【目的】

       结核病（Tuberculosis，TB）是全球重大公共卫生问题。TB防控的重点在于及时发现病例，给予有效治疗，然而，目前的诊断技术不能很好地满足临床需求，TB治疗面临着长疗程、不良反应、耐药以及特殊人群等诸多困难。宿主感染免疫应答是解决TB诊断、治疗和预后 相关问题的理论基石。血小板作为免疫细胞参与感染性疾病的发生发展已经得到了广泛认识。本研究旨在评估血小板和白细胞数及其衍生指标在TB诊断和发病风险评估中的临床价值，拟揭示TB患者血小板对淋巴细胞功能的调控作用，以期为TB诊断和治疗提供新思路。

【方法】

      使用受试者工作曲线评估诊断效能；使用回顾性巢式病例对照研究设计分析结核活动的风险因素；使用流式细胞术检测TB患者血小板-免疫细胞聚合体的频率，表型以及功能；使用超高速流式细胞分选仪分选细胞；使用q-PCR检测基因表达；使用体外共培养实验明确血小板对淋巴细胞功能的调控作用。

【结果】

       与健康人相比，活动性结核患者（Active tuberculosis，ATB）外周血血小板、单核细胞和中性粒细胞增加，淋巴细胞减少，出现血小板-淋巴细胞比值（Platelet-lymphocyte ratio，PLR）、单核细胞-淋巴细胞比值（Monocyte -lymphocyte ratio，MLR）、中性粒细胞-淋巴细胞比值（Neutrophil-lymphocyte ratio，NLR）升高。抗结核治疗2月后血小板、单核细胞、中性粒细胞、PLR、MLR和NLR回降，淋巴细胞回升。与结核活动前相比，发生结核活动后的ATB患者淋巴细胞减少，PLR、MLR和NLR增加。联合单核细胞、PLR和NLR三个指标对于区分ATB患者和健康人具有最优诊断效能。在一项全国多中心的风湿免疫病（Rheumatic disease，RD）随访队列中，发现基线合并ATB的RD患者相较于不合并ATB的RD患者血小板、PLR和NLR升高，淋巴细胞减少。RD人群基线PLR≥130.27时，其一年后发生ATB的风险是PLR＜130.27的23.761倍。

       ATB患者血小板与机体炎症反应指标呈正相关关系。血小板以不同比例粘附各类免疫细胞形成聚合体。ATB患者血小板-T细胞聚合体，血小板-NK细胞聚合体比例均高于潜伏结核感染者和健康对照。血小板-T细胞聚合体倾向于向终末分化效应记忆细胞、中心记忆T细胞、Th2和Th17细胞分化，高表达程序性死亡受体1、CD38/HLA-DR、P-选择素等活化耗竭信号。随血小板和T细胞胞膜表面P-选择素/P-选择素糖蛋白配体-1（P-selectin glycoprotein ligand-1，PSGL-1）和CD40配体（CD40 ligand，CD40L）/CD40受体配体对表达水平增加，ATB患者血小板-T细胞聚合体比例也同步增加。ATB患者血小板-T细胞聚合体比例与结核特异性抗原刺激引起的血浆γ-干扰素（Interferon-γ，IFN-γ）和白介素-2（Interleukin-2，IL-2）分泌水平呈正相关关系。ATB患者的血小板-T细胞聚合体和血小板-NK细胞聚合体相较于单个的T细胞和NK细胞高表达IFN-γ、IL-2、肿瘤坏死因子-α、穿孔素和颗粒酶B等细胞因子和溶细胞蛋白。血小板粘附T细胞和NK细胞形成聚合体具有促进其表达细胞因子和溶细胞蛋白的作用。

【结论】

       血小板和白细胞数及其衍生指标可作为辅助诊断ATB和观测抗结核治疗疗效的简易指标，PLR≥130.27有助于预测高危人群TB发病风险。TB患者血小板通过P-选择素/PSGL-1和CD40/CD40L介导粘附T细胞，形成高活化耗竭表型的血小板-T细胞聚合体，血小板粘附T细胞和NK细胞具有促进T细胞和NK细胞分泌细胞因子和溶细胞蛋白的作用，有望为研发靶向调节血小板的宿主免疫疗法提供新视角。

Objective

Tuberculosis (TB) is a major global public health problem. The key to TB prevention and control lies in timely detection of cases and effective treatment. However, TB diagnostic technology cannot meet clinical needs well, and TB treatment faces many difficulties such as long treatment courses, adverse reactions, drug resistance, and special population treatment. The host infection immune response is the theoretical cornerstone for solving the diagnosis, treatment, and prognosis of TB. It has been widely recognized that platelets, as immune cells, participate in the occurrence and development of infectious diseases. Therefore, this study is committed to evaluating the clinical value of platelet and leukocyte counts and their derived indicators in the diagnosis and risk assessment of TB, to reveal the regulatory ability of platelets on lymphocyte function in TB patients, thus providing new ideas for the diagnosis and treatment of TB.

Methods

The receiver operating characteristic curve was used to evaluate diagnostic efficacy. A retrospective nested case-control study design was used to analyze risk factors for TB activity. Flow cytometry was used to detect the frequency, phenotype, and function of platelet-immune cell aggregates in TB patients. A high-speed flow cytometer sorted cells and q-PCR detected gene expression. Co-culture experiments ex vivo clarified the regulatory ability of platelets on lymphocyte function.

Results

Active tuberculosis (ATB) patients demonstrated increased peripheral blood platelet count, monocyte count, neutrophil count, platelet-lymphocyte ratio (PLR), monocyte-lymphocyte ratio (MLR), and neutrophil-lymphocyte ratio (NLR), along with a decrease in lymphocyte count. Following anti-TB treatment for 2 months, ATB patients exhibited decreased platelet count, monocyte count, neutrophil count, PLR, MLR, and NLR, along with an increase in lymphocyte count. Moreover, when compared to those parameters before the onset of ATB, lymphocyte count decreased, and PLR, MLR, and NLR increased after the onset of ATB. A combination of monocyte count, PLR, and NLR, exhibited a good diagnostic performance for discriminating ATB from healthy control. Additionally, in a nationwide multicenter follow-up cohort for rheumatic disease (RD) study, compared to RD patients without concomitant ATB at baseline, RD patients with concomitant ATB demonstrated increased platelet count but decreased lymphocyte count. RD patients with a baseline PLR≥130.27 had a 23.761-fold higher risk of developing ATB within one year.

The platelet count of ATB patients is positively correlated with some inflammatory index. Platelets can bind to various immune cells and form aggregates in different proportions, and the proportion of platelet-T cell aggregates and platelet-NK cell aggregates in the peripheral blood of ATB patients is higher than that of latent tuberculosis infection and healthy control. Platelet-T cell aggregates tend to differentiate towards terminally differentiated effector memory T cells, central memory T cells, Th1 and Th17 cells and express more activation/exhaustion signals such as PD-1, CD38/HLA-DR, and P-selectin. In addition, with the increased expression levels of P-selectin/P-selectin glycoprotein ligand-1 (PSGL-1) and CD40 ligand (CD40L)/CD40 receptor-ligand pairs on the cell membrane of platelets and T cell, the proportion of platelet-T cell aggregates in ATB patients elevated synchronously. The proportion of platelet-T cell aggregates in ATB patients is positively correlated with Mtb-antigen stimulated plasma interferon-γ (IFN-γ) and IL-2. Importantly, platelet-T cell aggregates and platelet-NK cell aggregates in ATB patients were demonstrated to express more cytokines such as IFN-γ, IL-2 and tumor necrosis factor-α, and cytolytic proteins such as perforin and granzyme B. Moreover, platelets upregulate the production of cytokines and cytolytic proteins by forming aggregates with T cells and NK cells ex vivo.

Conclusions

The platelet and leukocyte-derived ratios could serve as some simple indicators for assisting in the diagnosis of ATB and assessing the efficacy of anti-TB treatment, and PLR≥130.27 can help to predict the risk of ATB in high-risk populations. The platelets of TB patients could bind to T cells mainly through P-selectin/PSGL-1 and CD40/CD40L receptor-ligand pairs, leading to an increase of platelet-T cell aggregates with high activation/exhaustion phenotype in the ATB patient. Platelet adhesion promotes cytokines and cytotoxic protein secretion of T cells and NK cells, thus providing a new perspective to develop host⁃directed therapy targeting platelets.