The cytokine interleukin (IL)-1β is a key mediator of antimicrobial immunity as well as autoimmune inflammation. Production of IL-1β requires transcription by innate immune receptor signaling and maturational cleavage by inflammasomes. Whether this mechanism applies to IL-1β production seen in T cell-driven autoimmune diseases remains unclear. Here, we describe an inflammasome-independent pathway of IL-1β production that was triggered upon cognate interactions between effector CD4+ T cells and mononuclear phagocytes (MPs). The cytokine TNF produced by activated CD4+ T cells engaged its receptor TNFR on MPs, leading to pro-IL-1β synthesis. Membrane-bound FasL, expressed by CD4+ T cells, activated death receptor Fas signaling in MPs, resulting in caspase-8-dependent pro-IL-1β cleavage. The T cell-instructed IL-1β resulted in systemic inflammation, whereas absence of TNFR or Fas signaling protected mice from CD4+ T cell-driven autoimmunity. The TNFR–Fas–caspase-8-dependent pathway provides a mechanistic explanation for IL-1β production and its consequences in CD4+ T cell-driven autoimmune pathology.
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The data that support the findings of this study are available from the corresponding author upon reasonable request. Source data for Figs. 1–4 are provided with this paper.
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We thank all the members of the Pasare laboratory and R. Bagirzadeh for helpful discussions. We thank A. Ma for sharing WT and D117A Il1b constructs. We thank M. Jordan for sharing Zbtb46-GFP reporter mice. This work was supported by grants from the National Institutes of Health (AI113125 and AI123176) to C.P. M.M.M. was supported by a National Science Foundation Graduate Research Fellowship under grant 2017220107.
The authors declare no competing interests.
Peer review information Ioana Visan was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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a, Gating strategy for flow cytometric analysis of intracellular pro-IL-1β in CD11c+ DCs following BMDC-T cell co-culture. b, Gating strategy for flow cytometric analysis of CD11b+ monocytes and neutrophils frequency in various tissues.
Extended Data Fig. 2 Induction of pro-IL-1β in BMDCs is independent of TLR activation but dependent on TNF.
(a and b) Expression of intracellular pro-IL-1β measured by flow cytometry in WT, Tlr2−/−Tlr4−/−, or Myd88−/− BMDCs (live,CD90−CD11c+) cultured with TH0 cells in the presence of anti-CD3 Ab for 6 h. Fold change indicates proportion of pro-IL-1β+ BMDCs, compared to PBS controls. Error bars indicate s.e.m. three (n = 3) independent experiments. c, Expression of intracellular pro-IL-1β measured by flow cytometry in WT BMDCs (live, CD11c+) stimulated in vitro with recombinant TNF (20 ng/mL) for 6 h. Data are representative of two independent experiments. d, TNFα, as quantified by ELISA, in the supernatants of WT or Tnf−/− TH0 cells cultured with WT or Tnf−/− BMDCs in the presence of anti-CD3 Ab for 6 h. Error bars indicate s.e.m. from n = 4 independent experiments. e, lL-1β was quantified by ELISA in the supernatants of WT TH0 cells cultured with BMDCs of the indicated genotypes in the presence of anti-CD3 Ab and neutralizing CD40L Ab (10 µg/mL) for 6 h. Error bars indicate s.e.m. from n = 2 technical replicates. Data are representative of two independent experiments. (a, b, d) Statistical analysis was performed by paired, two-tailed Student’s t-test. *p < 0.05.
Extended Data Fig. 3 T cell-induced IL-1β production is only partially impaired in gasdermin-D deficient BMDCs.
a, lL-1β was quantified by ELISA in the supernatants of WT TH0 cells cultured with Il1b−/− BMDCs transduced with retrovirus expressing WT or D117A pro-IL-1β in the presence of anti-CD3 Ab for 18 h. Error bars indicate s.e.m. from n = 2 technical replicates. Data are representative of two independent experiments. b, lL-1β was quantified by ELISA in the supernatants of WT TH0 cells cultured with WT or Gsdmd−/− BMDCs in the presence of anti-CD3 Ab for 12 h. Error bars indicate s.e.m. from n = 6 independent experiments. Statistical analysis was performed by paired, two-tailed Student’s t-test. **p < 0.01.
a, Expression of surface FasL measured by flow cytometry in in vitro polarized TH1, TH2 and TH17 cells (live, CD90.2+) cultured with WT BMDCs in the presence of anti-CD3 Ab for 1 h. Data are representative of two independent experiments. (b and c) Cell death as assayed by Zombie Yellow viability dye was measured by flow cytometry in WT, Ripk3−/−, or Ripk3−/−Casp8−/− live,CD90.2+CD11c+ BMDCs cultured with WT TH0 cells in the presence or absence of anti-CD3 Ab and neutralizing TNF Ab (20μg/mL) or FasL Ab (10μg/mL) for 6 h. Data are representative of two independent experiments.
Extended Data Fig. 5 Gating strategy and post sort purity of various myeloid cell populations using in Fig. 5.
(a and b) Pre-sort and post-sort purity of FACS sorted BMDC subsets: CD11b+, CD11b+CD11c+MHCIIhi, or CD11b+CD11c+MHCIIint BMDCs b, and CD11c+MHCIIintCSF1R+ (GM-Macs) or CD11c+MHCIIhiCSF1R− (GM-DCs). Data are representative of 4 independent experiments. c, Post-sort purity of CD11c+ splenic DCs showing lack of CSF1R+ cell contamination. Data are representative of 4 independent experiments. d, Pre-sort and post-sort purity of CD11c+Zbtb46-GFP+Ly6C− splenic cDCs that were FACS sorted from pre-sorted total CD11c+ Zbtb46-GFP splenocytes to obtain a pure cDC population. Data are representative of three independent experiments.
Extended Data Fig. 6 Anti-CD3 stimulation of T cells in vivo leads to IL-1β dependent inflammatory cell recruitment dictated by Fas expression on CD11c+ cells.
a, Expression of intracellular pro-IL-1β measured by flow cytometry in CD11b+Ly6C−CSF1R+ macrophages, CD11b+Ly6G−Ly6Cint monocytes, CD11b+Ly6Chi inflammatory monocytes, and CD11b+Ly6G+ granulocytes quantified from the spleens of WT mice 3-4 h post anti-CD3 Ab injection (50μg, i.v.). n = 3 independent experiments are quantified in the inset. Statistical analysis was performed by paired, one-tailed Student’s t-test. b, Neutrophil infiltration as measured by flow cytometry in the spleen (left panel) or SI-LP (right panel) of WT mice 18 h post anti-CD3 Ab injection (20μg, i.p.). Error bars indicate s.e.m. from n = 4 independent experiments. c, qPCR of Il1b mRNA in lysates of splenocytes collected from WT or Rag1-/- mice 4 h post anti-CD3 Ab injection (50μg, i.v.). Data are normalized to Hprt. Error bars indicate s.e.m. from n = 3 technical replicates. Data are representative of two independent experiments. d, Neutrophil infiltration in the spleen of Rag1-/- mice as measured by flow cytometry 3-4 h post anti-CD3 Abinjection (50μg, i.v.). Error bars indicate s.e.m. from n = 3 independent experiments. e, Expression of Foxp3 and f, ICOS measured by flow cytometry in splenic live, CD4+ T cells from WT mice, 18 h post anti-CD3 Ab injection (20μg, i.p.). Error bars indicate s.e.m. from n = 3 independent experiments. g, Infiltration of CD11b+ cells as measured by flow cytometry in the SI-LP or (h) spleen of WT and Il1b-/- mice, 18 h post anti-CD3 Ab injection (20μg, i.p.). Error bars indicate s.e.m. from n = 3 independent experiments. i, Expression of cell surface Fas measured by flow cytometry in CD11b+ or CD11c+ BMDCs from given genotypes. Data are representative of two independent experiments. j, Neutrophil infiltration (live, CD11b+F480−) as measured by flow cytometry in the SI-LP of Fasfl/fl or Fasfl/fl x CD11c-cre (FasΔDC) mice, 18 h post anti-CD3 Ab injection (20μg, i.p.). Error bars indicate s.e.m. from n = 5 independent experiments. Splenocytes were taken from WT mice immunized with MOG35-55, and stimulated for 4 d in vitro with MOG in the presence of IL-1β, IL-23, and anti-IFNγ. k, lL-1β was quantified by ELISA in the supernatants of WT BMDCs cultured with CD4+ T cells isolated from the stimulated splenocyte culture in the presence of MOG35-55 and neutralizing antibodies TNF Ab (20μg/mL) or FasL Ab (10μg/mL) for 24 h. Error bars indicate s.e.m. from n = 2 independent experiments. (b-h,j) Statistical analysis was performed by unpaired, one-tailed Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s. = not significant.
Extended Data Fig. 7 Illustration of “T cell-instructed” IL-1β production by MPs and its comparison to inflammasome induced IL-1β production by macrophages.
(Left) During inflammasome activation in monocytes and macrophages, TLR and NLR−caspase-1 activation leads to synthesis and cleavage of pro-IL-1β, respectively. Robust production of IL-1β as a result of inflammasome activation is critical for pathogen clearance. (Right) In contrast, “T cell-instructed” IL-1β production by antigen presenting MPs utilizes TNFR signaling for pro-IL1β synthesis while the cleavage signal is provided by Fas-caspase-8 axis. The IL-1β produced upon such T cell instruction drives cytokine production by effector CD4 T cells, systemic leukocyte recruitment, and autoimmunity. Figure was created using Biorender.
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Jain, A., Irizarry-Caro, R.A., McDaniel, M.M. et al. T cells instruct myeloid cells to produce inflammasome-independent IL-1β and cause autoimmunity. Nat Immunol 21, 65–74 (2020). https://doi.org/10.1038/s41590-019-0559-y
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