STING activation promotes autologous type I interferon-dependent development of type 1 regulatory T cells during malaria

Abstract

The development of highly effective malaria vaccines and improvement of drug-treatment protocols to boost antiparasitic immunity are critical for malaria elimination. However, the rapid establishment of parasite-specific immune regulatory networks following exposure to malaria parasites hampers these efforts. Here, we identified stimulator of interferon genes (STING) as a critical mediator of type I interferon production by CD4+ T cells during blood-stage Plasmodium falciparum infection. The activation of STING in CD4+ T cells by cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) stimulated IFNB gene transcription, which promoted development of IL-10- and IFN-γ-coproducing CD4+ T (type I regulatory [Tr1]) cells. The critical role for type I IFN signaling for Tr1 cell development was confirmed in vivo using a preclinical malaria model. CD4+ T cell sensitivity to STING phosphorylation was increased in healthy volunteers following P. falciparum infection, particularly in Tr1 cells. These findings identified STING expressed by CD4+ T cells as an important mediator of type I IFN production and Tr1 cell development and activation during malaria.

Keywords: Cellular immune response; Infectious disease.

Figure 1. Higher TMEM173 expression by Tr1 cells compared with Th1 cells during malaria.

(A) Schematic showing the experimental design for the RNA-Seq analysis of Tr1 and Th1 cells from volunteers participating in CHMI studies with P. falciparum. (B) List of the top 30 differentially upregulated genes between Tr1 and Tneg cells as well as Th1 and Tneg cells from the CHMI study. (C) Validation of higher TMEM173 mRNA expression by Tr1 cells compared with Th1 cells. Human CD4⁺ T cells were isolated from 5 healthy volunteers and then cultured with αCD3ε and αCD28 mAbs plus IL-2 for 3 days. Tr1 and Th1 cells were sorted based on IL-10 and IFN-γ expression, as shown in Figure 1A. TMEM173 mRNA was detected by qPCR and normalized to the housekeeping gene 18S rRNA. Data were log2-transformed for statistical analysis. Lines connect paired samples, and box shows extent of lower and upper quartiles plus median, while whiskers indicate minimum and maximum data points. n = 5 samples. Repeated measures 1-way ANOVA with Šídák’s multiple-comparisons test. **P < 0.01; ***P < 0.001; ****P < 0.0001. (D) IPA prediction of genes directly or indirectly associated with IL-10 as well as the extent of the predicted interaction, as indicated by pink to red coloring.

Figure 2. Modulation of CD4⁺ T cell STING expression by CRISPR/Cas9 gene editing.

(A) CD4⁺ T cells from 8 healthy volunteers were stimulated with αCD3ε and αCD28 mAbs plus IL-2 for 72 hours before nucleofection and then stimulated for another 72 hours under the same conditions. Cells were treated with or without cGAMP for 18 hours before analysis. (B) A diagram showing the gene structure of human TMEM173 and the CRISPR gRNA–targeting sites within exon 4. Domain structure of the human STING protein showing the 4 transmembrane domains of the N-terminal, responsible for ligand binding and protein dimerization. The C-terminal contains the cyclic dinucleotide domain and binding sites for TBK1 and IRF3. (C) qPCR validation of TMEM173 mRNA expression in control and CRISPR gRNA–treated samples. TMEM173 mRNA was normalized to 18S rRNA in each sample. Data were log2-transformed for statistical analysis. Lines connect paired samples, and box shows the extent of lower and upper quartiles plus median, while whiskers indicate minimum and maximum data points. n = 8 samples. Two-tailed paired t test. ***P < 0.001. (D) Representative Western blot showing the effect of CRIPSR gRNA modification of TMEM173 in response to cGAMP stimulation, as indicated. β-Actin was used as a protein-loading control, relative to STING protein levels. (E) Representative FACS plots showing loss of STING phosphorylation in control and CRISPR gRNA samples treated, as indicated.

Figure 3. CD4⁺ T cell STING activation promotes Tr1 cell development.

Human CD4⁺ T cells were cultured and subjected to CRISPR/Cas9 TMEM173 gene editing as shown in Figure 2. (A) Gating strategy used to assess changes in human CD4⁺ T cells. Cells were gated on single cells, live cells, and conventional CD4⁺ T cells (FoxP3^–) before further analysis. Representative plots and enumeration showing the frequency of LAG3⁺CD49b⁺ CD4⁺ T cells following CRISPR/Cas9-mediated modification of TMEM173 expression. Lines connect paired samples, and box shows extent of lower and upper quartiles plus median, while whiskers indicate minimum and maximum data points. (B) Representative histograms and enumeration showing the frequencies of p-STING–positive LAG3⁺CD49b⁺, LAG3⁺CD49b^–, LAG3^–CD49b⁺, and LAG3^–CD49b^– CD4⁺ T cell subsets. Box shows the extent of lower and upper quartiles plus median, while whiskers indicate minimum and maximum data points. (C–E) Expression of IL10, IFNG, and IFNB1 in the control and TMEM173-modified cells with and without cGAMP activation was measured by qPCR. Lines connect paired samples, and box shows the extent of lower and upper quartiles plus median, while whiskers indicate minimum and maximum data points. n = 8 (A, C–E); n = 5 (B). Repeated measures 2-way ANOVA with Šídák’s multiple-comparisons test. *P < 0.05; ***P < 0.001; ****P < 0.0001.

Figure 4. STING-dependent IFN-β1 production by CD4⁺ T cells drives Tr1 cell development.

(A) CD4⁺ T cells were stimulated with αCD3ε and αCD28 mAbs plus IL-2, as shown, prior to treating with an antibody against type I IFNR (αIFNR), isotype control mAb, or cGAMP for 18 hours before analysis, as indicated. (B) Cells were gated on conventional CD4⁺ T cells (FoxP3^–), as shown in Figure 3A. The frequency of LAG3⁺CD49b⁺ CD4⁺ T cells as well as IL10, IFNG, and IFNB1 mRNA levels in each treatment group was measured. qPCR data were normalized to the housekeeping gene 18S rRNA. (C) CRISPR/Cas9 modification of TMEM173 in CD4⁺ T cells stimulated with αCD3ε and αCD28 mAbs plus IL-2, as shown, prior to treating with 100 ng/μl recombinant IFN-β1 and/or 30 μg/ml cGAMP 18 hours before analysis, as indicated. (D) Frequency of LAG3⁺CD49b⁺ CD4⁺ T cells and IL10, IFNG, and IFNB1 mRNA were measured in CD4⁺ T cells treated as indicated. (B and D) Lines connect paired samples, and box shows extent of lower and upper quartiles plus median, while whiskers indicate minimum and maximum data points. n = 5. Repeated measures 2-way ANOVA with Šídák’s multiple-comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 5. CD4⁺ T cell STING is required for Tr1 cell development in experimental malaria.

(A) 5 × 10⁵ CD45.2⁺ PbTII^ΔSting and 5 × 10⁵ CD45.1⁺ CD45.2⁺ PbTII^WT cells were transferred into the Ptprca (CD45.1⁺) recipient mice at day –1. The mice were infected with P. berghei ANKA (PbA) on day 0 and were assessed on day 4. (B) Representative histograms and enumeration showing the IL-10– and IFN-γ–producing PbTII^WT and PbTII^ΔSting cells. (C and D) Representative plots and enumeration showing the frequencies of IL-10⁺IFN-γ⁺ and LAG3⁺CD49b⁺ CD4⁺ T cells, respectively. Data in each plot were pooled from 3 independent experiments. Lines connect paired samples, and box shows extent of lower and upper quartiles plus median, while whiskers indicate minimum and maximum data points. n = 24. Two-tailed paired t test. ***P < 0.001; ****P < 0.0001.

Figure 6. Type I IFN signaling to CD4⁺ T cells drives Tr1 cell development in experimental malaria.

(A) 5 × 10⁵ CD45.2⁺ PbTII^ΔIfnar and 5×10⁵ CD45.1⁺ CD45.2⁺ PbTII^WT cells were transferred into the Ptprca (CD45.1⁺) recipient mice at day –1. The mice were infected with P. berghei ANKA (PbA) on day 0 and were assessed on day 4. (B) Representative histograms and enumeration showing IL-10– and IFN-γ–producing PbTII^WT and PbTII^ΔIfnar cells. (C and D) Representative plots and enumeration showing the frequencies of IL-10⁺IFN-γ⁺ and LAG3⁺CD49b⁺ CD4⁺ T cells, respectively. Data are pooled from 2 independent experiments. Lines connect paired samples, and box shows extent of lower and upper quartiles plus median, while whiskers indicate minimum and maximum data points. n = 10. Two-tailed paired t test. ****P < 0.0001.

Figure 7. Tr1 cells from humans infected with P. falciparum are more sensitive to STING activation.

(A) PBMCs were isolated from volunteers participating in a CHMI study with P. falciparum at day 0 and 15 p.i. and stimulated with or without cGAMP for 1.5 hours before analysis. (B) Representative histogram and enumeration showing the expression of p-STING in different Th cell subsets on days 0 and 15 p.i. (C) PBMCs were stimulated with uRBCs or pRBCs for 18 hours with or without cGAMP before analysis of CD4⁺ T cell subset frequencies. (D) Tr1 (LAG3⁺CD49b⁺) cell frequencies as a percentage of CD4⁺ T cells are shown. (E) PBMCs were stimulated with uRBCs or pRBCs for 72 hours and stimulated with cGAMP 18 hours before analysis. (F) Frequency of p-STING⁺CD4⁺ T cells in the presence of pRBCs with or without cGAMP at days 0 and 15 p.i. (G) Expression of p-STING in different CD4⁺ T cell subsets in the presence of pRBCs and cGAMP at days 0 and 15 p.i. (H) IL-10 and IFN-γ produced in the cell-culture supernatant in the presence of pRBCs with or without cGAMP at days 0 and 15 p.i. Lines connect paired samples, and box shows extent of lower and upper quartiles plus median, while whiskers indicate minimum and maximum data points. (B, D, F, G and H) n = 8. Repeated measures 2-way ANOVA with Šídák’s multiple-comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.