Infectious Diseases Divison, icddr, b (International Centre for Diarrhoeal Disease Research, Bangladesh), Dhaka, Bangladesh.
Department of Microbiology and Immunology, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden.
Front Immunol. 2023 Sep 20;14:1220130. doi: 10.3389/fimmu.2023.1220130. eCollection 2023.
Infection with enterotoxigenic (ETEC) gives rise to IgA antibodies against both the heat labile toxin (LT) and colonization factors (CFs), which are considered to synergistically protect against ETEC diarrhea. Since the development of ETEC-specific long lived plasma cells and memory B cells is likely to be dependent on T helper (Th) cells, we investigated if natural ETEC diarrhea elicits ETEC-specific Th cells and their relation to IgA responses.
Th cell subsets were analyzed in adult Bangladeshi patients hospitalized due to ETEC diarrhea by flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) isolated from blood collected day 2, 7, 30 and 90 after hospitalization as well as in healthy controls. The LT- and CF-specific Th responses were determined by analysis of IL-17A and IFN-γ in antigen stimulated PBMC cultures using ELISA. ETEC-specific IgA secreted by circulating antibody secreting cells (plasmablasts) were analyzed by using the antibodies in lymphocyte supernatants (ALS) ELISA-based method and plasma IgA was also measured by ELISA.
ETEC patients mounted significant ALS and plasma IgA responses against LTB and CFs on day 7 after hospitalization. ETEC patients had significantly elevated proportions of memory Th cells with a Th17 phenotype (CCR6+CXCR3-) in blood compared to controls, while frequencies of Th1 (CCR6-CXCR3+) or Th2 (CCR6-CXCR3-) cells were not increased. Antigen stimulation of PBMCs revealed IL-17A responses to LT, most clearly observed after stimulation with double mutant heat labile toxin (dmLT), but also with LT B subunit (LTB), and to CS6 in samples from patients with LT+ or CS6+ ETEC bacteria. Some individuals also mounted IFN-γ responses to dmLT and LTB. Levels of LTB specific IgA antibodies in ALS, but not plasma samples correlated with both IL-17A (r=0.5, p=0.02) and IFN-γ (r=0.6, p=0.01) responses to dmLT.
Our results show that ETEC diarrhea induces T cell responses, which are predominantly of the Th17 type. The correlations between IL-17A and IFN-g and intestine-derived plasmablast responses support that Th responses may contribute to the development of protective IgA responses against ETEC infection. These observations provide important insights into T cell responses that need to be considered in the evaluation of advanced ETEC vaccine candidates.
肠产毒性大肠杆菌（ETEC）感染会引起针对不耐热毒素（LT）和定植因子（CFs）的 IgA 抗体，这些抗体被认为协同保护免受 ETEC 腹泻的侵害。由于 ETEC 特异性长寿浆细胞和记忆 B 细胞的产生可能依赖于辅助性 T 细胞（Th），因此我们研究了自然发生的 ETEC 腹泻是否会引发 ETEC 特异性 Th 细胞及其与 IgA 反应的关系。
通过流式细胞术分析住院的孟加拉国成年 ETEC 腹泻患者外周血单个核细胞（PBMC）中 Th 细胞亚群，从住院第 2、7、30 和 90 天采集血液。同时分析健康对照组的 Th 细胞亚群。通过酶联免疫吸附试验（ELISA）分析抗原刺激 PBMC 培养物中白细胞介素 17A（IL-17A）和干扰素-γ（IFN-γ）来确定 LT 和 CF 特异性 Th 反应。通过使用淋巴细胞上清液（ALS）ELISA 基于方法分析循环抗体分泌细胞（浆母细胞）分泌的 ETEC 特异性 IgA，并通过 ELISA 测量血浆 IgA。
ETEC 患者在住院后第 7 天对 LTB 和 CFs 产生明显的 ALS 和血浆 IgA 反应。与对照组相比，ETEC 患者血液中记忆性 Th 细胞（具有 Th17 表型的 CCR6+CXCR3-）的比例显著升高，而 Th1（CCR6-CXCR3+）或 Th2（CCR6-CXCR3-）细胞的频率并未增加。PBMC 的抗原刺激显示出对 LT 的 IL-17A 反应，在用双突变不耐热毒素（dmLT）刺激时最明显，但也用 LT B 亚单位（LTB）和 CS6 刺激时也可以观察到，对 ETEC 中含有 LT+或 CS6+的细菌进行了检测。一些个体还对 dmLT 和 LTB 产生 IFN-γ 反应。ALS 中 LTB 特异性 IgA 抗体水平，但不是血浆样本与 dmLT 诱导的 IL-17A（r=0.5，p=0.02）和 IFN-γ（r=0.6，p=0.01）反应均相关。
我们的研究结果表明，ETEC 腹泻会引发 Th 细胞反应，主要是 Th17 型反应。IL-17A 和 IFN-γ 与肠道衍生的浆母细胞反应之间的相关性表明，Th 反应可能有助于对 ETEC 感染产生保护性 IgA 反应。这些观察结果为评估先进的 ETEC 疫苗候选物时需要考虑的 T 细胞反应提供了重要的见解。
