Cell Reports, Volume 24 Supplemental Information IRF-5 Promotes Cell Death in T Cells during Chronic Infection Aymeric Fabié, Linh Thuy Mai, Xavier Dagenais-Lussier, Akil Hammami, Julien van Grevenynghe, and Simona Stäger
Supplemental information
Figure S1 A Isotype control Spleen 0,16 0.48 1.68 7.66 12.5 Liver IRF-5 0.56 8.54 8.38 9.63 9.83 C (4 - CD62L + ) 4 + CD62L - 4 + CD62L + Spleen 12.9 12.4 Isotype control 0.19 0.03 0.47 Liver Normalized frequency 6.84 8.79 0.42 1.71 1.63 DS Liver Spleen Normalized frequency 51.3 84.6 14.2 10.2 IRF-5 2.27 2.14 18.4 34.2 16.0 32.6 DS Figure S1. IRF-5 expression and nuclear translocation in hepatic and splenic T cell. Related to Figure 1. Mice were infected with 2x10 7 amastigotes intravenously and euthanized at various time points after infection. (A) Representative dot plots of splenic T cells expressing IRF5; () representative histograms of IRF5 nuclear translocation; and (C) representative dot plots of splenic effector (4 + CD62L - ), memory (4 + CD62L + ) and naïve (4 - CD62L + ) T cells expressing IRF5 over the course of L. donovani infection.
A Figure S2 1.96 87.4 1.39 86.7 Gated on Lin - ETP 0.30 0.50 DN2 ETP 0.30 0.60 DN2 CD8 C 2.55 5.81 3.52 5.95 CD11 7 CD25 30.7 DN3 29.5 DN3 11.7 8.92 16.0 17.6 CD8 0.19 0.25 D 0.01 0.03 0.12 0.15 1.30 1.45 0.16 0.04 0.33 0.34 2.08 2.01 0.47 0.51 1.07 1.11 1.31 4.66 5.62 5.09 1.22 1.31 1.32 1.42 IFN -γ 4.56 5.74 PD-1 TIM3 8.32 8.64 FoxP3 1.33 1.30 IFN -γ IL-10 3.45 3.76 Figure S2. Analysis of T cells in the thymus of T cell-specific Irf5 -/- mice and effector cell functions during VL. Related to Figure 2. (A) Representative dot plots of thymic T cells from naïve Irf5 f/f -Cre - and Irf5 f/f -Cre + mice, showing + CD8 + double positive (DP), + or CD8 + single positive and - CD8 - double negative (DN) T cells. () Gating strategy for ETP (early thymic progenitors) and DN2 and 3. T cell progenitors were first gated on Lin - (220, CD3ε, CD11b, GR1, Ter119, CD8α) and then separated according to c-kit (CD117) and CD25 expression as follows: c-kit + CD25 - ETP, c-kit + CD25 + DN2, and c-kit - CD25 + DN3. (C) Representative dot plots of splenic and CD8 T cells from naïve Irf5 f/f -Cre - and Irf5 f/f -Cre + mice. (D) Representative dot plots for IFNγ + CD3 + +, PD-1 + TIM3 + CD3 + +, IFNγ + IL-10 + CD3 + +, and FoxP3 + CD3 + + cells present in the spleen of L. donovani infected Irf5 f/f -Cre - and Irf5 f/f -Cre + mice.
Figure S3 0.75 A C D 12.4 16.4 30.6 74.6 CD3 % of CD3 + 25 20 15 10 5 DR5 % of DR5 + 40 30 20 10 % of AnnV + PI AnnV 40 30 20 10 23.0 % of 220 + 220 80 60 40 20 0 0 0 0 Figure S3. Expression of CD3, DR5, annexin V, and 220 in adoptively transferred lo/neg T cells. Related to Figure 4. 5.1 T cells were adoptively transferred into C57L/6 mice prior to L. donovani infection. Mice were euthanized at p.i. and adoptively transferred 5.1 cells that have down regulated were analysed for CD3, DR5, Annexin V, and 220 expression. Graphs show representative dot plots (upper panels) and percentages (lower panels) of CD3 (A), DR5 (), Annexin V (C), and 220 (D) positive cells.
Figure S4 A C 0.006 0.00 0.00 0.25 0.40 7.45 2.94 3.06 0.92 1.27 7.20 7.20 7.77 1.30 1.33 20.6 3.52 2.72 1.65 1.63 DR5 55.3 Fas 3.28 3.61 TNFR-I 2.36 3.04 D E 6.60 3.72 0.00 18.5 TNFR-I 4.90 1.30 CD3 4.93 3.02 3.14 1.17 7.20 6.10 17.4 14.1 13.4 2.32 PI 29.3 1.00 AnnV Figure S4. Expression of DR5, FasR, TNFR-I, and Annexin V during VL. Related to Figure 4. Representative dot plots for (A) DR5, () Fas and (C) TNFR-I expression in splenic T cells from naïve and L. donovani infected mice. (D) Positive control for TNFR-I staining; PMC from naïve mice. (E) Representative dot plots for Annexin V staining of T cells from L. donovani infected Irf5f/f-Cre - and Irf5f/f-Cre + mice.
Figure S5 A 3.67 7.10 6.85 5.85 0.60 1.76 1.00 0.70 5.58 5.67 6.52 4.23 0.40 0.71 0.85 1.50 8.19 30.8 8.61 10.5 3.12 20.9 5.93 4.65 10.1 7.98 9.85 27.2 4.43 6.37 4.69 17.1 20.7 43.4 23.3 22.1 10.2 26.5 5.18 6.85 DR5 21.4 23.5 23.1 30.8 IRF-5 9.17 10.7 6.22 23.0 Figure S5. DR5 and IRF-5 expression in purified T cells following treatment with various stimuli. Related to Figure 6. Mice were infected with 2x10 7 amastigotes intravenously and euthanized at various time points after infection. T cells were purified at, 12, and 28 p.i. from the spleen of infected mice and incubated for 30h with medium,,,, living parasites, parasite RNA, parasite DNA, or supernatant from apoptotic cells. Graphs show representative dot plots of splenic T cells expressing (A) DR5 and () IRF5 after treatments.
Figure S6 SN RNA SN DNA DNA 28s rrna 18s rrna Figure S6. RNA and DNA from SN of apoptotic cells. Related to Figure 7. Agarose gel electrophoresis of RNA (left panel) and DNA (right panel) purified from the supernatant of apoptotic cells.