Selection of an antibody library identifies a pathway to induce immunity by targeting CD36 on steady-state CD8 alpha+ dendritic cells

Improvement of the strategy to target tumor Ags to dendritic cells (DCs) for immunotherapy requires the identification of the most appropriate ligand/receptor pairing. We screened a library of Ab fragments on mouse DCs to isolate new potential Abs capable of inducing protective immune responses. The screening identified a high-affinity Ab against CD36, a multi-ligand scavenger receptor primarily expressed by the CD8alpha+ subset of conventional DCs. The Ab variable regions were genetically linked to the model Ag OVA and tested in Ag presentation assays in vitro and in vivo. Anti-CD36-OVA was capable of delivering exogenous Ags to the MHC class I and MHC class II processing pathways. In vivo, immunization with anti-CD36-OVA induced robust activation of naive CD4+ and CD8+ Ag-specific T lymphocytes and the differentiation of primed CD8+ T cells into long-term effector CTLs. Vaccination with anti-CD36-OVA elicited humoral and cell-mediated protection from the growth of an Ag-specific tumor. Notably, the relative efficacy of targeting CD11c/CD8alpha+ via CD36 or DEC205 was qualitatively different. Anti-DEC205-OVA was more efficient than anti-CD36-OVA in inducing early events of naive CD8+ T cell activation. In contrast, long-term persistence of effector CTLs was stronger following immunization with anti-CD36-OVA and did not require the addition of exogenous maturation stimuli. The results identify CD36 as a novel potential target for immunotherapy and indicate that the outcome of the immune responses vary by targeting different receptors on CD8alpha+ DCs.     

Expression regulation and function of NLRC5

The NOD like receptors (NLRs), a class of intracellular receptors that respond to pathogen attack or cellular stress, have gained increasing attention. NLRC5, the largest member of the NLR protein family, has recently been identified as a critical regulator of immune responses. While NLRC5 is constitutively and widely expressed, it can be dramatically induced by interferons during pathogen infections. Both in vitro and in vivo studies have demonstrated that NLRC5 is a specifi c and master regulator of major mistocompatibility complex (MHC) class I genes as well as related genes involved in MHC class I antigen presentation. The expression of MHC class I genes is regulated by NLRC5 in coordination with the RFX components through an enhanceosome-dependent manner. And the involvement of NLRC5 in MHC class I mediated CD8⁺ T cell activation, proliferation and cytotoxicity is proved to be critical for host defense against intracellular bacterial infections. Nevertheless, the role of NLRC5 in innate immunity remains to be further explored. Here, we review the research advances on the structure, expression regulation and function of NLRC5.     

Characterization of pancreatic somatostatin binding sites with a I-somatostatin 28 analog

Somatostatin binding to guinea pig pancreatic acinar cell plasma membranes was characterized with an iodinated stable analog of somatostatin 28 (S₂₈): ¹²⁵I-[Leu⁸, DTrp²²,Tyr²⁵] S₂₈. The binding was highly dependent on calcium ions. In 0.2 mM free Ca²⁺ medium, binding at 37°C was saturable, slowly reversible and exhibited a single class of high affinity binding sites (K_D=0.05±0.01 nM, B_max=157±33 fmol/mg protein). Dissociation of bound radioactivity occurred with biphasic kinetics. Rate of dissociation increased when dissociation was measured at a time before equilibrium binding was reached. In 30 nM free Ca²⁺ medium, binding affinity and maximal binding capacity were decreased by about 4-fold. Decreasing calcium concentrations increased the amount of rapidly dissociating form of the receptor. Somatostatin 14 antagonist, Des AA^1,2[AzaAla^4–5,DTrp⁸,Phe^12–13]-somatostatin was active at the membrane level in inhibiting the binding. We conclude that using ¹²⁵I-[Leu⁸,DTrp²²,Tyr²⁵]S₂₈ as radioligand allows us to characterize a population of specific somatostatin receptors which are not different from those we previously described with the radioligand ¹²⁵I-[Tyr¹¹]-somatostatin. Somatostatin receptors could exist in two interconvertible forms. Calcium ions are an essential component in the regulation of the conformational change of somatostatin receptors.     

Characterization of the novel CCK analogs JMV-180, JMV-320, and JMV-332 in H345 cells

The interaction of the novel CCK analogs JMV-180, JMV-320, and JMV-332 with CCK-B/gastrin receptors on small cell lung cancer (SCLC) cells was investigated. JMV-180, JMV-320, and JMV-332 potently inhibited specific binding of ¹²⁵I-CCK-8 to CCK-B/gastrin receptors expressed on the SCLC cell line NCI-H345 (H345) with IC₅₀ values of 4.9, 1.8, and 7.0 nM, respectively. JMV-320 and JMV-332 stimulated intracellular calcium ([Ca²⁺]_i) release in a dose-dependent manner in cells preloaded with indo-1. JMV-180 did not stimulate [Ca²⁺]_i but inhibited the [Ca²⁺]_i release elicited by 10 nM CCK-8 in a dose-dependent manner. These data indicate that JMV-320 and JMV-332 function as CCK-B/gastrin receptor agonists while JMV-180 functions as a CCK-B/gastrin receptor antagonist in H345 cells.     

小檗碱对大鼠脑缺血/再灌注损伤的保护作用及免疫机制*

目的:探讨小檗碱对大鼠脑缺血/再灌注损伤的保护作用及免疫机制。方法:50只SD大鼠随机分为假手术组(Sham group)、模型组(Model group)、小檗碱低剂量组(BBR-L,25 mg/kg)、小檗碱中剂量组(BBR-M,50 mg/kg)、小檗碱高剂量组(BBR-H,100 mg/kg),每组各10只。采用Longa线栓法建立脑缺血/再灌注大鼠模型,缺血2 h后再灌注24 h处理。于造模成功2 h后灌胃给药,假手术组和模型组组按上述方法同体积给予生理盐水。给药24 h后,测定各组大鼠神经功能缺损程度评分及脑梗死率;采用ELISA法检测抗氧化酶SOD和GSH-Px的活性、细胞因子TNF-α、IFN-β、IL-6和NO的含量;采用流式细胞术检测CD4⁺、CD8⁺及CD4⁺/CD8⁺血清含量;进一步采用RT-qPCR与Western blot技术检测大鼠脑组织内NF-κB-NLRP3信号轴关键基因及蛋白的表达情况。结果:与假手术组比较,模型组大鼠神经功能缺损程度、脑梗死率均升高(P＜0.05),且血清NO、TNF-α、IFN-β、IL-6含量和脑组织的NF-κB p65、NLRP3、ASC及caspase-1基因与蛋白表达水平均升高(P＜0.05),而血清中SOD、GSH-Px活性和CD4⁺、CD8⁺及CD4⁺/CD8⁺水平下降(P＜0.05);与模型组比较,BBR-H、BBR-M、BBR-L组大鼠神经功能缺损程度、脑梗死率均下降(P＜0.05),且血清NO、TNF-α、IFN-β、IL-6含量和脑组织的NF-κB p65、NLRP3、ASC及caspase-1基因与蛋白表达水平均降低(P＜0.05),而血清中SOD、GSH-Px活性和CD4⁺、CD8⁺及CD4⁺/CD8⁺水平升高(P＜0.05)。结论:小檗碱可通过减轻氧化应激,抑制炎症反应,增强免疫功能,减轻大鼠脑缺血/再灌注损伤,其机制可能与抑制NF-κB-NLRP3信号有关。     

口腔鳞状细胞癌患者外周血Naa10及淋巴细胞免疫分型的特点及临床意义

目的探讨口腔鳞状细胞癌（OSCC）患者外周血N-α-乙酰基转移酶10 （Naa10）及淋巴细胞免疫分型的特点,并分析其临床意义。 方法选取97例OSCC患者及50名健康体检者,分别纳入患者组、对照组。检测两组受试者外周血Naa10、淋巴细胞免疫分型,并比较不同TNM分期、不同淋巴结转移状态患者外周血Naa10、淋巴细胞免疫分型的差异,总结其特点与临床意义。不同性别人数采用χ²检验,计量资料行正态性检验,满足正态性且组间方差相等则采用t检验,不满足正态性则采用非参数Wilcoxon秩和检验,相关性分析采用Pearson法。 结果患者组血清Naa10为（62.91±17.15）pg/ml,高于对照组的（38.82±9.04）pg/ml,差异有统计学意义（t?= 9.280,P?< 0.05）。患者组CD3⁺、CD4⁺、CD4⁺/CD8⁺、NK细胞百分比分别为（71.63±9.11）﹪、（34.34±4.26）﹪、（1.03±0.28）、（14.23±4.61）﹪,低于对照组的（76.25±2.87）﹪、（45.41±7.08）﹪、（1.77± 0.39）、（19.96±5.13）﹪,CD8⁺、B淋巴细胞百分比分别为（14.23±4.61）﹪、（14.91±3.29）﹪,高于后者的（19.96±5.13）﹪、（9.11±2.60）﹪,差异有统计学意义（P?< 0.05）。TNM分期Ⅰ~Ⅱ期患者42例,其血清Naa10为（64.08±15.26）?pg/?ml,与Ⅲ~Ⅳ期患者的（61.71±13.15）pg/ml比较,差异无统计学意义（t?= 0.820,P?> 0.05）。淋巴结转移者34例,其血清Naa10为（65.15±14.07）pg/ml,与无淋巴结转移者的（61.45±12.66）?pg/?ml比较,差异无统计学意义（t?= 1.321,P?> 0.05）。Ⅰ~Ⅱ期患者CD3⁺、CD4⁺、CD4⁺/CD8⁺、NK细胞百分比低于Ⅲ~Ⅳ期患者,其CD8⁺、B淋巴细胞百分比高于后者,差异有统计学意义（P?< 0.05）。Pearson相关性分析示,外周血Naa10与CD3⁺ （-0.631）、CD4⁺ （-0.529）、CD4⁺/CD8⁺ （-0.587）、NK细胞百分比（-0.603）呈负相关,与CD8⁺ （0.558）、B淋巴细胞百分比（0.670）呈正相关（P?< 0.05）。 结论OSCC患者外周血Naa10明显升高但与TNM分期、淋巴结转移无关;其淋巴细胞免疫分型存在明显改变,且TNM分期的上升、淋巴结转移的发生伴随着CD3⁺、CD4⁺、CD4⁺/?CD8⁺、NK细胞百分比的下降,以及CD8⁺、B淋巴细胞百分比的上升。     

唾液酸免疫球蛋白型凝集素-15促进结直肠癌细胞增殖和迁移并抑制肿瘤组织中CD4+与CD8+T细胞浸润

唾液酸免疫球蛋白型凝集素-15(sialic acid-binding immunoglobulin-type lectin-15,Siglec-15)属于Siglecs家族的一员,是一种新型免疫抑制分子。Siglec-15在多种人类肿瘤细胞和肿瘤相关巨噬细胞中高表达,但Siglec-15在结直肠癌(colorectal cancer,CRC)中的生物学功能及其对免疫微环境的影响尚不明确。本文旨在分析Siglec-15异常表达对CRC细胞功能及CD4⁺T细胞、CD8⁺T细胞浸润的影响。首先,分析TCGA数据库中结直肠癌与正常组织中Siglec-15 mRNA表达水平,并对52例人CRC与配对癌旁组织进行免疫组织化学染色(IHC),发现Siglec-15在CRC中的表达水平高于癌旁组织(P<0.01)。CCK8和划痕愈合结果显示,敲低Siglec-15能抑制人CRC细胞SW480增殖(P<0.01)和迁移(P<0.05)。磁珠分选小鼠脾的CD8⁺T细胞并与小鼠CRC细胞MC38共培养,发现MC38细胞过表达Siglec-15能抑制CD8⁺T细胞对其的杀伤以及IFN-γ和TNF-α的分泌(P<0.01)。小鼠荷瘤结果表明,过表达Siglec-15可以促进小鼠肿瘤生长(P<0.05)。单样本基因集富集分析、荷瘤小鼠肿瘤组织及人结直肠癌组织IHC分析均表明,Siglec-15高表达时,肿瘤微环境中CD4⁺T细胞、CD8⁺T细胞浸润减少(P<0.05)。综上所述,Siglec-15可能通过促进CRC细胞增殖迁移以及抑制CD4⁺T细胞、CD8⁺T细胞浸润促进结直肠癌进展。本文为探究Siglec-15在CRC中的免疫抑制作用提供了一些新的实验依据。     

Mapping the binding site on CD8 beta for MHC class I reveals mutants with enhanced binding

In an effective immune response, CD8+ T cell recognition of virally derived Ag, bound to MHC class I, results in killing of infected cells. The CD8alphabeta heterodimer acts as a coreceptor with the TCR, to enhance sensitivity of the T cells to peptide/MHC class I, and is two orders of magnitude more efficient as a coreceptor than the CD8alphaalpha. To understand the important interaction between CD8alphabeta and MHC class I, we created a panel of CD8beta mutants and identified mutations in the CDR1, CDR2, and CDR3 loops that decreased binding to MHC class I tetramers as well as mutations that enhanced binding. We tested the coreceptor function of a subset of reducing and enhancing mutants using a T cell hybridoma and found similar reducing and enhancing effects. CD8beta-enhancing mutants could be useful for immunotherapy by transduction into T cells to enhance T cell responses against weak Ags such as those expressed by tumors. We also addressed the question of the orientation of CD8alphabeta with MHC class I using CD8alpha mutants expressed as a heterodimer with wild-type CD8alpha or CD8beta. The partial rescuing of binding with wild-type CD8beta compared with wild-type CD8alpha is consistent with models in which either the topology of CD8alphaalpha and CD8alphabeta binding to MHC class I is different or CD8alphabeta is capable of binding in both the T cell membrane proximal and distal positions.     

恶性血液病患者外周血淋巴细胞亚群变化及其临床意义的研究

目的: 探讨恶性血液病外周血淋巴细胞亚群变化特征及临床意义。方法: 采用流式细胞仪检测64例初诊的血液系统恶性肿瘤患者的外周血淋巴细胞亚群。病种包括急性髓系白血病(acute myeloid leukemia,AML)、急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)、霍奇金淋巴瘤(Hodgkin’s lymphoma,HL)、非霍奇金淋巴瘤(Non-Hodgkinlymphoma,NHL)。分析比较30例正常人的外周血淋巴细胞亚群与实验组的差异,并对64例恶性血液病患者中连续动态监测的21例急性白血病患者外周血淋巴细胞亚群结果变化与预后关系进行分析。结果： 不同成人恶性血液病患者年龄分组淋巴细胞亚群变化无明显差异;恶性血液病患者中CD3 ⁺CD8 ⁺ T淋巴细胞百分比、Treg细胞百分比均增加;CD16 ⁺/CD56 ⁺NK细胞百分比及CD4 ⁺/CD8 ⁺比值均下降;CD3 ⁺T淋巴细胞数量、CD3 ⁺CD4 ⁺淋巴细胞数、CD3 ⁺CD8 ⁺淋巴细胞数量、CD3 ^-CD19 ⁺淋巴细胞数量、CD16 ⁺/CD56 ⁺NK淋巴细胞数量及CD4 ⁺/CD8 ⁺比值均减少;急性白血病及恶性淋巴瘤患者外周血淋巴细胞亚群与正常对照组比较存在一定的差异;急性白血病未缓解组的Treg细胞比例明显高于急性白血病首疗程缓解组及对照组;急性白血病复发组Treg细胞比例明显高于急性白血病持续缓解组以及对照组;对21例急性白血病患者动态监测的淋巴细胞亚群发现,化疗缓解的患者Treg在化疗过程中逐渐下降,至第3~6个疗程逐渐接近正常对照,化疗未缓解的患者Treg细胞在化疗过程中逐渐上升或持续大于10%,明显高于完全缓解组,复发患者Treg在化疗过程中先下降后明显上升。 结论： 恶性血液病患者免疫功能显著低于健康人,且伴随免疫功能紊乱,且不同疾病类型、不同的疾病状态免疫紊乱的程度不一,Treg细胞比例可以用来预测急性白血病患者疗效及复发,可以为患者的临床治疗方案及用药强度提供指导依据。     

成人t(8;21)急性髓系白血病患者初诊Ki-67的表达特征及预后意义

目的:研究成人t(8; 21)急性髓系白血病(AML)初诊Ki-67抗原的表达特征及预后意义。方法:采集2012年7月至2019年2月本院57例成人初诊t(8; 21) AML患者的新鲜骨髓标本,采用流式细胞术(FCM)检测CD34和Ki-67抗原,分析Ki-67表达与患者初诊生物学特征、疗效及复发的关系。结果:全部患者中,CD34~+Ki-67~+细胞比例的中位值为30. 5%(范围:10. 0%～65. 8%);通过受试者工作特征(ROC)曲线确定CD34~+Ki-67~+细胞比例的最适分界阈值,CD34~+Ki-67~+细胞高比例与初诊c-KIT基因突变阳性及WT1转录本低水平均明显相关(P=0. 001; P=0. 042)。随访的36例患者中,CD34~+Ki-67~+高比例比低比例患者具有明显更高的1年累积复发(CIR)率(P=0. 035);此外,初诊WT1转录本低水平和微小残留病(MRD)高水平(2个疗程巩固治疗后RUNX1-RUNX1T1转录本水平下降3-log)均与更高的1年CIR率明显相关(P 0. 0001;P=0. 041),初诊c-KIT基因突变阳性和白细胞计数 10×109/L的患者分别有较高的1年CIR率趋势(P=0. 091; P=0. 054)。联合分组显示,MRD高水平同时CD34~+Ki-67~+细胞高比例的患者比其他患者具有明显更高的1年CIR率(P 0. 0001)。结论:初诊骨髓高比例的CD34~+Ki-67~+可能是成人t(8; 21) AML患者预后不良因素,MRD联合初诊CD34~+Ki-67~+细胞比例可能比单纯MRD更好地预测复发。     

The beta1 and beta3 integrins promote T cell receptor-mediated cytotoxic T lymphocyte activation

Recognition by CD8+ cytotoxic T lymphocytes (CTLs) of antigenic peptides bound to major histocompatibility class (MHC) I molecules on target cells leads to sustained calcium mobilization and CTL degranulation resulting in perforin-dependent killing. We report that beta1 and beta3 integrin-mediated adhesion to extracellular matrix proteins on target cells and/or surfaces dramatically promotes CTL degranulation. CTLs, when adhered to fibronectin but not CTL in suspension, efficiently degranulate upon exposure to soluble MHC.peptide complexes, even monomeric ones. This adhesion induces recruitment and activation of the focal adhesion kinase Pyk2, the cytoskeleton linker paxillin, and the Src kinases Lck and Fyn in the contact site. The T cell receptor, by association with Pyk2, becomes part of this adhesion-induced activation cluster, which greatly increases its signaling.     

Specific receptors for adrenomedullin in cultured rat vascular smooth muscle cells

The effects of synthetic rat adrenomedullin (rAM), a novel vasorelaxant peptide originally isolated from human pheochromocytoma, on receptor binding and cAMP generation were studied in cultured rat vascular smooth muscle cells (VSMC). A binding study using [¹²⁵I]rAM revealed the presence of a single class of high-affinity (K_d1.3 × 10⁻⁸ M) binding sites for rAM in VSMC. The apparent K_i of rat calcitonin gene-related peptide (rCGRP) was 3 × 10⁻⁷ M. Affinity labeling of VSMC membranes with [¹²⁵I]rAM revealed two distinct labeled bands with apparent molecular weights of 120 and 70 kDa, both of which were abolished by excess unlabeled rAM or rCGRP. rAM stimulated cAMP formation with an approximate EC₅₀ of 10⁻⁸ M, the effect of which was additive with isoproterenol, but not with rCGRP. The rAM-induced cAMP response was unaffected by propranalol, indomethacin, or quinaerine, but inhibited by a CGRP receptor antagonist, human CGRP[8–37]. These data suggest that VSMC possesses specific AM receptors functionally coupled to adenylate cyclase with which CGRP interacts.     

Different T cell receptor affinity thresholds and CD8 coreceptor dependence govern cytotoxic T lymphocyte activation and tetramer binding properties

T cells have evolved a unique system of ligand recognition involving an antigen T cell receptor (TCR) and a coreceptor that integrate stimuli provided by the engagement of peptide-major histocompatibility complex (pMHC) antigens. Here, we use altered pMHC class I (pMHCI) molecules with impaired CD8 binding (CD8-null) to quantify the contribution of coreceptor extracellular binding to (i) the engagement of soluble tetrameric pMHCI molecules, (ii) the kinetics of TCR/pMHCI interactions on live cytotoxic T lymphocytes (CTLs), and (iii) the activation of CTLs by cell-surface antigenic determinants. Our data indicate that the CD8 coreceptor substantially enhances binding efficiency at suboptimal TCR/pMHCI affinities through effects on both association and dissociation rates. Interestingly, coreceptor requirements for efficient tetramer labeling of CTLs or for CTL activation by determinants displayed on the cell surface operated in different TCR/pMHCI affinity ranges. Wild-type and CD8-null pMHCI tetramers required monomeric affinities for cognate TCRs of KD < approximately 80 microM and approximately 35 microM, respectively, to label human CTLs at 37 degrees C. In contrast, activation by cellular pMHCI molecules was strictly dependent on CD8 binding only for TCR/pMHCI interactions with KD values >200 microM. Altogether, our data provide information on the binding interplay between CD8 and the TCR and support a model of CTL activation in which the extent of coreceptor dependence is inversely correlated to TCR/pMHCI affinity. In addition, the results reported here define the range of TCR/pMHCI affinities required for the detection of antigen-specific CTLs by flow cytometry.     

Murine CD160, Ig-like receptor on NK cells and NKT cells, recognizes classical and nonclassical MHC class I and regulates NK cell activation

Human and mouse NK cells use different families of receptors to recognize MHC class I (MHC I) on target cells. Although human NK cells express both Ig-like receptors and lectin-like receptors specific for MHC I, all the MHC I-specific receptors identified on mouse NK cells to date are lectin-like receptors, and no Ig-like receptors recognizing MHC I have been identified on mouse NK cells. In this study we report the first MHC I-specific Ig-like receptor on mouse NK cells, namely, murine CD160 (mCD160). The expression of mCD160 is restricted to a subset of NK cells, NK1.1+ T cells, and activated CD8+ T cells. The mCD160-Ig fusion protein binds to rat cell lines transfected with classical and nonclassical mouse MHC I, including CD1d. Furthermore, the level of mCD160 on NK1.1+ T cells is modulated by MHC I of the host. Overexpression of mCD160 in the mouse NK cell line KY-2 inhibits IFN-gamma production induced by phorbol ester plus ionomycin, whereas it enhances IFN-gamma production induced by NK1.1 cross-linking or incubation with dendritic cells. Cross-linking of mCD160 also inhibits anti-NK1.1-mediated stimulation of KY-2 cells. Anti-mCD160 mAb alone has no effect. Thus, mCD160, the first MHC I-specific Ig-like receptor on mouse NK cells, regulates NK cell activation both positively and negatively, depending on the stimulus.     

外周血T淋巴细胞亚群及早期炎症标志物表达对老年非小细胞肺癌化疗预后的影响分析

目的评估外周血T淋巴细胞亚群及早期炎症标志物表达对老年非小细胞肺癌(NSCLC)化疗预后的影响。方法入选2015年1月至2018年12月在武汉科技大学附属孝感医院接受化疗的NSCLC患者86例为NSCLC组,另选本院常规体检的健康人群82名作为对照组,分析两组人群外周血T淋巴细胞亚群与炎症细胞的分布差异。采用Kaplan-Meier单因素生存分析不同临床参数及化疗后不同T细胞亚群和炎症细胞水平患者的生存期,采用Cox比例回归风险模型分析影响NSCLC患者预后的独立影响因素。结果与对照组比较,NSCLC组患者CD3^+(71.31±6.02比68.22±7.09)、CD4^+(40.20±5.79比36.61±7.11)、CD4^+/CD8+(1.49±0.37比1.30±0.56),CD8+(28.43±6.37比31.79±9.88)均降低,而PLT(229.73±58.84比211.32±54.18)、淋巴细胞计数(LY)(1.67±0.61比30.01±8.45)及淋巴细胞百分比(LY﹪)(25.65﹪±6.87﹪比30.01﹪±8.45﹪)均升高,差异具有统计学意义(P均<0.05)。Kaplan-Meier单因素生存分析结果显示,是否淋巴转移、远处转移及不同TNM分期的患者生存期相比,差异具有统计学意义(P均<0.05);CD8+T细胞≥31.8﹪、CD4/CD8<1.28、中性粒细胞与淋巴细胞比值(NLR)<3.16及血小板与淋巴细胞比值(PLR)<197的患者生存期较长,差异具有统计学意义(P均<0.05);Cox多因素生存分析结果显示,伴远处转移(HR=9.310)、TNM分期ⅢB-Ⅳ期(HR=1.059)、CD8+T细胞<31.8﹪(HR=2.697)、NLR≥3.16(HR=1.887)及PLR≥197(HR=2.869)是影响老年NSCLC患者预后的独立影响因素(P均<0.05)。结论外周血CD8+T细胞、CD4/CD8比值、NLR及PLR水平是影响老年NSCLC预后的独立影响因素,可作为评估老年NSCLC患者化疗预后的简易生物标志物。     

Novel exosome-targeted CD4+ T cell vaccine counteracting CD4+25+ regulatory T cell-mediated immune suppression and stimulating efficient central memory CD8+ CTL responses

T cell-to-T cell Ag presentation is increasingly attracting attention. In this study, we demonstrated that active CD4+ T (aT) cells with uptake of OVA-pulsed dendritic cell-derived exosome (EXO(OVA)) express exosomal peptide/MHC class I and costimulatory molecules. These EXO(OVA)-uptaken (targeted) CD4+ aT cells can stimulate CD8+ T cell proliferation and differentiation into central memory CD8+ CTLs and induce more efficient in vivo antitumor immunity and long-term CD8+ T cell memory responses than OVA-pulsed dendritic cells. They can also counteract CD4+25+ regulatory T cell-mediated suppression of in vitro CD8+ T cell proliferation and in vivo CD8+ CTL responses and antitumor immunity. We further elucidate that the EXO(OVA)-uptaken (targeted)CD4+ aT cell's stimulatory effect is mediated via its IL-2 secretion and acquired exosomal CD80 costimulation and is specifically delivered to CD8+ T cells in vivo via acquired exosomal peptide/MHC class I complexes. Therefore, EXO-targeted active CD4+ T cell vaccine may represent a novel and highly effective vaccine strategy for inducing immune responses against not only tumors, but also other infectious diseases.     

Characterization of functional VIP/PACAP receptors in the human erythroleukemic HEL cell line

R. LEMA-KISOKA, N. HAYEZ, I. LANGER, P. ROBBERECHT, E. SARIBAN AND C. DELPORTE. Characterization of functional VIP/PACAP receptors in the human erythroleukemic HEL cell line. PEPTIDES. The presence of VIP/PACAP receptors was investigated on the human erythroleukemic cell line HEL. Specific binding of [¹²⁵I]-PACAP or [¹²⁵I]-VIP on HEL cells or membranes was very low and did not allow to perform competition curves. At 37°C PACAP transiently increased cAMP levels in the presence of the non-specific phosphodiesterase inhibitor IBMX, suggesting rapid desensitization. Kinetic studies revealed that optimal conditions to measure the EC₅₀ of PACAP(1–27) were 10 min at 20°C. Under those conditions, PACAP-related peptides increased cAMP levels with EC₅₀ in agreement with the pharmacological profile of the VPAC₁ receptor subtype: PACAP = VIP > [K¹⁵, R^16, L²⁷]VIP(1–7)/GRF(8–27) = [R¹⁶]ChSn (two VPAC₁ agonists) HELODERMIN = secretin. RO 25–1553, a selective activator of VPAC₂ receptor was inactive at 1 μM. Dose-response curves of VPAC₁ agonist molecules (PACAP, VIP, [K¹⁵, R¹⁶, L²⁷]VIP(1–7)/GRF(8–27), [R¹⁶]ChSn) were shifted to the right by the VPAC₁ receptor antagonist [AcHis¹, D-Phe², Lys¹⁵, Leu¹⁷]VIP(3–7)/GRF(8–27), with a K_i of 3 ± 1 nM (n = 3). The presence of VPAC₁ receptor mRNA was confirmed by RT-PCR. Preincubation with PACAP or PMA showed that VPAC₁ receptors underwent homologous and heterologous desensitization.

This study provides the first evidence for the expression of functional VPAC₁ receptors undergoing rapid desensitization in HEL cells.