Immune profiling in subclinical secondary dengue-infected cases reveals adaptive immune signatures correlated to protection from severe dengue

Abstract

Development of strategies to prevent severe dengue has been challenging, partly by our incomplete understanding of a protective immune response after dengue virus (DENV) infection. To define adaptive immune signatures associated with protection from hospitalized dengue, we performed in-depth single-cell immunoprofiling and quantified DENV-specific T cells in subclinical or hospitalized dengue-infected children. Individuals with subclinical infection exhibit clonally expanded CD4⁺ TEMRA cells, increased frequency of DENV-specific CD4⁺ T cells, and demonstrate a gene expression signature of increased Treg functionality. Across all T cell subsets, subclinical cases upregulated a type I IFN response gene signature. In contrast, expanding CD8⁺ EM cells from hospitalized patients express more inhibitory markers and fewer cytotoxic proteins. In addition, hospitalized dengue is characterized by high frequencies and clonally expanded immunoglobulin G (Ig)G1-expressing plasmablasts. These findings identify candidate correlates of protection and support a rationale for T cell-directed interventions for dengue disease.

Keywords: DENV-specific T cells; antibody-dependent enhancement; correlates of protection; subclinical dengue; type I IFN.

Figure 1:. Higher frequency of CD4+ TEMRA cells and MAIT cells in blood of acute DENV-infected patients with subclinical infection.

A: Overview of the study design and patient cohorts. B: UMAP plot of T cells gene expression profiles, determined by scRNA-seq, colored by cell types, in subclinical (left) and hospitalized patients (right). C: Cell type proportions (top) and their differences (bottom) in subclinical and hospitalized samples, observed in the scRNA-seq analysis. Log fold-differences (FD) are positive for cells types more frequent in hospitalized patients, negative if more frequent in subclinical patients. Red: significant values (|log2(FD)| ≥ 0.58; FDR < 0.05). Cell type proportions: median and interquartile range are shown, and dots representing each sample are colored by disease severity. D: Position of the CD4+ TEMRA cells (blue) in the scRNA-seq UMAP plot. E: Position of the MAIT cells (blue) in the scRNA-seq UMAP plot. F: Position of the γδ T cells (blue) in the scRNA-seq UMAP plot. G: Frequency of TEMRA cells (CD45RA+CCR7−) in CD4+ T cells, MAIT cells (TCR Vα7.2+) in CD3+CD8+ T cells and γδ T cells (CD4−CD8− γδ TCR+) in CD3+ T cells in 8 subclinical (S, blue) and 19 hospitalized (H, red) dengue patients, as measured by flow cytometry. Median and interquartile range are shown. P values are calculated by Mann-Whitney test. Also see Figure S1, S2, S5.

Figure 2:. Metabolic activity, cell cycle dynamics, and sub-cluster analysis of CD8+ EM cells.

A: Dotplot of the activity scores of selected metabolic pathways, computed by scMetabolism. B: Position of the cells expressing MKI67 (blue) on the scRNA-seq UMAP plot. C: Cell cycle phase assessment computed by Seurat; thereby phase G2 and M are not distinguished. D: UMAP plot of the CD8+ EM cells isolated from the scRNA-seq analysis results: unsupervised clustering (resolution 0.4) distinguishes five distinct sub-clusters among these cells. E: Cell type proportions (right) and their differences (left) in the CD8+ EM sub-clusters between the subclinical and hospitalized patients. Log fold-differences (FD) are positive for cells types more frequent in hospitalized patients, negative if more frequent in subclinical patients. Differences for clusters 1-4 are statistically significant (FDR < 0.05). Cell type proportions: median and interquartile range are shown, and dots representing each sample are colored by disease severity. F: Expression profile of MAP3K8 in the CD8+ EM cells, in subclinical and hospitalized patients.

Figure 3:. Higher frequencies of DENV-specific CD4+ T cells in the acute phase of secondary infection in patients with subclinical infection.

A: graphical representation of the activation-induced marker (AIM) assay to identify DENV-specific T cells. B: representative dot plots of CD8+ T cells upregulating CD69 and CD137 and CD4+ T cells upregulating OX40 and CD25 after stimulation with the DENV megapools. C: Percentage of individuals with CD8+ T cells responding to the CD8 peptide megapool. D: Percentages of CD69+CD137+ T cells in total CD8+ and CD8+ EM cells in 8 subclinical (S, blue) and 19 hospitalized (H, red) dengue patients. Median and interquartile range are shown. E: Percentage of individuals with CD4+ T cells responding to the CD4 peptide megapool. F: Percentages of OX40+CD25+ T cells in total CD4+ and CD4+ TEMRA cells in 8 subclinical (S, blue) and 19 hospitalized (H, red) dengue patients. Median and interquartile range are shown. P values are calculated by Mann-Whitney test. Also see Figure S3.

Figure 4:. Gene expression profiles and expression of functional markers in CD4+ and CD8+ T cell subsets associated with protection after secondary infection.

A: Gene expression levels with statistically significant differences between subclinical and hospitalized patients in T cells subtypes, computed by DESeq2 on pseudo-bulked counts. Genes expressed in less than 5% of the cells in both groups are not visualized. B: Heatmap of the log2 fold differences in expression of selected genes in T cell subtypes between subclinical and hospitalized patients. Statistically significant differences according to DESeq2 are indicated by stars: adj. p-value: *** < 0.001, ** 0.01, * 0.05. C: Percentages of cells expressing proliferation, activation, inhibitory receptors and functional markers as assessed by flow cytometry in 8 subclinical (S, blue) and 19 hospitalized (H, red) dengue patients. Median and interquartile range are shown. P values are calculated by Mann-Whitney test. Also see Figure S4.

Figure 5:. Identification of TCR sequences associated to protection after secondary infection.

A: Frequencies of the T cell clonotypes visualized on the scRNA-seq UMAP plot. B: Proportion of clonotype sizes among the CD4+ TEMRA and in proliferating CD8+ EM cells. C: Cell type proportions in subclinical and hospitalized samples in cells of non-expanded clonotypes, and in cells belonging to expanded clonotypes consisting of 2 to 5, 6 to 20 or more than 20 cells. D: Pairings between the V genes of the TCRβ and TCRα in CD4+ TEMRA and CD8+ EM T cells. Values for non-expanded clonotypes (size 1) are not visualized. Pairs with significantly different usage between subclinical and hospitalized patients are colored (adjusted p-value < 0.05; gray: non-significant differences). E: Most common TCRβ CDR3 sequences in CD4+ TEMRA and CD8+ EM T cells of subclinical and hospitalized patients. Also see Figure S6, S7, S8.

Figure 6:. Identification of a cluster of proliferating plasmablasts in hospitalized dengue patients.

A: UMAP plot of B cells gene expression profiles, determined by scRNA-seq, colored by cell types, in subclinical (left) and hospitalized patients (right). B: Cell type proportions (left) and their differences (right) in subclinical and hospitalized samples, observed in the scRNA-seq analysis. Log fold-differences (FD) are positive for cells types more frequent in hospitalized patients, negative if more frequent in subclinical patients. Red: significant values (|log2(FD)| ≥ 0.58; FDR < 0.05). Cell type proportions: median and interquartile range are shown, and dots representing each sample are colored by disease severity. C: Results of unsupervised clustering (resolution 0.6) by Seurat visualized on the UMAP plot. D: Cell cycle phase assessment computed by Seurat; thereby phase G2 and M are not distinguished. E: Expression profile of MKI67 visualized on the UMAP plot. F: Gene expression levels with statistically significant differences between subclinical and hospitalized patients in B cells subtypes, computed by DESeq2 on pseudo-bulked counts. Genes expressed in less than 5% of the cells in both groups are not visualized. Also see Figure S9.

Figure 7:. BCR features associated to hospitalized dengue.

A: Frequency of the B cell clonotypes visualized on the scRNA-seq UMAP plot. B: Cell type proportions in subclinical and hospitalized samples in cells of non-expanded clonotypes, and in cells belonging to expanded clonotypes consisting of 2 to 5, 6 to 20 or more than 20 cells. C: Ig isotype visualized on the scRNA-seq UMAP plot. D: Frequencies of Ig isotypes in selected cell types. E: Honeycomb plot of the clonotypes in the proliferating and non-proliferating subsets of the plasmablasts of hospitalized patients, colored by Ig isotype. F: Number of somatic hypermutations observed in non-proliferating and proliferating plasmablasts of hospitalized patients. Differences observed between the two groups of plasmablasts are not statistically significant. G: Pairings between the V genes of the heavy and light chain in plasmablasts of hospitalized patients. Values for non-expanded clonotypes (size 1) are not visualized. Pairs with significantly different usage between proliferating and non-proliferating plasmablasts are colored (adjusted p-value < 0.05; gray: non-significant differences). H: Most common heavy chain CDR3 sequences in subclinical and hospitalized patients, colored by cell type. Also see Figure S10.