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RIPK1 and NF-κB signaling in dying cells determines cross-priming of CD8+ T cells

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Science  16 Oct 2015:
Vol. 350, Issue 6258, pp. 328-334
DOI: 10.1126/science.aad0395
  • Fig. 1 Necroptotic cells release DAMPs and induce dendritic cell maturation.

    (A to C) NIH 3T3 cells expressing the death constructs were stimulated with dimerizer. Cells were harvested at the indicated times and stained with Annexin-V and Live/Dead reagent (A); cleaved caspase-3 antibody and Live/Dead reagent (B); or CRT antibody (C). Cells that are Annexin V+ Live/Dead (indicating phosphatidylserine exposure before membrane permeabilization) or cleaved caspase-3+ (indicating the activation of executioner caspases) are undergoing apoptosis. At later times (24 hours), staining with Live/Dead reagent indicates a loss of plasma membrane integrity and characterizes secondary necrotic cells. Rapid membrane permeabilization without activation of executioner caspases (caspase-3Live/Dead+) is a feature of necroptosis. Results are from one representative experiment (n = 2 independent experiments). (D and E) ATP and HMGB1 were quantified from dying cell culture supernatants. Results are reported as means (± SEM) of triplicates of one representative experiment (n = 3 independent experiments). (F) BMDCs were cocultured with dimerizer-treated acC8-, acR3-, and acR3ΔC-expressing cells for 24 hours. DC maturation phenotypes were assessed by flow cytometry. Results are reported as in (D) and (E) (n = 4 independent experiments). (G) 2 × 106 dimerizer-treated cells were injected into the peritoneal cavity of WT C57BL/6 mice. Forty-eight hours later, peritoneal cells were collected, and immune cells were enumerated by cytometry. Bars indicate means of two pooled independent experiments with four to five mice per group [except in the phosphate-buffered saline (PBS) group]. Each circle represents one mouse. P values for (F) were determined by the one-way ANOVA test; P values for (G) were determined by the Kruskal-Wallis test (multigroup comparision), followed by Dunn’s post-test, comparing each group to the PBS group. *P < 0.05; ****P < 0.0001; NS, not significant. acC8, caspase-8 apoptosis; acR3, RIPK3 necroptosis; acR3ΔC, RHIM-less RIPK3 necroptosis.

  • Fig. 2 Necroptotic cells are immunogenic and require RHIM-dependent ripoptosome formation for efficient cross-priming of CD8+ T cells.

    To elicit cross-priming, we intradermally injected OVA-expressing dying cells (H-2q) into mice (H-2b), and we analyzed them on day 9 postimmunization (p.i.). (A and B) Using Kb-SIINFEKL tetramers, OVA-specific CD8+ T cells were quantified and plotted as a percentage of total CD8+ T cells. Bars indicate medians, and results are pooled from three independent experiments with three to six mice per group (each circle represents one mouse). NI, nonimmunized. (C and D) We quantified the production of IFN-γ, tumor necrosis factor–α (TNF-α), and IL-2 in response to ex vivo SIINFEKL peptide restimulation. In (C), representative fluorescence-activated cell sorting (FACS) plots are shown, with a pseudo-colored density plot in the left panel. Numbers indicate the percentage of gated cells. In (D), the frequency of IFN-γ–expressing and polyfunctional cells is plotted. Results are pooled from three independent experiments with three to six mice per group and reported as individual mice (each circle represents one mouse). The left panel shows medians as horizontal bars (IFN-γ); the right panel shows means with SEM (error bars) (TNF-α and IL-2). (E and F) In vivo cytotoxicity assays were performed in acR3-OVA–immunized mice. At day 8 p.i., mice were adoptively transferred with carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled splenocytes. The frequency of CSFEhi (irrelevant peptide control) and CFSElow (SIINFEKL-loaded) splenocytes (injected at a 1:1 ratio) was determined at day 9. Representative FACS plots are shown in (E), and the percent of specific killing is plotted in (F). In (F), bars indicate medians, and results are pooled from three independent experiments with four mice per group (each circle represents one mouse). (G) Tumor challenge experiments were performed, involving the injection of 5 × 105 B16F10-OVA cells on day 12 p.i. Results are reported as a survival curve from one representative experiment with 8 to 11 mice per group (n = 2 independent experiments). For (A) and (B), P values were determined by the Kruskal-Wallis test, followed by Dunn’s post-test; for (D) and (F), P values were determined by the Mann-Whitney test; and in (G), mice survival percentages were compared using the log-rank test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

  • Fig. 3 RIPK3 oligomerization results in RIPK1-dependent activation of NF-κB–mediated gene expression.

    (A to D) acC8-, acR3-, and acR3ΔC-expressing NIH 3T3 cells were treated with dimerizer. At the indicated times, protein extracts were analyzed by Western blot (n = 2 independent experiments) (A); RNA was extracted for transcriptional profiling (n = 3 independent experiments) (B), or culture supernatants were collected for luminex analysis [(C) and (D)]. (E to H) We quantified the release of IL-6 release upon addition of dimerizer, after treating the cells as follows: In (E), 2 μg/ml of ActD or 2.5 μg/ml of CHX were added at the indicated times, after the addition of dimerizer (0 hours), and IL-6 was measured at 6 hours. In (F), cells were preteated with 10 μM of Wedelolactone (NFκBi), and IL-6 was measured at the indicated times. In (G), acR3 cells that were stably expressing the control vector (acR3-vector) or the mutant SR IκB (acR3-SR) were used. In (H), control NIH 3T3 cells (Tet-acR3), and cells lacking RIPK1 and expressing RIPK3 2xFv under a tetracycline promoter (Tet-acR3-ripk1−/−), were treated overnight with 500 ng/ml of doxycycline before the addition of dimerizer. In (C) to (H), the data are presented as means (± SEM) of triplicates from one representative experiment (n ≥ 2 independent experiments). The heat map indicates the relative expression of the indicated transcript (red indicates high levels and green indicates low levels of expression).

  • Fig. 4 RIPK1 expression and NF-κB activation during cell death are required for efficient cross-priming and anti-tumor immunity.

    (A) Mice were immunized with Tet-acR3-OVA and Tet-acR3-OVA-ripk1−/− NIH 3T3 cells. Data represent one experiment with six mice per group; bars indicate medians. (B) acR3-OVA NIH 3T3 cells were pretreated with dimethyl sulfoxide (DMSO) or BAY 11-7085 (NFκBi, 10 μM) for 10 min before the addition of dimerizer and immunization. (C) Mice were immunized with acR3-OVA cells expressing NF-κB SR or the control vector. In (D), acR3-OVA were pretreated with DMSO or ActD for 45 min before immunization. (E and F) OVA-expressing MEFs were transfected with 10 μg/ml of poly I:C and, after 6 hours, used for immunization. WT or ripk1−/− cells were used in (E); cells expressing the control vector or NF-κB SR were used in (F). Cross-priming was assessed on day 9 p.i. In (B) to (F), results shown are pooled from three independent experiments with three to six mice per group. (G and H) CT26 control (Ctrl) cells or cells from a CRISPR/cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9)–modified line that lacks RIPK1 expression (CT26 ripk1−/−) were poly I:C–transfected and injected into Balb/cByJ mice. Seven days later, spleens and lymph nodes were harvested, and IFN-γ production was quantified (G). In (H), mice were challenged with 5 × 105 WT CT26 cells injected in the opposite flank, and tumor growth was monitored every 3 days. Results are from one representative experiment with six mice per group (n = 3 independent experiments). P values were determined by the Mann-Whitney test [(A) to (G)] or the two-way ANOVA test (multiple group comparison, comparing each group to the NI group) (H). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Supplementary Materials

  • RIPK1 and NF-κB signaling in dying cells determines cross-priming of CD8+ T cells

    Nader Yatim, Hélène Jusforgues-Saklani, Susana Orozco, Oliver Schulz, Rosa Barreira da Silva, Caetano Reis e Sousa, Douglas R. Green, Andrew Oberst, Matthew L. Albert

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S11
    • Tables S1
    • Captions for Movies S1 to S3

    Images, Video, and Other Other Media

    Movie S1
    acC8-expressing NIH-3T3 cells were treated with 100nM dimerizer in the presence of Yo- Pro3 (1μM) and cell death was imaged every 30 minutes for 24 hours using the IncuCyte imaging system
    Movie S2
    acR3-expressing NIH-3T3 cells were treated with 100nM dimerizer in the presence of Yo- Pro3 (1μ) and cell death was imaged every 5 minutes for 3 hours using the IncuCyte imaging system
    Movie S3
    acR3ΔC-expressing NIH-3T3 cells were treated with 100nM dimerizer in the presence of Yo- Pro3 (1μM) and cell death was imaged every 5 minutes for 3 hours using the IncuCyte imaging system

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