奥拉替尼对犬T细胞增殖和细胞因子产生的体外免疫调节作用（2019 ...

王帆

Immunomodulatory in vitro effects of oclacitinib on canine T-cell proliferation and cytokine production

奥拉替尼对犬T细胞增殖和细胞因子产生的体外免疫调节作用

Frane Banovic , Jaime Tarigo, Hannah Gordon, James P. Barber and Robert M. Gogal Jr

 

 

翻译：王帆

 

Abstract

Background – Oclacitinib is a Janus kinase inhibitor used to control pruritus and skin lesions in canine allergic skin disease; its effect on canine T cells is not well-characterized.

Hypothesis/Objectives – To evaluate the impact of oclacitinib on cultured T cells using peripheral blood mononuclear cells from dogs.

Animals – Six bluetick coonhounds.

Methods and materials – Lymphocyte-enriched cells were incubated with or without the T-cell mitogen concanavalin A (Con A), oclacitinib (0.5, 1 or 10 μM), ciclosporin (200 ng/mL), Con A + oclacitinib 1 μM and Con A + ciclosporin. We assessed both T-cell proliferation and the secretion of cytokines.

Results – Ciclosporin and oclacitinib both inhibited the spontaneous proliferation of T cells; this effect was significant only after incubation with oclacitinib at 10 μM. At this concentration, oclacitinib significantly reduced the spontaneous secretion of clonal activator cytokines [interleukin (IL)-2, IL-15], pro-inflammatory cytokines [interferon-gamma (IFN-γ), IL-18] and the regulatory cytokine IL-10; tumour necrosis factor alpha (TNF-α) and IL-6 cytokine production was mildly inhibited. After Con A stimulation, only T cells co-treated with ciclosporin achieved a significant proliferation inhibition and reduction of IL-2, IL-10, IL-15, IL-18, IFN-γ and TNF-α. Surprisingly, oclacitinib at 1 μM (337 ng/mL, corresponding to the oral dosage of 0.4–0.6 mg/kg) did not significantly affect Con A-stimulated T-cell proliferation nor cytokine production (IL-2, IL-10, IL-15, IL-18, IFN-γ and TNF-α).

Conclusions – Although a limited number of dogs were investigated, these preliminary results suggest that oclacitinib appears to have immunosuppressive properties, but only at dosages above those used to treat allergic pruritus in dogs.

摘要

背景 – 奥拉替尼是一种Janus激酶抑制剂，用于控制犬过敏性皮肤病的瘙痒和病变; 它对犬T细胞的作用尚未被充分了解。

假设/目的 - 评估奥拉替尼对犬外周血单核细胞中T细胞的影响。

动物 - 6只蓝斑猎浣熊猎犬。

方法与材料 - 分别用伴刀豆球蛋白A（Con A）、奥拉替尼（0.5、1或10μM）、环孢素（200ng/mL）、Con A+奥拉替尼1μM和Con A+环孢素孵育富集淋巴细胞，其中的T细胞有/无有丝分裂。我们评估T细胞的增殖和细胞因子的分泌。

结果 - 环孢素和奥拉替尼均能抑制T细胞的自发增殖; 只有达到10μM的奥拉替尼孵育才明显有效。在此浓度下，奥拉替尼能够显著减少克隆激活细胞因子[白介素（IL）-2、IL-15]、促炎性细胞因子[干扰素-γ（IFN-γ）、IL-18]和调节细胞因子IL-10的自发分泌; 同时轻度抑制肿瘤坏死因子α（TNF-α）和IL-6细胞因子的分泌。在Con A刺激后，仅用环孢素处理的T细胞的增殖得到了显著抑制，并且IL-2、IL-10、IL-15、IL-18、IFN-γ和TNF-α的分泌量减少。令人惊讶的是，1μM的奥拉替尼（337 ng / mL，相当于0.4-0.6 mg / kg的口服剂量）在Con A的刺激下，T细胞的增殖和细胞因子的产生没有显著变化（IL-2、IL-10、IL-15、IL-18、IFN-γ和TNF-α）。

结论 - 尽管进行研究的犬数量有限，但这些结果初步表明，奥拉替尼似乎具有免疫抑制特性，但仅在高于犬过敏性瘙痒剂量时才有这种特性。

 

Introduction

介绍

Janus kinases (JAKs) are nonreceptor tyrosine kinases activated by various cytokine receptors that regulate the expression of multiple inflammatory genes through phosphorylation of signal transducers and activators of transcription (STAT) proteins. In mammals, four JAK families of enzymes (JAK1, JAK2, JAK3 and tyrosine kinase 2) and seven STAT family members (STAT1, STAT2, STAT3,STAT4, STAT5a, STAT5b and STAT6) are employed by over 50 cytokines and growth factors to mediate intracellular signalling. Janus kinase inhibitors are being developed and utilized for the treatment of a variety of inflammatory, autoimmune and haematological disorders in humans.

Janus激酶(JAKs)是由多种细胞因子受体激活的非受体酪氨酸激酶，通过信号转导因子和转录激活因子(STAT)蛋白磷酸化调控多种炎症基因的表达。在哺乳动物中，50多种细胞因子和生长因子利用4个JAK酶家族(JAK1、JAK2、JAK3和酪氨酸激酶2)和7个STAT家族成员(STAT1、STAT2、STAT3、STAT4、STAT5a、STAT5b和STAT6)介导细胞内信号传递。Janus激酶抑制剂正在开发和应用于治疗人类的各种炎症、自身免疫性和血液病。

 

Oclacitinib (Apoquel, Zoetis Inc.; Florham Park, NJ,USA) is a JAK1 inhibitor that has been approved for the control or treatment of pruritus associated with flea allergic dermatitis and atopic dermatitis in dogs at least 12 months of age. The JAK1 has a broad role for signalling of numerous type I cytokines through association with several receptor chains such as the common γ-chain [interleukin (IL)-2, IL-4, IL-7, IL-15], gp130 subunit (IL-6) and IL-4Ra (IL-13) subunit, as well as type II cytokines interferon-gamma (IFN-c) and IL-10. During initial safety studies, oclacitinib induced – dose-dependently –viral skin papillomas, interdigital furunculosis, demodicosis and bacterial pneumonia in several 6- and 12-month-old dogs; such adverse effects suggested that this drug exerts substantial immunosuppressive activity. Currently, it is not known whether oclacitinib is an immunosuppressive agent and if it could be administered in place of, or in conjunction with, other immunosuppressive therapy, such as glucocorticoids or azathioprine, for treating T-cell-mediated diseases or cancer chemotherapy in dogs.

奥拉替尼（爱波克）是一种JAK1抑制剂，已被批准用于控制或治疗与大于12月龄犬的跳蚤过敏性皮炎和特应性皮炎有关的瘙痒症。JAK1通过与多种受体链结合，像是常见的γ-链[白细胞介素(IL)-2、IL-4、IL-7、IL-15]、gp130亚基(IL-6)和IL- 4Ra (IL-13)亚基、以及II型细胞因子干扰素-γ(IFN-γ)和IL-10，在众多I型细胞因子的信号传递中发挥着广泛的作用。在最初的安全性研究中，奥拉替尼在几只6月龄和12月龄的犬上诱导了-剂量依赖的-病毒性皮肤乳头瘤、趾间疖病、蠕形螨病和细菌性肺炎。这些不良反应表明该药具有明显的免疫抑制活性。目前，还不清楚奥拉替尼是否是一种免疫抑制剂，是否可以替代或与其他免疫抑制疗法(如糖皮质激素或硫唑嘌呤)联合使用，用于治疗T细胞介导的疾病或犬的癌症化疗。

 

Given that the JAK–STAT pathway plays an essential role in regulating the function of T cells, the effect of JAK inhibitors on T-cell signalling is frequently evaluated in human and murine studies through in vitro drug exposure of peripheral blood mononuclear cells (PBMCs). The isolation of canine PBMCs is an established technique; there is a large amount of variability in the rate and time of centrifugation, the type of density separation medium used and the contamination with red blood cells (RBCs) and platelets, which can influence lymphocyte proliferation and cytokine secretion.

鉴于JAK - stat通路在调节T细胞功能方面发挥着重要作用，JAK抑制剂对T细胞信号传导的影响在人类和小鼠研究中经常通过体外药物暴露外周血单核细胞(PBMCs)来评估。犬PBMCs的分离是一项成熟的技术;离心速度和时间、所用密度分离介质的种类、红细胞和血小板的污染等方面存在较大多变性，影响淋巴细胞的增殖和细胞因子的分泌。

 

The purpose of this paper was to evaluate the influence of oclacitinib, at concentrations that are comparable to in vivo therapeutic levels, on T-cell proliferation and cytokine production using canine PBMCs; ciclosporin, a frequently used immunomodulator in dogs, served as a control.11 Furthermore, we examined the advantages of enhanced RBC removal on T-cell responses using an erythrolysis step in the preparation of PBMCs in comparison to untreated PBMCs.

本研究的目的是评估在与体内治疗水平相当的浓度下，奥拉替尼对T细胞增殖和细胞因子产生的影响;环孢素是一种犬常用的免疫调节剂。此外，我们还研究了与未经处理的PBMC相比，采用红细胞裂解法制备PBMC时，增强红细胞去除率对T细胞反应的影响。

 

Methods and materials

方法和材料

Animals and reagents

试验动物和试剂

6只健康的蓝斑猎浣熊猎犬——3只已去势雄性、2只已绝育雌性和1只未绝育雌性——被纳入这项研究;它们的年龄从2.5岁到3岁不等(中间值为3岁)。曾用药停用时间为12周，包括全身和/或外用抗生素、抗真菌药物、非甾体类抗炎症药物、外用(皮肤和耳朵)和口服糖皮质激素。在研究期间，没有一只犬接受任何额外的药物治疗。我们大学动物保护与使用委员会批准了这项研究。

Six healthy bluetick coonhound dogs – three neutered males, two spayed females and one intact female – were enrolled in this study;their ages ranged from 2.5- to 3-year-old (median, 3-year-old). Withdrawal times from previous medications were 12 weeks for systemic and/or topical antibiotics, antifungals, nonsteroidal antiinflammatory medications, and topical (skin and ear) and oral glucocorticoids. None of the dogs received any additional drugs during the study. Our University Institutional Animal Care and Use Committee approved the study.

 

Oclacitinib purchased from Adooq Bioscience (Irvine, CA, USA)was diluted with dimethyl sulfoxide (DMSO) and RPMI media (Cellgro; Herndon, VA, USA) and used at 0.5, 1 or 10 micromolar (lM)concentrations, which correspond to 168.5, 337 and 3,370 ng/mL.The 0.5 and 1 lM concentrations were chosen as comparable to the therapeutic levels of oclacitinib based on pharmacokinetic and pharmacodynamic data in dogs; the mean maximum concentration(Cmax) of oclacitinib in plasma was 324 ng/mL (range 255–333 ng/mL) following oral oclacitinib administration at approved dosages of0.4–0.6 mg/kg once or twice daily to 24 dogs. The 10 lM concentration would correspond to Cmax achieved through oral administration of 3–4 mg/kg oclacitinib twice daily to dogs. Ciclosporin A (SigmaAldrich; St Louis, MO, USA) was diluted with DMSO and RPMI media; final concentration per well was 200 ng/mL. Target trough ciclosporin blood levels in dogs vary widely depending on the method and sample tested, and range from 100 to 600 ng/mL. A ciclosporin concentration of 200 ng/mL for PBMC in media and serum modulated canine PBMCs cytokine responses and was postulated to be comparable to therapeutic levels in vivo. The DMSO concentrations used to dilute both drugs for treatments never exceeded0.001%; this concentration does not inflfluence T-cell proliferation.

从Adooq生物科学购买的奥拉替尼用二甲基亚砜(DMSO)和RPMI培养基稀释;并在0.5、1或10微摩尔(lM)浓度下使用，相当于168.5、337和3,370 ng/mL。根据犬的药代动力学和药效学数据，选择0.5和1 lM浓度作为与奥拉替尼治疗水平相当的药物;口服奥拉替尼后，24只犬的血浆中奥拉替尼的平均最大浓度(Cmax)为324 ng/mL(范围为255-333 ng/mL)，每日一至两次，批准剂量为0.4 - 0.6 mg/kg。10 lM浓度与每日两次口服3-4 mg/kg奥拉替尼给犬所达到的Cmax相对应。环孢素用DMSO和RPMI培养基稀释;每个培养基的最终浓度为200ng /mL。根据检测方法和样本的不同，犬血环孢素波谷的目标水平差异很大，范围在100到600 ng/mL之间。环孢素浓度为200 ng/mL，用于PBMC的培养基和血清调节的犬PBMCs细胞因子反应，并被假定与体内治疗水平相当。用于稀释两种药物治疗的DMSO浓度从未超过0.001%14;这种浓度不会影响T细胞的增殖。

 

Blood collection and leucocyte enrichment

血液采集和白细胞富集

In order to investigate the variability in canine T-cell response due to other cell contamination (RBCs, platelets), the experiment was run in two phases; leucocyte enrichment in phase 2 involved cell lysis buffer as an additional step. Briefly, in each experimental phase (pre- versus post-lysis), a total of eight peripheral blood samples of approximately 20 mL were collected from jugular veins of six dogs(four dogs collected once and two dogs collected twice) into EDTA vacutainer tubes. The blood was thoroughly mixed via gentle and repetitive inversion and centrifuged at 283g for 30 min at 23°C. The plasma was carefully removed, whereas the nonplasma cell fraction was diluted with 23°C phosphate buffered saline (PBS) and layered over a density gradient medium (Histopaque 1.077, Sigma-Aldrich). After centrifugation (400g for 30 min at 23°C), the buffy coat layer was carefully collected, washed twice with room temperature PBS and re-suspended in fresh complete media consisting of 10% fetal bovine serum, 2% penicillin-streptomycin and 1% nonessential amino acids in Ca+ and Mg+ free RPMI-1,640 media supplemented with L-glutamine (Cellgro). In phase 2, the lysis buffer (AKC Lysing Buffer, Lonza; Walkersville, MD, USA) was added in the first wash cycle to remove any remaining RBCs. The cells were resuspended in cold complete media and enumerated with a Nexcelom electronic cell counter (Nexcelom Bioscience; Lawrence, MA, USA).

为了研究其他细胞(红细胞、血小板)污染对犬T细胞反应的影响，本实验分两阶段进行。白细胞在第2阶段的富集涉及细胞裂解缓冲液作为一个额外的步骤。简单来说，在每个实验阶段(裂解前和裂解后)，从6只犬(4只犬收集一次，2只犬收集两次)的颈静脉中收集大约20 mL的8个外周血样本，放入EDTA真空管中。通过温和、重复的反转使血液充分混合，并在23℃下以283g离心30 min。仔细去除血浆，而非浆细胞部分用23℃磷酸盐缓冲盐水(PBS)稀释，并在密度梯度培养基(Histopaque 1.077, Sigma-Aldrich)上分层。离心(400g在23℃30 min)后，仔细收集白膜层，用室温PBS洗涤两次，并在含10%胎牛血清、2%青霉素-链霉素和1%非必需氨基酸的新鲜完全培养基中重新悬浮，培养基中添加有l -谷氨酰胺(Cellgro)的Ca+和Mg游离rpmi - 1640培养基中。在第二阶段，裂解缓冲液(AKC裂解缓冲液，Lonza;在第一个洗涤循环中添加Walkersville, MD, USA)以去除任何剩余的红细胞。将细胞重悬于冷的完全培养基中，并利用Nexcelom电子细胞计数器对细胞进行计数(Nexcelom Bioscience;劳伦斯，马萨诸塞州，美国)。

 

 

Packed cell volume and PBMCs cytospins

堆积的细胞体积和PBMC的细胞自旋

A small amount of whole blood (c. 50 lL) from the initial collection was used for blood smears, total protein and packed cell volume(PCV) analyses. Blood was drawn up into heparinized micro-haematocrit capillary tubes, centrifuged for 3 min and the PCV was determined using the micro-capillary reader. For cell purity evaluation,cytospins of the leucocyte-rich fraction were centrifuged in a Wescor Aerospray slide stainer-cytocentrifuge (7150 Hematology, Japan) at 28g for 3 min. All slides were stained using the Wright-Giemsa stain(Sigma-Aldrich). Cell counts of 2 9 200 leucocytes were performed by two investigators to compare the concentrations of leucocyte subsets present, as well as to monitor for contamination of other cells after the lymphocyte extraction process.

从最初采集的少量全血(约50 lL)用于血液涂片、总蛋白和填充细胞体积(PCV)分析。将血液抽取到肝素化微红细胞压积毛细管中，离心3 min，用微毛细管阅读器测定PCV。为评价细胞纯度，用Wescor气溶胶载玻片染色细胞离心机(7150血液学，日本)以28g离心3分钟，分离富含白细胞的部分的细胞质。所有载玻片均使用right- giemsa染色(Sigma-Aldrich)染色。两名研究人员对2 9200个白细胞进行计数，以比较白细胞亚群的浓度，并监测淋巴细胞提取过程后对其他细胞的污染。

 

Canine lymphocyte proliferation assay

犬淋巴细胞增殖试验

A commercially available bioreductive flfluorometric assay (Alamar BlueTM, Molecular Probes, Invitrogen; Carlsbad, CA, USA) was used to assess lymphocyte proliferation. Duplicate sets of 96 well tissue culture plates were set up; one for evaluation of proliferation and the other for evaluation of cytokine expression levels. Each set of plates was cultured at 37°C and 5% CO2 in a humidifified tissue culture incubator. Approximately 4*105 cells per well were plated and incubated for 64 h in fresh complete RPMI media in the presence of the T-cell mitogen concanavalin A (Con A; Sigma-Aldrich) at 10 mg/mL, oclacitinib (0.5, 1 or 10 lM) and ciclosporin (200 ng/mL); cells in media served only as a control.

一种商业上可用的生物还原荧光测定法(Alamar BlueTM，分子探针，Invitrogen;采用卡尔斯巴德(美国加利福尼亚州)淋巴细胞增殖试验。建立96孔组织培养板复套;一个用于评估增殖，另一个用于评估细胞因子的表达水平。每组板在37℃和5% CO2的湿化组织培养箱中培养。在新鲜完整的RPMI培养基中，在T细胞丝裂原伴刀豆蛋白 A (Con A;在10 mg/mL，奥拉替尼(0.5,1或10 lM)和环孢素(200 ng/mL);培养基中的细胞仅作为对照。

 

In order to study the action of oclacitinib with ciclosporin on lymphocytes in conjunction with in vitro mitogen stimulation, two additional treatments, oclacitinib (1 lM) and ciclosporin (200 ng/mL),were additionally investigated. At 64 h, each well from the proliferation plate received 20 lL of the Alamar BlueTM dye. The plate was returned to the incubator for an additional 8 h. At 72 h, the proliferation plate was removed and the cells evaluated for both degree of spontaneous (media only), Con A-induced proliferation and oclacitinib/ciclosporin inhibition measuring the change in fluorescence of the cells using a Synergy4 microplate reader (Biotek; Winooski, VT,USA). All sample wells were assayed in quadruplicate, and results were reported as the mean optical density of the four replicate wells absorbed at 570 nm or 600 nm wavelengths. The values were reported as a Δ = 570–600 nm.

为了研究奥拉替尼与环孢素联合体外有丝分裂原刺激对淋巴细胞的作用，另外研究了奥拉替尼(1lm)和环孢素(200ng /mL)两种治疗方法。在64 h时，增殖板上的每孔接受20 lL的Alamar BlueTM染料。在72 h时，去除增殖板，评估细胞的自发程度(仅培养基)、Con a诱导的增殖程度和奥拉替尼/环孢素的抑制程度，使用Synergy4酶标仪(Biotek;美国佛蒙特Winooski)。所有样品孔均检测四份，结果报告为在570 nm或600 nm波长吸收的4个重复孔的平均光密度。将值报告为Δ = 570-600 nm。

 

Supernatant cytokine assay

细胞因子上清试验

At 48 h of incubation, the cell culture supernatants were collected from the cytokine plate and stored in microcentrifuge tubes at~80°C. Supernatant analysis was performed using a 13-plex canine cytokine/chemokine MILLIPLEXTM MAP Kit assay (EMD Millipore Corporation; Billerica, MA, USA). The following cytokines of interest were evaluated: (i) cell proliferation cytokines IL-2 and IL-15; (ii) pro-inflammatory cytokines IFN-c, tumour necrosis factor alpha (TNF-a),IL-6 and IL-18; and (iii) anti-inflammatory cytokine IL-10. The method was performed according to the manufacturer’s instructions. The observed concentration of each analyte for each sample was calculated using a standard curve generated from the seven standards and a blank provided by the manufacturer. For each cytokine, multiplex data recorded out of range were assigned the respective lowest or highest extrapolated values; samples were not diluted and retested because of cost restraints.16 The values were reported in pg/mL.

于孵育48 h时，从细胞因子平板收集细胞培养上清液，保存于约80℃的微离心管中。上清分析使用13倍犬细胞因子/趋化因子MILLIPLEXTM MAP试剂盒检测(EMD Millipore Corporation;Billerica, MA, USA)。评估以下感兴趣的细胞因子:(i)细胞增殖细胞因子IL-2和IL-15;(ii)促炎细胞因子IFN-c、TNF-a、IL-6和IL-18;和(iii)抗炎细胞因子IL-10。根据制造商的说明执行该方法。使用由7个标准品生成的标准曲线和制造商提供的空白来计算每个样品中每个分析物的观察浓度。对于每个细胞因子，我们为超出范围的多重数据分配了相应的最低或最高外推值;由于成本限制，未对样品进行稀释和复测数值以pg/mL报告。

 

Statistical analysis

统计分析

Statistical analyses were performed using GraphPad Prism v6.0 for Windows (GraphPad Software; La Jolla, CA, USA). Red blood cell and platelet contamination as evaluated using Wilcoxon matched paired signed rank test. One-way (T-cell proliferation and cytokines)or two-way (pre-lysis versus post-lysis groups) repeated measures ANOVA was used to determine the statistical significance of differences between groups with Bonferroni or Dunn’s post-tests. Data are expressed as mean±standard mean (SEM). Findings were considered statistically significant when P-values were ≤0.05, as indicated with asterisks (*P ≤ 0.05, P < 0.01, *P < 0.001)

采用GraphPad Prism v6.0 for Windows (GraphPad Software;美国加州拉霍亚)。采用Wilcoxon配对符号秩检验评价红细胞和血小板污染情况。采用单因素(t细胞增殖和细胞因子)或双因素(裂解前和裂解后组)重复测量方差分析确定组间差异的统计学意义，采用Bonferroni或Dunn 's后检验。数据以均数±标准均数(SEM)表示。当P值≤0.05时，用星号表示，认为结果有统计学意义(*P≤0.05，**P <0.01， ***P <0.001)

 

Discussion

讨论

These results demonstrate the immunomodulatory effect of ciclosporin and oclacitinib on spontaneously proliferating and Con A-stimulated canine T cells; the spontaneous resting and mitogen (Con A)-induced activation were chosen to mimic the T-cell environment in conditions of health and disease activation. Ciclosporin and oclacitinib both showed weak inhibition of the spontaneous proliferation of T cells, but this effect was severe and significant only after incubation with oclacitinib at 10 lM (corresponding to an oral administration of 3–4 mg/kg oclacitinib twice daily). At this high concentration, oclacitinib significantly reduced the spontaneous secretion of clonal activator cytokines (IL-2, IL-15) and pro-inflammatory cytokines (IFN-c, IL-18). Our data show that oclacitinib exerts strong immunosuppressive activity at high dosages, which is in line with previous safety studies where several dogs dose-dependently developed cutaneous viral papillomas, demodicosis and bacterial pneumonia.

这些结果表明环孢素和奥拉替尼对自发增殖和Con A刺激的犬T细胞具有免疫调节作用;选择自发增殖和丝裂原(Con A)诱导的激活模拟健康和疾病激活条件下的T细胞环境。环孢素和奥拉替尼对T细胞的自发增殖均表现出较弱的抑制作用，但这种抑制作用较强，仅在10 lM时与奥拉替尼孵育后才显著(相当于口服3-4 mg/kg奥拉替尼，每日两次)。在这种高浓度下，奥拉替尼显著降低了克隆激活因子(IL-2, IL-15)和促充盈因子(IFN-c, IL-18)的自发分泌。我们的数据显示，奥拉替尼在高剂量时具有较强的免疫抑制活性，这与之前的安全性研究一致，在之前的研究中，有几只犬的皮肤病毒乳头状瘤、蠕形螨和细菌性肺炎都是剂量依赖性的。

 

Investigations of several JAK inhibitors reveal a dosedependent decrease in T-cell cytokine production with a predicted inhibitory potency frequently proposed based on a high concentration of 10 lM.17–19 Both oclacitinib at 1 lM concentration and ciclosporin (200 ng/mL) reduced the spontaneous IL-2, IL-15 and IFN-c secretion from spontaneously growing T cells; however, the treatments did not demonstrate significant changes. Our findings indicate that ciclosporin and oclacitinib at therapeutic concentrations do not significantly affect spontaneous T-cell proliferation in vitro and future drug evaluations should involve measurements of inhibition after specific and nonspecific T-cell activation.

对几种JAK抑制剂的研究表明，T细胞细胞因子的产生呈剂量依赖性下降，通常基于10 lM的高浓度提出预测抑制效力。奥拉替尼在1lm浓度和环孢素(200ng /mL)均可降低自发生长的T细胞分泌的IL-2、IL-15和IFN-c。然而，治疗并没有显示出明显的变化。我们的研究结果表明，环孢素和奥拉替尼在治疗浓度下对体外自T细胞增殖没有显著影响，未来的药物评估应包括特异性和非特异性T细胞活化后抑制作用的测量。

 

Traditionally, in veterinary medicine, the mitogenic lectins such as phytohemagglutinin (PHA) and Con A are utilized for polyclonal T-cell stimulation, whereas phorbol 12- myristate 13-acetate (PMA) stimulates T cells by direct activation of protein kinase C. A more physiologically specific approach that partially mimics stimulation by antigen-presenting cells in humans and murine models utilizes anti-CD3 and anti-CD28 antibodies to stimulate T-cell activation, including IL-2 gene transcription.20 Currently, the anti-canine CD3/CD28-induced T-cell expansion model in dogs is not optimized, validated and commercially available. In the present study, we used commercially available mitogen Con A to stimulate canine T cells. Concanavalin A activates T cells by binding not only to the T-cell receptor/CD3 complex, but also to other surface glycoproteins providing an accessory signal. The activation resembles specific antigen-induced lymphocyte stimulation through TCR with calciumions (Ca2+) influx through different types of nonselective cation channels. The activation of different transcriptional pathways is dependent upon distinct Ca2+ signalling patterns and prolonged elevation of intracellular Ca2+ is required for Tcell functions, including proliferation, differentiation, gene transcription and cytokine production.

传统上，在兽医中，植物血凝素(PHA)和Con A等有丝分裂凝集素被用于多克隆T细胞刺激，而佛波醇12-肉豆醇13-乙酸酯(PMA)通过直接激活蛋白激酶c刺激T细胞。在人类和小鼠模型中，部分模拟抗原呈递细胞刺激的更具有生理学特异性的方法利用抗cd3和抗cd28抗体刺激T细胞活化。包括IL-2基因转录目前，抗犬CD3/ cd28诱导的犬T细胞扩增模型尚未得到优化、验证和商品化。在本研究中，我们使用市售有丝分裂原Con A刺激犬T细胞。刀豆蛋白A不仅通过与T细胞受体/CD3复合物结合来激活T细胞，而且通过与提供辅助信号的其他表面糖蛋白结合来激活T细胞。这种激活类似于特异性抗原诱导的淋巴细胞刺激，TCR通过不同类型的非选择性阳离子通道内流钙离子(Ca2+)。不同转录通路的激活依赖于不同的Ca2+信号传导模式，细胞内Ca2+的长期升高是t细胞功能所必需的，包括增殖，分化，基因转录和细胞因子产生。

 

There is extensive literature on optimizing collection, processing and storage protocols for PBMCs in dogs using the Histopaque 1.077 density gradient; however, the significance of contamination with RBCs and platelets has not been investigated. Similar to investigations of human PBMC isolation and in vitro culture behaviour, the contamination with RBCs and platelets confounded the canine PMBC proliferation and cytokine results in this study. In humans, stimulated lymphocytes in whole blood produce more IL-2 than purified lymphocytes in culture and platelet–PBMC interaction enhances production of cytokines from PMBCs. Results of this investigation indicate that further depletion of RBCs using lysis buffer during PBMC enrichment is essential for optimal in vitro proliferation and consistent cytokine response in dogs.

关于使用Histopaque 1.077密度梯度优化犬体内PBMCs的收集、处理和存储协议，已有大量文献;然而，尚未研究红细胞和血小板污染的意义。类似于对人PBMC分离和体外培养行为的研究，红细胞和血小板的污染混淆了本研究中犬PMBC增殖和细胞因子的结果。在人类中，全血中受刺激的淋巴细胞比培养的纯化淋巴细胞产生更多的IL-2，血小板与PBMC的相互作用增强了PBMC细胞因子的产生。本研究的结果表明，在PBMC富集过程中使用裂解缓冲液进一步消耗红细胞对于犬的体外最佳增殖和细胞因子反应的一致性至关重要。

 

As expected, exposure of canine T cells to ciclosporin abrogated Con A-induced canine T-cell proliferation and the expression of multiple evaluated cytokines, including the secretion of pro-inflammatory cytokines IFN-c, TNF-a and IL-18. Ciclosporin suppresses the first phase of T-cell activation by binding to cyclophilin to form complexes that then bind to and inhibit the activated calcineurin. Calcineurin, a serine-threonine phosphatase, plays an important role in activating the nuclear factor of activated T cells (NFAT), which binds to the promoter regions of various cytokines, such as interleukin IL-2 and activates T-cell proliferation. In humans, ciclosporin equally suppressed Con A- and anti-CD3-induced T-cell proliferation, resulting in approximately 60% inhibition depending on the ciclosporin concentration.

正如预期的那样，将犬T细胞暴露于环孢素诱导的Con A诱导的犬T细胞增殖和多种评估细胞因子的表达，包括促炎细胞因子IFN-c、TNF-a和IL-18的分泌。环孢素通过与亲环细胞素结合形成复合物，然后结合并抑制活化的钙调神经磷酸酶，来抑制T细胞活化的第一阶段。钙调神经磷酸酶是一种丝氨酸-苏氨酸磷酸酶，在激活活化的T细胞核因子(NFAT)中发挥重要作用。NFAT与多种细胞因子的启动子区域(如白细胞介素-2)结合，激活T细胞增殖。在人类中，环孢素对Con A-和抗CD3诱导的T细胞增殖均有抑制作用，根据环孢素浓度的不同，抑制率约为60%。

 

In contrast to the ciclosporin effect, oclacitinib at plasma concentrations corresponding to the clinically approved therapeutic oral dosage of 0.4–0.6 mg/kg(1 lM; 337 ng/mL) failed to inhibit Con A-induced T-cell activation, proliferation and expression of clonal activator cytokines (IL-2, IL-15) and pro-inflammatory cytokines(IFN-c, TNF-a, IL-18). The activation of JAK1 is important for the downstream signalling of IL-2 and IFN-c; an in vitro screening study of the specificity of oclacitinib on the targeted kinases using whole canine blood revealed high inhibitory specificity for canine IL-2.Therefore, we hypothesized that oclacitinib as a JAK1 high-affinity inhibitor would block the autocrine and paracrine cytokine signalling pathway of JAK1-dependent cytokines on T cells, after the Con A-induction of IL-2 and IFN-c. Previous studies have shown that IFN-c enhances clonal expansion and survival of T cells.30 However, the results obtained in this study suggest that oclacitinib may impair Con A-induced T-cell activation, but only at dosages above those used to treat pruritus in allergic dogs. A simple explanation would be a lack of correlation between the in vitro inhibitory kinase specificity of oclacitinib and the direct in vivo effects on T cells; another possible explanation involves lack of JAK–STAT pathway specificity for mitogen Con Ainduced T-cell activation and oclacitinib to induce inhibition. Further investigations using plate-bound anti-canine CD3 alone, or in combination with anti-CD28 and recombinant canine IL-2, are necessary to define the molecular mechanisms mediating the immunosuppressive effects of JAK inhibitors in veterinary medicine.

与环孢素效应相反，符合临床批准的口服治疗剂量为0.4-0.6mg/kg的奥拉替尼的血浆浓度(1 lM;337 ng/mL)未能抑制Con A诱导的T细胞活化、增殖及克隆激活因子(IL-2、IL-15)、促炎因子(IFN-c、TNF-a、IL-18)的表达。JAK1的活化对IL-2和IFN-c的下游信号传导具有重要作用;用犬全血进行奥拉替尼对靶向激酶特异性的体外筛选研究显示，奥拉替尼对犬IL-2具有较高的抑制特异性。因此，我们推测奥拉替尼作为JAK1高亲和力抑制剂，在诱导IL-2和IFN-c后，会阻断JAK1依赖细胞因子在T细胞上的自分泌和旁分泌细胞因子信号通路。以往的研究表明IFN-c能增强T细胞的克隆扩增和存活。然而，本研究的结果表明，奥拉替尼可能损害ConA诱导的T细胞活化，但仅当剂量高于用于治疗过敏犬瘙痒的剂量时。一个简单的解释是奥拉替尼的体外抑制激酶特异性与体内对T细胞的直接影响之间缺乏相关性;另一种可能的解释是缺乏JAK-STAT通路特异性，导致丝裂原诱导T细胞活化和奥拉替尼诱导抑制。为了阐明JAK抑制剂在兽医学中介导免疫抑制作用的分子机制，有必要进一步研究单独使用血小板结合的抗犬CD3，或联合抗cd28和重组犬IL-2。

 

Our studies indicate that IL-10 production was markedly decreased with ciclosporin indicating that T-cell production of IL-10 strongly depends on the calcium/ calcineurin pathway, whereas IL-10 was not signifificantly reduced except at the high concentration of oclacitinib. However, the activation of the IL-10 receptor involves the JAK–STAT signalling pathway through JAK1 and TYK2. Thus, further investigations of IL-10 receptor induced JAK–STAT signal transduction blockage are essential for understanding how JAK inhibitors modulate IL-10 biological functions, because our study design did not inform us of changes in the immunomodulatory response to this or other cytokines when signalling through the JAK–STAT signalling pathway.

我们的研究表明，环孢素显著降低IL-10的产生，说明T细胞IL-10的产生强烈依赖于钙/钙调神经素途径，而IL-10的产生除高浓度奥拉替尼外没有显著下降。然而，IL-10受体的激活涉及通过JAK1和TYK2的JAK-STAT信号通路。因此,进一步研究IL- 10受体诱导的JAK-STAT信号转导阻断，对于理解JAK抑制剂如何调节IL- 10的生物功能至关重要,因为我们的研究设计并没有通知我们,当通过JAK-STAT信号通路传递信号时，该细胞因子或其他细胞因子的免疫调节反应的变化。

 

Interleukin-10 is a potent anti-inflammatory cytokine that plays a crucial, and often essential, role in preventing inflammatory and autoimmune pathologies. In particular, IL-10 regulates tolerance to allergens during allergen immunotherapy (AIT) and serum IL-10 concentration increased in dogs successfully treated with AIT. Currently, the long-term effect of adjuvant immunomodulatory medications on AIT outcome in canine atopic dermatitis is unknown. However, veterinary clinicians should bear in mind that blocking IL-10 pathways through systemic ciclosporin and potentially oclacitinib, during immunotherapy administration, may influence the long-term effectiveness of AIT treatment modality.

白细胞介素-10是一种强效抗炎细胞因子，在预防炎症和自身免疫性疾病中发挥着至关重要的作用。特别是，在过敏原免疫治疗(AIT)过程中，IL-10调节对过敏原的耐受性，而成功应用AIT治疗的犬血清IL-10浓度升高。目前，辅助免疫调节药物对犬特应性皮炎AIT预后的长期影响尚不清楚。然而，兽医临床医生应记住，在免疫治疗期间，通过全身环孢素和可能的奥拉替尼阻断IL-10通路，可能会影响AIT治疗方式的长期有效性。

 

In conclusion, we used an integrated modelling approach by pairing potencies and clinical oclacitinib concentrations based on plasma pharmacokinetics to provide a better understanding of the T-cell inhibition profile. Our results confirm that the current in vitro whole blood and cell model kinase systems utilized to test the potency and selectivity of JAK inhibitors may not be adequate. They support the importance of studying a broad range of JAK potencies and clinically meaningful concentrations on cultured T cells when evaluating JAK-inhibitor compounds. Although a limited number of dogs were enrolled, these preliminary results suggest that oclacitinib exerts immunosuppressive properties, but only at dosages above those used to treat allergic pruritus in dogs. Future studies should assess JAK and other signalling inhibitors in veterinary medicine using specific T-cell activation with anti-canine CD3/CD28 and IL-2.

综上所述，我们采用了一种综合建模方法，基于血浆药代动力学效力与临床奥拉替尼浓度配对，以更好地了解T细胞抑制特性。我们的结果证实，目前体外全血和细胞模型激酶系统用于测试JAK抑制剂的效力和选择性可能不够。他们支持在评估JAK抑制剂化合物时，研究广泛的JAK效应和对培养的T细胞具有临床意义的浓度的重要性。虽然纳入的犬只数量有限，但这些初步结果表明，奥拉替尼具有免疫抑制特性，但仅在剂量高于治疗犬过敏性瘙痒的剂量时出现。未来的研究应该利用抗犬CD3/CD28和IL-2的特异性T细胞活化来评估JAK和其他信号抑制剂在兽医学中的作用。

 

 

 

杨先生

刘双元

南海

！！！！！