Journal: bioRxiv

Article Title: Temporal mixture modelling of single-cell RNA-seq data resolves a CD4 + T cell fate bifurcation

doi: 10.1101/074971

Figure Lengend Snippet: Single-cell mRNA-sequencing of activated antigen-specific PbTII CD4 + T cells. ( A ) Experimental setup. PbTII cells were transferred from a single donor to multiple recipients. “n” refers to the number of recipient mice per time point. Also shown are the numbers of single cells from which high-quality mRNA-seq data was successfully recorded. The numbers in parentheses refer to the experiment presented in . p.i., post-infection. ( B-C ) Representative FACS plots showing bifurcation of splenic Th1 (IFNγ + T-bet + ) and Tfh (CXCR5 + Bcl6 + ) PbTII CD4 + T cells at day 7 post-infection with Pc AS. ( D ) Flow cytometry data indicate concurrent differentiation of Th1 (IFNγ + ) and Tfh (CXCR5 + ) PbTII CD4 + T cells within the spleen of Pc AS-infected mice (n=4). Index expression is the product of MFIand proportion IFNγ + or CXCR5 + . These data are representative of two independent experiments. MFI, mean fluorescence intensity. ( E ) PCA of single PbTII cells at 7 days post-infection with Pc AS. The arrows represent the Pearson correlation with PC1 and PC2. Cell size refers to the number of detected genes. The size of the data points also represents cell size. “Th1 signature” and “Tfh signature” refer to cumulative expression of genes associated with Th1 or Tfh phenotypes . PC, Principal Component. (F) Expression of top 50 genes with largest PC2 loadings of day 7 cells (D). The genes were annotated as Th1-or Tfh-associated based on public datasets ( , , , ). * Cdk2ap2 appears twice because two alternative genomic annotations exist. PC, Principal Component

Article Snippet: T cells were stained with the following antibodies (Biolegend): CD4-APC (GK1.5), TCRβ-APC-Cy7 (H57-597), CD45.1-FITC (A20), Vα2-FITC (B20.1), Vβ12-eFluor710 (MR11-1) (eBioscience), CD69-PE (H1.2F3), PD-1-APCCy7 (29F.1A12), CXCR5-Biotinylated (2G8) (BD Pharmigen), Streptavidin-PeCy7, CD183 (CXCR3)-PE (CXCR3-173), IFNγ-BV421 (XMG1.2), Bcl6-PercpCy5.5 (K112-91) (BD Pharmigen), Ki-67-PE (16A8), T-bet-eFluor660 (4-B10) (eBioscience) and TCF-1-PE (CD63D9) (Cell Signaling Technology).

Techniques: Sequencing, Infection, Flow Cytometry, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Temporal mixture modelling of single-cell RNA-seq data resolves a CD4 + T cell fate bifurcation

doi: 10.1101/074971

Figure Lengend Snippet: The bifurcation of T cell fates is accompanied by changes in transcription, proliferation and metabolism. ( A ) The relationship of Th1-Tfh bifurcation point and the number of detected genes per cell. ( B ) The expression level of the proliferative marker Ki-67 (encoded by the Mki67 gene) across pseudotime. ( C ) Representative FACS plots showing kinetics of CellTrace ™ Violet (CTV) dilution and Ki67, IFNγ or CXCR5 expression, with summary graphs showing % of PbTII cells expressing these (after 10 6 PbTII cells transferred) in uninfected (Day 0) and PcAS -infected mice at indicated days post-infection (n=4 mice/timepoint, with individual mouse data shown in summary graphs; solid line in summary graphs indicates results from third order polynominal regression analysis.) Data are representative of two independent experiments. ( D ) Experimental and computational analysis of cell cycle speed of PbTII CD4 + T cells activated in response to Pc AS. The allocation of cells to cell cycle phases was performed by flow cytometry using Hoechst staining and computationally using the Cyclone algorithm . The relative cell-cycle speed was determined by measuring the fraction of cells in S, G2, or M phases. ( E ) Cell size estimation using FSC (Forward Scatter) measurements of PbTII cells. ( F ) Cellular metabolic activity of PbTII cells in naive mice (n=3) and at days 4 and 7 post-infection (n=6) as determined by flow cytometric assessment of ribosomal protein S6 phosphorylation (p-S6). Histogram and proportions are representative of two independent experiments. Statistics are one-way ANOVA and Tukey's multiple comparisons tests ***p<0.001.

Article Snippet: T cells were stained with the following antibodies (Biolegend): CD4-APC (GK1.5), TCRβ-APC-Cy7 (H57-597), CD45.1-FITC (A20), Vα2-FITC (B20.1), Vβ12-eFluor710 (MR11-1) (eBioscience), CD69-PE (H1.2F3), PD-1-APCCy7 (29F.1A12), CXCR5-Biotinylated (2G8) (BD Pharmigen), Streptavidin-PeCy7, CD183 (CXCR3)-PE (CXCR3-173), IFNγ-BV421 (XMG1.2), Bcl6-PercpCy5.5 (K112-91) (BD Pharmigen), Ki-67-PE (16A8), T-bet-eFluor660 (4-B10) (eBioscience) and TCF-1-PE (CD63D9) (Cell Signaling Technology).

Techniques: Expressing, Marker, Infection, Flow Cytometry, Staining, Activity Assay

Journal: bioRxiv

Article Title: Temporal mixture modelling of single-cell RNA-seq data resolves a CD4 + T cell fate bifurcation

doi: 10.1101/074971

Figure Lengend Snippet: Mechanisms underlying the differentiation of Th1 and Tfh cells. ( A ) Parallel Th1 and Tfh differentiation within cells of a single CD4 + T cell clone. The colours represent clones determined by sequence analysis of secondary T cell receptor genes (Supplementary Tables 2 and 3). ( B ) Identification of genes associated with the differentiation of Th1 or Tfh cells. For every gene, the correlation of its expression with pseudotime (x-axis) and Tfh trend assignment (y-axis) are shown. Statistical significance was determined using the bifurcating score (methods). Genes satisfying the significance threshold of FDR<0.002 are represented in colours according to the functional classification of the genes (methods and Supplementary Table 4). FDR, False Discovery Rate, estimated by performing the same analysis with permuted data. ( C ) The genes with strongest association with Th1 (left) or Tfh differentiation (right). The genes were filtered using the bifurcation score as in (B). The genes were then ranked in descending order of association with either Th1 or Tfh trend. Cdk2ap2 appears twice because two alternative genomic annotations exist. ( D ) The expression of Id2 (upper panel) and Tcf7 (lower panel) across the pseudotime. The curves represent the Th1 (red) and Tfh (blue) trends when weighing the information from data points according to trend assignment. The colour of the data points represents the strength of the relationship with the two alternative trends. ( E ) The correlation of Id2 and Tcf7 expression at single-cell level. Using a rolling window method, Spearman rho was calculated in windows of 100 cells. The pseudotime values are mean values within each window. ( F ) A model depicting known interactions of Id2 and Tcf7 . The colours represent the Pearson correlation of gene expression and the Th1 trend assignment in single cells. The numbers in parentheses refer to the original publications ( , , , , , – ). ( G ) The expression kinetics of the chemokine receptor genes Cxcr5, Cxcr3, Ccr4, Ccr2, Ccr5, and Cxcr6 across pseudotime. The curves represent the expression patterns associated with the Th1 (red) and the Tfh (blue) trends. ( H ) A model summarizing the expression patterns of Id2 , Tcf7 , and the chemokine receptors during Th1-Tfh cell fate determination. The size of the cell represents proliferative capacity ( . The colour of receptors and transcription factors represent differences in expression level.

Article Snippet: T cells were stained with the following antibodies (Biolegend): CD4-APC (GK1.5), TCRβ-APC-Cy7 (H57-597), CD45.1-FITC (A20), Vα2-FITC (B20.1), Vβ12-eFluor710 (MR11-1) (eBioscience), CD69-PE (H1.2F3), PD-1-APCCy7 (29F.1A12), CXCR5-Biotinylated (2G8) (BD Pharmigen), Streptavidin-PeCy7, CD183 (CXCR3)-PE (CXCR3-173), IFNγ-BV421 (XMG1.2), Bcl6-PercpCy5.5 (K112-91) (BD Pharmigen), Ki-67-PE (16A8), T-bet-eFluor660 (4-B10) (eBioscience) and TCF-1-PE (CD63D9) (Cell Signaling Technology).

Techniques: Clone Assay, Sequencing, Expressing, Functional Assay

Journal: bioRxiv

Article Title: Temporal mixture modelling of single-cell RNA-seq data resolves a CD4 + T cell fate bifurcation

doi: 10.1101/074971

Figure Lengend Snippet: Correlation of expression of Ifng with Tcf7 and Id2 across pseudotime. (A-B) The correlation of the expression Ifng with Tcf7 (A) and with Id2 (B) at single-cell level. Using a rolling window method, Spearman rho was calculated in windows of 100 cells. The pseudotime values are mean values within each window. (C) Representative FACS plots showing TCF-1 (gene product of Tcf7 ) expression in CXCR5+ (blue gate) and IFNγ+ (red gate) PbTIIs, compared to naïve PbTIIs (gray) (isotype control shown in black in FACS histogram) at 7 days post-infection. Summary graph shows mean & standard deviations for geometric mean fluorescence intensity of TCF-1 expression in gated PbTII populations (n=4 mice) Statistics: Mann-Whitney U test *p<0.05.

Article Snippet: T cells were stained with the following antibodies (Biolegend): CD4-APC (GK1.5), TCRβ-APC-Cy7 (H57-597), CD45.1-FITC (A20), Vα2-FITC (B20.1), Vβ12-eFluor710 (MR11-1) (eBioscience), CD69-PE (H1.2F3), PD-1-APCCy7 (29F.1A12), CXCR5-Biotinylated (2G8) (BD Pharmigen), Streptavidin-PeCy7, CD183 (CXCR3)-PE (CXCR3-173), IFNγ-BV421 (XMG1.2), Bcl6-PercpCy5.5 (K112-91) (BD Pharmigen), Ki-67-PE (16A8), T-bet-eFluor660 (4-B10) (eBioscience) and TCF-1-PE (CD63D9) (Cell Signaling Technology).

Techniques: Expressing, Infection, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Temporal mixture modelling of single-cell RNA-seq data resolves a CD4 + T cell fate bifurcation

doi: 10.1101/074971

Figure Lengend Snippet: Flow cytometric validation of select marker genes in PbTII cells prior to and after bifurcation. (A) Representative FACS plots showing kinetics of CellTraceTM Violet (CTV) dilution and CXCR6 expression, with summary graphs showing % of PbTII cells expressing this (after 106 PbTII cells transferred) in un-infected (Day 0) and PcAS -infected mice at indicated days post-infection (n=4 mice/timepoint, with individual mouse data shown in summary graphs; solid line in summary graphs indicates results from third order polynominal regression analysis.) Data are representative of two independent experiments. (B) Representative FACS plots showing CXCR6 expression in Tbethi (red gate) and Bcl6hi (blue gate) PbTII cells, compared to naïve PbTIIs (grey) at 7 days post-infection. Summary graph shows mean & standard deviations for geometric mean fluorescence intensity of CXCR6 expression in gated PbTII populations (n=4 mice) Statistics: Mann-Whitney U test *p<0.05. (C) Representative FACS plots and proportions of splenic PbTII cells co-expressing CXCR5 and CXCR3 in naive (gray; n=3) or infected mice (green; n=6) at 4 days post-infection with P.chabaudi chabaudi AS ( Pc AS). Results are representative of two independent experiments. Statistics: Mann-Whitney U test *p<0.05.

Article Snippet: T cells were stained with the following antibodies (Biolegend): CD4-APC (GK1.5), TCRβ-APC-Cy7 (H57-597), CD45.1-FITC (A20), Vα2-FITC (B20.1), Vβ12-eFluor710 (MR11-1) (eBioscience), CD69-PE (H1.2F3), PD-1-APCCy7 (29F.1A12), CXCR5-Biotinylated (2G8) (BD Pharmigen), Streptavidin-PeCy7, CD183 (CXCR3)-PE (CXCR3-173), IFNγ-BV421 (XMG1.2), Bcl6-PercpCy5.5 (K112-91) (BD Pharmigen), Ki-67-PE (16A8), T-bet-eFluor660 (4-B10) (eBioscience) and TCF-1-PE (CD63D9) (Cell Signaling Technology).

Techniques: Marker, Expressing, Infection, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Temporal mixture modelling of single-cell RNA-seq data resolves a CD4 + T cell fate bifurcation

doi: 10.1101/074971

Figure Lengend Snippet: B cells are essential for Tfh responses in PbTII cells during Pc AS infection. Representative FACS plots (gated on CD4+ TCRβ+ CD45.1+ live singlets) of splenic PbTII cells, showing proportions exhibiting Tfh (Bcl6+ CXCR5+) and Th1 (Tbet+ IFNγ+) phenotypes in WT mice (receiving 104 PbTII cells), treated with anti-CD20 monoclonal antibodies (0.25mg) to deplete B-cells, or control IgG, and infected for 7 days with Pc AS. Individual mice data (n=5) shown in summary graph. Mann-Whitney U test *p<0.05; **p<0.01. Results are representative of two independent experiments.

Article Snippet: T cells were stained with the following antibodies (Biolegend): CD4-APC (GK1.5), TCRβ-APC-Cy7 (H57-597), CD45.1-FITC (A20), Vα2-FITC (B20.1), Vβ12-eFluor710 (MR11-1) (eBioscience), CD69-PE (H1.2F3), PD-1-APCCy7 (29F.1A12), CXCR5-Biotinylated (2G8) (BD Pharmigen), Streptavidin-PeCy7, CD183 (CXCR3)-PE (CXCR3-173), IFNγ-BV421 (XMG1.2), Bcl6-PercpCy5.5 (K112-91) (BD Pharmigen), Ki-67-PE (16A8), T-bet-eFluor660 (4-B10) (eBioscience) and TCF-1-PE (CD63D9) (Cell Signaling Technology).

Techniques: Infection, MANN-WHITNEY

Journal: bioRxiv

Article Title: Temporal mixture modelling of single-cell RNA-seq data resolves a CD4 + T cell fate bifurcation

doi: 10.1101/074971

Figure Lengend Snippet: Myeloid cells influence Th bifurcation in uncommitted PbTII cells. ( A-C ) 131 single splenic CD8α + and CD11b + CD8α − cDCs from a naïve mouse, mixed cDCs from a day 3-infected mouse, and (D-F) 154 single Ly6C hi monocytes from naïve and infected mice were analysed by scRNAseq, with mRNA reads filtered by minimum expression of 100 TPM in at least 2 cells. ( A & D ) Principal Component Analyses, showing PC combinations best separating populations of (A) cDCs, and (D) Ly6C hi monocytes from naïve and infected mice. ( B & E ) Volcano plots showing fold-change and confidence for differentially expressed genes between (B) cDCs or (E) monocytes in infected versus naïve mice - genes filtered on expression in >10 cells; genes satisfying qval < 0.05 are represented in colours according to functional classification displayed. Full gene lists are provided in and , respectively. ( C & F ) Expression heatmaps for significantly (qval<0.05) differentially expressed genes in (C) cDCs and (F) Ly6C hi monocytes, between naive and infected mice: cells and genes are ordered according to PC score and loading respectively, using PC6 for cDCs, and PC2 for Ly6C hi monocytes. The 12 common genes between cDCs and monocyte heatmaps are annotated in (F). ( G ) Representative FACS histograms and proportions of splenic CD8α + cDCs, CD8α − cDCs and Ly6C hi monocytes expressing CXCL9 in naive and infected mice between 2-7 days post-infection - individual mouse data plotted with line at mean; data representative of two independent experiments (n=4 mice/time point/experiment). ( H ) Scheme depicting experimental design: PbTII cells were transferred into LysM Cre x iDTR mice 1 day prior to infection. At 3 days p.i., mice were treated with diphtheria toxin (DT) or control saline, with PbTII Th1/Tfh responses assessed at 7 days p.i.. Representative FACS plots (gated on splenic PbTII cells) showing Th1 proportions (T-bet hi IFNγ + ) and Tfh proportions (CXCR5 + ) in DT or saline-treated LysM Cre x iDTR mice; data pooled from two independent experiments. Numbers depict proportions within respective gates. Statistics: Mann-Whitney U Test. ****p<0.001; NS, not significant. ( I ) Summary model proposing chemokine interactions between non-bifurcated PbTII cells and myeloid cells support a Th1 fate, while Tfh fates are sustained by B cells.

Article Snippet: T cells were stained with the following antibodies (Biolegend): CD4-APC (GK1.5), TCRβ-APC-Cy7 (H57-597), CD45.1-FITC (A20), Vα2-FITC (B20.1), Vβ12-eFluor710 (MR11-1) (eBioscience), CD69-PE (H1.2F3), PD-1-APCCy7 (29F.1A12), CXCR5-Biotinylated (2G8) (BD Pharmigen), Streptavidin-PeCy7, CD183 (CXCR3)-PE (CXCR3-173), IFNγ-BV421 (XMG1.2), Bcl6-PercpCy5.5 (K112-91) (BD Pharmigen), Ki-67-PE (16A8), T-bet-eFluor660 (4-B10) (eBioscience) and TCF-1-PE (CD63D9) (Cell Signaling Technology).

Techniques: Infection, Expressing, Functional Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Temporal mixture modelling of single-cell RNA-seq data resolves a CD4 + T cell fate bifurcation

doi: 10.1101/074971

Figure Lengend Snippet: Single-cell mRNA-sequencing of activated antigen-specific PbTII CD4 + T cells. ( A ) Experimental setup. PbTII cells were transferred from a single donor to multiple recipients. “n” refers to the number of recipient mice per time point. Also shown are the numbers of single cells from which high-quality mRNA-seq data was successfully recorded. The numbers in parentheses refer to the experiment presented in . p.i., post-infection. ( B-C ) Representative FACS plots showing bifurcation of splenic Th1 (IFNγ + T-bet + ) and Tfh (CXCR5 + Bcl6 + ) PbTII CD4 + T cells at day 7 post-infection with Pc AS. ( D ) Flow cytometry data indicate concurrent differentiation of Th1 (IFNγ + ) and Tfh (CXCR5 + ) PbTII CD4 + T cells within the spleen of Pc AS-infected mice (n=4). Index expression is the product of MFIand proportion IFNγ + or CXCR5 + . These data are representative of two independent experiments. MFI, mean fluorescence intensity. ( E ) PCA of single PbTII cells at 7 days post-infection with Pc AS. The arrows represent the Pearson correlation with PC1 and PC2. Cell size refers to the number of detected genes. The size of the data points also represents cell size. “Th1 signature” and “Tfh signature” refer to cumulative expression of genes associated with Th1 or Tfh phenotypes . PC, Principal Component. (F) Expression of top 50 genes with largest PC2 loadings of day 7 cells (D). The genes were annotated as Th1-or Tfh-associated based on public datasets ( , , , ). * Cdk2ap2 appears twice because two alternative genomic annotations exist. PC, Principal Component

Article Snippet: T cells were stained with the following antibodies (Biolegend): CD4-APC (GK1.5), TCRβ-APC-Cy7 (H57-597), CD45.1-FITC (A20), Vα2-FITC (B20.1), Vβ12-eFluor710 (MR11-1) (eBioscience), CD69-PE (H1.2F3), PD-1-APCCy7 (29F.1A12), CXCR5-Biotinylated (2G8) (BD Pharmigen), Streptavidin-PeCy7, CD183 (CXCR3)-PE (CXCR3-173), IFNγ-BV421 (XMG1.2), Bcl6-PercpCy5.5 (K112-91) (BD Pharmigen), Ki-67-PE (16A8), T-bet-eFluor660 (4-B10) (eBioscience) and TCF-1-PE (CD63D9) (Cell Signaling Technology).

Techniques: Sequencing, Infection, Flow Cytometry, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Temporal mixture modelling of single-cell RNA-seq data resolves a CD4 + T cell fate bifurcation

doi: 10.1101/074971

Figure Lengend Snippet: The expression of Tbx21 (left) and Bcl6 (right) across pseudotime. The curves represent the Th1 (red) and Tfh (blue) trends when weighing the information from data points according to trend assignment. The color of the data points represents the strength of the relationship with the Th1 trend.

Article Snippet: T cells were stained with the following antibodies (Biolegend): CD4-APC (GK1.5), TCRβ-APC-Cy7 (H57-597), CD45.1-FITC (A20), Vα2-FITC (B20.1), Vβ12-eFluor710 (MR11-1) (eBioscience), CD69-PE (H1.2F3), PD-1-APCCy7 (29F.1A12), CXCR5-Biotinylated (2G8) (BD Pharmigen), Streptavidin-PeCy7, CD183 (CXCR3)-PE (CXCR3-173), IFNγ-BV421 (XMG1.2), Bcl6-PercpCy5.5 (K112-91) (BD Pharmigen), Ki-67-PE (16A8), T-bet-eFluor660 (4-B10) (eBioscience) and TCF-1-PE (CD63D9) (Cell Signaling Technology).

Techniques: Expressing

Journal: bioRxiv

Article Title: Temporal mixture modelling of single-cell RNA-seq data resolves a CD4 + T cell fate bifurcation

doi: 10.1101/074971

Figure Lengend Snippet: B cells are essential for Tfh responses in PbTII cells during Pc AS infection. Representative FACS plots (gated on CD4+ TCRβ+ CD45.1+ live singlets) of splenic PbTII cells, showing proportions exhibiting Tfh (Bcl6+ CXCR5+) and Th1 (Tbet+ IFNγ+) phenotypes in WT mice (receiving 104 PbTII cells), treated with anti-CD20 monoclonal antibodies (0.25mg) to deplete B-cells, or control IgG, and infected for 7 days with Pc AS. Individual mice data (n=5) shown in summary graph. Mann-Whitney U test *p<0.05; **p<0.01. Results are representative of two independent experiments.

Article Snippet: T cells were stained with the following antibodies (Biolegend): CD4-APC (GK1.5), TCRβ-APC-Cy7 (H57-597), CD45.1-FITC (A20), Vα2-FITC (B20.1), Vβ12-eFluor710 (MR11-1) (eBioscience), CD69-PE (H1.2F3), PD-1-APCCy7 (29F.1A12), CXCR5-Biotinylated (2G8) (BD Pharmigen), Streptavidin-PeCy7, CD183 (CXCR3)-PE (CXCR3-173), IFNγ-BV421 (XMG1.2), Bcl6-PercpCy5.5 (K112-91) (BD Pharmigen), Ki-67-PE (16A8), T-bet-eFluor660 (4-B10) (eBioscience) and TCF-1-PE (CD63D9) (Cell Signaling Technology).

Techniques: Infection, MANN-WHITNEY