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).