BTLA: a new inhibitory receptor with a B7-like ligand

The B and T lymphocyte attenuator, BTLA, is a recently discovered Ig superfamily member. BTLA engagement results in downregulation of T-cell activation, and mice deficient in BTLA show increased incidence and severity of autoimmune disorders. A novel B7 family member, B7x (B7S1, B7-H4), has been proposed as the ligand for BTLA. A B7x-Ig fusion protein inhibits T-cell activation and interleukin-2 (IL-2) production. Thus, cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1) and BTLA comprise an expanding set of inhibitory receptors expressed on lymphocytes.     

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.     

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.     

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 regulation of lymphocyte activation by inhibitory receptors

The ability of activating immune recognition receptors on lymphocytes to regulate cellular activation and function can be profoundly altered by co-stimulation with inhibitory receptors. Inhibitory receptors, such as the MHC-recognizing inhibitory receptors expressed on NK cells and subpopulations of activated T cells, can fully block the generation of any cytotoxic function by targeting proximal signals. Inhibitory Fc receptors on B cells, macrophages and mast cells can influence their threshold for activation, but the induction of inhibitory Fc receptors also appears to play a major role in the attenuation of ongoing responses. The three identified groups of inhibitory B7-recognizing receptors (CTLA-4, PD-1 and BTLA) are only expressed on activated hematopoietic cells, thus exclusively regulating ongoing immune responses in lymphoid organs and the periphery. In each case, the integrated positive and negative regulatory events determine the nature of the functional response.     

Cell surface expression of channel catfish leukocyte immune-type receptors (IpLITRs) and recruitment of both Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 and SHP-2

Channel catfish leukocyte immune-type receptors (IpLITRs) are immunoglobulin superfamily (IgSF) members believed to play a role in the control and coordination of cellular immune responses in teleost. Putative stimulatory and inhibitory IpLITRs are co-expressed by different types of catfish immune cells (e.g. NK cells, T cells, B cells, and macrophages) but their signaling potential has not been determined. Following cationic polymer-mediated transfections into human cell lines we examined the surface expression, tyrosine phosphorylation, and phosphatase recruitment potential of two types of putative inhibitory IpLITRs using ‘chimeric’ expression constructs and an epitope-tagged ‘native’ IpLITR. We also cloned and expressed the teleost Src homology 2 domain-containing protein tyrosine phosphatases (SHP)-1 and SHP-2 and examined their expression in adult tissues and developing zebrafish embryos. Co-immunoprecipitation experiments support the inhibitory signaling potential of distinct IpLITR-types that bound both SHP-1 and SHP-2 following the phosphorylation of tyrosine residues within their cytoplasmic tail (CYT) regions. Phosphatase recruitment by IpLITRs represents an important first step in understanding their influence on immune cell effector functions and suggests that certain inhibitory signaling pathways are conserved among vertebrates.     

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。     

Negative regulation of FcepsilonRI signaling by FcgammaRII costimulation in human blood basophils

BACKGROUND: Signaling through the antigen receptors of human B and T cells and the high-affinity IgE receptor FcepsilonRI of rodent mast cells is decreased by cross-linking these receptors to the low-affinity IgG receptor FcgammaRII. The inhibition is thought to involve the tyrosine phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the FcgammaRIIB cytoplasmic tail, creating binding sites for SH2-containing protein (Src homology domain containing protein tyrosine phosphatase 1 and 2 [SHP-1, SHP-2]) and/or lipid (SH2 domain-containing polyphosphatidyl-inositol 5-phosphatase) phosphatases that oppose activating signals from the costimulated antigen receptors. OBJECTIVE: In human basophils and mast cells FcepsilonRI signaling generates mediators and cytokines responsible for allergic inflammation. We proposed to determine whether FcepsilonRI signaling is inhibited by FcgammaRII costimulation in human basophils and to explore the underlying mechanism as an approach to improving the treatment of allergic inflammation. METHODS: FcgammaR expression on human basophils was examined using flow cytometry and RT-PCR analysis. FcgammaRII/FcepsilonRI costimulation was typically accomplished by priming cells with anti-dinitrophenol (DNP) IgE and anti-DNP IgG and stimulating with DNP-BSA. Phosphatases were identified by Western blotting, and their partitioning between membrane and cytosol was determined by cell fractionation. Biotinylated synthetic peptides and phosphopeptides corresponding to the FcgammaRIIB ITIM sequence were used for adsorption assays. RESULTS: We report that peripheral blood basophils express FcgammaRII (in both the ITIM-containing FcgammaRIIB and the immunoreceptor tyrosine-based activation motif-containing FcgammaRIIA forms) and that costimulating FcgammaRII and FcepsilonRI inhibits basophil FcepsilonRI-mediated histamine release, IL-4 production, and Ca(2+) mobilization. The inhibition of basophil FcepsilonRI signaling by FcgammaRII/FcepsilonRI costimulation is linked to a significant decrease in Syk tyrosine phosphorylation. Human basophils express all 3 SH2-containing phosphatases. CONCLUSIONS: Evidence that FcgammaRII/FcepsilonRI costimulation induces SHP-1 translocation from the cytosolic to membrane fractions of basophils and that biotinylated synthetic peptides corresponding to the phosphorylated FcgammaRIIB ITIM sequence specifically recruit SHP-1 from basophil lysates particularly implicates this protein phosphatase in the negative regulation of FcepsilonRI signaling by costimulated FcgammaRII.     

The effect of phosphatases SHP-1 and SHIP-1 on signaling by the ITIM- and ITAM-containing Fcgamma receptors FcgammaRIIB and FcgammaRIIA

Inositol and tyrosine phosphatases have been implicated in inhibitory signaling by an Fc receptor for immunoglobulin G, FcgammaRIIB, in B cells, mast cells, and monocytes. Here, we propose a role for the Src homology 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1) in FcgammaRIIB-mediated inhibition of FcgammaR signaling. Coexpression of SHP-1 enhances FcgammaRIIB-mediated inhibition of FcgammaRIIA phagocytosis in COS-1 cells. SHP-1 also enhances the reduction in FcgammaRIIA tyrosine phosphorylation that accompanies this inhibition. Significantly, tyrosine phosphorylation of Syk kinase is substantially inhibited by SHP-1. Furthermore, the activation of SHP-1 tyrosine phosphorylation is observed following stimulation of FcgammaRII in COS-1 cells and in human monocytes. The SH2 domain containing inositol phosphatase (SHIP), SHIP-1 also enhances FcgammaRIIB-mediated inhibition of FcgammaRIIA, indicating that FcgammaRIIB can use more than one pathway for its inhibitory action. In addition, SHP-1 and SHIP-1 can inhibit FcgammaRIIA phagocytosis and signal transduction in the absence of FcgammaRIIB. The data support emerging evidence that SH2-containing phosphatases, such as SHP-1 and SHIP-1, can modulate signaling by "activating" receptors.     

Biliary glycoprotein (BGPa, CD66a, CEACAM1) mediates inhibitory signals

Biliary glycoprotein (BGP, CD66a, CEACAM1) is a member of the carcinoembryonic antigen family (CEA, CD66), a group of transmembrane proteins belonging to the immunoglobulin superfamily. The structural features surrounding the tyrosine residues in the cytoplasmic domain of BGP share similarity with the consensus sequence of the immunoreceptor tyrosine-based inhibition motif (ITIM), the docking site for SHIP, SHP-1, and SHP-2 molecules. Using the well-characterized inhibitory receptor, FcgammaRIIB, we constructed a FcgammaRIIB-BGPa chimeric molecule that contained the extracellular and transmembrane domain of FcgammaRIIB and the cytoplasmic tail of BGPa and expressed it in DT40 B cells. Our results showed that FcgammaRIIB-BGPa, just like the unmodified FcgammaRIIB molecule, inhibited calcium influx in activated DT40 B cells. Substitution of tyrosine with phenylalanine (Y459F) in FcgammaRIIB-BGPa completely abrogated its ability to inhibit calcium influx, indicating that the motif surrounding Y459 is ITIM. The presence of ITIM was also supported by showing that the FcgammaRIIB-BGPa-mediated inhibitory effect was reduced in SHP-1and SHP-2 mutant DT40 B cells and further diminished in a SHP-1/-2 double-deficient mutant line. The results suggest that SHP-1 and SHP-2 are required for the FcgammaRIIB-BGPa-mediated inhibitory signals.     

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.     

BTLA-HVEM通路阻断对树突状细胞功能影响的体内外研究

目的 探讨阻断BTLA-HVEM(B/T淋巴细胞弱化因子疱疹病毒进入介质)通路对树突状细胞功能的影响和相关免疫学机制.方法 构建小鼠BTLA胞外功能区的真核表达载体psBTLA,转染CHO细胞;HSP70-TC-1肿瘤抗原肽刺激小鼠骨髓来源DCs,流式细胞仪检测处理后DCs表面BTLA、HVEM的表达,同时给予转染了psBTLA质粒的CHO细胞的培养上清处理后,检测DCs表面B7-1的表达,ELISA检测上清中IL-12的分泌;处理后的DCs刺激脾细胞,检测淋巴细胞增殖和细胞因子分泌;检测psBTLA体内转染对宫颈癌细胞系TC-1成瘤小鼠DCs表达B7-1和肿瘤生长的影响.结果 成功构建小鼠BTLA胞外段的真核表达载体psBTLA,获得了稳定转染psBTLA的CHO细胞,在其培养上清检测到BTLA胞外段(sBTLA)的表达.DCs经抗原肽刺激后BTLA、HVEM表达均上调,加入含sBTLA的上清处理后上调B7-1,上清中分泌的IL-12增加,与脾细胞共培养时促进细胞增殖和IL-2、IFN-γ的分泌;体内基因转染psBTLA促进DCs表达B7-1以及抑制肿瘤生长.结论 通过sBTLA阻断BTLA-HVEM共抑制通路,可以进一步促进DCs的功能,更好地激活淋巴细胞,促进抗肿瘤免疫应答.     

一株鼠抗人BTLA功能性单克隆抗体的研制及其生物学特性鉴定

为了研制鼠抗人BTLA功能性单克隆抗体,以高表达人BTLA分子的基因转染细胞L929/BTLA为免疫原,常规免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术进行细胞融合,并以基因转染细胞293T/BTLA和293T/mock作为抗原,筛选阳性杂交瘤克隆,经间接免疫荧光标记和流式细胞术分析、反复鉴定和多次克隆化培养,筛选获得分泌特异性鼠抗人BTLA分子单克隆抗体的杂交瘤细胞株;采用Ig亚型快速定性试纸法、细胞核染色体计数、竞争结合抑制试验和T增殖抑制试验等对单抗进行生物学特性的鉴定。结果表明,成功获得一株持续、稳定分泌鼠抗人BTLA单克隆抗体的杂交瘤细胞株8H9,该单抗可特异性识别基因转染细胞以及静止与活化T淋巴细胞上表达的BTLA分子,单抗8H9和BTLA交联后能显著抑制鼠抗人CD3单抗对T细胞激发的增殖作用。     

IL-10 inhibits CD28 and ICOS costimulations of T cells via src homology 2 domain-containing protein tyrosine phosphatase 1

BACKGROUND: Specific T-cell activation requires T-cell receptor stimulation and the generation of costimulatory signals. Major costimulatory signals are delivered to T cells by the interaction of CD28 and inducible costimulator (ICOS). OBJECTIVE: To investigate the molecular pathways involved in direct T-cell suppression by IL-10. METHODS: T-cell proliferation analysis, immunoprecipitations, and Western blots were performed after T-cell receptor and CD28 and ICOS stimulations in the absence or presence of IL-10. Dominant-negative src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) overexpression, small inhibitory RNA, and SHP-1-deficient and IL-10-deficient mice were used. RESULTS: IL-10 receptor-associated tyrosine kinase Tyk-2 acts as a constitutive reservoir for SHP-1 in resting T cells, and then tyrosine phosphorylates SHP-1 on IL-10 binding. SHP-1 rapidly binds to CD28 and ICOS costimulatory receptors and dephosphorylates them within minutes. In consequence, the binding of phosphatidylinositol 3-kinase to either costimulatory receptor no longer occurs, and downstream signaling is inhibited. Accordingly, spleen cells from SHP-1-deficient mice showed increased proliferation with CD28 and ICOS stimulation in comparison with wild-type mice, which was not suppressed by IL-10. Generation of dominant-negative SHP-1-overexpressing T cells or silencing of the SHP-1 gene by small inhibitory RNA both altered SHP-1 functions and abolished the T-cell suppressive effect of IL-10. CONCLUSION: The rapid inhibition of the CD28 or ICOS costimulatory pathways by SHP-1 represents a novel mechanism for direct T-cell suppression by IL-10. CLINICAL IMPLICATIONS: Molecular mechanisms of direct T-cell suppression by IL-10 may provide a novel target for therapy of allergy/asthma and autoimmune disease.     

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。     

Allergen‐specific immunotherapy alters the expression of B and T lymphocyte attenuator,a co‐inhibitory molecule,in allergic rhinitis

Background B7/CD28 family co‐signalling molecules play a key role in regulating T cell activation and tolerance. Allergen‐specific immunotherapy (SIT) alters allergen‐specific T cell responses. However, the effect of SIT on the expression of various co‐signalling molecules has not been clarified. Objective We sought to determine whether SIT might affect the expression of three co‐inhibitory molecules, programmed death (PD)‐1, B7‐H1 and B and T lymphocyte attenuator (BTLA), in Japanese cedar pollinosis (JCP). Methods Peripheral blood mononuclear cells (PBMCs) were isolated from JCP patients who had or had not received SIT. PBMC were cultured in the presence or absence of Cry j 1, after which the cell surface expression of PD‐1, B7‐H1 and BTLA, as well as IL‐5 production, were determined. In addition, the effect of BTLA cross‐linking on IL‐5 production was examined. Results After Cry j 1 stimulation, no significant differences in PD‐1 and B7‐H1 expression were observed between SIT‐treated and SIT‐untreated patients. BTLA expression was down‐regulated in untreated patients after Cry j 1 stimulation and up‐regulated in SIT‐treated patients. Up‐regulation of BTLA in SIT‐treated patients was particularly apparent in a CD4⁺ T cell subset. IL‐5 production was clearly reduced among SIT‐treated patients, and the observed changes in BTLA expression correlated negatively with IL‐5 production. Moreover, immobilization of BTLA suppressed IL‐5 production in JCP patients. Conclusion These results suggest that both IL‐5 production and down‐regulation of BTLA in response to allergen are inhibited in SIT‐treated patients with JCP. BTLA‐mediated co‐inhibition of IL‐5 production may contribute to the regulation of allergen‐specific T cell responses in patients receiving immunotherapy.     

Leukocyte-associated Ig-like receptor-1 has SH2 domain-containing phosphatase-independent function and recruits C-terminal Src kinase

Most inhibitory receptors in the immune system contain one or several immunoreceptor tyrosine-based inhibitory motifs (ITIM) and recruit the SH2 domain-containing phosphatases SHP-1, SHP-2 and/or SHIP, which are generally believed to be essential for the inhibitory function. However, it has not been systematically investigated whether ITIM-bearing receptors exert their function through alternative interactions. Here we describe that leukocyte-associated Ig-like receptor (LAIR)-1 has inhibitory function in DT40 chicken B cells that lack both SHP-1 and SHP-2. In addition, we found that LAIR-1 did not recruit SHIP upon phosphorylation. Thus, LAIR-1 can function independently from SH2 domain-containing phosphatases and must recruit at least one other signaling molecule. Using a yeast-tri-hybrid system, we found that phosphorylated LAIR-1 bound the C-terminal Src kinase (Csk). The interaction required the SH2 domain of Csk and phosphorylation of the tyrosine in the N-terminal ITIM of LAIR-1. We propose that Csk is an additional player in the regulation of the immune system by ITIM-bearing receptors.     

SHP-2 phosphatase negatively regulates the TRIF adaptor protein-dependent type I interferon and proinflammatory cytokine production

The Toll-like receptor 3 (TLR3) and TLR4-signaling pathway that involves the adaptor protein TRIF activates type I interferon (IFN) and proinflammatory cytokine expression. Little is known about how TRIF pathway-dependent gene expression is regulated. SH2-containing protein tyrosine phosphatase 2 (SHP-2) is a widely expressed cytoplasmic tyrosine phosphatase. Here we demonstrate that SHP-2 negatively regulated TLR4- and TLR3-activated IFN-beta production. SHP-2 inhibited TLR3-activated but not TLR2-, TLR7-, and TLR9-activated proinflammatory cytokine IL-6 and TNF-alpha production. SHP-2 inhibited poly(I:C)-induced cytokine production by a phosphatase activity-independent mechanism. C-terminal domain of SHP-2 directly bound TANK binding kinase (TBK1) by interacting with the kinase domain of TBK1. SHP-2 deficiency increased TBK1-activated IFN-beta and TNF-alpha expression. TBK1 knockdown inhibited poly(I:C)-induced IL-6 production in SHP-2-deficient cells. SHP-2 also inhibited poly(I:C)-induced activation of MAP kinase pathways. These results demonstrate that SHP-2 specifically negatively regulate TRIF-mediated gene expression in TLR signaling, partially through inhibiting TBK1-activated signal transduction.