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The expanded regulatory T cell population in chronic filarial infection is highly heterogeneous, activated and suppresses dendritic cell function indirectly through effector T cells.

ABSTRACT: Natural regulatory T cells (nTregs) are known to increase during chronic infection or at the site tumor, though the exact mechanisms that contribute to their accumulation and mode of action remain unclear. We used flow cytometry and microarray analyses to delineate the phenotype of nTregs and their role in modulating T cell and antigen presenting cell (APC) function in the setting of chronic infection. Using cells from 18 filaria-infected (Fil+) and 19 filaria-uninfected (Fil-) subjects, we found that the frequencies of nTreg expressing CTLA-4, GITR, LAG-3, and IL-10 were significantly higher in Fil+ compared with Fil- subjects. Microarray analysis revealed that, compared with those from Fil-, nTreg populations in Fil+ subjects were more heterogeneous and had higher expression of IL-10, CCL-4, IL-29 and CTLA-4, molecules that have been implicated in immune suppression. Moreover, the nTregs from Fil+ subjects had markedly upregulated activation-induced apoptotic genes with concomitant down regulation of cell survival genes. However, these activated nTregs from Fil+ subjects did not significantly affect cytokine production by APCs. To determine if nTregs directly modulate APC function, we modeled an in vitro culture system with cells from normal individuals. In this system, in response to antigen or anti-CD3, nTregs did inhibit APC production of IL-12-, CXCL-9, and CXCL-10, but did so indirectly through effector T cells. Taken together, our results suggest that in filarial infection, the expanded nTreg populations are heterogeneous, short-lived, activated and express regulatory molecules that modulate cytokine production by APC indirectly through an effector T cell-dependent mechanism.

ORGANISM(S): Homo sapiens

PROVIDER: GSE44767 | GEO | 2016/02/25

SECONDARY ACCESSION(S): PRJNA191609

REPOSITORIES: GEO

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Project description:Introduction: Expansion of antigen (Ag)-specific natural occurring regulatory T cells (nTregs) is required to obtain sufficient numbers of cells for cellular immunotherapy. In this study, different allogeneic stimuli were studied for their capacity to generate functional alloAg-specific nTregs. Methods: A highly enriched nTreg-fraction (CD4+CD25brightCD127- T cells) was alloAg-specific expanded using HLA-mismatched immature, mature monocyte-derived dendritic cells (moDC) or peripheral blood mononuclear cells (PBMC). The allogeneic mature moDC-expanded nTregs were fully characterized by analysis of the demethylation status within the TSDR of the FOXP3 gene and the expression of both protein and mRNA of FOXP3, HELIOS, CTLA4 and cytokines. In addition, the antigen-specific suppressive capacity of these expanded nTregs was tested. Results: Allogeneic mature moDC and skin-derived DC were superior in inducing nTreg-expansion compared to immature moDC or PBMC in an HLA-DR and CD80/CD86-dependent way. Remarkably, the presence of exogenous IL-15 without IL-2, could facilitate optimal mature moDC-induced nTreg-expansion. Allogeneic mature moDC-expanded nTregs were at low ratios (<1:320), potent suppressors of alloAg-induced proliferation without significant suppression of completely HLA-mismatched-Ag-induced proliferation. Mature moDC-expanded nTregs were highly demethylated at the TSDR within the FOXP3 gene and highly expressed of FOXP3, HELIOS and CTLA4. A minority of the expanded nTregs produced IL-10, IL-2, IFN-g and TNF-a but very few IL-17 producing nTregs were found. Next generation sequencing of mRNA of moDC-expanded nTregs revealed a strong induction of Treg-associated mRNAs. Conclusions: Human allogeneic mature moDC are highly efficient stimulator cells, in presence of exogenous IL-15, for expansion of stable alloAg-specific nTregs with superior suppressive function. Four different batches of highly pure regulatory T cells (all from the same donor) were expanded in two different ways, and compared to non-expanded samples.

Project description: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.

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The expanded regulatory T cell population in chronic filarial infection is highly heterogeneous, activated and suppresses dendritic cell function indirectly through effector T cells.

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