Research ArticleT CELLS

IL-23 costimulates antigen-specific MAIT cell activation and enables vaccination against bacterial infection

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Science Immunology  15 Nov 2019:
Vol. 4, Issue 41, eaaw0402
DOI: 10.1126/sciimmunol.aaw0402
  • Fig. 1 Both BM-derived and non–BM-derived APCs can drive MAIT cell accumulation in the lungs in response to bacterial infection.

    (A) MAIT cell frequency in the lungs of C57BL/6 mice uninfected or at day 7 after intranasal infection with 106 S. Typhimurium BRD509 (Salm.) or 104 L. longbeachae (Leg.). Data show individual mice and means ± SEM. **P < 0.01, ****P < 0.0001, one-way ANOVA with Dunnett’s multiple comparisons. (B) Schematic of BM chimeras for (C) and (D). (C) Frequency and (D) absolute number of MAIT cells of WT → WT or WT → Mr1−/− chimeric mice uninfected or on day 7 p.i. intranasally infected with 106 S. Typhimurium BRD509 or 104 L. longbeachae. *P < 0.05, ***P < 0.001, ****P < 0.0001, multiple unpaired t test. (E) Lung CD45.2+TCRβ+ cells from Mr1−/− → WT chimeric mice with or without 105 adoptively transferred C57BL/6 MAIT cells 2 weeks after transfer showing reconstitution of MAIT cells in the lungs. (F) Schematic of protocol and BM chimeras for (G) and (H). (G) Frequency and (H) absolute number of transferred MAIT cells on day 7 p.i. in Mr1−/− → WT chimeric mice, adoptively transferred with 105 WT MAIT cells and then intranasally infected with 106 S. Typhimurium BRD509 or 104 L. longbeachae. ns, nonsignificant. ****P < 0.0001, one-way ANOVA with Dunnett’s multiple comparisons. Also see fig. S1.

  • Fig. 2 Active invasion by S. Typhimurium is vital for MAIT cell stimulation in vivo.

    (A) CD69 expression on Jurkat.MAIT cells after coculture (for 16 hours) with C1R.MR1 cells and 5-OP-RU or filtered culture supernatant from S. Typhimurium SL1344, SL1344ΔinvA, or media control (LB). Data show mean fluorescence intensity (MFI) of gated Jurkat.MAIT cells, with SEM of triplicate samples as error bars. ***P < 0.001, ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons. The experiment was performed twice with similar results. (B) Bacterial counts from human and murine cells (106 per well) of either epithelial [human: BSB-II; mouse: lung epithelial type 1 (LET1) (96)] or macrophage [human: THP-1; mouse: bone marrow–derived macrophages (BMDM)] origin after 1-hour infection with SL1344 (open circle) or SL1344ΔinvA (closed circle) Salmonella (MOI, 10). (C and D) MAIT cells as a percentage of αβ T cells (C) and bacterial load (CFU) (D) from the lungs on day 6 p.i. of C57BL/6 mice intranasally infected with 105 S. Typhimurium SL1344 or SL1344ΔinvA. Pooled data show means ± SEM of seven to nine mice per group. ***P < 0.001, ****P < 0.0001, unpaired t test. The experiments were carried out independently two times with similar results.

  • Fig. 3 ICOS is highly expressed on MAIT cells and is important for driving their response to infection.

    (A) Representative FACS histograms showing expression of CD154, CD28, CD137, CD27, and ICOS on MAIT cells, non-MAIT T cells, and B cells from the lungs of uninfected C57BL/6 WT mice. (B) MAIT cell frequency in the lungs at day 7 p.i. of Cd80−/−Cd86−/−, Icos−/−, and C57BL/6 WT mice, intranasally infected with 106 S. Typhimurium BRD509 or 104 L. longbeachae. Data show individual mice and means ± SEM. *P < 0.05, ****P < 0.0001, one-way ANOVA with Dunnett’s multiple comparisons. (C) Representative FACS plots and (D) stacked plots showing proportion of MAIT cells expressing T-bet and RORγt from naïve, S. Typhimurium BRD509–infected, or L. longbeachae–infected Cd80−/−Cd86−/−, Icos−/−, and C57BL/6 WT mice (7 days p.i.), means ± SEM of five (naïve) or eight to nine (infected) mice, pooled from two experiments. See also fig. S2.

  • Fig. 4 IL-23 is required for MAIT cell accumulation and activation during pulmonary infection with S. Typhimurium.

    (A) Percentage and (B) absolute numbers of MAIT cells isolated from the lungs of gene KO mice lacking indicated cytokines after intranasal infection with 106 S. Typhimurium BRD509 (day 7 p.i.). Data show individual mice and means ± SEM of 3 to 15 mice per group. ****P < 0.0001 (all uninfected mice non-significant), one-way ANOVA with Dunnett’s multiple comparisons for infected mice. (C) Representative flow cytometry plots and stacked plots showing intracellular staining of T-bet and RORγt in MAIT cells from S. Typhimurium–infected Il-12p35−/−, Il-12p40−/−, Il-23p19−/−, and WT (C57BL/6) mice (day 7 p.i.). (D) Flow cytometry plots and (E) percentages of pulmonary TCRβ+ lymphocytes (non-MAIT T cells and MAIT cells) producing IL-17A by intracellular staining, directly ex vivo from the lungs of WT (C57BL/6), Il-23p19−/−, and Il-6−/− mice infected with 106 S. Typhimurium BRD509 (day 7 p.i.). Data show means ± SEM and individual mice for eight to nine mice per group. **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA with Dunnett’s multiple comparisons. See also fig. S3.

  • Fig. 5 Infused IL-23–Ig plasmid restores MAIT cell accumulation in Il-23p19−/− mice after infection.

    (A) Absolute numbers and (B) MAIT cell percentage of T cells isolated from the lungs of WT and Il-23p19−/− mice intranasally infected with 106 S. Typhimurium BRD509 (day 7 p.i.). Il-23p19−/− mice were treated with 10 μg of plasmid encoding recombinant IL-23–Ig or control Ig by hydrodynamic injection 1 day before infection. Data show means ± SEM and individual mice. ***P < 0.001, unpaired two-tailed Student’s t test. (C) Expression of T-bet and RORγt in MAIT cells from lungs of mice described above. Plots show gated MAIT cells from one representative mouse per group. (D) Relative ICOS expression (compared with WT C57BL/6 mice) on pulmonary MAIT cells from untreated (nil) WT, Il-23p19−/− or Icos−/− mice, or WT mice treated with 10 μg of IL-23–Ig or control-Ig plasmid for 24 hours. Data show means ± SEM and individual mice. ***P < 0.001, ****P < 0.0001, one-way ANOVA with Dunnett’s multiple comparisons test. See also fig. S4.

  • Fig. 6 MAIT cells express IL-23R and respond directly to IL-23.

    (A) GFP expression (indicating IL-23R expression) in TCRβ+ cells isolated from the lungs of IL-23rgfp/+ uninfected mice or after intranasal infection with 106 S. Typhimurium BRD509 (day 7 or day 100 p.i.). (B) Schematic of MAIT cell transfer and tracking using CD45 congenic markers. Plots show MAIT cells (CD45+TCRβ+MR1–5-OP-RU–Tetramer+) isolated from the lungs of recipient mice color-coded to match the donor/recipient cells. Fold increase of MAIT cell number after infection is shown. (C) Total MAIT cells (CD45.2+ endogenous and CD45.1+ adoptively transferred) isolated from the lungs of Il-23p19−/− or Il-23r−/− mice that received 105 MAIT cells (sorted from CD45.1 congenically labeled mice primed for 7 days with S. Typhimurium BRD509) and, 2 weeks later, were intranasally infected with 106 S. Typhimurium BRD509. Data show means ± SEM and individual mice fold change of normalized data at day 7 p.i. **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, P > 0.05, one-way ANOVA with Tukey’s multiple comparisons test. See also fig. S5.

  • Fig. 7 IL-23 plus synthetic 5-OP-RU antigen is sufficient to induce MAIT cell activation and accumulation in vivo and increase protection against infectious challenge.

    (A) Absolute numbers of MAIT cells and (B) absolute number of non-MAIT TCRβ+ cells isolated from the lungs on day 8 of naïve WT (C57BL/6) mice or WT mice treated with 10 μg of plasmid encoding recombinant IL-23–Ig or control plasmid by hydrodynamic injection (day 0) and inoculated intranasally with one to four doses (as indicated) of 5-OP-RU on days 0, 1, 2, and 4. Data show means ± SEM and individual mice. (C) Expression of T-bet and RORγt in MAIT cells from the lungs of mice described in (A). Plots show data from one representative mouse from each group. (D) Experimental scheme for vaccination experiments shown in (E). d.p.i., days post infection. (E) Bacterial CFU counts of Legionella from the lungs at indicated time points after intranasal infection with 104 CFU of mice previously untreated (open diamonds) or primed with IL-23–Ig (hydrodynamic injection) alone (open squares) or IL-23–Ig or control-Ig with 5-OP-RU (four doses intranasally) (closed and open circles, respectively). Data show means ± SEM and individual mice. **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA with Dunnett’s multiple comparisons test. See also fig. S4.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/41/eaaw0402/DC1

    Fig. S1. Validation of BM chimeras for assessment of MR1-dependent MAIT cell activation.

    Fig. S2. MAIT cell expansion after bacterial infection is impaired in Icos−/− mice.

    Fig. S3. IL-23 is required for optimal MAIT cell accumulation and activation during pulmonary infection with L. longbeachae.

    Fig. S4. IL-23 is expressed in the lungs from hydrodynamically delivered plasmid DNA and expands MAIT cells in the lungs in combination with 5-OP-RU.

    Fig. S5. MAIT cells respond directly to IL-23.

    Fig. S6. IL-23 drives IL-17 production by human MAIT cells in response to 5-OP-RU.

    Table S1. Raw data in Excel spreadsheet.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Validation of BM chimeras for assessment of MR1-dependent MAIT cell activation.
    • Fig. S2. MAIT cell expansion after bacterial infection is impaired in Icos−/− mice.
    • Fig. S3. IL-23 is required for optimal MAIT cell accumulation and activation during pulmonary infection with L. longbeachae.
    • Fig. S4. IL-23 is expressed in the lungs from hydrodynamically delivered plasmid DNA and expands MAIT cells in the lungs in combination with 5-OP-RU.
    • Fig. S5. MAIT cells respond directly to IL-23.
    • Fig. S6. IL-23 drives IL-17 production by human MAIT cells in response to 5-OP-RU.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Raw data in Excel spreadsheet.

    Files in this Data Supplement:

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