BTLA:一个新发现的CD28超家族共刺激分子

BTLA是新近发现的一个CD8超家族共刺激分子,它与CTLA-4、PD-1有相似的结构和功能,对T细胞活化起负性调控作用,同时还可作为胸腺细胞阳性选择的早期标志。研究发现BTLA可与HVEM直接结合,目前多认为HVEM是BTLA的配体。BTLA—HVEM相互作用沟通了CD28及TNFR两个共刺激分子超家族,是一个比较特别的．CD28超家族分子。     

Distinct expression and inhibitory function of B and T lymphocyte attenuator on human T cells

B and T lymphocyte attenuator (BTLA) has been recently identified as a new inhibitory receptor of the CD28 superfamily, with similarities to cytotoxic T lymphocyte activation antigen (CTLA)-4 and programmed death (PD)-1. Engagement of BTLA on T lymphocytes can profoundly reduce the T cell receptor (TCR)-mediated activation. In this study, we generated four monoclonal antibodies (mAbs) against human BTLA. Using the produced mAb 8H9, the BTLA molecule was found to distinctly express on many subgroups of immunocytes and show a regulatory expression, which was in accordance with its unique ligand herpes virus entry mediator (HVEM) in the process of T cell activation. In addition, the expression of BTLA was increased in the CD4(+) and CD8(+) T cells of pleural fluid in lung cancer patients. Furthermore, we showed that the BTLA-induced negative signals could be triggered by mAb 7D7. Cross-linking of BTLA with mAb 7D7 suppressed T lymphocyte proliferation, downregulated the expression of T cell activation marker CD25, and inhibited the production of interferon (IFN)-gamma, interleukin (IL)-2, IL-4, and IL-10.     

人共信号分子HVEM在外周血PBMC表达特性及其对T细胞的生物学作用

HVEM既可作为受体与LIGHT作用传递正性共刺激信号,又能作为配体作用于BTLA介导负性共抑制信号。为深入探讨HVEM对T细胞复杂而又独特的调控作用,本文研究了HVEM在免疫细胞上的表达特性,初步探讨了T细胞表达的HVEM分子所介导的生物学作用。采用LPS刺激人新鲜PBMC,以及PHA或PMA/IM刺激活化T细胞;间接免疫荧光标记和流式细胞术检测HVEM表达;MTT法分析T细胞增殖作用。结果显示,HVEM在不同条件刺激活化的T细胞表面均呈现先上调后下调表达;T细胞增殖试验表明,基因转染细胞L929/LIGHT能够明显促进T细胞的增殖及IL-2和IFN-γ的分泌,而以抗人BTLA单抗在一定程度上模拟HVEM所介导的BTLA/HVEM信号能够明显抑制T细胞增殖作用及细胞因子IL-2、IFN-γ和IL-10的产生。     

The CD160, BTLA, LIGHT/HVEM pathway: a bidirectional switch regulating T-cell activation

Summary:  CD160 is a newly identified ligand for HVEM (herpes virus entry mediator). Previously identified HVEM ligands include BTLA (B- and T-lymphocyte attenuator), LIGHT (lymphotoxin-like, exhibits inducible expression, and competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes) and LTα (lymphotoxin-α). The binding of LIGHT or LTα to HVEM delivers a costimulatory signal, whereas the binding of BTLA or CD160 to HVEM delivers a coinhibitory signal. Thus, HVEM is a bidirectional switch regulating T-cell activation in a costimulatory or coinhibitory fashion whose outcome depends on the ligand engaged. The cysteine-rich domain 1 (CRD1) of HVEM is essential for the binding of coinhibitory ligands CD160 and BTLA but not costimulatory ligand LIGHT. Deletion or blockade of HVEM CRD1 abolishes the binding of CD160 and BTLA, but not LIGHT, and converts HVEM to a dominant costimulatory molecule, possibly through the loss of negative signaling by CD160/BTLA. Therapies targeting the CRD1 of HVEM to block BTLA and CD160 binding are being developed to enhance immune responses and vaccination.     

B和T淋巴细胞衰减因子(BTLA)在类风湿关节炎滑膜组织的表达

目的:探讨CD28家族共抑制分子B和T淋巴细胞衰减因子在类风湿关节炎(RA)患者滑膜组织内的表达。方法:免疫组化法检测RA患者滑膜组织BTLA的表达;并使用免疫荧光法检测BTLA的细胞定位及分布。结果:免疫组化结果证实,RA患者滑膜组织中有大量的BTLA阳性细胞,形态观察提示这些阳性的细胞主要是淋巴结处的淋巴细胞及巨噬细胞;免疫荧光分析进一步表明这些BTLA+细胞主要为CD3+T细胞及CD68+巨噬细胞,少数CD31+内皮细胞也表达BTLA。此外,对比其他B7家族共刺激分子在滑膜组织中的分布,免疫荧光发现BTLA共表达于B7-H1+,B7-H4+及HVEM+细胞,但不表达于B7-DC+及B7-H3+细胞。结论:关节炎滑膜组织内有大量BTLA阳性细胞,提示BTLA有可能参与并调节了关节炎的病理进程。     

Monoclonal antibodies to B and T lymphocyte attenuator (BTLA) have no effect on in vitro B cell proliferation and act to inhibit in vitro T cell proliferation when presented in a cis, but not trans, format relative to the activating stimulus

B and T lymphocyte attenuator (BTLA) is an immunoglobulin superfamily member surface protein expressed on B and T cells. Its ligand, herpesvirus entry mediator (HVEM), is believed to act as a monomeric agonist that signals via the CRD1 of HVEM to inhibit lymphocyte activation: HVEM is also the receptor for lymphotoxin-α and LIGHT, which both bind in the CRD2 and CRD3 domains of the HVEM molecule, and for CD160 which competes with BTLA. We have shown that recombinant HVEM and a panel of different monoclonal antibodies specifically bind murine BTLA on both B and T cells and that some antibodies inhibit anti-CD3ε-induced T cell proliferation in vitro, but only when constrained appropriately with a putatively cross-linking reagent. The antibodies had no significant effect on in vitro T cell proliferation in a mixed lymphocyte reaction (MLR) assay nor on in vitro DO11.10 antigen-induced T cell proliferation. None of these antibodies, nor HVEM-Fc, had any significant effect on in vitro B cell proliferation induced by anti-immunoglobulin M antibodies (±anti-CD40) or lipopolysaccharide. We further elucidated the requirements for inhibition of in vitro T cell proliferation using a beads-based system to demonstrate that the antibodies that inhibited T cell proliferation in vitro were required to be presented to the T cell in a cis, and not trans, format relative to the anti-CD3ε stimulus. We also found that antibodies that inhibited T cell proliferation in vitro had no significant effect on the antibody captured interleukin-2 associated with the in vivo activation of DO11.10 T cells transferred to syngeneic recipient BALB/c mice. These data suggest that there may be specific structural requirements for the BTLA molecule to exert its effect on lymphocyte activation and proliferation.     

TNF Superfamily Networks: bidirectional and interference pathways of the herpesvirus entry mediator (TNFSF14)

The herpesvirus entry mediator (HVEM; TNFRSF14) can activate either proinflammatory or inhibitory signaling pathways. HVEM engages two distinct types of ligands, the canonical TNF-related cytokines, LIGHT and Lymphotoxin-α, and the Ig-related membrane proteins, BTLA (B and T lymphocyte attenuator) and CD160. Recent evidence indicates that the signal generated by HVEM depends on the context of its ligands expressed in trans or in cis. HVEM engagement by all of its ligands in trans initiates bidirectional signaling. In contrast, na?ve T cells coexpress BTLA and HVEM forming a cis-complex that interferes with the activation of HVEM by extraneous ligands in the surrounding microenvironment. The HVEM Network is emerging as a key survival system for effector and memory T cells in mucosal tissues.     

BTLA expression contributes to septic morbidity and mortality by inducing innate inflammatory cell dysfunction

A proper innate inflammatory response is essential for prevention of the systemic inflammation associated with sepsis. BTLA is an immune-regulatory receptor demonstrated to be expressed not only on adaptive immune populations and have potent inhibitory effects on CD4(+) T cells but is also expressed on innate cell populations (CD11c(+) and CD11b(+) cells) and has been shown to diminish pathogen clearance following bacterial and parasite infection. The role of BTLA in sepsis and the mechanisms by which BTLA alters pathogen clearance, however, have not been addressed clearly. Here, we show that following acute experimental sepsis induction in mice (CLP), the number of infiltrating BTLA- and HVEM (the ligand for BTLA)-expressing macrophages, inflammatory monocytes, mature and immature DCs, and neutrophils increased in the peritoneum compared with sham surgery, suggesting that a high level of HVEM:BTLA interactions occurs between these cells at the site of septic insult. Given this, we evaluated BTLA(-/-) mice, 24 h post-CLP, and observed a marked increase in the degree of activation on these cell populations, as well as a reduction in peritoneal bacterial burden and IL-10 induction, and most importantly, BTLA(-/-) mice exhibited a higher rate of survival and protection from organ injury when compared with WT mice. Such changes were not restricted to experimental mice, as circulating BTLA+ and HVEM+ monocytes and HVEM+ granulocytes were increased in septic ICU patients, supporting a role for BTLA and/or HVEM as potential, novel diagnostic markers of innate immune response/status and as therapeutic targets of sepsis.     

The expression and anatomical distribution of BTLA and its ligand HVEM in rheumatoid synovium

Co-inhibitory signaling from B and T lymphocyte attenuator (BTLA) can suppress lymphocyte activation and maintain peripheral tolerance. However, the expression and anatomical distribution of BTLA and its ligand, herpesvirus entry mediator (HVEM), in rheumatoid arthritis (RA) synovium have not been reported. In this study, we analyzed the expression of HVEM and BTLA in RA synovium by immunohistochemistry, and our results showed that both factors were observed in all four cases of RA samples. At the cellular level, both HVEM and BTLA were found on the cell membrane and in the cytoplasm. Fluorescence dual staining demonstrated that HVEM was chiefly on CD3⁺ T cells, CD68⁺ macrophages, and to a lesser extent was found on CD31⁺ endothelial cells. Similarly, the expression of BTLA was observed on infiltrated CD3⁺ T cells and CD68⁺ macrophages. The co-expression of HVEM and BTLA with some members of the B7 family in these sections was also analyzed, and the results showed that HVEM antigen was also found on B7-H3⁺ capillaries, while it was absent on B7-H1⁺, B7-DC⁺, B7-H4⁺, and Z39Ig⁺ cells. Interestingly, BTLA was observed on B7-H1⁺, B7-H4⁺, and HVEM⁺ cells in the synovium. The characteristic expression and distribution of BTLA/HVEM in the synovium indicated that their signaling probably affects the pathogenesis of RA, and a clear understanding of their functional roles may further elucidate the pathogenesis of this disease.     

B and T lymphocyte attenuator regulates T cell activation through interaction with herpesvirus entry mediator

B and T lymphocyte attenuator (BTLA) provides an inhibitory signal to B and T cells. Previously, indirect observations suggested that B7x was a ligand for BTLA. Here we show that BTLA does not bind B7x; instead, we identify herpesvirus entry mediator (HVEM) as the unique BTLA ligand. BTLA bound the most membrane-distal cysteine-rich domain of HVEM, distinct from regions where the ligands LIGHT and lymphotoxin-alpha bound HVEM. HVEM induced BTLA tyrosine phosphorylation and association of the tyrosine phosphatase SHP-2 and repressed antigen-driven T cell proliferation, providing an example of reverse signaling to a non-tumor necrosis factor family ligand. The conservation of the BTLA-HVEM interaction between mouse and human suggests that this system is an important pathway regulating lymphocyte activation and/or homeostasis in the immune response.     

B and T lymphocyte attenuator regulates CD8+ T cell-intrinsic homeostasis and memory cell generation

B and T lymphocyte attenuator (BTLA) is a negative regulator of T cell activation, but its function in vivo is not well characterized. Here we show that mice deficient in full-length BTLA or its ligand, herpesvirus entry mediator, had increased number of memory CD8(+) T cells. The memory CD8(+) T cell phenotype resulted from a T cell-intrinsic perturbation of the CD8(+) T cell pool. Naive BTLA-deficient CD8(+) T cells were more efficient than wild-type cells at generating memory in a competitive antigen-specific system. This effect was independent of the initial expansion of the responding antigen-specific T cell population. In addition, BTLA negatively regulated antigen-independent homeostatic expansion of CD4(+) and CD8(+) T cells. These results emphasize two central functions of BTLA in limiting T cell activity in vivo.     

The influence of cyclosporin A on lymphocyte attenuator expression

B and T lymphocyte attenuator (BTLA), a recently identified immune inhibitory receptor, has been demonstrated to have the ability to maintain self-tolerance and transplant-tolerance in mice. However, little is known about the effects of immunosuppressive drugs on the expression of BTLA. In the present study, we observed that the immunosuppressive drug cyclosporin A (CsA) could significantly reduce BTLA but not CD25 and CD69 expression on CD4+ T cells during activation in vitro, while rapamycin (RPM) had little effect on it. Exogenous interleukin-2 (IL-2) failed to reverse the inhibitory effect that CsA had on BTLA expression. Furthermore, phorbol 12-myristate 13-acetate (PMA) or ionomycin alone could efficiently induce BTLA protein expression on CD4+ and CD8+ T cells, while CsA significantly suppressed BTLA expression in this system. The present data indicate that the regulation of BTLA expression on CD4+ T cells does not depend on IL-2 and T cell activation but depends on calcineurin-dependent and calcineurin-independent pathways. The observation that CsA significantly inhibits BTLA expression on CD4+ T cells during activation, suggests that CsA might block the immune tolerance induced by BTLA and potentially increase the susceptibility to autoimmune diseases and graft rejection.     

CpG-ODN-induced sustained expression of BTLA mediating selective inhibition of human B cells

BTLA (B- and T-lymphocyte attenuator) is a prominent co-receptor that is structurally and functionally related to CTLA-4 and PD-1. In T cells, BTLA inhibits TCR-mediated activation. In B cells, roles and functions of BTLA are still poorly understood and have never been studied in the context of B cells activated by CpG via TLR9. In this study, we evaluated the expression of BTLA depending on activation and differentiation of human B cell subsets in peripheral blood and lymph nodes. Stimulation with CpG upregulated BTLA, but not its ligand: herpes virus entry mediator (HVEM), on B cells in vitro and sustained its expression in vivo in melanoma patients after vaccination. Upon ligation with HVEM, BTLA inhibited CpG-mediated B cell functions (proliferation, cytokine production, and upregulation of co-stimulatory molecules), which was reversed by blocking BTLA/HVEM interactions. Interestingly, chemokine secretion (IL-8 and MIP1β) was not affected by BTLA/HVEM ligation, suggesting that BTLA-mediated inhibition is selective for some but not all B cell functions. We conclude that BTLA is an important immune checkpoint for B cells, as similarly known for T cells.     

BTLA associates with increased Foxp3 expression in CD4+ T cells in dextran sulfate sodium-induced colitis

Ulcerative colitis (UC) is an inflammatory bowel disease, and its pathogenesis involves a variety of genetic, environmental, and immunological factors such as T helper cells and their secreted cytokines. B and T lymphocyte attenuator (BTLA) is an immunoregulatory receptor that has a strong suppressive effect on T-cell function. However the role of BTLA in UC remains poorly understood. Here we demonstrated that the frequency of BTLA-expressing CD3⁺ T cells, especially CD4⁺ T cells, increased in blood and mucosa in mice with DSS-induced colitis. The frequency of Foxp3-expressing cells in BTLA+ CD4⁺ T cell from lamina propria mononuclear cells (LPMCs) was much higher in DSS-treated mice than that in controls. Similarly, the proportion of IL-17+ cells in BTLA+ CD4⁺ T cells from LPMCs in DSS-treated mice is much higher than that in controls, while no perceptible difference for the proportion of IFN-γ+ cells in BTLA+ CD4⁺ T cells was noted between DSS-treated mice and controls. Treatment of mesalazine, an anti-ulcerative colitis drug, down-regulated Foxp3 and IL-17 expression in BTLA positive T cells along with attenuated severity for colitis. Our findings indicate that BTLA may be involved in the control of inflammatory responses through increasing Foxp3 expression, rather than attenuating IL-17 production, in DSS-induced colitis.     

HIV 感染者调节性T 细胞表面B、T 淋巴细胞衰减因子的表达研究

目的:对HIV 感染者调节性T 细胞(Regulatory T cell,Treg)上B 、T 淋巴细胞衰减因子(B and T lymphocyte at-tenuator,BTLA)的表达水平进行检测,并探讨其在HIV 感染进程中的作用。方法:选取24 例感染在一年之内的HIV 早期感染者(Early HIV infected patients,EHI 组)、14 例感染超过一年的HIV 慢性感染者(CD4+ T 计数>200 cells/ μl,HIV 组)、6 例AIDS患者(CD4+ T 计数<200 cells/ μl,AIDS 组)和9 例健康人作为对照,应用流式细胞仪检测不同时期感染者及健康对照者Treg 细胞BTLA 的表达水平,分析其与疾病进展及免疫活化的相关性。结果:随着HIV 疾病进展,EHI 组、HIV 组及AIDS 组Treg 细胞BTLA 表达水平依次升高,其中HIV 组与AIDS 组Treg 细胞BTLA 表达水平显著高于EHI 组(P<0.05 及P<0.01),AIDS 组Treg 细胞BTLA 的表达水平高于健康对照(P<0.05);Treg 细胞BTLA 表达水平与CD4+ T 淋巴细胞计数呈负相关(P<0.001),与病毒载量呈正相关(P<0.01);Treg 细胞BTLA 表达水平与活化CD4+ CD38+ T 淋巴细胞及CD4+ HLA-DR+ T 淋巴细胞呈正相关(P<0.001,P<0.001)。结论:HIV 感染者Treg 细胞BTLA 表达升高,与疾病进展显著相关,提示其可能通过加强Treg 细胞的抑制功能加速疾病进展,并为未来HIV 感染的干预提供信息。     

Activated CD4+ and CD8+ T Cell Proportions in Multiple Sclerosis Patients

The goal of this study was to trace the course of multiple sclerosis (MS) by evaluating the lymphocyte subpopulation counts and the levels of CD4+ and CD8+ T cell activation using flow cytometry. Samples obtained from healthy subjects (N?=?40) and patients with MS (N?=?290) were analyzed. Lymphocytes were labeled for the surface markers CD4+, CD8+, CD3+, CD16+, CD19+, CD45+, and CD53+ and the activation marker HLA-DR+. Cell counts were then determined using flow cytometry. A high degree of inter-individual variability was observed in the counts of all lymphocyte subtypes in the MS group. A significantly lower proportion of CD3+ T cells (69?±?14 % in healthy subjects and 60?±?17 % as a percent of total lymphocytes in MS patients), CD4+ T cells (41?±?11 and 28?±?18 %, respectively), and a significantly higher proportion of NK T cells (12?±?5 and 25?±?21 %, respectively) were observed in patients with MS than in healthy subjects. These differences led to a lowered CD4+/CD8+ T cell ratio. Furthermore, a significantly lower proportion of activated CD4+ T cells (HLA-DR+ CD4+; from 48?±?10 to 38?±?15 % as a percent of CD4+ cells) was observed in patients with MS than in healthy subjects. The high level of inter-individual variability in lymphocyte cell counts and the counts of activated T cells suggest that MS is a complex and heterogeneous disease.     

LIGHT-HVEM—BTLA共信号分子的研究进展

BTLA是新近发现的一个CD28超家族共抑制分子,它的配体不是B7家族成员而是TNF受体超家族成员HVEM。HVEM同时还存在一个TNF超家族的配体,即T细胞上可诱导表达的与HSV的糖蛋白D竞争结合HVEM的淋巴毒素类似物（LIGHT）。HVEM可以作为一个分子开关,通过结合LIGHT或BTLA7E免疫调节中发挥不同的作用。     

Targeting lymphocyte activation through the lymphotoxin and LIGHT pathways

Summary: Cytokines mediate key communication pathways essential for regulation of immune responses. Full activation of antigen-responding lymphocytes requires cooperating signals from the tumor necrosis factor (TNF)-related cytokines and their specific receptors. LIGHT, a lymphotoxin-β (LTβ)-related TNF family member, modulates T-cell activation through two receptors, the herpesvirus entry mediator (HVEM) and indirectly through the LT-β receptor. An unexpected finding revealed a non-canonical binding site on HVEM for the immunoglobulin superfamily member, B and T lymphocyte attenuator (BTLA), and an inhibitory signaling protein suppressing T-cell activation. Thus, HVEM can act as a molecular switch between proinflammatory and inhibitory signaling. The non-canonical HVEM–BTLA pathway also acts to counter LTβR signaling that promotes the proliferation of antigen-presenting dendritic cells (DCs) within lymphoid tissue microenvironments. These results indicate LTβ receptor and HVEM–BTLA pathways form an integrated signaling circuit. Targeting these cytokine pathways with specific antagonists (antibody or decoy receptor) can alter lymphocyte differentiation and activation. Alternately, agonists directed at their cell surface receptors can restore homeostasis and potentially reset immune and inflammatory processes, which may be useful in treating autoimmune and infectious diseases and cancer.     

BTLA/HVEM抑制信号通路研究进展

BTLA是新近发现的Ig家族的抑制性受体,其唯一配体HVEM,来自TNF受体超家族,可与TNF家族的LIGHT结合提供正向刺激信号.而BTLA与HVEM的相互作用传递抑制信号下调淋巴细胞的免疫应答.这两个来自不同家族的受体间的负向调节作用,改变了对传统抑制性信号通路作用方式的看法.通过介绍目前对其生物学特性、信号传导机制及与相关疾病关系的研究,为进一步深入了解免疫应答的调控机制及寻找治疗自身免疫病、移植排斥、肿瘤等疾病的新方法提供参考.