Functional cloning of Src-like adapter protein-2 (SLAP-2), a novel inhibitor of antigen receptor signaling.

In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.     

Distinct signal thresholds for the unique antigen receptor-linked gene expression programs in mature and immature B cells.

Although it is well established that immature B lymphocytes are exquisitely sensitive to tolerance induction compared with their mature counterparts, the molecular basis for this difference is unknown. We demonstrate that signaling by B cell antigen receptors leads to distinct and mutually exclusive biologic responses in mature and immature B cells: upregulation of CD86, CD69, and MHC class II in mature cells and receptor editing in immature cells. These responses can be induced simply by elevation of intracellular free calcium levels, as occurs after receptor aggregation. Importantly, induction of immature B cell responses requires much smaller increases in intracellular free calcium than does induction of mature B cell responses. These differences in biologic response and sensitivity to intracellular free calcium likely contributes to selective elimination at the immature stage of even those B cells that express low affinity for self-antigens.     

A B cell receptor with two Igalpha cytoplasmic domains supports development of mature but anergic B cells

B cell receptor (BCR) signaling is mediated through immunoglobulin (Ig)alpha and Igbeta a membrane-bound heterodimer. Igalpha and Igbeta are redundant in their ability to support early B cell development, but their roles in mature B cells have not been defined. To examine the function of Igalpha-Igbeta in mature B cells in vivo we exchanged the cytoplasmic domain of Igalpha for the cytoplasmic domain of Igbeta by gene targeting (Igbetac-->alphac mice). Igbetac-->alphac B cells had lower levels of surface IgM and higher levels of BCR internalization than wild-type B cells. The mutant B cells were able to complete all stages of development and were long lived, but failed to differentiate into B1a cells. In addition, Igbetac-->alphac B cells showed decreased proliferative and Ca2+ responses to BCR stimulation in vitro, and were anergic to T-independent and -dependent antigens in vivo.     

A role for lipid rafts in B cell antigen receptor signaling and antigen targeting

The B cell antigen receptor (BCR) serves both to initiate signal transduction cascades and to target antigen for processing and presentation by MHC class II molecules. How these two BCR functions are coordinated is not known. Recently, sphingolipid- and cholesterol-rich plasma membrane lipid microdomains, termed lipid rafts, have been identified and proposed to function as platforms for both receptor signaling and membrane trafficking. Here we show that upon cross-linking, the BCR rapidly translocates into ganglioside G(M1)-enriched lipid rafts that contain the Src family kinase Lyn and exclude the phosphatase CD45R. Both Igalpha and Lyn in the lipid rafts become phosphorylated, and subsequently the BCR and a portion of G(M1) are targeted to the class II peptide loading compartment. Entry into lipid rafts, however, is not sufficient for targeting to the antigen processing compartments, as a mutant surface Ig containing a deletion of the cytoplasmic domain is constitutively present in rafts but when cross-linked does not internalize to the antigen processing compartment. Taken together, these results provide evidence for a role for lipid rafts in the initial steps of BCR signaling and antigen targeting.     

T淋巴细胞激活与凋亡的信号转导及其分子机制

研究发现,人T淋巴细胞抗原受体复合物(TCR/CD3)中,TCR负责接受抗原刺激,CD3负责转导抗原刺激信号。抗TCR或抗CD3抗体的刺激不仅可以激活T淋巴细胞,也可以导致胸腺细胞、激活后的或转化的T淋巴细胞凋亡,但其分子机制和信号转导途径还不清楚。本文着重介绍了近年来这方面的研究工作进展。首先,利用人CD3ε过量表达的转基因小鼠发现,CD3ε分子的过量表达可阻断胎鼠的胸腺细胞发育,而在小鼠出生后,促进小鼠胸腺细胞的凋亡,随着年龄增加,胸腺细胞凋亡的数目增多。进而采用基因定点突变和基因转移等分子生物学研究技术,构建了CD8胞外区与跨膜区和CD3ε胞内区的融合分子及其突变分子,转染CD8~-的T淋巴细胞后,建立了细胞凋亡模型。利用此细胞凋亡模型,发现T细胞激活和凋亡的信号转导均是通过CD3ε分子的胞内区免疫酪氨酸受体激活基序(ITAM)中的2个酪氨酸转导的,其中任何1个或2个酪氨酸突变,均可阻断细胞信号的转导;在T细胞凋亡的过程中,立早基因Nur77和细胞凋亡基因fas表达显著增加,但未发现fas配体表达增加;在T细胞激活过程中,3-磷脂酰肌醇激酶P85α亚基与CD3ε-ITAM中的2个酪氨酸特异性地结合,其中任何1个酪氨酸突变,均大大减低P85α与CD3ε-ITAM的结合能力;使用合成的CD3ε-ITAM多肽及其酪氨酸突变了的突变体     

Expression of Recombination Activating Genes in Germinal Center B Cells: Involvement of Interleukin 7 (IL-7) and the IL-7 Receptor

Mouse germinal center (GC) B cells have been shown to undergo secondary V(D)J (V, variable; D, diversity; J, joining) recombination (receptor editing) mediated by the reexpressed products of recombination activating gene (RAG)-1 and RAG-2. We show here that interleukin (IL)-7 as well as IL-4 was effective in inducing functional RAG products in mouse IgD⁺ B cells activated via CD40 in vitro. Blocking of the IL-7 receptor (IL-7R) by injecting an anti– IL-7R monoclonal antibody resulted in a marked suppression of the reexpression of RAG-2 and subsequent V(D)J recombination in the draining lymph node of immunized mice, whereas RAG-2 expression was not impaired in immunized IL-4–deficient mice. Further, these peripheral B cells activated in vitro or in vivo were found to express IL-7R. These findings indicate a novel role for IL-7 and IL-7R in inducing receptor editing in GC B cells.     

嵌合抗原受体T细胞用于系统性红斑狼疮治疗的策略与研究进展

系统性红斑狼疮(SLE)是一种以产生一系列自身抗体为特征的慢性自身免疫性疾病。目前,SLE尚不能根治,糖皮质激素等药物治疗是临床治疗SLE的主要方法,但会产生严重的不良反应。近年来,嵌合抗原受体T细胞(CAR-T)免疫疗法作为血液肿瘤的新型治疗方法已被证实效果显著。CAR-T细胞通过识别结合SLE中分泌自身抗体的B细胞的特异性表面标志进行研究设计,以精准杀伤上述“坏”B细胞,达到减少甚至避免自身抗体产生的目的,从而改善患者病情。本文将从B细胞表面标志物出发,探讨CAR-T细胞用于治疗SLE的机制及研究进展,为探索SLE治愈性方法提供新的思路。     

A dual role for Src homology 2 domain-containing inositol-5-phosphatase (SHIP) in immunity: aberrant development and enhanced function of b lymphocytes in ship -/- mice

In this report, we demonstrate that the Src homology 2 domain-containing inositol-5-phosphatase (SHIP) plays a critical role in regulating both B cell development and responsiveness to antigen stimulation. SHIP(-/-) mice exhibit a transplantable alteration in B lymphoid development that results in reduced numbers of precursor B (fraction C) and immature B cells in the bone marrow. In vitro, purified SHIP(-/)- B cells exhibit enhanced proliferation in response to B cell receptor stimulation in both the presence and absence of Fcgamma receptor IIB coligation. This enhancement is associated with increased phosphorylation of both mitogen-activated protein kinase and Akt, as well as with increased survival and cell cycling. SHIP(-/)- mice manifest elevated serum immunoglobulin (Ig) levels and an exaggerated IgG response to the T cell-independent type 2 antigen trinitrophenyl Ficoll. However, only altered B cell development was apparent upon transplantation into nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice. The in vitro hyperresponsiveness, together with the in vivo findings, suggests that SHIP regulates B lymphoid development and antigen responsiveness by both intrinsic and extrinsic mechanisms.     

谷氨酰胺对梗阻性黄疸大鼠可溶性白细胞介素2受体的影响

目的探讨免疫营养剂谷氨酰胺(Glutamine,Gln)对梗阻性黄疸大鼠可溶性白细胞介素2受体(sIL-2R)的影响。方法90只大鼠随机分成对照组(C组)、梗阻性黄疸组(J组)和谷氨酰胺干预组(G组)各30只,于术后5周抽尽腔静脉血测定血清sIL-2R。结果J组与G组sIL-2R水平较C组明显升高(P<0·01),G组sIL-2R水平较J组明显降低,差异显著(P<0·01)。结论谷氨酰胺能够降低梗阻性黄疸大鼠sIL-2R水平,改善机体免疫状态。     

血液透析对慢性肾功能衰竭患者血清SIL2R、IL6和TNFα的影响

目的：探讨慢性肾功能不全（ＣＲＦ）患者细胞免疫功能状况,以及血液透析（血透）的影响。方法：应用酶联免疫吸附（ＥＬＩＳＡ）双抗体夹心法检测３０例ＣＲＦ未透析患者和６２例血透患者血透前后及对照组血清可溶性白介素－α受体（ＳＩＬ－２Ｒ）、白介素－６（ＩＬ－６）和肿瘤坏死因子－α（ＴＮＦ－α）水平。结果：与对照组相比,ＣＲＦ未透析患者和血透患者血清ＳＩＬ－２Ｒ、ＩＬ－６和ＴＮＦ－α水平无显著差异。血清ＳＩＬ－２Ｒ较透前显著增高。结论：ＣＲＦ患者均存在显著的细胞免疫功能紊乱,单次透析对细胞免疫功能无显著影响。     

T helper type 2 cell differentiation occurs in the presence of interleukin 12 receptor beta2 chain expression and signaling

The differentiation of CD4(+) T cells into T helper type 1 (Th1) cells is driven by interleukin (IL)-12 through the IL-12 receptor beta2 (IL-12Rbeta2) chain, whereas differentiation into Th2 cells is driven by IL-4, which downregulates IL-12Rbeta2 chain. We reexamined such differentiation using IL-12Rbeta2 chain transgenic mice. We found that CD4(+) T cells from such mice were able to differentiate into Th2 cells when primed with IL-4 or IL-4 plus IL-12. In the latter case, the presence of IL-4 suppressed interferon (IFN)-gamma production 10-100-fold compared with cells cultured in IL-12 alone. Finally, in studies of the ability of IL-12 to convert Th2 cells bearing a competent IL-12R to the Th1 cells, we showed that: (a) T cells bearing the IL-12Rbeta2 chain transgene and primed under Th2 conditions could not be converted to Th1 cells by repeated restimulation under Th1 conditions; and (b) established Th2 clones transfected with the IL-12Rbeta2 chain construct continued to produce IL-4 when cultured with IL-12. These studies show that IL-4-driven Th2 differentiation can occur in the presence of persistent IL-12 signaling and that IL-4 inhibits IFN-gamma production under these circumstances. They also show that established Th2 cells cannot be converted to Th1 cells via IL-12 signaling.     

急性中毒患者血清可溶性白细胞介素2受体检测的临床意义

目的 探讨血清可溶性白细胞介素2受体(sIL-2R)在急性中毒中的临床意义。方法采用 ELISA 法检测80例急性中毒患者(镇静催眠药、乙醇、一氧化碳及农业杀虫剂各20例)及20例正常人血清 sIL-2R。结果 急性乙醇中毒组(AL 组)与急性一氧化碳组(CO 组)血清 sIL-2R 均高于正常对照组(P<0.01),急性镇静催眠药中毒组(SH 组)、急性农业杀虫剂中毒组(FI 组)与对照组比较差异无显著性(P>0.05);AL 组与 CO 组血清 sIL-2R 均高于 SH 组(P<0.05),余各中毒组间差异无显著性(P>0.05);AL 组治疗后 sIL-2R 低于治疗前(P<0.05),CO 组治疗后 sIL-2R明显低于治疗前(P<0.01),SH 组与 FI 组治疗后 sIL-2R 与治疗前差异无显著性(P>0.95)。结论急性乙醇中毒与急性一氧化碳中毒均可致 sIL-2R 增高。经用糖皮质激素治疗后 sIL-2R 降低较非糖皮质激素治疗更明显。故糖皮质激素治疗有免疫异常的急性中毒尤其是无特效解毒剂治疗的急性中毒是切实可行而有效的。     

T cell receptor engagement leads to phosphorylation of clathrin heavy chain during receptor internalization

T cell receptor (TCR) internalization by clathrin-coated vesicles after encounter with antigen has been implicated in the regulation of T cell responses. We demonstrate that TCR internalization after receptor engagement and TCR signaling involves inducible phosphorylation of clathrin heavy chain (CHC) in both CD4+ and CD8+ human T cells. Studies with mutant Jurkat T cells implicate the Src family kinase Lck as the responsible enzyme and its activity in this process is influenced by the functional integrity of the downstream signaling molecule ZAP-70. CHC phosphorylation positively correlates with ligand-induced TCR internalization in both CD4+ and CD8+ T cells, and CHC phosphorylation as a result of basal Lck activity is also implicated in constitutive TCR endocytosis by CD4+ T cells. Remarkably, irreversible CHC phosphorylation in the presence of pervanadate reduced both constitutive and ligand-induced TCR internalization in CD4+ T cells, and immunofluorescence studies revealed that this inhibition affected the early stages of TCR endocytosis from the plasma membrane. Thus, we propose that CHC phosphorylation and dephosphorylation are involved in TCR internalization and that this is a regulatory mechanism linking TCR signaling to endocytosis.     

血清可溶性白细胞介素-2受体水平在预测高血压急性脑出血手术治疗中的意义

目的：探讨血清可溶性白细胞介素２受体（ＳＩＬ２Ｒ）水平在预测高血压急性脑出血（ＨＡＣＨ）患者手术治疗中的意义。方法：采用双抗体夹心酶联免疫吸附法测定２０例正常人及４９例ＨＡＣＨ急性期手术和非手术患者血清ＳＩＬ２Ｒ含量。结果：ＨＡＣＨ患者入院当日血清中ＳＩＬ２Ｒ含量〔（０．５９２±０．０３７）Ｕ／Ｌ〕显著高于正常对照组〔（０．２３７±０．０６９）Ｕ／Ｌ〕；手术组患者术前与术后第１天和第１０天血清ＳＩＬ２Ｒ含量比较分别为无显著性差异（Ｐ＞０．０５）和有显著性差异（Ｐ＜０．０１）；手术组患者术后第１日和第１０日与非手术组患者血清ＳＩＬ２Ｒ含量同步比较分别为无显著性差异（Ｐ＞０．０５）和有显著性差异（Ｐ＜０．０５）；死亡患者入院当日血清ＳＩＬ２Ｒ含量〔（０．７４３±０．０４９）Ｕ／Ｌ〕高于存活组〔（０．５９８±０．０４４）Ｕ／Ｌ，Ｐ＜０．０１〕，且显著高于对照组〔０．２３７±０．０６８）Ｕ／Ｌ，Ｐ＜０．０１〕。结论：ＳＩＬ２Ｒ与ＨＡＣＨ患者免疫功能紊乱有关，动态测定血清ＳＩＬ２Ｒ含量，可作为观察ＨＡＣＨ病情发展变化的一项指标，同时对判断ＨＡＣＨ的预后亦有一定临床意义。     

B7-1 Engagement of Cytotoxic T Lymphocyte Antigen 4 Inhibits T Cell Activation in the Absence of CD28

Ligation of cytotoxic T lymphocyte antigen 4 (CTLA4) appears to inhibit T cell responses. Four mechanisms have been proposed to explain the inhibitory activity of CTLA4: competition for B7-1 and B7-2 binding by CD28; sequestration of signaling molecules away from CD28 via endocytosis; delivery of a signal that antagonizes a CD28 signal; and delivery of a signal that antagonizes a T cell receptor (TCR) signal. As three of these potential mechanisms involve functional antagonism of CD28, an experimental model was designed to determine whether CTLA4 could inhibit T cell function in the absence of CD28. TCR transgenic/recombinase activating gene 2–deficient/CD28–wild-type or CD28-deficient mice were generated and immunized with an antigen-expressing tumor. Primed T cells from both types of mice produced cytokines and proliferated in response to stimulator cells lacking B7 expression. However, whereas the response of CD28^+/+ T cells was augmented by costimulation with B7-1, the response of the CD28^−/− T cells was strongly inhibited. This inhibition was reversed by monoclonal antibody against B7-1 or CTLA4. Thus, CTLA4 can potently inhibit T cell activation in the absence of CD28, indicating that antagonism of a TCR-mediated signal is sufficient to explain the inhibitory effect of CTLA4.     

Hepatitis B surface antigen escape mutations: Indications for initiation of antiviral therapy revisited

Approximately 240 million people are chronically infected with hepatitis B. The implementation of rigorous vaccination programs has led to an overall decrease in the prevalence of this disease worldwide but this may also have led to emergence of viral mutations that can escape the protection of hepatitis B surface antibody. As this phenomenon is increasingly recognized, concern for transmission to vaccinated individuals has also been raised. Herein, we describe two cases where the suspected presence of a hepatitis B surface antigen escape mutation impacted the decision to initiate early antiviral therapy, as well as provide a brief review of these mutations. Our findings described here suggest that a lower threshold for initiating therapy in these individuals should be considered in order to reduce the risk of transmission, as vaccination does not provide protection.