RSOB-16-0098.R2 - Supplementary Figures and legends from Physico-chemical properties that control protein aggregation also determine whether a protein is retained or released from necrotic cells

Supplementary material for "Physicochemical properties that control protein aggregation also determine whether a protein is retained or released from necrotic cells" by Samson et al., 2016

Supplementary Table S3 from Physico-chemical properties that control protein aggregation also determine whether a protein is retained or released from necrotic cells

Complete list of enriched cellular compartments in NCC-participating and NCC-resistant protein subsets

Role of TRAIL-mediated tumor cell apoptosis.

<p>(A) DR5 expression on AB1-HA tumor cells <i>in vitro</i>. (B) Tumor growth curves in CY-treated and untreated TRAIL-deficient mice, compared with immunocompetent mice and nude mice. Data shown are mean±SEM (<i>n</i> = 10) from two combined experiments. Tumor cells were inoculated at day 0 and treated with CY at day 10. ** P<0.005 when CY in BALB/c is compared with CY in TRAIL-deficient mice.</p

Role of CD8 T cells and type-I IFN in CY-driven anti-tumor efficacy.

<p>(A) Anti-tumor responses depend on CD8⁺ T cells. Tumor-bearing mice were treated with CY at day 10 after tumor inoculation (day 0) and anti-CD8 mAbs (150 µg) were injected at days −1, 0, 2, 4, 6 and 8 with respect to CY treatment. Data shown are mean±SEM (<i>n</i> = 5) from one representative experiment from a total of three experiments with a total of 15 mice. * P<0.05 when CY is compared with CY + anti-CD8. (B) <i>In vivo</i> IFN-α/β neutralization marginally affects tumor growth in CY-treated mice. BALB/c mice bearing AB1-HA tumors were given IFN-α/β blocking antibody on days −1, +2, +4 with respect to CY treatment. Tumors were inoculated at day 0 and were treated with CY at day 9. Data shown are mean±SEM (<i>n</i> = 5) from one experiment. <i>ns</i> = not-significant.</p

CD8 T cell effector mechanisms.

<p>(A) Tumor growth and Kaplan-Meier survival curves in CY-treated IFN-γ-deficient mice and control mice. Data shown are mean±SEM (<i>n</i> = 5) from one experiment (growth curve, left panel) or total data from two experiments (survival curve, right panel). Tumor cells were inoculated at day 0, treated with CY at day 9. * P<0.05 when CY in immunocompetent mice and IFN-γ deficient mice is compared. (B) Tumor growth curves and Kaplan-Meier survival curves in CY-treated perforin-deficient and normal control mice. <i>ns</i> = not significant when CY in immuno-competent and perforin-deficient mice are compared. Tumor cells were inoculated at day 0 and treated with CY at day 8. <i>ns</i>, not significant. (C) Tumor growth and Kaplan-Meier survival curves after CY treatment in perforin/IFN-γ double-deficient mice, compared to immunocompetent mice. Data shown are mean±SEM (<i>n</i> = 5) from one experiment (growth curve, left panel) or total data from two experiments (survival curve, right panel). Tumor cells were inoculated at day 0 and treated with CY at day 9. * P<0.05, *** P<0.001 when BALB/c <i>+</i> CY is compared with IFN-γ/<i>pfp</i>-ko + CY.</p

Rescue of CY anti-tumor efficacy in nude mice by anti-DR5 antibodies.

<p>Tumor-bearing athymic nude mice (inoculated at day 0) were treated with CY or with PBS at day 9 (a) or day 7 (b) after tumor cell inoculation and were treated with anti-DR5 antibody (clone MD5-1) or PBS at day 8, 12 and 16 after tumor cell inoculation. Data shown are mean±SEM (<i>n</i> = 5/group for each experiment). * P<0.05 when CY is compared with CY + anti-DR5. The effect of anti-TRAIL Ab without CY is shown in (b). Two mice were disqualified from the graph as they were found dead early in the experiment.</p

Antibody-mediated IFNAR1 blockade boosts humoral immune responses during blood-stage infection.

<p>WT mice (n = 5/group) were treated with anti-IFNAR1 blocking antibody (α-Ifnar1) or control IgG prior to and during infection with <i>Pc</i>AS. (A) Representative FACS plots (gated on CD4⁺ TCRβ⁺ live singlets), proportions and absolute numbers of splenic ICOS⁺ CD4⁺ T cells in naïve and infected mice on days 6 and 7 <i>p</i>.<i>i</i>. (B) Representative FACS plots (gated on CD4⁺ TCRβ⁺ live singlets), proportions and numbers of splenic Tfh cells (as PD1⁺CXCR5⁺ CD4⁺ T cells) in naïve and infected and antibody-treated mice, 7 days <i>p</i>.<i>i</i>. Bcl-6 expression is also shown in histograms for Tfh (PD1⁺CXCR5⁺; red gate) and non-Tfh cells (PD1^-CXCR5^-; blue gate), alongside Geometric Mean Bcl-6 expression by these populations in individual mice. (C and D) Numbers of splenic (C) plasmablasts and (D) GC B cells in naïve, and infected and treated mice, 7 days <i>p</i>.<i>i</i>. Data representative of 2 independent experiments. Mann-Whitney U test *P<0.05; **P<0.01.</p

IFNAR1-signalling simultaneously limits splenic Th1 and Tfh cell responses.

<p>(A & B) Representative FACS plots (gated on CD4⁺ TCRβ⁺ live singlets), proportions and absolute numbers of splenic (A) Th1 (IFNγ⁺ Tbet⁺) and (B) emerging Tfh (PD1⁺ CXCR5⁺) cells in WT <i>and Ifnar1</i>^<i>-/-</i> mice (n = 6/group), 6 days <i>p</i>.<i>i</i> with <i>Pc</i>AS. Data representative of two independent experiments. (C) Numbers of splenic ICOS⁺ Th1 cells (Tbet⁺ IFNγ⁺ CD4⁺ T cells) and Tfh cells (PD1⁺CXCR5⁺ CD4⁺ T cells) 6 days <i>p</i>.<i>i</i>. with <i>Pc</i>AS. (D) Numbers of ICOS⁺ Tfh cells (PD1⁺CXCR5⁺ CD4⁺ T cells), 16 days <i>p</i>.<i>i</i>. with <i>Py</i>17XNL infection. (E) Proportions and absolute numbers of splenic CD4⁺ T-cells expressing Ki-67 in naïve, WT and <i>Ifnar1</i>^<i>-/-</i> mice 6 days <i>p</i>.<i>i</i>. with <i>Pc</i>AS. (F) Proportions and absolute numbers of splenic CD4⁺ T-cells expressing Ki-67 in naïve mice, and WT mice, 6 days <i>p</i>.<i>i</i>. with <i>Py</i>17XNL and treatment with α-IFNAR1 or Control IgG. (G) Absolute numbers of splenocytes, CD4⁺ T-cells and B-cells, in WT naïve, infected WT and <i>Ifnar1</i>^<i>-/-</i> mice 6 days (n = 17–18, pooled from three independent experiments (n = 5–6 per expt)) and 8 days (n = 29, pooled from five experiments (n = 5–6 per expt)) <i>p</i>.<i>i</i>. with <i>Pc</i>AS. (H) Absolute numbers of splenocytes, CD4⁺ T-cells and B-cells in WT naïve, infected WT and <i>Ifnar1</i>^<i>-/-</i> mice, 16 days <i>p</i>.<i>i</i>. with <i>Py</i>17XNL (n = 17–18, pooled from three experiments (n = 5–6 per expt)) Mann-Whitney U-test **P<0.01, *P<0.05.</p

IFNAR1-signalling obstructs B-cell and parasite-specific antibody responses during blood-stage infection.

<p>(A) Time-course analysis of parasitemia in WT and <i>Ifnar1</i>^<i>-/-</i>mice (n = 5/group) infected with <i>Py</i>17XNL. (B) ELISA quantitation of <i>Py</i>17XNL-specific IgM, total IgG, IgG1, IgG2b, IgG3 in serum diluted from 1 in 400 in two-fold sequential dilutions to 1 in 3200, and (C) IgG2c at 1 in 400 in the sera of naïve and infected WT and <i>Ifnar1</i>^<i>-/-</i> mice, 16 days <i>p</i>.<i>i</i>. (D&E) Representative FACS plots (gated on B220⁺ CD19⁺ live singlets), proportions and absolute numbers of (D) splenic GC B-cells (GL7⁺ Fas⁺) and (E) Ig-class-switched B-cells (IgD^lo IgM^lo) in naïve and infected WT and <i>Ifnar1</i>^<i>-/-</i> mice, 16 days <i>p</i>.<i>i</i>. (F) Representative FACS plots (gated on B220⁺ CD19⁺ live singlets), proportions and absolute numbers of splenic plasmablasts (IgD^loCD138^hi) in naïve and infected WT and <i>Ifnar1</i>^<i>-/-</i> mice (n = 6), 6 days <i>p</i>.<i>i</i>. (G) Time course analysis of IgM, total IgG, IgG2b and IgG3 levels in naïve and infected WT and <i>Ifnar1</i>^<i>-/-</i> mice (n = 6). Statistics: Mann-Whitney U test (A & C-G), Two way ANOVA and Tukey’s test for multiple comparisons in (B), *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Data representative of three independent experiments for (A), (D) and (E), two for (B) and (F) and one for (C) and (G).</p

IFNAR1-signalling via cDCs limits Tfh and GC B-cell responses.

<p>(A) Schematic for the mixed bone marrow chimeric approach: 50:50 mixed WT (<i>Ifnar1</i>^<i>+/+</i>; CD45.1) and <i>Ifnar1</i>^<i>-/-</i> (CD45.2) bone marrow was transferred into lethally-irradiated <i>rag1</i>^<i>-/-</i> mice (to avoid potential issues with residual radio-resistant T- and B-cells), and subsequently left for 8–12 weeks prior to infection studies. (B) Gating strategy for splenic WT (CD45.1⁺) and <i>Ifnar1</i>^<i>-/-</i> (CD45.2⁺) CD8⁺ (TCRβ^- B220^- CD11c^hi MHC-II^hi CD8α⁺) and CD8^- (TCRβ^- B220^- CD11c^hi MHC-II^hi Sirpα⁺ CD8^-) conventional DCs. (C) Paired analysis between splenic WT and <i>Ifnar1</i>^<i>-/-</i> cDC subsets in individual mice for cell-surface expression of CD86, PD-L1, PD-L2, CD40 and ICOS-L, 2 days <i>p</i>.<i>i</i>. with <i>Py</i>17XNL. Data representative of two independent experiments. Statistics: Wilcoxon test, *P<0.05. (D) Representative FACS plots and proportions for WT (CD45.1⁺) <i>Ifnar1</i>^<i>+/+</i> and <i>Ifnar1</i>^<i>-/-</i> plasmablasts, 7 days <i>p</i>.<i>i</i>. with <i>Pc</i>AS infection. Data representative of 2 independent experiments. Statistics: Wilcoxon test. (E) Representative FACS plots and proportions for WT (CD45.1⁺) <i>Ifnar1</i>^<i>+/+</i> and <i>Ifnar1</i>^<i>-/-</i> CD4⁺ T cells expressing ICOS and CXCR5, 7 days <i>p</i>.<i>i</i>. with <i>Pc</i>AS infection. Experiment performed once. Statistics: Wilcoxon test, *P<0.05. (F) Representative FACS plots (gated on CD4⁺ TCRβ⁺ live singlets), proportions and absolute numbers of splenic Tfh cells and (G) splenic GC B-cells (gated on B220⁺ CD19⁺ live singlets) in CD11cCre^+/- <i>ifnar1</i>^<i>f/f</i> and CD11cCre^-/- <i>ifnar1</i>^<i>f/f</i> littermates (n = 6) (WT naïve mice as staining controls), on day 6 <i>p</i>.<i>i</i>. with <i>Py</i>17XNL; Statistics: Mann-Whitney U-test **P<0.01, *P<0.05. Data representative of 2 independent experiments.</p