Journal: EMBO Molecular Medicine

Article Title: IL ‐27 produced during acute malaria infection regulates Plasmodium ‐specific memory CD4 + T cells

doi: 10.15252/emmm.202317713

Figure Lengend Snippet: B6 mice were transferred with PbT‐II cells, infected with Pcc, and treated with control (IgG; n = 1 biological replicate) or anti‐IL‐27 mAb (α‐IL‐27; n = 1 biological replicate) between −1 and 5 days of infection. PbT‐II cells were purified from these mice 7 days after infection and single‐cell RNA sequencing (scRNA‐seq) analysis was performed. Details of the experiments are shown in Fig . UMAP plots of PbT‐II cells from IgG control ( n = 4,030) and anti‐IL‐27 mAb‐treated mice ( n = 7,476) after unsupervised clustering of pooled single‐cell data from the two groups, with clusters colored by gene expression profiles. UMAP clustering of PbT‐II cells colored by cell cycle profiles. Summary graph of proportions of PbT‐II cells in each cluster for IgG and anti‐IL‐27 mAb‐treated mice in (A). Dot plots showing the expression of Th1‐, Tfh‐, Tcmp‐related genes (Ciucci et al , ), and other genes of interest in each UMAP cluster of PbT‐II cells from IgG and anti‐IL‐27 mAb‐treated mice. Dot colors represent the intensity of expression, while dot size represents the proportion of cells with the corresponding expression. Violin plots showing the expression of Th1‐, Tfh‐, Tcmp‐, and proliferation‐associated genes in PbT‐II cells from IgG (light blue) and anti‐IL‐27 mAb (blue) treated mice. Ridge plots showing the expression of published Th1, Tfh, Tmem, and Tcmp CD4 + T cell signatures in each of the UMAP clusters in (A) based on (Ciucci et al , ). Source data are available online for this figure.

Article Snippet: Stained CD4 + T cells were washed using the recommended Cell Wash Protocol 1 in preparation for 10X Single Cell RNA sequencing (Chromium Next GEM Single Cell 5′ v2 Reagent kits (Dual Index); 10X Genomics).

Techniques: Infection, Control, Purification, RNA Sequencing, Gene Expression, Expressing

Journal: EMBO Molecular Medicine

Article Title: IL ‐27 produced during acute malaria infection regulates Plasmodium ‐specific memory CD4 + T cells

doi: 10.15252/emmm.202317713

Figure Lengend Snippet: B6 mice were transferred with PbT‐II cells, infected with Pcc, and were treated with either IgG or anti‐IL‐27 mAb between −1 and 7 days after infection ( n = 1 biological replicate per timepoint). PbT‐II cells were prepared from spleen at day 28 pi, stained for CD4/TCR/CD45.1 and for CD127, KLRG1, and CD49d with TotalSeq antibodies, sort purified, and processed for scRNA‐seq and CITE‐Seq analysis. Details of the experiment are found in Fig . A–G Comparative analysis of scRNA‐seq data from IgG and anti‐IL‐27 mAb‐treated PbT‐II cells. (A) UMAP plot colored of day 28 PbT‐II cells from IgG control ( n = 7,491) and anti‐IL‐27 mAb‐treated mice ( n = 4,944) after unsupervised clustering of pooled single cell data from the two groups, with clusters colored by gene expression profiles. Cluster labels were harmonized to reflect similar gene expression patterns in the clusters at day 7 pi (Fig ) and anti‐IL27 mAb day 7–28 PbT‐II analysis (Fig ). (B) UMAP clustering of PbT‐II cells colored by cell cycle profiles. (C) CITE‐seq analysis of PbT‐II cells for IgG2a (isotype control), CD127, KLRG1, and CD49d, shown in the same UMAP clustering as (A). (D) Proportions (%) of each cluster within PbT‐II cells, with bar graph sizes shown relative to the total number of PbT‐II cells in IgG (36.8 × 10 ) and anti‐IL‐27 mAb treated (265.7 × 10 ) mice. (E) Ridge plots of PbT‐II cells showing the expression of published CD4 + T cell signature genes (Ciucci et al , ). (F) Violin plots comparing the expression of the CD4 + T cell signature genes. (G) Dot plots showing the expression of Th1‐, Tfh‐, Tcmp‐, and proliferation‐associated genes in each cluster. Dot colors represent the intensity of expression, while dot size represents the proportion of cells with the corresponding expression. H Volcano plot of differentially expressed genes between major clusters 1* and 1** within PbT‐II cells from anti‐IL‐27‐treated mice and corresponding Gene Ontology enrichment analysis for the upregulated genes in each group using Metascape. Source data are available online for this figure.

Article Snippet: Stained CD4 + T cells were washed using the recommended Cell Wash Protocol 1 in preparation for 10X Single Cell RNA sequencing (Chromium Next GEM Single Cell 5′ v2 Reagent kits (Dual Index); 10X Genomics).

Techniques: Infection, Staining, Purification, Control, Gene Expression, Expressing

Journal: EMBO Molecular Medicine

Article Title: IL ‐27 produced during acute malaria infection regulates Plasmodium ‐specific memory CD4 + T cells

doi: 10.15252/emmm.202317713

Figure Lengend Snippet: B6 mice were transferred with PbT‐II cells, treated with IgG or anti‐IL‐27 mAb on day −1, 2 and 5 for day 7 analysis, while mice were treated with anti‐IL‐27 mAb on day −1, 2, 5, and 7 for day 14 and 28 analysis ( n = 1 biological replicate per timepoint). PbT‐II cells were purified and subjected to single‐cell RNA sequencing (scRNA‐seq) and CITE‐seq analysis. The ProjecTILs algorithm (Andreatta et al , ) was used to analyze CD4 + T cell states of PbT‐II cells based on a published reference atlas (Andreatta et al , ). A Experimental scheme. B Gating strategy for the sorting of PbT‐II cells for the scRNA‐seq experiments: Spleen cells were stained for CD4, TCRβ, and CD45.1 to distinguish PbT‐II cells and for TotalSeq IgG2a, CD127, KLRG1, and CD49d for CITE‐seq analysis. C Flow cytometry profiles for each PbT‐II sample analyzed for single‐cell transcriptomics. D, E Predicted distribution of the projected PbT‐II cells in IgG and anti‐IL‐27 mAb‐treated mice on day 7 (D) and day 28 (E) after Pcc infection as density contours in a UMAP plot of a CD4 + T cell reference map (Andreatta et al , ). The bar graphs represent the proportions of the PbT‐II cells projected in the indicated reference subtype.

Article Snippet: Stained CD4 + T cells were washed using the recommended Cell Wash Protocol 1 in preparation for 10X Single Cell RNA sequencing (Chromium Next GEM Single Cell 5′ v2 Reagent kits (Dual Index); 10X Genomics).

Techniques: Purification, RNA Sequencing, Staining, Flow Cytometry, Single-cell Transcriptomics, Infection

Journal: EMBO Molecular Medicine

Article Title: IL ‐27 produced during acute malaria infection regulates Plasmodium ‐specific memory CD4 + T cells

doi: 10.15252/emmm.202317713

Figure Lengend Snippet: scRNA‐seq data of PbT‐II cells from Pcc‐infected anti‐IL‐27 mAb‐treated mice (day7, 14, and 28) were pooled, and unsupervised clustering was performed. UMAP plot colored by gene expression clustering. Proportions (%) of each cluster for each time point. Feature plots of indicated genes across cell clusters as distributed in UMAP plots. Dot plots showing the expression of Th1‐, Tfh‐, Tmem‐, and proliferation‐associated genes in each cluster. Dot colors represent the intensity of expression, while dot size represents the proportion of cells with the corresponding expression. Ridge plots of PbT‐II cell clusters showing the expression of published CD4 + T cell signature genes (Ciucci et al , ).

Article Snippet: Stained CD4 + T cells were washed using the recommended Cell Wash Protocol 1 in preparation for 10X Single Cell RNA sequencing (Chromium Next GEM Single Cell 5′ v2 Reagent kits (Dual Index); 10X Genomics).

Techniques: Infection, Gene Expression, Expressing

Journal: EMBO Molecular Medicine

Article Title: IL ‐27 produced during acute malaria infection regulates Plasmodium ‐specific memory CD4 + T cells

doi: 10.15252/emmm.202317713

Figure Lengend Snippet:

Article Snippet: Stained CD4 + T cells were washed using the recommended Cell Wash Protocol 1 in preparation for 10X Single Cell RNA sequencing (Chromium Next GEM Single Cell 5′ v2 Reagent kits (Dual Index); 10X Genomics).

Techniques: Marker, Staining, FACS, Purification, Software, Cell Isolation