetic deletion of BTLA was associated with impaired ALL cell proliferation and colony formation in vitro. By contrast, BTLA deficiency had no functional consequences in a murine model of CML.Conclusions. Taken together these data suggest for the first time that BTLA expression may contribute to metastasis and CNS progression of activated B-cell lymphoma and to resistance in ALL and Ph+ ALL. Our data suggest that BTLA may not only be a useful biomarker in these aggressive neoplasms, but also a potential therapeutic target. Additional studies are needed to deduce the mechanisms by which BTLA contributes to resistance and metastasis in ABC DLBCL and in ALL.Supported by the Leukemia and Lymphoma Society and the National Cancer Institute DisclosuresRubenstein:Celgene: Research Funding; Genentech: Research Funding.

Alterative Expression Pattern of T Cell Immunosuppressive Receptors in Peripheral Blood of Patients with ITP

Immune Thrombocytopenia (ITP) is an autoimmune disorder characterized by production of auto-antibodies against platelet antigens. Dysregulation of T cells is a major role involved in this disease, while anti-platelet antibodies induce platelets destruction due to an imbalanced immune response. Increasing data showed that abnormal expression of T cell immunosuppressive receptors such as Programmed death 1 (PD-1) and Cytotoxic T lymphocyte antigen-4 (CTLA-4) may relate to autoimmune disease pathogenesis. In this study, we analyzed the expression levels of T cell immunosuppressive receptors including PD-1, PD-L1, CTLA-4, T cell immunoglobulin mucin 3 (Tim-3), lymphocyte activation gene-3 (LAG-3) and B and T lymphocyte attenuator (BTLA) in RNA from peripheral blood mononuclear cells (PBMCs) of ITP patients (18 cases, male: 12, female: 6, median age: 38.5 years, range 24-68 years), PBMCs from 20 healthy individuals were served as control group, by real-time PCR. The results showed that significant lower expression levels of PD-1 (median: 0.0015) and PD-L1 (median: 0.0572) were showed in ITP group in comparison with healthy group (PD-1, median: 0.0117, p < 0.0001; PD-L1, median: 0.5428, p < 0.0001), while the expression levels of Tim-3 (median: 0.1352), LAG-3 (median: 0.1403) and BTLA (median: 0.1403) were significant higher than that from healthy group (Tim-3, median: 0.0751, p < 0.007; Lag-3, median: 0.0155, p < 0.0001; BTLA, median: 0.0315, p < 0.0001). There was not significant different on the CTLA-4 expression level between ITP group (median: 0.0818) and healthy group (median: 0.1667) (p = 0.219). In conclusion, we firstly described the alterative expression pattern of T cell immunosuppressive receptors in ITP, which may have a role in ITP pathogenesis. DisclosuresLi:The National Natural Science Foundation of China (81370605): Research Funding.

Clinical significance of tumor-infiltrating immune cells focusing on BTLA and Cbl-b in patients with gallbladder cancer.

The host immune system plays a significant role in tumor control, although most cancers escape immune surveillance through a variety of mechanisms. The aim of the present study was to evaluate the clinicopathological significance of a novel co-inhibitory receptor, B and T lymphocyte attenuator (BTLA), the anergy cell marker Casitas-B-lineage lymphoma protein-b (Cbl-b), and clinical implications of tumor-infiltrating immune cells in gallbladder cancer (GBC) tissues. We investigated 211 cases of GBC, 21 cases of chronic cholecystitis (CC), and 11 cases of xanthogranulomatous cholecystitis (XGC) using immunohistochemistry to detect tissue-infiltrating immune cells and their expression of BTLA and Cbl-b, and carried out correlation and survival analyses. The density of infiltrating T cells was significantly higher in CC and XGC than in GBC. The density ratio of BTLA(+) cells to CD8(+) T cells (BTLA/CD8) and that of Cbl-b(+) cells to CD8(+) T cells (Cbl-b/CD8) were significantly higher in GBC than in CC and XGC. The FOXP3/CD4, BTLA/CD8, and Cbl-b/CD8 ratios were significantly correlated with each other, and also with malignant phenotypes. Survival analyses revealed that a lower density of tumor-infiltrating CD8(+) cells, and higher Foxp3/CD4, BTLA/CD8, and Cbl-b/CD8 ratios were significantly associated with shorter overall survival and disease-free survival in GBC patients. Multivariate analyses showed that M factor, perineural invasion, BTLA/CD8, and Cbl-b/CD8 were closely associated with shorter overall survival. These findings suggest that higher ratios of BTLA/CD8 and Cbl-b/CD8 are independent indicators of unfavorable outcome in GBC patients, and that upregulation of BTLA in cancer tissues is involved in inhibition of antitumor immunity.

Lymphoma-associated mutant Herpesvirus entry mediator selects for B and T lymphocyte attenuator inhibitory signaling (TUM9P.1009)

Abstract Innate and adaptive lymphocytes respond to and manipulate their microenvironment through diverse cellular communication pathways including tumor necrosis factor (TNF) network signaling. A frequent target of cancers and viruses is the TNF receptor herpesvirus entry mediator (HVEM, TNFRSF14) and its ligand-receptor network because of the central role of this signaling system in maintaining immune homeostasis, and in regulating inflammatory responses. Notably, the biophysical basis for altered agonistic activity by pathogen and tumor-associated receptor variants remains unclear. Here we show that HVEM mutations associated with human follicular and diffuse large B cell lymphoma uniformly disrupt binding to CD160, while subsets of mutations retain binding to B and T lymphocyte attenuator (BTLA) and LIGHT (TNFSF14). We show that while HVEM activation of the inhibitory receptor BTLA is restricted by the expression of CD160 in lymphocytes, selectivity for BTLA by mutated HVEM or a viral paralog results in unhindered inhibition of ERK signaling and activation of IL-2 transcription. Together, the acquisition of ligand selectivity for BTLA by tumors and viruses illustrates a common mechanism of immune modulation utilized by intracellular pathogens to thwart inflammatory signaling.

BTLA marks a less-differentiated tumor-infiltrating lymphocyte subset in melanoma with enhanced survival properties

In a recent adoptive cell therapy (ACT) clinical trial using autologous tumor-infiltrating lymphocytes (TILs) in patients with metastatic melanoma, we found an association between CD8+ T cells expressing the inhibitory receptor B- and T-lymphocyte attenuator (BTLA) and clinical response. Here, we further characterized this CD8+BTLA+ TIL subset and their CD8+BTLA− counterparts. We found that the CD8+ BTLA+ TILs had an increased response to IL-2, were less-differentiated effector-memory (TEM) cells, and persisted longer in vivo after infusion. In contrast, CD8+BTLA− TILs failed to proliferate and expressed genes associated with T-cell deletion/tolerance. Paradoxically, activation of BTLA signaling by its ligand, herpes virus entry mediator (HVEM), inhibited T-cell division and cytokine production, but also activated the Akt/PKB pathway thus protecting CD8+BTLA+ TILs from apoptosis. Our results point to a new role of BTLA as a useful T-cell differentiation marker in ACT and a dual signaling molecule that curtails T-cell activation while also conferring a survival advantage for CD8+ T cells. These attributes may explain our previous observation that BTLA expression on CD8+ TILs correlates with clinical response to adoptive T-cell therapy in metastatic melanoma.