Identification of a super-functional Tfh-like subpopulation in murine lupus by pattern perception
- German Rheumatism Research Center (DRFZ), A Leibniz Institute, Germany
- University of Potsdam, Germany
- Charité, Campus Mitte, Germany
Figures
Figure 1
PRI allows the visualization of the combinatorial protein expression.
Flow cytometry data from stimulated (PMA/ionomycin) splenic T cells from NZBxW mice were analyzed either conventionally or by bin plots. (A) Overlay of IFN-γ+ cells on all T cells. (B) FlowJo‘s …
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Figure 1—source data 1
Figure 1C,D: Frequencies of double and single producers.
- https://cdn.elifesciences.org/articles/53226/elife-53226-fig1-data1-v2.xlsx
Figure 2 with 3 supplements
The cytokine co-expression is altered in PD-1+ T cells in young and old mice.
(A) Disease status of NZBxW mice was scored according to their age and proteinuria (PU). The disease score was correlated with the frequencies of PD-1+ T cells and serum levels of anti-ds-DNA-IgG …
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Figure 2—source data 1
Figure 2A: Serum levels of anti-ds-DNA-IgG vs. PD-1 frequency.
- https://cdn.elifesciences.org/articles/53226/elife-53226-fig2-data1-v2.xlsx
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Figure 2—source data 2
Figure 2B: Frequencies of protein expression of mice in disease score 1 and 5, respectively.
Data represent two independent experiments with n = 6 or seven mice per group.
- https://cdn.elifesciences.org/articles/53226/elife-53226-fig2-data2-v2.xlsx
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Figure 2—source data 3
Figure 2D: Frequencies of IL-2, TNF-α, IFN-γ and IL-10 producers in PD-1 subpopulations of mice in disease score 1 and 5, respectively.
Data represent two independent experiments with n = 7 mice per group.
- https://cdn.elifesciences.org/articles/53226/elife-53226-fig2-data3-v2.xlsx
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Figure 2—source data 4
Figure 2E: Frequencies of TNF-α cells of the PD-1+ IFN-γ+ subpopulation in mice of disease score 1 and 5, respectively.
Data represent two independent experiments with n = 5 mice per group.
- https://cdn.elifesciences.org/articles/53226/elife-53226-fig2-data4-v2.xlsx
Figure 2—figure supplement 1
The combinatorics of the co-expression of IFN-γ, TNF-α and IL-2 in CD44+ T cells is altered with disease progression.
(A) Gating strategy to identify CD44+ CD4+ T cells. (B–D) Quantification of cytokine co-expression in disease stages by pie charts (B), bar plots (C) and bin plots (D). Bin plots visualize density …
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Figure 2—figure supplement 1—source data 1
Figure 2—figure supplement 1B: Frequencies of boolean combinations of the co-expression of cytokines in mice of disease score 1 to 5.
Data represent one experiment with PMA/ionomycin stimulated splenic T cells: n = 2 mice per group.
- https://cdn.elifesciences.org/articles/53226/elife-53226-fig2-figsupp1-data1-v2.xlsx
Figure 2—figure supplement 2
PD-1+ T cells exhibit differential cytokine expression levels compared to PD-1- T cells in young and old diseased mice.
(A) Representative histogram overlays for disease score 1 (top) and 5 (bottom) showing the cytokine expression in the PD-1 subgroups. Controls are colored in grey. (B) Barplots with mean …
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Figure 2—figure supplement 2—source data 1
Figure 2—figure supplement 2B: Mean fluorescence intensity in PD-1 subpopulations of mice in disease score 1 and 5, respectively.
Data represent two independent experiments with n = 4 mice per group.
- https://cdn.elifesciences.org/articles/53226/elife-53226-fig2-figsupp2-data1-v2.xlsx
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Figure 2—figure supplement 2—source data 2
Figure 2—figure supplement 2E: Frequencies of boolean combinations of the co-expression of IL-2, TNF-α, IFN-γ and IL-10 in PD-1 subpopulations in disease score 1 and 5, respectively.
Data represent two independent experiments with n = 7 mice per group.
- https://cdn.elifesciences.org/articles/53226/elife-53226-fig2-figsupp2-data2-v2.xlsx
Figure 2—figure supplement 3
Bin plot patterns are reproducible and statistically robust.
(A) Representative contour plots for the co-production of IL-10 with other cytokines by PMA/ionomycin stimulated T cells of diseased mice. (B–C) Comparison of the IL-10 cell expression patterns of …
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Figure 2—figure supplement 3—source data 1
Figure 2—figure supplement 3D: Frequencies shown in the upper right quadrant in the bin plots of mice in disease score 1 and 5, respectively.
Data represent two independent experiments with n = 4 mice per group. Samples were compared using an unpaired two-tailed t-test.
- https://cdn.elifesciences.org/articles/53226/elife-53226-fig2-figsupp3-data1-v2.xlsx
Figure 3 with 1 supplement
Most IL-21 producers in the NZBxW model strongly co-express Th1 cytokines but no CXCR5.
(A) Co-production of IL-21 with cytokines and the Tfh markers PD-1 and CXCR5 in concatenated CD4+ CD44+ cells from young and old diseased mice. (B) Tfh cells were identified based on their high …
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Figure 3—source data 1
Figure 3C: Proportion of different CD4+CD44+ T cell subsets in young score 1-diseased mice versus old score 5-diseased mice.
Data from two pooled experiments involving n = 1–5 mice per group.
- https://cdn.elifesciences.org/articles/53226/elife-53226-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
The majority of IL-21 is produced by non-Tfh cells.
(A) Co-production of IFN-γ, IL-2, IL-10, IL-21 and TNF-α was analyzed by a pie chart. (B, C) PRI-based statistical analysis of marker co-expression in young and old mice. (D) Bin plots of PD-1 …
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Figure 3—figure supplement 1—source data 1
Figure 3—figure supplement 1A: Raw data to determine the frequencies of boolean combinations of coexpression of five cytokines.
- https://cdn.elifesciences.org/articles/53226/elife-53226-fig3-figsupp1-data1-v2.xlsx
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Figure 3—figure supplement 1—source data 2
Figure 3—figure supplement 1B, C: Frequencies from IL-21+ subpopulations extracted from PRI bin plots.
Data as in Figure 3—source data 1.
- https://cdn.elifesciences.org/articles/53226/elife-53226-fig3-figsupp1-data2-v2.xlsx
Figure 4 with 1 supplement
Super-functional T cells exhibit Th1 characteristics and are CD40Lhi ICOS+.
(A) Bin plots visualize the co-expression of PD-1 and IFN-γ with various proteins in stimulated (PMA/ionomycin) splenic T cells of old mice. (B) Distribution of the top 50% Z+ cells of selected …
Figure 4—figure supplement 1
Super-functional T cells are CD40Lhi ICOS+.
(A) Bin plots visualize the relative expression level of parameter Z (MFI+) per bin of PMA/ionomycin stimulated splenic T cells. Grey bins contain less than 10 Z+ cells. (B) Histograms as example …
Figure 5
Super-functional T cells in peripheral organs exceed extrafollicular T cells and Tph cells in terms of frequency.
(A) Absolute numbers of PD-1 subsets in spleens. (B) Frequency of IL-21 producers in spleens. (C, D) Frequency of IL-21 producers in terms of localization and in terms of PD-1 subset. Data represent …
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Figure 5—source data 1
Figure 5A: Frequencies of PD-1 subpopulation.
Data represent two independent experiments with n = 4 mice per organ.
- https://cdn.elifesciences.org/articles/53226/elife-53226-fig5-data1-v2.xlsx
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Figure 5—source data 2
Figure 5B: Frequencies of IL-21 producers in spleens.
Data represent two independent experiments with n = 4 mice per organ.
- https://cdn.elifesciences.org/articles/53226/elife-53226-fig5-data2-v2.xlsx
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Figure 5—source data 3
Figure 5C: Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset.
Data represent two independent experiments with n = 4 mice per organ.
- https://cdn.elifesciences.org/articles/53226/elife-53226-fig5-data3-v2.xlsx
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Figure 5—source data 4
Figure 5D: Frequencies of IL-21 producers in terms of localization and in terms of PD-1 subset.
Data represent two independent experiments with n = 4 mice per organ.
- https://cdn.elifesciences.org/articles/53226/elife-53226-fig5-data4-v2.xlsx
Figure 6 with 1 supplement
Comparison of Tfh and Tfh-like cell subpopulations by bin patterns.
(A) Subdivision of Tmem cells into nine categories based on their PD-1 and IFN-γ expression (compare to D). (B) Barplots depicting the frequencies of CXCR5, Bcl6 and IL-21 producers in respective …
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Figure 6—source data 1
Figure 6B: Frequencies of CXCR5, Bcl6 and IL-21 producers in respective subpopulations.
Data represent three independent experiments with n = 3–9 mice.
- https://cdn.elifesciences.org/articles/53226/elife-53226-fig6-data1-v2.xlsx
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Figure 6—source data 2
Figure 6C: Frequencies of IL-21 of CD44+ producers in respective subpopulations.
Data represent three independent experiments with n = 3–9 mice.
- https://cdn.elifesciences.org/articles/53226/elife-53226-fig6-data2-v2.xlsx
Figure 6—figure supplement 1
PRI results can be confirmed with viSNE and conventional analysis.
(A) Bar plots of subpopulation frequencies sub-divided into regions as described in Figure 6 (A, D). (B) viSNE plots displaying cell density and MFI of different markers. Grey circles mark the …
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Figure 6—figure supplement 1—source data 1
Figure 6—figure supplement 1A: Frequencies of protein expressions sub-divided into regions.
Data represent three independent experiments with n = 3–11 mice.
- https://cdn.elifesciences.org/articles/53226/elife-53226-fig6-figsupp1-data1-v2.xlsx
Figure 7 with 2 supplements
Super-functional T cells provide help for IgG production in co-cultures with B cells and expand B cells with activated GL7+ Fas+Germinal Center-like phenotype.
(A) Splenocytes were FACS-sorted for naïve B cells (CD19+ B220+ CD44low) and Th1 cells (CD4+ CXCR5- CD44+ CD25- CXCR3+). Both fractions were co-cultured for 5 days in presence or absence of …
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Figure 7—source data 1
Figure 7B: Frequencies of IgG concentrations in co-cultures with different antibody settings.
Data represent one of three independent experiments with n = 3–4 mice.
- https://cdn.elifesciences.org/articles/53226/elife-53226-fig7-data1-v2.xlsx
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Figure 7—source data 2
Figure 7C: Cells per well and frequencies of GC-like and CD138+ cells in co-cultures with different T cell subsets.
Data on B cell count and viability are representative of one out of two experiments involving six replicates per condition. Data on GL7+ Fas+ GC-like B cells and CD138+ antibody-producing cells are pooled data from two independent experiments involving 1–7 replicates.
- https://cdn.elifesciences.org/articles/53226/elife-53226-fig7-data2-v2.xlsx
Figure 7—figure supplement 1
Sorting strategy and purity control of naive B cells and Th1 cells.
FACS sorting scheme for naive B cells (CD19+ CD44low and Th1 cells (CD4+ CXCR5- CD44+ CD25- CXCR3+). Fractions before (left) and after sort (right) are shown.
Figure 7—figure supplement 2
Functional comparison of CXCR3+ PD-1lo Tsh, CXCR3- PD-1lo CD4+ T cells and PD-1hi cells in B and T cell co-cultures.
(A) Gating strategy used to sort CXCR3+ PD-1lo Tsh, CXCR3- PD-1lo CD4+ T cells and PD-1hi CXCR5+/-. Upper row (pseudo-color plots) shows pre-sorted cells prepared from pooled splenocytes of …
Author response image 1
Author response image 2
Additional files
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Source code 1
A notebook to show step by step how to create the bin plots (in HTML).
- https://cdn.elifesciences.org/articles/53226/elife-53226-code1-v2.zip
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Source code 2
A notebook how to create bin functions (in R).
- https://cdn.elifesciences.org/articles/53226/elife-53226-code2-v2.zip
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Supplementary file 1
Key resources table.
- https://cdn.elifesciences.org/articles/53226/elife-53226-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/53226/elife-53226-transrepform-v2.docx
