IL-10-producing Th1 cells possess a distinct molecular signature in malaria

Abstract

Control of intracellular parasites responsible for malaria requires host IFN-γ+T-bet+CD4+ T cells (Th1 cells) with IL-10 produced by Th1 cells to mitigate the pathology induced by this inflammatory response. However, these IL-10-producing Th1 (induced type I regulatory [Tr1]) cells can also promote parasite persistence or impair immunity to reinfection or vaccination. Here, we identified molecular and phenotypic signatures that distinguished IL-10-Th1 cells from IL-10+Tr1 cells in Plasmodium falciparum-infected people who participated in controlled human malaria infection studies, as well as C57BL/6 mice with experimental malaria caused by P. berghei ANKA. We also identified a conserved Tr1 cell molecular signature shared between patients with malaria, dengue, and graft-versus-host disease. Genetic manipulation of primary human CD4+ T cells showed that the transcription factor cMAF played an important role in the induction of IL-10, while BLIMP-1 promoted the development of human CD4+ T cells expressing multiple coinhibitory receptors. We also describe heterogeneity of Tr1 cell coinhibitory receptor expression that has implications for targeting these molecules for clinical advantage during infection. Overall, this work provides insights into CD4+ T cell development during malaria that offer opportunities for creation of strategies to modulate CD4+ T cell functions and improve antiparasitic immunity.

Keywords: Immunology; Infectious disease; Th1 response.

Figure 1. A molecular signature for human Tr1 cells during P. falciparum malaria.

A schematic showing a brief outline of the work flow (A) for isolating peripheral blood Th1 (IFN-γ⁺IL-10^–) and Tr1 cells (IFN-γ⁺IL-10⁺) (B). Mean-difference plot from differential expression analysis between Tr1 and Th1 cells (C). The 521 upregulated DEGs are colored red, the 794 downregulated DEGs are colored blue, and nonsignificant genes are colored grey. Genes of interest are labelled and outlined in black. (D) The top 10 up (right) and downregulated (left) canonical pathways identified in Tr1 cells relative to Th1 cells are listed, as well as significantly different predicted upstream transcription factors, cytokines and transmembrane receptors between the 2 cell populations (E). The analysis was performed on 5 paired Th1 and Tr1 cell samples isolated from the blood of volunteers participating in CHMI studies with P. falciparum.

Figure 2. Common DEGs associated with human Tr1 cells in malaria, dengue fever and GVHD.

DEGs from malaria Tr1 and Th1 cell comparisons (from Figure 1) were compared to the top 250 genes in IL-10⁺IFN-γ⁺ versus IL-10^–IFN-γ⁺ dengue virus-specific CD4⁺T cells (ref. 35) and to 289 DEGs in alloantigen-specific Tr1 cells versus non-Tr1 cells (ref. 36). The Venn diagram shows 11 Tr1 cell DEGs were common to all 3 diseases.

Figure 3. A molecular signature for mouse Tr1 cells during experimental malaria caused by PbA.

A schematic showing the workflow for isolating splenic Th0 (FoxP3^–IFN-γ^–IL-10^–), Th1 (FoxP3^–IFN-γ⁺IL-10^–), and Tr1 cells (FoxP3^–IFN-γ⁺IL-10⁺) for RNA-Seq analysis (A). Mean-difference plot from differential expression analysis between Tr1 and Th1 cells (B). The 1,025 upregulated DEGs are colored red, the 1,006 downregulated DEGs are colored blue, and nonsignificant genes are colored grey. Genes of interest are labelled and outlined in black. A heat map with the top 1,000 upregulated DEGs between Tr1 and Th1 cells is shown with previously identified Tr1 cell-associated gene signatures labelled on the right (C). Th0, Th1, and Tr1 cells from the spleens of the same animals (n = 5) were compared.

Figure 4. Common DEGs and pathways in human and mouse Tr1 cells relative to Th1 cells during Plasmodium infection.

All upregulated DEGs and the top 10 downregulated DEGs in Tr1 cells relative to Th1 cells are shown (A). The interactions between these genes were assessed using the STRING: protein-protein interaction networks functional enrichment analysis. Genes found with no interactions were removed (B). Gene over-representation analysis was used to identify enriched biological processes based on the common DEGs in both human and mouse Tr1 cells, relative to Th1 cells. Selected processes with a positive enrichment ratio are shown in (C).

Figure 5. Development of coinhibitory receptor rich Tr1 cells during CHMI studies.

Peripheral blood Tr1 cells defined by high levels of CD49b and LAG3 expression were assessed by FACS from volunteers participating in CHMI studies prior to infection (Day 0) and 8 days after antiparasitic drug treatment (Day 16) (A). The expression of cMAF, BLIMP-1, PD1, ICOS, CTLA4, TIGIT, TIM3, CCR5, Tbet, and CCR2 on Tr1 cells was measured, and MFI of staining presented as a heat map (B). Clustering of Tr1 cells based on MFI of all molecules (C) and individual molecule expression in the tSNE plot (D) was performed. Coinhibitory–receptor rich clusters among Tr1 cells can be visualized in the heatmap,and clusters that are significantly different between day 0 and 16 after infection are indicated (E). Arcsinh-scaled MFI values used to generate heat maps are shown in Supplemental Table 5. n = 8 paired volunteer samples; **P < 0.01; significance assessed by Mann-Whitney test (A) and using edgeR for all clusters at day 0 and day 16 after infection, indicated by red circles (E).

Figure 6. Development of coinhibitory–receptor rich Tr1 cells in the spleen during experimental malaria caused by infection of triple reporter (Il10gfp × Ifngyfp × Foxp3rfp) C57BL/6 mice with PbA.

Splenic CD4⁺T cells were identified by flow cytometry at day 5 after infection (A). Clustering of cells based on expression of IFN-γ, IL-10, FoxP3, CD49b, LAG3, PD1, TIGIT, TIM3, CCR2, and CCR5 (B) and individual molecule expression by tSNE plot (C) was performed. The MFI of staining for all 20 cell clusters identified was presented as a heat map, along with the relative frequency of each cluster (D). Selected clusters were then overlayed on all CD4⁺T cells and the expression of IL-10 and IFN-γ, LAG3 and CD49b, or PD1 and TIGIT is shown (E). n = 5 individual mice (B–E). Arcsinh scaled MFI values used to generate heat maps are shown in Supplemental Table 5.

Figure 7. Distinct roles for cMAF and BLIMP-1 in human Tr1 cell development and functions.

CRISPR/Cas9 editing of cMAF, PRDM1, and IL10 was conducted in primary human CD4⁺T cells, followed by activation and assessment (A). IL-10 and IFN-γ levels were measured in cell culture supernatants after 72 hours of activation (B). The expression of cMAF and BLIMP-1 is shown in tSNE plots, as well as in violin plots for each sgRNA-treated group (C). The expression (as MFI) and percentage (as positive events) is summarized in dot-plot format (D). Clustering based on the MFI of all molecules in each treatment group (upper tSNE) and individual molecules (lower heat map) was performed, and clusters significantly different between control and PRDM1 sgRNA groups are indicated by red circles (E). n = 4 paired volunteer samples in each treatment group; *P < 0.05 and **P < 0.01 were assessed by paired 1-way ANOVA (B) and significance was assessed using edgeR for clusters, indicated by red circles (E). Arcsinh scaled MFI values used to generate heat maps are shown in Supplemental Table 5.