Decoding at single cell resolution the molecular and functional program of pathogenic Th2 cells subsets in humans

Unraveling the diversity of Th2 effector cells subsets may help us to understand their pathogenic role in Type-2 associated inflammatory disorders, including allergic diseases and helminth infections. To characterize the heterogeneity and function of these effector Th2 cells, we analyzed 47 subjects' PBMCs [23 filarial-infected (Fil+) with or without coincident HDM sensitization (Fil+HDM+ n=12; Fil+HDM-, n=11) and 24 subjects without filarial infection (Fil-) (Fil-HDM+ n=12; and Fil-HDM-, n=12) using multiparameter flow cytometry and two-level FlowSOM analysis. The frequency of 3 memory CD4+ T cell clusters, including CCR4+CCR6+CRTH2- (subset 1), CCR4+CCR6-CRTH2+ (subset 2), and CCR6+CCR4+CRTH2+ (subset 3) were markedly enriched among Fil+ subjects. The functional characterization indicated subset 2 and 3 as distinct Th2 cytokine producers, which together were responsible for the majority of IL-4, IL-5, or IL-13 produced among the Fil+ subjects. These subsets were sorted and analyzed by multiomic single cell RNA profiling. Indeed, both subset 2 and subset 3 presented features of pathogenic Th2 effector cells based on their single cell molecular signature, including downregulation of cd27 and high expression levels of itga4, il-17rb, hpgds, klrb1, ptgdr2, il-9r, il-4, il5 and il-13 genes when compared with conventional Th2 cells from healthy controls. When the Fil+ subjects were divided based on allergic status, the Fil+HDM+ subjects had an expansion of both subset 2 (3.19% vs 1.28%, p=0.001) and subset 3 (0.39% vs 0.15%, p=0.002) Th2 cells when compared with Fil+HDM-. Gene expression analysis further demonstrated that concomitant HDM sensitization in the presence of filarial infection reshaped the molecular program of these pathogenic effector Th2 subsets by even further upregulating the expression of gata3, il17rb, clrf2, and klrb1. Notably, Fil+HDM+ patients' cells showed higher responsiveness to filarial or HDM antigenic stimulation, producing even higher levels of IL-4, IL-5 and IL-13 when compared with Fil+HDM- patients. This distinct molecular and functional program of Th2 effector cells subsets sheds new light on the Th2 cell plasticity and their contribution to immune regulation in helminth infection and allergic diseases. Overall design: From a previously described cohort of 49 subjects used to characterize the nature of the T cell response induced by parasite antigens and aeroallergens in the context of filarial-infected individuals with or without coincident allergic sensitization, 47 north American individuals, with no history of helminthic infection prior to this study were included in the current study population. Out of the 47 subjects, 24 patients were diagnosed with filarial infection, including either Loa loa, Wuchereria bancrofti or O. volvulus by clinical, parasitological, and molecular analysis. Moreover, 23 healthy volunteers (NIH blood bank donors) comprised the control group. Later, these individuals were divided in 4 groups based on their allergic status presenting or not concomitant HDM sensitization. Individuals with allergen-specific IgE levels >0.35 kUA/L by ImmunoCAPTM Phadiatop assay were considered allergic/atopic. Group 1: HDM-Filarial- (n=12), group 2: HDM+Filarial- (n=12), group 3: HDM-Filarial+ (n=11), and group 4: HDM+Filarial+ (n=12).

阐明Th2效应细胞亚群的多样性，有助于理解其在2型相关炎症性疾病（包括过敏性疾病与蠕虫感染）中的致病作用。为表征此类效应性Th2细胞的异质性与功能，本研究对47名受试者的外周血单个核细胞（PBMCs）开展分析：其中23名丝虫感染阳性（Fil+）受试者，伴或不伴屋尘螨（HDM）致敏（Fil+HDM+组n=12；Fil+HDM-组n=11），以及24名丝虫感染阴性（Fil-）受试者（Fil-HDM+组n=12；Fil-HDM-组n=12），分析方法为多参数流式细胞术与双级FlowSOM分析。

3种记忆性CD4+T细胞簇的频率在Fil+受试者中显著富集，分别为CCR4+CCR6+CRTH2-（亚群1）、CCR4+CCR6-CRTH2+（亚群2）与CCR6+CCR4+CRTH2+（亚群3）。功能表征结果显示，亚群2与亚群3为特异性Th2细胞因子产生细胞，二者共同贡献了Fil+受试者体内绝大多数的IL-4、IL-5与IL-13表达。研究人员对上述亚群进行分选后，采用多组学单细胞RNA谱分析进行解析。

相较于健康对照的经典Th2细胞，基于单细胞分子特征，亚群2与亚群3均表现出致病性Th2效应细胞的特征，包括cd27基因下调，以及itga4、il-17rb、hpgds、klrb1、ptgdr2、il-9r、il-4、il5与il-13基因的高表达。按过敏状态对Fil+受试者分组后发现，与Fil+HDM-组相比，Fil+HDM+组的亚群2（3.19% vs 1.28%，p=0.001）与亚群3（0.39% vs 0.15%，p=0.002）Th2细胞均出现扩增。

基因表达分析进一步证实，在丝虫感染背景下伴随HDM致敏，可通过进一步上调gata3、il17rb、clrf2与klrb1的表达，重塑此类致病性效应性Th2亚群的分子程序。值得注意的是，与Fil+HDM-患者相比，Fil+HDM+患者的细胞对丝虫或HDM抗原刺激的应答更强，产生的IL-4、IL-5与IL-13水平更高。Th2效应细胞亚群的这种独特分子与功能特征，为阐明Th2细胞可塑性及其在蠕虫感染与过敏性疾病中对免疫调控的贡献提供了新视角。

整体实验设计：
本研究队列源自此前一项已发表的研究，该研究旨在表征丝虫感染伴或不伴过敏性致敏个体中，寄生虫抗原与气源性过敏原诱导的T细胞应答特征；本研究纳入47名北美受试者，且所有受试者在本研究开展前均无蠕虫感染史。47名受试者中，24名经临床、病原学与分子生物学分析确诊为丝虫感染，感染病原体包括罗阿丝虫（Loa loa）、班氏吴策线虫（Wuchereria bancrofti）或旋盘尾丝虫（O. volvulus）。另有23名健康志愿者（美国国立卫生研究院血库捐献者，NIH blood bank donors）作为对照组。

随后，研究人员根据受试者是否伴随HDM致敏的过敏状态，将所有受试者分为4组：通过ImmunoCAPTM Phadiatop检测，过敏原特异性IgE水平>0.35 kUA/L的个体被判定为过敏/特应性体质。分组如下：组1：HDM-Filarial-（n=12）；组2：HDM+Filarial-（n=12）；组3：HDM-Filarial+（n=11）；组4：HDM+Filarial+（n=12）。