Increased frequencies of highly activated regulatory T cells skewed to a T helper 1-like phenotype with reduced suppressive capacity in dengue patients

Abstract

The pathogenesis of dengue involves a complex interplay between the viral factor and the host immune response. A mismatch between the infecting serotype and the adaptive memory response is hypothesized to lead to exacerbated immune responses resulting in severe dengue. Here, we aim to define in detail the phenotype and function of different regulatory T cell (Treg) subsets and their association with disease severity in a cohort of acute dengue virus (DENV)-infected Cambodian children. Treg frequencies and proliferation of Tregs are increased in dengue patients compared to age-matched controls. Tregs from dengue patients are skewed to a Th1-type Treg phenotype. Interestingly, Tregs from severe dengue patients produce more interleukin-10 after in vitro stimulation compared to Tregs from classical dengue fever patients. Functionally, Tregs from dengue patients have reduced suppressive capacity, irrespective of disease severity. Taken together, these data suggest that even though Treg frequencies are increased in the blood of acute DENV-infected patients, Tregs fail to resolve inflammation and thereby could contribute to the immunopathology of dengue.

Importance: According to the World Health Organization, dengue is the fastest-spreading, epidemic-prone infectious disease. The extent of dengue virus infections increased over the years, mainly driven by globalization-including travel and trade-and environmental changes. Dengue is an immunopathology caused by an imbalanced immune response to a secondary heterotypic infection. As regulatory T cells (Tregs) are essential in maintaining immune homeostasis and dampening excessive immune activation, this study addressed the role of Tregs in dengue immunopathology. We show that Tregs from dengue patients are highly activated, skewed to a Th1-like Treg phenotype and less suppressive compared to healthy donor Tregs. Our data suggest that Tregs fail to resolve ongoing inflammation during dengue infection and hence contribute to the immunopathology of severe dengue disease. These data clarify the role of Tregs in dengue immunopathogenesis, emphasizing the need to develop T cell-based vaccines for dengue.

Keywords: FOXP3; dengue virus; regulatory T cells; severe dengue disease.

Fig 1

Frequency of Tregs in pediatric dengue patients. (A) Representative dot plots of Tregs (CD4⁺CD25⁺FOXP3⁺) and Tacts (CD4⁺CD25⁺FOXP3⁻) in total CD4⁺ T cells from age-matched HD (left) and dengue patients (right). (B–D) The frequency of Tregs and Tacts in total CD4⁺ T cells and the ratio of Tregs/Tacts comparing HD with acute DENV-infected patients. (E) Representative dot plots of Tregs (CD4⁺CD25⁺FOXP3⁺) and Tacts (CD4⁺CD25⁺FOXP3⁻) in total CD4⁺ T cells from DF (left) and DHF/DSS patients (right). (F–H) The frequency of Tregs and Tacts in total CD4⁺ T cells and the ratio of Treg/Tacts comparing DF with DHF/DSS patients. Seventeen age-matched HD, 35 pediatric dengue patients consisting of 19 DF, and 16 DHF/DSS patients were included. The bars indicate median with interquartile range. Mann–Whitney U test was used for statistical analysis (*P < 0.05; ****P < 0.0001).

Fig 2

Activation and proliferation state of Tregs from pediatric dengue patients. (A and B) The frequencies of CD45RA⁺ Treg and HLA-DR⁺ Treg in HD and dengue patients. (C and D) The frequencies of CD45RA⁺ Treg and HLA-DR⁺ Treg in DF and DHF/DSS patients. (E) Representative dot plots of the percentages of Ki-67⁺ in CD3⁺CD8⁻FOXP3⁺ in HD (left) and dengue patients (right). (F) Summary of the frequencies of Ki-67⁺ in CD3⁺CD8⁻FOXP3⁻ and CD3⁺CD8⁻FOXP3⁺ population. (G) Representative dot plots of the percentages of Ki-67⁺ in CD3⁺CD8⁻FOXP3⁺ in DF (left) and DHF/DSS (right). (H) Summary of the frequencies of Ki-67⁺ in CD3⁺CD8⁻FOXP3⁻ and CD3⁺CD8⁻FOXP3⁺ population. Seventeen age-matched HD, 35 pediatric dengue patients consisting of 19 DF, and 16 DHF/DSS patients were included. The bars indicate median and interquartile range of the data. Mann–Whitney U test was used for statistical analysis (*P < 0.05; ****P < 0.0001).

Fig 3

Treg subsets based on chemokine receptors. (A–C) Frequency of Th1-like, Th2-like, and Th17-like Treg in HD and dengue patients. (D–F) Frequency of Th1-like, Th2-like, and Th17-like Treg in DF and DHF/DSS patients. Seventeen age-matched HD, 35 pediatric dengue patients consisting of 19 DF, and 16 DHF/DSS patients were included. The graphs display median with interquartile range of the data. Mann–Whitney U test was used for statistical analysis (**P < 0.01).

Fig 4

Expression of functional markers in Tregs. (A) Representative dot plots of CD39⁺ Tregs in HD (left) and dengue patients (right). (B–D) Frequency of CD39⁺ Tregs, CTLA-4⁺ Tregs and ICOS⁺ Tregs in HD and dengue patients. (E) Representative dot plots of CD39⁺ Tregs in DF (left) and DHF/DSS patients (right). (F–H) Frequency of CD39⁺ Tregs, CTLA-4⁺ Tregs and ICOS⁺ Tregs in DF and DHF/DSS patients. Seventeen age-matched HD, 35 pediatric dengue patients consisting of 19 DF, and 16 DHF/DSS patients were included. The graphs display median and interquartile range of the data. Mann–Whitney U test was used for statistical analysis (*P < 0.05).

Fig 5

Cytokine production in Helios⁺ Tregs. (A–C) Frequency of IFN-γ⁺Helios⁺ Tregs, IL-10⁺Helios⁺ Tregs, and IL-17⁺Helios⁺ Tregs in HD and dengue patients. (D–F) Frequency of IFN-γ⁺Helios⁺ Tregs, IL-10⁺Helios⁺ Tregs, and IL-17⁺Helios⁺ Tregs in DF and DHF/DSS patients. Fifteen age-matched HD, 34 pediatric dengue cases consisting of 19 DF, and 15 DHF/DSS patients were included. The graphs show median with interquartile range of the data. Mann–Whitney U test was used for statistical analysis (*P < 0.05).

Fig 6

In vitro suppression assay measuring Treg functionality. (A–C) Representative plots showing gating strategy for cell proliferation in HD samples, dengue patient samples, and a cross-culture between Tregs from HD and Tresps from DENV patients. A total of 10,000 Tresp cells were used for positive control, and 5,000 Treg cells with 5,000 Tresp cells were used for co-culture and cross-culture with 1:1 ratio to Treg suppressive inspector. (D and E) Summary of percentages of each suppression assay show low suppressive function of Tregs in pediatric dengue patients compared to age-matched HD (HD: n = 10; DENV: n = 10; DF: n = 5; and DHF/DSS: n = 5). Bars indicate median with interquartile range. Kruskal–Wallis test followed by Dunn’s multiple comparison test was used for multiple comparisons (D), and Mann–Whitney U test was used for two groups (E).